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PRODUCTION OF NOVEL LIPASE INHIBITOR

FROM STREPTOMYCES SP

A thesis submitted to the University of Mysore for the award of the degree of

DOCTOR OF PHILOSOPHY

IN

BIOTECHNOLOGY

By

NAVEEN BABU KILARU

Department of Fermentation Technology and Bioengineering

Central Food Technological Research Institute Mysore - 570 020, India

July 2005

2

Dr. A. P. Sattur, Date:

Scientist,

Fermentation Technology and

Bioengineering Department,

CERTIFICATE I hereby certify that the thesis entitled “ PRODUCTION OF NOVEL LIPASE

INHIBITOR FROM STREPTOMYCES SP” submitted to the University of Mysore for the

award of the degree of DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY by Mr.

NAVEEN BABU KILARU, is the result of the research work carried out by him in the

Department of Fermentation Technology and Bioengineering, Central Food Technological

Research Institute, Mysore, India, under my guidance during the period 2001-2005.

(A. P. Sattur)

3

NAVEEN BABU KILARU, Date: Senior Research Fellow, Fermentation Technology and Bioengineering Department, Central Food Technological Research Institute, Mysore – 570 020.

DECLARATION

I hereby declare that the thesis entitled “PRODUCTION OF NOVEL LIPASE INHIBITOR

FROM STREPTOMYCES SP” submitted to the University of Mysore for the award of the

degree of DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY is the result of the

research work carried out by me in the Department of Fermentation Technology and

Bioengineering, Central Food Technological Research Institute, Mysore, India, under the

guidance of Dr. Avinash P Sattur during the period 2001-2005.

I further declare that the work embodied in this thesis had not been submitted for the award of

degree, diploma or any other similar title.

(NAVEEN BABU KILARU)

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CONTENTS

Page No.

List of Tables VIII

List of Figures XII

CHAPTER 1. INTRODUCTION 1.1. Actinomycetes 1

1.2. Lipases 2

1.2.1. Reaction mechanism 3

1.2.2. Specificity of lipases 5

1.2.3. Pancreatic lipase 6

1.3. Obesity a medical complication caused by lipase 7

1.3.1.a. Reduction of food intake 8

1.3.1.a.1. Noradrenergic receptors 8

1.3.1.a.2. Serotonergic receptors 8

1.3.1. b. Increased energy expenditure 9

1.3.1.c. Altered metabolism 9

1.4. Sources of lipase inhibitors 10

1.4. a. Lipase inhibitors from microbial origin 10

1.4.b. Lipase inhibitors from plant sources 12

1.5. Scope of the present investigation 15

CHAPTER 2. MATERIALS AND METHODS

2.1. Screening of actinomycetes 17

2.1.A. Collection of different terrestrial substrates for selective

isolation of actinomycetes 17

2.1.B. Pretreatment of soil samples 17

2.1.B.1. Calcium carbonate treatment 17

2.1.B.2. Dry heat treatment 17

2.1.B.3. Phenol treatment 18

2.1.B.4. Centrifugation method 18

2.1.B.5. Control (no pretreatment) 18

5

2.2. Media composition 18

2.2.A. Actinomycetes 18

2.2.A.1. Medium for screening of actinomycetes for pancreatic

lipase inhibitor 19

2.2.A.2. Culture maintenance medium 20

2.2.A.3. Morphology and pigmentation 20

2.2.A.4. Cultural Characteristics media 21

2.2.A.4.1. Enzyme activity test media 27

2.2.A.4.2. Degradation tests media 29

2.2.A.4.3.Test for resistance to antibiotics 33

2.2.A.4.4.Effect of temperature on growth of isolate N2 33

2.2.A.4.5. Effect of pH on growth of isolate N2 34

2.2.A.4.6.Growth in the presence of inhibitory compounds 34

2.2.A.4.7.Test for carbon utilization medium 34

2.2.A.4.8.Test for nitrogen utilization medium 35

2.2.A.4.9.Test for production of acid and gas 36

2.2.A.4.10. Media for cultivation of cells for determination of cell

wall composition 36

2.2.A.5. Primary screening medium 37

2.2.A.6.Secondary screening media 37

2.2.A.7. Selection of inoculum media for production of streptolipin 38

2.2.A.8. Standard media for screening streptolipin production 39

2.2.B. Fungi 43

2.2.B.1. Culture maintenance medium for fungi 43

2.2.B.2. Screening of fungal cultures for the production of lipase

inhibitor 43

2.2.B.3. Medium for submerged fermentation (SmF) of fungi 44

2.2.B.4. Medium for solid state fermentation (SSF) of fungi 44

2.3. General fermentation conditions

2.3.1. Inoculum development for screening 44

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2.3.2. Submerged fermentation for actinomycetes cultures 45

2.3.3. Solid state fermentation for fungal cultures (SSF) 45

2.3.4. Submerged fermentation for fungal cultures (SmF) 45

2.4. General extraction conditions

2.4. 1. Extraction of inhibitor from SmF broth 46

2.4.2. Extraction of inhibitor from SSF bran 46

2.5. Analytical methods

2.5.1. Lipase assays 46

2.5.2. Tests for cell wall composition of isolate N2 48

2.5.2.1. Amino acid analysis 48

2.5.2.2. Sugar analysis 49

2.5.2.3. Analysis of menaquinones 49

2.5.2.4. Analysis of fatty acids 50

2.5.2.5. Analysis of phospholipids 50

2.5.2.6. Analysis of mycolic acids 51

2.5.2.7. Phylogenetic analysis of isolate N2 51

2.5.3.1. Column chromatography 52

2.5.3.2. Thin layer chromatography (TLC) 52

2.5.3.3. High pressure liquid chromatography (HPLC) method 53

2.5.3.4. Melting point determination 53

2.5.3.5. Elemental analysis 53

2.5.3.6. Infrared spectroscopy 54

2.5.3.7. Nuclear magnetic resonance spectroscopy (NMR) 54

2.5.3.8. Liquid chromatography mass spectroscopy (LC-MS) 54

2.5.3.9. Detection of elements by chemical method 55

2.6. Microorganisms used for comparative studies 56

2.7. In vivo efficacy of streptolipin on experimental animal models 56

2.7.1. Effect of single and multiple doses of streptolipin on fat

absorption 56

2.7.2. Faecal triglycerides 57

2.7.3. Lipid peroxides 58

2.7.4. Antioxidant enzymes 58

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2.7.4.1. Catalase assay 58

2.7.4.2. Estimation of Superoxide dimutase 59

2.7.4.3. Estimation of Glutathione peroxidase 59

2.7.5. Plasma non specific enzymes 59

2.7.6. Lipid analysis 60

CHAPTER. 3. RESULTS AND DISCUSSION

CHAPTER. 3, SECTION A. Screening and selection of actinomycetes for the

production isolation of pancreatic lipase inhibitor

3.A.1.Screening of different terrestrial substrates for selective isolation

of actinomycetes 61

3.A.2.Techniques for the isolation of actinomycetes 61

3.A.3. Screening of microorganisms for pancreatic lipase inhibitor

production 70

CHAPTER 3, SECTION B. Culture characterization and identification of isolate N2 as

Streptomyces vayuensis

3.B.1. Morphological characters of isolate N2 87

3.B.2. Cultural characteristics of isolate N2 on different media 87

3.B.3. Antimicrobial activity of isolate N2 92

3.B.4. Enzyme activity tests for isolate N2 93

3.B.5. Degradation tests of isolate N2 94

3.B.6. Antibiotics resistance of isolate N2 94

3.B.7. Effect of temperature and pH on growth of isolate N2 94

3.B.8. Growth of isolate N2 in the presence of inhibitory compounds 94

3.B.9. Test for carbon source utilization by isolate N2 95

3.B.10. Test for nitrogen utilization by isolate N2 101

3.B.11. Test for production of acid and gas by isolate N2 101

3.B.12. Chemotaxonomic characteristics of isolate N2 101

3.B.12.1. Test for sugars and amino acids 101

3.B.12.2. Test for menaquinones 102

3.B.12.3. Test for mycolic acid 102

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3.B.12.4.Test for fatty acids 102

3.B.12.5. Test for phospholipids 102

3.B.13. Phylogenetic analysis of isolate N2 105

3.B.14. Comparative studies of isolate N2 112

3.B.15. Final and brief description of isolate N2 as Streptomyces

vayuensis sp.nov 112

CHAPTER. 3, SECTION C. Isolation, purification and structure elucidation of Streptolipin, a

pancreatic lipase inhibitor from Streptomyces vayuensis

3.C.1.Isolation and purification of pancreatic lipase inhibitor 117

3.C.2. Adaptation of the PNPB spectrophotometric assay to

TLC system 117

3.C.3. Physico-chemical properties of streptolipin 119

3.C.4.Structure elucidation of the inhibitor 125

3.C.5. Kinetic studies on inhibition of streptolipin against pancreatic

lipase 127

3.C.5.1. Lineweaver-Bulk (LB) plot of pancreatic lipase inhibition

by streptolipin 127

3.C.5.2. Determination of irreversibility of the pancreatic lipase

enzyme inhibition 131

3.C.6. Other biological activities of streptolipin 131

CHAPTER. 3, SECTION D. Studies on media optimization for streptolipin production

3.D.1. Standard HPLC curve for streptolipin 136

3.D.2.Time course fermentation for streptolipin production 136

3.D.3. Optimization of physical parameters for production of streptolipin

3.D.3.1. Selection of inoculum media for production of streptolipin 137

3.D.3.2. Effect of temperature on production of streptolipin 139

3.D.3.3. Effect of initial pH of the medium on production of streptolipin 139

3.D.3.4. Effect of aeration on production of streptolipin 142

3.D.4. Optimization of nutritional parameters for the production

of streptolipin

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3.D.4.1. Screening of standard media for the production of streptolipin 142

3.D.4.2. Effect of different carbon sources on production of streptolipin 144

3.D.4.3. Effect of inorganic nitrogen sources on production of streptolipin 149

3.D.4.4. Effect of organic nitrogen sources on production of streptolipin 149

3.D.4.5.Effect of trace elements on production of streptolipin 151

3.D.4.6. Effect of lipids on production of streptolipin 154

3.D.4.7. Effect of molasses on production of streptolipin 156

3.D.4.8. Formulation of medium for the production of streptolipin 156

3.D.4.9. Effect of different glucose concentrations on the kinetics 157

of streptolipin production

3.D.4.10. Effect of different concentrations of yeast extract on

of streptolipin production 169

3.D.4.11. Production of streptolipin with optimum conditions

on shake flask 171

3.D.4.12. 10 L Fermentor study 176

3.D.5. Optimization of down stream processing conditions for

streptolipin extraction

3.D.5.1. Choice of the extraction solvent 179

3.D.5.2. Biomass to solvent ratio 180

3.D.5.3. Effect of static and agitated conditions during extraction 180

3.D.5.4. Repeated extraction 181

3.D.5.5. Sequential extraction 181

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CHAPTER. 3, SECTION E. In vivo efficacy of streptolipin on experimental animal

models

3.E.1. Influence on lipase activity and dietary triglyceride absorption 185

3.E.2. Influence on blood and liver lipid profile 192

3.E.3. Influence on antioxidant status 199

CHAPTER .4. Summary and highlights of the investigation 203

Recommendation for further work 206

References 207

List of publications 225

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LIST OF TABLES

Table No. Title Page No.

Table 3.1: Different soil samples for screening of actinomycetes 62

Table 3.2: Different pretreatments of various soil samples 64

Table 3.3: Isolation of actinomycetes on different media 65

Table 3.4: Distribution of actinomycete isolates in various soil samples 68

Table 3.5: Distribution of actinomycete isolates from various samples 69

Table 3.6: Primary screening of fungal cultures from CFTRI Culture

Collection Center for pancreatic lipase inhibition 71

Table 3.7: Primary screening of actinomycetes isolates for pancreatic

lipase inhibition 75

Table 3.8: Classification of actinomycete isolates based on inhibition

(%) of pancreatic lipase 82

Table 3.9: Distribution of actinomycetes isolates showing inhibition

greater than 20% on soil sample type 83

Table 3.10: Distribution of actinomycetes isolates showing inhibition

greater than 20% on soil pretreatment basis 83

Table 3.11: Distribution of active actinomycetes isolates on the basis

of colour 84

Table 3.12: Secondary screening of actinomycetes cultures for

reproducibility and consistent production of lipase inhibitor 86

Table 3.13: Morphological characters of isolate N2 89

Table 3.14: Cultural characteristics of isolate N2 on different media 90

Table 3.15: Antimicrobial activity of isolate N2 92

Table 3.16: Enzyme activities of isolate N2 93

Table 3.17: Degradation of various compounds by isolate N2 96

Table 3.18: Resistance of isolate N2 to antibiotics 97

Table 3.19: Growth of isolate N2 at different temperature 98

Table 3.20: Growth of isolate N2 at different pH 98

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Table 3.21: Growth of isolate N2 in the presence of inhibitory compounds 99

Table 3.22: Growth of isolate N2 on different carbon sources 100

Table 3.23: Growth of isolate N2 on nitrogen source 103

Table 3.24: Production of acid and gas by isolate N2 104

Table 3.25: rRNA gene sequence similarity of Streptomyces vayuensis

sp. nov. (strain N2) with species of Streptomyces exhibiting

95% similarity at the rRNA gene level as determined by

BLAST 110

Table 3.26: Phenotypic and chemotaxonomic characteristics that

differentiate Streptomyces vayuensis sp. nov. (strain N2)

from the closely related species of the genus Streptomyces 114

Table 3.27: Differences in the phenotypic and chemotaxonomic

features of isolate N2 and S. violaceusniger, the

phylogenetically nearest neighbour 116

Table 3.28.A: 1H and 13C NMR chemical shifts of compound 128

Table 3.28.B: Mass spectrometry; fragmentation of inhibitor in EI-MS (m/z) 129

Table 3.29: Physico-chemical properties of the inhibitor 130

Table 3.30: Selection of inoculum media for production of streptolipin 139

Table 3.31: A list of standard media for the production of antibiotics 145

Table 3.32: Screening of standard media for the production of

streptolipin 146

Table 3.33: Effect of different carbon sources on production of

streptolipin 147

Table 3.34: Effect of inorganic nitrogen sources on production

of streptolipin 150

Table 3.35: Effect of organic nitrogen sources on production of

streptolipin 150

Table 3.36: Effect of different amino acids on production of streptolipin 150

Table 3.37: Effect of trace elements on production of streptolipin 151

Table 3.38: Effect of lipids on production of streptolipin 154

Table 3.39: Production of streptolipin on different nitrogen sources in

presence of 15 g/L Galactose 156

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Table 3.40: Production of streptolipin on different nitrogen sources in

presence of 15 g/L sodium pyruvate 157

Table 3.41: Effect of initial glucose concentration on biomass synthesis 158

Table 3.42: Effect of initial glucose concentration on streptolipin synthesis 166

Table 3.43: Effect of initial glucose concentration on glucose utilization pattern 166

Table 3.44: Effect of initial yeast concentration on biomass formation 170

Table 3.45: Effect of initial yeast extract concentration on streptolipin synthesis 170

Table 3.46: Comparison of medium studies before and after

optimization in shake flask 174

Table 3.47: Comparison between shake flask and fermentor studies

after optimization of media 176

Table 3.48: Choice of the extraction solvent 180

Table 3.49: Effect of streptolipin on faecal excretion of triglycerides

(Single dose study) 187

Table 3.50: Effect of streptolipin on faecal excretion of triglycerides

(Multiple dose study) 188

Table 3.51: Effect of streptolipin on serum lipid profile


Table 3.52: Effect of streptolipin on serum lipid profile


Table 3.53: Effect of streptolipin on liver lipid profile


Table 3.54: Effect of streptolipin on liver lipid profile


Table 3.55: Effect of streptolipin on liver and perirenal fat weight


Table 3.56: Effect of streptolipin on liver and perirenal fat weight


Table 3.57: Effect of streptolipin on activities of hepatic antioxidant

enzymes (Multiple dose study) 201

Table 3.58: Effect of streptolipin on the activities of plasma non-specific enzymes (Multiple dose study). 202

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LIST OF FIGURES

Figure No. Title Page No.

Figure 1.1: Lipase-catalyzed hydrolysis of p-nitrophenyl acetate. 4

Figure 1.2: Proposed reaction mechanism showing acylation and

deacylation via the formation of an acylenzyme intermediate. 5

Figure 2.1: Sample preparation for scanning electron microscope 22

Figure 3.1: Screening of actinomycetes on starch casein agar 66

Figure 3.2: Isolated culture slants of actinomycetes 67

Figure 3.3: Isolation and screening of actinomycetes isolates 73

Figure 3.4: Scanning electron micrograph showing N2 warty spores

and spiral spore chains 88

Figure 3.5: Culture growth on glycerol aspargine agar 91

Figure 3.6: Mass spectra of menaquinones isolated from N2 106

Figure 3.7: Cellular fatty acids of the isolate N2 107

Figure 3.8: Neighbour-joining tree based on 16S rRNA gene sequences

showing the phylogenetic relationship between Streptomyces

vayuensis sp. nov. and other species of the genus Streptomyces

and related reference microorganisms. Bootstrap values

(expressed as percentages of 1000 replications) greater than

50% are given at the nodes 111

Figure 3.9: Purification protocol of lipase inhibitor 118

Figure 3.10: Adaptation of the PNPB (p-nitrophenylbutyrate) spectrophotometric

assay to TLC system 120

Figure 3.11: IR-spectrum of inhibitor, isolated from Streptomyces vayuensis 121

Figure 3.12: 1H NMR spectrum of inhibitor from Streptomyces vayuensis 122

Figure 3.13: 13C NMR spectrum of inhibitor from Streptomyces vayuensis 123

Figure 3.14: LCMS of inhibitor from Streptomyces vayuensis 124

Figure 3.15: Chemical structure of streptolipin 127

Figure 3.16: Concentration dependent inhibition of streptolipin on

pancreatic lipase 132

Figure 3.17: [V] versus [S] plot in the presence of different fixed

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concentrations of streptolipin 133

Figure 3.18: L-B plot of 1/[V] vs1/[S] in the presence of different fixed

concentrations of streptolipin 134

Figure 3.19: The slope (km/Vmax) of the lines described from the double

reciprocal plot are plotted against the streptolipin concentration

in order to derive the Ki value for the inhibitor 135

Figure 3.20: Standard curve for streptolipin 137

Figure 3.21: Time course fermentation of streptolipin production 138

Figure 3.22: Effect of temperature on production of streptolipin 140

Figure 3.23: Effect of initial pH on production of streptolipin 141

Figure 3.24: Effect of aeration on production of streptolipin 143

Figure 3.25: Effect of dipotassium hydrogen sulphate on production of

streptolipin 152

Figure 3.26: Effect of zinc sulphate on production of streptolipin 153

Figure 3.27: Effect of molasses on production of streptolipin 155

Figure 3.28: Variation of total biomass with initial glucose concentration 160

Figure 3.29: Semilogarithmic plot of biomass with time 161

Figure 3.30: Monod's plot of initial specific growth rates vs substrate

concentration 162

Figure 3.31: Double reciprocal plot of specific growth rate against the initial

glucose concentration 163

Figure 3.32: Variation of total streptolipin with initial glucose concentration 164

Figure 3.33: Rates of streptolipin formation at different initial glucose

concentration 165

Figure 3.34: Time course of initial glucose consumption during fermentation 167

Figure 3.35: Rates of glucose utilization at different initial glucose concentration 168

Figure 3.36: Influence of initial yeast extract concentration on cell growth 172

Figure 3.37: Influence of initial yeast extract concentration on streptolipin

production 173

Figure 3.38: Production of streptolipin with optimum conditions on shake flask 175

Figure 3.39A: Production of streptolipin in 10L laboratory fermentor 177

Figure 3.39.B: 10 L Fermentor study 178

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Figure 3.40: Effect of biomass to ethyl acetate ratio on extraction efficiency of

streptolipin 182

Figure 3.41: Effect of the extraction time of streptolipin during static and

agitated conditions 183

Figure 3.42: Effect of sequential extraction on streptolipin recovery from

the biomass 184

Figure 3.43: Effect of streptolipin on absorption of dietary triglyceride

(Single dose) 189

Figure 3.44: Effect of streptolipin on absorption of dietary Triglyceride

(Multiple dose) 190

Figure 3.45: Effect of streptolipin on activity of intestinal lipase 191

Figure 3.46: Effect of streptolipin in serum lipid peroxides in serum

(circulation) and liver (Multiple dose study) 200

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1.1. Actinomycetes:

Microorganisms are among man’s best friends and worst enemies. Exploitation of

microorganisms for useful purpose is known since 6000 B.C. History reveals many

applications of microbial processes for the production of desirable materials. The harnessing of

the activities of microorganisms represent one of the most fascinating aspects of man’s

scientific and technological development. From the stand point of industrial microbiology,

microorganisms can be considered as chemical factories in miniature. They have the potential

to produce novel and new therapeutic agents and to convert relatively inexpensive raw

materials into end products of value for human use, thus becoming attractive for commercial

exploitation.

Actinomycetes are that group of intracellular branching organisms which reproduce

either by fission or by means of spores or conidia. From an ecological point of view,

actinomycetes stand in an intermediate position between the fungi and the bacteria in terms of

numerical frequency of occurrence in various biotypes. They are closely related to the true

bacteria and frequently they are considered as higher filamentous bacteria. The outstanding

fungal characteristic of actinomycetes is morphological- possession of a true branching

mycelium. In addition actinomycetes may also show strong parallels with the true fungi in their

production of sporangia and motile spores. However, mycelium diameter and spore size is of a

lower order of magnitude in actinomycetes averaging 1µm only as compared with fungi. They

usually form a mycelium, which may be of a single kind designated as substrate (vegetative),

or of two kinds, substrate (vegetative) and aerial mycelium. They produce a wide variety of

spore types which includes the endospore, long regarded as the typical spore structure of

eubacterials. Some genera such as Streptomyces and Micromonospora form an extensive

branched mycelium composed of individual hyphae, subdivided by frequent cross walls. The

actinomycetes are predominantly aerobic, heterotropic and saprophytic. They have a cell wall

structure characteristic of bacteria and frequently show the presence of lytic viruses

(actinophages).

It is estimated that 85% of the antibiotics from microbial sources are being produced by

actinomycetes. Since the discovery of streptomycin by Waksman in 1943, interest of many

18

microbiologists was switched over to explore naturally occurring substrates for isolation of

novel actinomycete strains and novel metabolites with resounding commercial success. This is

the main reason for prominence of conventional screening programmes even in this age.

Many biological molecules are known to inhibit specific enzymes. As several diseases

are associated with abnormal enzyme activities, this concept has yielded valuable

pharmaceutical compounds. They can be called also as “target-enzyme” inhibitors and display

distinct pharmaceutical action depending on the target enzyme. The screening of microbial

culture filtrates for low molecular mass enzyme inhibitors was initiated by Hamao Umezawa in

1966. The first inhibitor, leupeptin, was discovered in 1969 as a metabolite of Streptomyces

strain. Since then many inhibitors have been discovered in culture filtrates of actinomycetes,

bacteria and fungi (Aoyagi and Takeuchi (1989) and some have been introduced commercially

(Umezawa, 1982).

1.2. Lipases:

Lipases (EC 3.1.1.3, triacylglycerol hydrolases) are ubiquitous enzymes playing a

pivotal role in all aspects of fat and lipid metabolism in variety of organisms. In humans and

other vertebrates, a variety of lipases control the digestion, absorption and reconstitution of fat

as well as lipoprotein metabolism (Desnuelle, 1986). In plants, during post germination, the

metabolism of oil reserves provide energy and carbon skeleton for embryonic growth and is

controlled by the action of lipases (Huang, 1987). Microorganisms such as bacteria and fungi

are also known to produce a wide spectrum of extracellular lipid degrading enzymes to

breakdown the insoluble lipid into soluble polar components to facilitate absorption (Lie et al,

1991).

Lipases belong to the hydrolase group of enzymes, which catalyze the hydrolysis of

glyceride ester bonds. They are also termed as acylglycerolases, acyl hydrolases or

triacylglycerol hydrolases. Their substrate specificities are wide and compounds other than

acylglycerols are also hydrolyzed. These enzymes can also be considered as transferases since

the fatty acids released is transformed to water or some other compound having a free hydroxyl

group related moiety (nucleophile).

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One major characteristic of lipases is that they are more active with insoluble ester

substrates as against esterases that act only on soluble ester substrates. Lipases also hydrolyze

water soluble substrates which is characteristic of esterases. Thus lipases can be considered as

a special class of esterases, that is, esterases with high activity towards water insoluble

substances (Sarda and Desnuelle, 1958; Entressangles and Desnuelle, 1968). Until recently,

little was known about the molecular basis of lipid hydrolysis. A number of hypotheses have

been put forward with respect to the mechanism of interfacial activation of lipases. These

involve-(a) increased substrate availability by displacement of water shell around the ester

molecules (Brockerhof, 1968): (b) increase in substrate concentration at the interface

(Brockman et al, 1973): (c) better orientation of the ester bond to be cleaved (Wells, 1974;

Sarda and Desnuelle, 1958): (d) conformational change in the enzyme could be responsible for

the enhancement of activity at the oil-water interface.

1.2.1. Reaction mechanism:

Lipases and esterases contain an active Ser in a consensus sequence G-X-S-X-G which is

reminiscent of the pentapeptides in serine proteases (Gly-Asp-Ser-Gly-Gly in the trypsin

family, and Gly-X-Ser-X-Ala in subtilisin) (Brenner, 1988). Most lipases are susceptible to

inactivation with classic serine potease inhibitors, such as diisopropylphosphofluoridate and

diethyl-p-nitrophenyl phosphate indicating that lipases belong to the mechanistic class of serine

proteases. Recent structural analyses have been shown conclusively that lipases contain the

same constellation of the catalytic triad, Ser … His… Asp, present in all serine proteases,

although the topological position of the individual residues side chain varies (Winkler et al,

1990; Brady et al, 1990). It has also been demonstrated that the hydrolysis of dissolved p-

nitrophenyl acetate by pancreatic lipase proceeds via an acylenzyme intermediate (Figure 1.1).

20

O2N OCOCH3k-1

k1 O2N OCOCH3E------E +

[Enzyme-Substrate] Complex

O2N O-Acylation k2

O

CH3E

Acyl-enzyme

k3H2O

Deacylation

E+CH3COOH

Figure 1.1: Lipase-catalyzed hydrolysis of p-nitrophenyl acetate

The following mechanism is suggested to occur based on the catalytic activity pathway of

serine proteases (Figure 1.2).

1. The enzyme first binds the substrate to form a Michaelis-Menten adsorption complex.

2. Nucleophilic attack by the essential Ser-OH on the acyl carbon of the substrate yields a

covalent tetrahedral intermediate. This step is facilitated by general base catalysis by

the His in the triad.

3. A colipase of the intermediate to an acylenzyme involves the His-catalyzed protonation

of the ester oxygen of the leaving group.

4. In the following step, deacylation of the acylenzyme occurs with H2O assisted by

general base catalysis involving the His, resulting in the formation of tetrahedral

intermediate.

5. Finally protonation of the Ser causes the break down of the intermediate,

resulting in liberation of the product as a carboxylic acid.

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coo-

Asp

......... NH N+

His

.......H-O:

Ser

coo-

Asp

........ NH N+

His

.....H.....R1

O

O

R2

R1O

:O-

R2

O

Ser R1OH

coo-

Asp

........ NH N+

His

R2

O

O

Ser

ACYLATION

R2

O

O

Ser

coo-

Asp

......... NH N+

His

OH

H

:

DEACYLATION....

NH NH+

His

.....H.....

HO

:O-

R2

O

Ser R2 COOH

.... coo-

Asp

NH N

His

......coo-

Asp

............OH

Ser

. . .. ..

..... .

ACYL ENZYME

Figure 1.2: Proposed reaction mechanism showing acylation and deacylation via the formation of an acylenzyme intermediate

1.2.2. Specificity of lipases:

The substrate specificity of lipase is defined by its positional specificity or

stereospecificity or its preference for short or long chain saturated or unsaturated fatty acids

(Brockerhoff and Jensen, 1975). Some lipases have affinity for short chain fatty acids (acetic,

butyric, capric, caproic, caprylic etc), some for unsaturated fatty acids (oleic, linoleic, linolenic

etc), while many others are non specific and randomly split the fatty acids from the

triglycerides. From the glycerol side of the triglycerides, the lipases often show positional

specificity and attack the fatty acids at 1 or 3 carbon position of glycerol or at both the

positions, but not the fatty acid at the 2 position of the glycerol molecule. However, through

random acyl migration, the 2 fatty acid monoglyceride undergoes rearrangement pushing the

fatty acid to the 1 or 3 position of the glycerol molecule: as acyl migration is a slow process

and as the available lipases do not act on glycerol 2 mono fatty acid esters, the hydrolysis

22

slows down and awaits the acyl migration to complete for enabling the lipase to attack the

glyceride at 1 or the 3 position (Saxena et al, 1999).

1.2.3. Pancreatic lipase:

Pancreatic lipase (PL) hydrolyses the water insoluble triacylglycerols in the intestinal

lumen and thereby plays an important role in the dietary fat absorption. Pancreatic lipase has a

molecular weight of approximately 50 kDa and has been isolated from a number of

mammalian species. Triglyceride hydrolysis by PL is inhibited by physiological concentrations

of bile salts. This inhibition can be overcome by the addition of co-lipase, a small pancreatic

protein that binds to the lipase and to lipid micelles. The crystal structure of human pancreatic

lipase has been refined to a resolution of 2.3 A0 (Winkler et al, 1990; Gubernator et al, 1991).

Its amino-acid sequence, comprises 449 residues for the mature enzyme. The protein is folded

into two domains, a larger N-terminal domain (N domain) comprising residues 1-335 and a

smaller C-terminal domain (C domain). The core of the N domain is formed by nine-stranded

β-pleated sheet in which most of the strands run parallel to one another. Seven α-helical

segments of varying length occur in the strand connections and six of them pack against the

two faces of the core sheet. The C domain is formed by two layers of antiparallel sheet, the

strands of which are connected by loops of varying length. The N domain contains the active

site, a glycosylation site a Ca2+- binding site and possibly a heparin-binding site. In the

crystalline form, the active site is buried beneath a surface loop, termed the flap. In this form

the enzyme cannot be enzymically active.

The enzyme consists of six disulfide bridges, Cys4-Cys10, Cys91-Cys102, Cys238-

Cys262, Cys286-Cys297, Cys300-Cys305 and Cys434-Cys450 with two free cysteines, Cys

104 and Cys 182. The porcine pancreatic enzyme is N-glycosylated at Asn167. The

glycosylation site Asn167-Gly168-Thr 169 in the porcine enzyme is conserved in the human

and canine lipases, and the glycan structure of porcine pancreatic lipase has been elucidated

(Caro et al, 1983; Benkouka et al, 1982, Hermoso et al, 1996; Bourne et al, 1994; Fournet at al,

1987). In contrast to the human pancreatic lipase, porcine and canine ovine and bovine

enzymes are not glycosylated. Unlike most of the pancreatic enzymes which are secreted as

proenzymes and further activated by proteolytic cleavage in the small intestine, pancreatic

23

lipase is directly secreted as a 50 kDa active enzyme. More information is available in reviews

on lipases (Rovery et al, 1978; Brady et al, 1990; Brazozowski et al, 1991; Uppenberg et al,

1994; Grochulski et al, 1994; Derewenda et al, 1994a; van Tilbeurgh et al, 1993; Bourne et al,

1994; Lawson et al, 1992).

1.3. Obesity a medical complication caused by lipase:

Excessive intake of dietary fat contributes to the development and maintenance of both

obesity and hyperlipidemia. It is generally accepted that obesity is not merely a cosmetic

problem, but that it also represents an unhealthy state, currently accepted as an illness. Obesity

is defined as an abnormal increase in body fat and is associated with a high basal metabolic

rate, low levels of physical activities, low rate of fat oxidation, increased insulin sensitivity,

low sympathetic nervous system activity and low plasma leptin concentration, heart disease

and stroke leading to an increased rate of mortality (Guzelhan et al, 1991; Drent and

Vanderveen, 1993; Guzelhan et al, 1994; Drent and Vanderveen, 1995; Drent et al, 1995).

Although obesity may be advantageous during starvation, excessive body fat is

associated with a long list of serious medical complications. The evidence surrounding this

concept reinforces the notion that effective obesity therapy must be directed towards limiting

the intake or absorption of dietary energy, whether through behavioral modification of eating

habits, effective pharmaceutical suppression of appetite or selective disruption of the normal

processes of digestion, whereas it is nearly axiomatic that behavioral modification is effective

only for those few whose time, motivation and financial resources permit intensive training in

eating and exercise habits and where as anorectic drugs pose unacceptable or potential risks,

the focus now turns to selective blockade of dietary fat absorption through inhibition of the

pancreatic enzyme lipase.

Conventional treatment for obesity has focused largely on strategies to control energy

intake. As obesity results from an imbalance between energy intake and energy expenditure,

methods to treat obesity can be divided into three groups; (a) reducing food intake (b) altering

metabolism and (c) increasing thermogenesis or increase energy expenditure.

24

1.3.1.a. Reduction of food intake:

1.3.1.a.1. Noradrenergic receptors:

A number of monoamines and neuropeptides are known to modulate food intake. Both

noradrenergic receptors and serotonergic receptors have been used as the site for clinically

useful compounds to decrease food intake (Bray, 1997; Bray et al, 1995; Bray and Inoue, 1992;

Bray, 1998; Frankish et al, 1995; Arch et al, 1981). Activation of the α1- and β2- adrenoceptors

decreases food intake. In animals however, stimulation of the α2-adrenoceptor increases food

intake. Direct agonists and drugs that release or block norepinephrine is released.

Phenylpropanolamine is an α1-agonist that decreases food intake by acting on α1-adrenergic

receptors in the paraventricular nucleus. The weight gain observed in patients treated for

hypertension or prostatic hypertrophy with α1 adrenergic antagonist indicates that the α1

adrenoceptor is clinically important in regulation of body weight. Stimulation of the β2-

adrenoxceptor by agonists such as terbutaline, clenbuterol or salbutamol reduces food intake.

1.3.1.a.2. Serotonergic receptors:

The serotonin 5-HT receptor system consists of seven families of receptors. Stimulation

of receptors in the hydroxytryptamine 5-HT1 and 5-HT2 families have the major effects on

feeding. Activation of the 5-HT1A receptor increases food intake but this acute effect is rapidly

down-regulated and is not clinically significant in regulation of body weight. Activation of 5-

HT2C and possibily 5-HT1B receptor increases food intake. Direct aganosits (quipazine) or

drugs that block serotinin reuptake (fluxetine, sibutramine, sertraline, fenfluramine) will reduce

food intake by acting on these receptors or by providing serotinin that modulates these

receptors (Toubro and Astrup, 1997; Davis and Faulds, 1996; Levitrsky and Troiano, 1992;

Mayer and Walsh, 1998).

1.3.1. b. Increased energy expenditure:

Increased energy expenditure through exercise would be an ideal approach to treating

obesity. Drugs that have same physiologic consequences as exercise could provide useful

25

pharmaceutical ways of treating obesity (Astrup et al, 1990; Dulloo and Miller, 1986; Vansal

and Feller, 1999; Colker et al, 1999).

1.3.1.c. Altered metabolism:

Excess fat is the visible sign of obesity. Metabolite strategies have been directed to pre

absorptive and post absorptive mechanisms of modifying fat absorption or metabolism (Zhi et

al, 1994). Pre absorptive mechanisms that influence digestion and absorption of macronutirents

have been studied which inhibit intestinal digestion of fat and lowers body weight. The second

strategy is to effect intermediary metabolism such as enhancing lipolysis, inhibiting

lipogenesis, and affecting fat distribution between subcutaneous and visceral sites.

Pancreatic lipase is a key enzyme for lipid breakdown that leads to the absorption of

fatty acids. Pancreatic lipase, one of the exocrine enzymes of pancreatic juice, catalyzes the

hydrolysis of emulsified esters of glycerol and long chain fatty acids. Short chain fatty acids

can be directly absorbed into the blood, while long-chain fatty acids and monoglycerides

combine with bile salts to form water soluble micelles. The micelles are absorbed into the

mucosal cells of the intestine and the fatty acids and monoglycerides are resynthesized into

triglycerides. Dietary triglyceride is usually stored in the adipose tissue. Pharmacological

agents that reduce the absorption of dietary triglycerides, reduce the probability of the

formation of atherosclerotic plaque.

26

Potent and specific lipase inhibitors are of special interest for four reasons:

1. They find applications as antiobesity agents.

2. They contribute to a better understanding of the mechanisms of lipase action.

3. They help to understand the non-catalytic functions of lipases.

4. They may find applications for the treatment of infectious diseases.

1.4. Sources of lipase inhibitors:

1.4.a. Lipase inhibitors of microbial origin:

1.4.a. 1. Lipstatin:

Lipstatin, a novel and very potent inhibitor of pancreatic lipase has been isolated from

Streptomyces toxytricini. Its hydrogenated analogue, tetrahydolipstatin (THL) ((s)-1-{[(1s, 2s,

3s)-3-hexyl-4-oxo-2-oxetanyl] methyl} dodecyl-(s)-1-fomamido-4-methylvalerate) has

selective inhibitory action for pancreatic lipase, where as phospholipase A2, amylase and

trypsin activity was not altered by THL. Lipstatin contains beta lactone structure carrying two

aliphatic residues with a chain length of 6 – 13 carbon, that probably accounts for the

irreversible lipase inhibition. The inhibition is due to covalent binding of THL to ser 152,

which is one of the residues in the catalytic trial of this enzyme (Hadvary et al, 1991). Lipstatin

is closely related to the esterase inhibitor esterastin, which contains a n-acetyl asparagine side

chain instead of N-formyl leucine. Lipoprotein lipase was rapidly inactivated by low

concentration of the inhibitor tetrahydrolipstatin (Weibel et al, 1987).

o ooo

NH CHO

Tetrahydrolipstatin

27

1.4.a.2. Panclicins:

Panclicins A, B, C, D and E are novel pancreatic lipase analogues of tetrahydrolipstatin,

which contain a β - lactone and a N- formyl leucine ester, isolated from Streptomyces sp. The

potency of the inhibitory activity of each compound is attributed to the amino acid moiety of

each structure. The panclicins are either glycine- type compounds such as Panclicin A, C, D, E

which are two or three fold more potent than THL or alanine type compounds such as

Panclicin A and B, which are less potent than the glycine type compounds. They irreversibly

inhibit pancreatic lipase. However, the compounds do not irreversibly inhibit the enzyme as

strongly as THL (Mutoh et al, 1994).

1.4.a.3. Marine algae:

The presence of an inhibitor of pancreatic lipase (triacylglycerol acylhydrolase) was

screened in 54 marine algae. An active inhibitor, caulerpenyne, was purified from an extract

Caulerpa taxifolia using ethylacetate extraction. Caulerpenyne competitively inhibited lipase

activities using emulsified tirolein and dispersed 4-methylumbelliferyl oleate (4-MU oleate) as

substrates. The concentrations producing 50% inhibition against triolein and 4-MU oleate

hydrolysis were 2mM and 13 µM respectively (Bitou et al, 1999).

1.4.a.4. Ebelactone B:

NHCHO

CO

O OO

CH3(CH2)6 (CH2)7CH(CH 3)2

3HC

Panclicin A

AcO

OAcOAcCaulerpenyne

28

Ebelactone A and B, natural products from Streptomyces aburaviensis are potent

inhibitors of pancreatic lipase. Ebelactone B inhibited, in a dose dependent manner, the

intestinal absorption of fat in animals. The most effective inhibition was observed when the

inhibitor was administered 60 min prior to fat feeding. When ebelactone B was administered

at 10mg/kg, the serum level of TG and cholesterol were decreased by 58 and 35 %

respectively. Since ebelactone B is effective inhibitor for fat absorption, it may be a promising

molecule for therapy of hyperlipidemia and obesity (Umezawa, 1980).

1.4.b. Lipase inhibitors from plant sources:

1.4.b.1. Grape seed extracts:

Grape seed extract, rich in bioactive phytochemicals, showed inhibitory activity on the

fat-metabolizing enzymes pancreatic lipase and lipoprotein lipase, thus suggesting that grape

seed extracts might be useful as a treatment to limit dietary fat absorption and the accumulation

of fat in adipose tissue. The reduction in intracellular lipolytic activity of cultured 3T3-L1

adipocytes indicated reduced levels of circulating free fatty acids linked to insulin resistance in

obese patients (Moreno et al, 2003).

1.4.b.2. Carnosic acid:

The methanolic extract from the levels of Salvia officinalis L (sage) showed inhibitory

effect on serum triglyceride elevation in olive oil fed mice (500 and 1000 mg/kg) and

inhibitory activity (IC50 94 µg/mL) against pancreatic lipase. Through bioassay-guided

separation using inhibitory activity against pancreatic lipase activity, 4

OH OO

O

R

Ebelactone A(R=Me)B(R=Et)

29

abietan-type diterpenes (carnosic acid, carnosol, royleanonic acid, 7-methoxyrosmanol) and a

triterpene (oleanolic acid) were isolated from the active fraction. Among these compounds,

carnosic acid and carnosol substantially inhibited pancreatic lipase activity with IC50 values of

36 µM and 13 µM respectively. Carnosic acid significantly inhibited triglyceride elevation in

olive oil fed mice at doses of 5-20 mg/kg. However, other constituents (carnosol, rorleanonic

acid, oleanoic acid) did not show any effects even at a dose of 200 mg/kg. Furthermore,

carnosic acid (20 mg/kg/day) reduced the gain of body weight and the accumulation of

epididymal fat weight in high fat diet-fed mice after 14 days (Ninomiya, et al, 2004).

1.4.b.3. Flavan dimmers:

Flavan dimmers which showed lipase-inhibiting effects were isolated from fruits of

Cassia nomame (Leguminosae). Structures of two new compounds among them were

determined to be (2S)-31, 41, 7-trihydroxyflavan-(4-8)-catechin. Four flavan dimers structurally

related to these two compounds were also synthesized for spectral comparision. Among 10

flavan dimmers tested for lipase-inhibitory activity, (2S)-31, 41, 7-trihydroxyflavan-(2S)-

catechin showed the most potent inhibitory effect (Hatano et al, 1997).

1.4.b.4. Dioscorea nipponica:

COOHHO

OH

Carnosic acid

OH

OH

OH

OH

O HO

HO O

OH H

H H OH

Flavan dimmer

30

The methanol extract of Dioscorea nipponica Makino powder appeared to have potent

inhibitory activity against pancreatic lipase with an IC50 value 5-10 µg/ml. Further purification

of active components present in the herb generated dioscin that belongs to the saponin family.

Dioscin and its aglycone, diosgenin, both suppressed the time dependent increase of blood

triacylglycerol level when orally injected with corn oil to mice, suggesting their inhibitory

potential against fat absorption. Sorauge-Dawley rats fed on a high-fat diet containing 5%

Dioscorea nipponica Makino and 40% beef tallow gained significantly less body weight and

adipose tissue. The IC50 of the dioscin (20 µg/ml), diosgenin (28), gracillin (28.9), pro-

sapogenin A (1.8), pro-sapogenin C (42.2) were reported (Kwon et al, 2003).

O

Me

Me

Me

O

O

Me

α-D-Rha(1 4)

α-L-Rha(1 2)

β-D-Glc

Dioscin

1.4.b.5. 5-hydroxy-7-(41-hydroxy-31-methoxyphenyl)-1-phenyl-3-heptone:

A pancreatic lipase inhibitor, 5-hydroxy-7-(41-hydroxy-31-methoxyphenyl)-1-phenyl-3-

heptone (HPH), from the rhizome of Alpinia officinarum was isolated and its

antihyperlipidemic activity was measured. HPH inhibited pancreatic lipase with an IC50 value

of 1.5 mg/ml. HPH significantly lowered the serum triglyceride in corn oil feeding-induced

triglyceridemic mice and reduced serum triglyceride and cholesterol in Triton-WR-1339-

induced hyperlipidemic mice. However, HPH did not show hypolipidemic activity in high

cholesterol diet-induced hyperlipidemic mice (Shin et al, 2004).

OCH

OH5-hydroxy-7-(4 1-hydroxy-3 1-methoxyphenyl)-1-phenyl-3-heptanone

31

1.4.b.6. 3-methylethergalangin:

The pancreatic lipase inhibitory activity of rhizome of Alpinia officinarum and its

antihyperlipidemic activity were measured. 3-methylethergalangin was isolated as an inhibitor

of pancreatic lipase with an IC50 of 1.3 mg/ml in the mice. It inhibited the serum triglyceride

level in corn oil feeding-induced triglyceridemic mice and serum triglyceride and cholesterol in

triton WR-1339-induced hyperlipidemic mice. However, it did not show hypolipidemic

activity in cholesterol diet-induced hyperlipidemic mice (Shin et al, 2003).

1.5. Scope of the present investigation:

In the area of microbial metabolites, a new horizon was opened about 3 decades ago

when the systematic search for small molecular enzyme inhibitors and other bioactive

metabolites from fermentation broths started.

Although a number of investigations have been carried out on the isolation of

pancreatic lipase inhibitors from synthetic, plant and microbial sources, only a few compounds

were reported from actinomycetes. Moreover, there are only a few systematic studies on the

screening, isolation, characterization and media optimization for the production of pancreatic

lipase inhibitors.

O

O

OCH3

3-methylethergalangin

32

In spite of the development of synthetic compounds for pancreatic lipase inhibition,

only a few compounds have gone to the marketing stage. Further, there is a need for the

isolation of new and novel metabolites from the available natural sources, thus leading to the

development of powerful inhibitors.

The present work is an attempt to screen, isolate and characterize pancreatic lipase

inhibitor from actinomycetes. The work carried out in this investigation has been presented in

four chapters. The first chapter surveys the existing information on natural sources.

Actinomycetes and their importance in producing bioactive metabolites and various aspects of

the role of pancreatic lipase and its inhibitors. The second chapter deals with various

experimental methods and analytical procedures used in the investigation. The third chapter

deals with the experimental results and discussion, is further sub-divided into five sections.

Section 1 deals with the screening and selection of actinomycetes for the production of

pancreatic lipase inhibitor. Section 2 deals with characterization of the selected culture. Section

3 deals with isolation and characterization of a new inhibitor designated as streptolipin along

with its biological activities and kinetic parameters. Section 4 deals with studies on

optimization of; physical, nutritional and downstream process parameters for the maximum

production of streptolipin. Section 5 deals with the invitro studies of streptolipin on pancreatic

lipase inhibition along with the hepatoprotective activity and comparative studies with a

commercially available inhibitor, Orlistat. The last chapter gives the summary and highlights

of the present investigation.

33

2.1. Screening of actinomycetes:

2.1.A. Collection of different terrestrial substrates for selective isolation of

actinomycetes:

Fifteen soil samples from different ecosystems like tea plantations, forest, lake, garden,

desert, hill station, cow barn yard, sugarcane, coconut and rice fields areas were collected and

air dried.

2.1.B. Pretreatment of soil samples:

The terrestrial soil samples were pretreated by the following methods:

2.1.B.1. Calcium carbonate treatment:

10 g of the soil sample was ground in a mortar with calcium carbonate in the ratio (1:1)

and incubated for 10 days at 28oC in a closed inverted sterile petri dish with water saturated

filter paper discs (Tsao et al, 1960). From this 2 g of sample was mixed with 100 ml of sterile

distilled water and agitated by shaking on a rotary shaker at 220 rpm for 30 minutes at 280C

and the supernatant was then serially diluted with sterile distilled water.

2.1.B.2. Dry heat treatment:

10g of soil sample was treated at 100oC for one hour (Nonomura and Ohara, 1969). Of

this, 1 g of sample was then mixed with 100 ml of sterile distilled water, agitated by shaking on

a rotary shaker at 220 rpm for 30 minutes at 280C and the supernatant was then serially diluted

with sterile distilled water.

2.1.B.3. Phenol treatment:

34

10 g soil was mixed with 100 ml of sterile distilled water in a 500 ml Erlenmeyer flask

and agitated on a rotary shaker at 220 rpm for 5 minutes at 280C. This suspension was then

mixed with 100 ml of 1.4 % (w/v) phenol solution and incubated for 10 minutes. The

supernatant was serially diluted with sterile distilled water (Lawrence 1956).

2.1.B.4. Centrifugation method:

10 g of the sample was mixed with 100 ml of sterile distilled water in a 500 ml

Erlenmeyer flask and agitated on a rotary shaker for 5 min at 280C. The soil suspension was

centrifuged for 20 minutes at 6000 rpm (Rehacek 1959). The supernatant was then serial

diluted with sterile distilled water.

2.1.B.5. Control (no pretreatment):

1 g of soil sample was mixed with 100 ml of sterile distilled water, and agitated on a

rotary shaker at 220 rpm for 30 minutes at 280C and the supernatant was then serial diluted

with sterile distilled water.

2.2. Media Composition:

2.2.A. Actinomycetes:

Soil samples were serially diluted up to 10-6 level. Of this, one ml was added to each of

50 ml of the sterile molten media maintained at 37 to 400C, thoroughly mixed and plated on

Starch Casein Agar (SCA) isolation medium and incubated at 280C for two weeks (Kuster and

Williams. 1964).

2.2.A.1. Medium for screening of actinomycetes for pancreatic lipase inhibitor:

Starch Casein Agar (g/L)

Soluble starch 10.0

Casein 0.03

35

Potassium nitrate 2.0

Di-potassium hydrogen ortho phosphate 2.0

MgSO4 7H2O 0.05

Calcium carbonate 0.02

Ferrous sulphate 0.01

Bacto agar 20.0

pH 7.0

The media was supplemented with rifampicin (antibacterial) 2.5µg/ml and fluconazole

(antifungal) 75µg/mL.

Potassium tellurite agar medium (g/L):

Potassium tellurite 0.1

Peptone 5.0

Yeast extract 2.5

Bacto-agar 20.0

pH 6.4

Half-strength nutrient agar medium (g/L):

Oxoid nutrient agar nutrients 14.0

Bacto-agar 10.0

pH 7.0

Oat meal agar (g/L):

Oat meal 20.0

Bacto-agar 20.0

20 g oatmeal was cooked or steamed in 1000 mL of distilled water for 20 minutes, filtered

through cheesecloth. Distilled water was added to make the volume of filtrate to 1000 mL.

Trace salt solution 1.0 mL

Bacto-agar 20.0

pH 7.2

All the above media were autoclaved at 1210C for 15 minutes

36

2.2.A.2. Culture maintenance medium:

Glycerol aspargine agar medium (g/L):

L-aspargine (anhydrous) 1.0

Glycerol 10.0

K2HPO4 (anhydrous) 1.0

Trace salt solution 1 mL

Bacto-agar 20

pH 7.0-7.4

Autoclaved at 1210C for 15 minutes. The cultures were subcultured on glycerol

aspargine agar slants. The slants incubated at 280C for two weeks were used for further

experiments.

2.2.A.3. Morphology and pigmentation:

For scanning electron microscopy studies, 14 day old culture of strain N2 grown on ISP

4 medium was used. Spore chain morphology, the presence of sclerotia, substrate spores and

fragmentation of substrate mycelium was examined by scanning electron microscopy LV 435

VP (Lieo Electron Microscopy, England). Sample preparation for scanning electron

microscope is given in Figure 2.1(Groth et al, 1997).

The macro and micro morphological features of the colonies developed on various

media and the colour determinations of the aerial mycelium, substrate mycelium and soluble

pigment were examined after 14 days of incubation. Macro morphology was noted by the

naked eye. The colours of aerial mycelium and substrate mycelium and soluble pigment when

grown on different media were observed and recorded.

Micromorphology was observed by placing the sterile cover slips at an angle of 450C in

solidified agar medium in a petri dish such that half of the cover slip was dipped in medium.

Inoculum was spread along the line where the upper surface of the cover clips meets the

medium. After complete sporulation, the cover slips were removed and examined directly

under the phase contrast microscope (Dietz and Mathews, 1971). The cell morphology of strain

37

N2 was examined by phase contrast microscope with a model BH-2 microscope (Olympus,

Tokyo, Japan). Colours were matched with one of the seven colour wheels of Tresner and

Backus (1963). Motility was studied by hanging drop method.

2.2.A.4. Cultural characteristics media:

The following media were used for studying the cultural characteristics of isolate N2

(Shirling and Gottlieb, 1966):

ISP 1: Tryptone- yeast extract broth

Bacto-Tryptone 5.0 g

Bacto-Yeast extract 3.0 g

Distilled water 1.0 liter

pH 7.0 - 7.2

ISP 2: Yeast extract-malt extract agar

Yeast extract 4.0 g

Malt extract 10.0 g

Dextrose 4.0 g

Agar 20.0 g


pH 7.3 - 7.5

Figure 2.1: Sample preparation for scanning electron microscope: (Groth et al, 1997)

Biomass Treated with 2.5% glutaraldehyde in 0.1 M

acetate buffer (pH 4-5) Centrifuge

Pellet washed with 0.1 M phosphate buffer pH 7.0 For 15 minutes, at 40C (4 times)

Centrifuge Wash pellet with 50% acetone

Wash pellet with 70% acetone

38





Dry the sample under vacuum

Mounted on the SEM stumps

ISP 3: Oatmeal Agar

Oatmeal 20.0 g

Agar 18.0 g


pH 7.2.

Cook or steam 20.0 g of oatmeal in 1.0 liter distilled water for 20 min. Filter through cheese

cloth. Add 18.0 g agar and make up to 1.0 liter. Add 1 ml of trace salts solution.

ISP 4: Inorganic salts-starch agar

39

Solution I: Soluble starch, 10.0 g. Make a paste of the starch with a small amount of cold

distilled water and bring to a volume of 500 ml.

Solution II:

CaCO3 2.0 g

K2HPO4 (anhydrous) 1.0 g

MgSO4. 7 H2O 1.0 g

NaCl 1.0 g

(NH4)2SO4 2.0 g

Distilled water 500.0 ml

Trace salt solution 1.0 ml

Agar 20.0 g

The pH should be between 7.0 and 7.4. Do not adjust if it is within this range. Mix solutions II

and I together. Add 20.0 g agar. Liquefy agar by steaming at 100°C for 10 to 20 min.

Trace salt solution:

CuSO4.5H2O 0.0064 g FeSO4.7H2O 0.0011 g MnCl2.4H2O 0.0079 g ZnSO4.7H2O 0.0015 g Distilled water 1.0 liter

ISP 5: Glycerol-aspargine agar

L-aspargine (anhydrous basis) 1.0 g

Glycerol 10.0 g

K2HPO4 (anhydrous basis) 1.0 g


Trace salts solution 1.0 ml

Agar 20.0 g

The pH should be between 7.0 and 7.4. Do not adjust if it is within this range. Liquefy agar by

steaming at 100°C for 15-20 minutes. Sterilize in flasks for pouring into petri dishes.

40

ISP 6: Peptone-yeast extract-iron agar

Bacto-peptone iron agar 36.0 g

Bacto-yeast extract 1.0 g


36.58 g of the dehydrated Bacto-peptone-iron agar was reconstituted into 1 L of distilled water,

sterilized at 1210C for 20 minutes and poured onto plates. The dehydrated media contained the

following ingredients.

Bacto-peptone 15.0 g

Protease peptone 5.0 g

Ferric ammonium citrate 0.5 g

Dipotassium phosphate 1.0 g

Sodium thiosulphate 0.08 g

Bacto-agar 15.0 g

pH 7.0

ISP 7: Tyrosine agar

Glycerol 15 g

L- tyrosine 0.5 g

L- aspargine 1.0 g

K2HPO4 (anhydrous) 5.0 g

MgSO4. 7H2O 5.0 g

NaCl 5 g

FeSO4. 7H2O 0.1 g

Trace salt solution 1 ml

Agar 20 g


pH 7.0

Bennett’s agar

Beef extract 1.0 g

41

Glucose 10.0 g

N-Z amine A (enzymatic digest of casein) 2.0 g

Yeast extract 1.0 g

Agar 15.0 g


pH 7.3

Nutrient agar

Peptone 5.0 g

Meat extract 3.0 g

Agar 15.0 g


pH 7.0.

Czapek-dox agar

Sucrose 30.0 g

NaNO3 3.0 g

MgSO4.7 H2O 0.5 g

KCl 0.5 g

FeSO4.7 H2O 0.01 g

K2HPO4 1.0 g

Agar 15.0 g


pH 7.2.

Modified Bennets Agar

Glycerol 10.0 g

Yeast extract 1.0 g

Beef extract 1.0 g

NZ- Amine 2.0 g

Agar 20.0 g


42

pH 7.3

Glycerol-arginine agar:

Glycerol 12.5 g

Arginine 1.0 g

Sodium chloride 1.0 g

K2HPO4 1.0 g

MgSO4.7H2O 0.5 g

Fe2(SO4)3. 6H2O 0.01 g

CuSO4.5H2O 0.001 g

ZnSO4.7H2O 0.001 g

MnSO4.H2O 0.001 g


Agar 20.0 g

pH 7.3

All the media were sterilized at 1210C for 20 minutes.

2.2.A.4.1. Enzyme activity test media:

Test for lipolysis and lecithinase: (Nitsch and Kutzner, 1969)

Modified Egg yolk medium (EY)

Bacteriological peptone 10.0 g

Glucose 1.0 g

NaCl 10.0 g

Yeast extract 5.0 g

Agar 12.0 g

Egg yolk emulsion 50.0 g

pH 7.0

Autoclaved at 1210C for 15 minutes

43

Spores were streaked in the center of the plate (white precipitate) to get a band like growth and

incubated at 280C for 14 days. Clearance of the precipitate was the measure of the activity.

Test for proteolysis: (Shirling and Gottlieb, 1966)

Milk casein agar:

Peptone 1.0 g

Agar 20.0 g

Sterile skimmed milk (10 %) 100ml


pH 7.6


The proteolytic activity was studied with milk-casein agar by clearing of the precipitate after

incubating the inoculated plates at 280C for 7 days.

Test for pectinolytic activity: (Hankin et al, 1971)

Pectinolytic activity was determined by using the following medium.

KH2PO4 4.0 g

Na2HPO4 6.0 g

Pectin 5.0 g

(NH4)2 SO4 2.0 g

Yeast extract 1.0 g

MgSO4. 7H2O 2.0 g

FeSO4.7H2O 0.001 g

CaCl2 0.001 g

Agar 10.0 g

pH 7.4

Autoclaved at 1210C for 15 minutes.

44

Spores were streaked in the center of the plate to get a band like growth and incubated

at 280C for 6 days. Hydrolysis zones were detected after 6 days by flooding plates with an

aqueous solution of hexadecyltrimethylammonium bromide (1% w/v).

Test for coagulation and peptonization of milk: (Shirling and Gottlieb, 1966)

Milk coagulation and peptonization test was carried out using 10% (w/v) skimmed

milk. The skimmed milk tubes were inoculated and incubated at 280C. The extent of

coagulation and peptonization was recorded on 3rd and 8th day.

Test for catalase production: (Williams et al, 1983)

Catalase production was detected by adding a few drops of 20% (v/v) H2O2 on to 7 day

old colonies grown on modified Bennets agar. Evolution of oxygen was detected under a

binocular microscope.

2.2.A.4.2. Degradation tests media:

Test for nitrate reduction: (Shirling and Gottlieb, 1966)

Nitrate broth:

Meat extract 3.0 g

Peptone 5.0 g

Potassium nitrate 1.0 g


pH 7.2


Reagents:

α-naphthylamine test solution:

α-naphthylamine 5.0 g

Conc.H2SO4 8.0 ml


To the diluted sulphuric acid, α-naphthylamine was added and stirred until solution was mixed.

45

Sulphanilic acid test solution:

Sulphonic acid 8.0 g

Conc.H2SO4 48 ml


Sulphuric acid was added to 500 ml of water. Then sulphanilic acid was added followed by

water to make up the volume.

Procedure:

5ml of nitrate broth medium was inoculated with a loopful of spores and incubated at

280C for seven days. Controls were also run without inoculation. On 7th day, the broth was

tested for the presence of nitrite. To 1 ml of the broth or control, two drops of sulphanilic acid

solution was added followed by two drops of α-naphthylamine solution were added. The

presence of nitrite was indicated by a pink, red or orange colour and absence of colour change

was considered as nitrite negative. In latter case presence or absence of nitrate in the broth

under examination was confirmed by adding a pinch of zinc dust after the addition of the

reagents, when the unreduced nitrate, if present, gave a pink, red or orange colour.

Test for H2S production: (Shirling and Gottlieb, 1966)

The inoculated peptone-yeast extract-iron agar (ISP 6) slants were incubated for 15

days at 280C. The slant was observed every 12 hrs upto 4 days and thereafter at 24 hrs

intervals, up to 15 days. Observation for the presence of the characteristic greenish brown,

brown, blackish brown, bluish black or black colour of the substrate was indicative of H2S

production.

Test for starch hydrolysis: (Cowan, 1974)

The organism was grown for seven days on 1% (w/v) starch agar plates. At the end of

the incubation period, the plates were flooded with iodine solution. Hydrolyzed zone around

the growth was observed.

46

Test for melanin formation: (Shirling and Gottlieb, 1966)

This test was carried out in tryrptone–yeast extract broth (ISP 1), yeast extract-iron agar

(ISP 6) and tyrosine agar (ISP 7) media. The inoculated tubes were observed every 12 hrs for 4

days. The inoculated tubes were compared with uninoculated controls. Deep brown, greenish

brown, greenish black or black colours were recorded as melanin positive. Absence of brown

to black colours or total absence of diffusible pigment was considered as negative for melanoid

pigment production.

Test for urea decomposition: (Gordon, 1968)

Media composition:

Peptone 20.0 g

NaCl 5.0 g

Mono potassium phosphate 2.0 g

Phenol red 0.012 g

Agar 20.0 g


pH 7.0


The media were inoculated heavily by spreading spores over the medium. The decomposition

was observed by characteristic red- violet colour on the plate

Test for arbutin degradation: (Kutzner, 1976)

Media composition:

Yeast extract 3.0 g

Arbutin 1.0 g

Ferric ammonium citrate 0.5 g

Agar 7.5 g

47


pH 7.2


Spores were spread over the medium and observed for brown to black pigment after 21 days.

Absence of brown-black pigment was taken as a negative test. A comparison with controls was

essential to avoid confusion with melanin production.

Test for allantoin degradation: (Gordon, 1966)

Media composition:

Yeast extract 0.1 g

KH2PO4 9.1 g

NaHPO4 9.5 g

Allantoin 3.3 g

Phenol red 0.01 g

Agar 7.5 g


pH 6.8


The organism was inoculated on the above media. The results were recorded after 28 days and

observed for orange yellow to pink or purple colour.

Test for gelatin hydrolysis: (Waksman, 1961)

Media composition:

Peptone 5.0 g

Beef extract 3.0 g

Gelatin powder 4.0 g

Agar 15.0 g


48

pH 7.0


Reagent:

Mercuric chloride 15 mg

Conc. HCl 20 ml

Distilled water 100 ml

The isolate was grown on gelatin agar plated for six days at 280C. After the incubation period,

the plates were flooded with 1 ml of the reagent. Presence of hydrolyzed zones was observed

after 6 days around the culture.

Test for tyrosine reaction: (Williams et al, 1983)

This test was carried out on tyrosine agar (ISP 7) medium slants. The inoculated slants

were incubated at 280C and observations were made at every 12 hrs up to 4 days and at 24 hrs

thereafter up to 15 days. The results were recorded after 15 days and observed for bluish black

or black diffusible pigment.

Other degradation tests: (Williams et al, 1983)

Hypoxanthine, xanthine, guanine, elastin, adenine and xylan (all at 0.5 %, w/v)

degradation tests were carried out on modified Bennets agar medium. Clearing of the insoluble

compounds around areas of growth was observed after 14 days.

2.2.A.4.3. Test for resistance to antibiotics: (Goodfellow and Orchard, 1974)

The isolate was tested for its ability to grow in the presence of antibiotics, using the

freeze-dried filter paper disc method. Discs previously soaked and air dried in ampicillin

(10µg), chloramphenicol (30µg), erythromycin (15µg), neomycin (30µg), oxytetracycline

(30µg), penicillin G (10 IU), rifamycin (10µg), gentamycin (10µg), streptomycin (10µg)and

kanamycin (30µg) were placed on modified Bennets agar inoculated with 0.1 ml of glycerol

spore suspension and incubated at 280C. Growth was noted on 2, 3, 4 and 7 days for all strains

49

and the resistance was scored as positive result. The first readable results were scored for

computation.

2.2.A.4.4. Effect of temperature on growth of isolate N2: (Williams et al, 1983)

Ability of the isolate to grow at different temperatures was studied at temperatures in

the range of 4 to 450C. The organism was inoculated on modified Bennets agar slants and

incubated at the different temperatures as mentioned above. Results were recorded on 7th and

14th day.

2.2.A.4.5. Effect of pH on growth of isolate N2: (Williams et al, 1983)

The pH of modified Bennet’s agar was adjusted to pH in the range of 2.0 to 11.0 and

spores were inoculated and incubated for 15 days. Then the tubes were examined for the extent

of growth of the organism.

2.2.A.4.6. Growth in the presence of inhibitory compounds: (Shirling and Gottlieb,

1966)

A range of potential inhibitors were added to Bennet’s agar medium and their effect on

growth of selected isolates was studied. (%, w/v): Crystal violet (0.00001), phenol (0.1),

thallous acetate (0.001 and 0.01), sodium chloride 1-13), thallous acetate (0.01), sodium azide

(0.01and 0.02) and potassium tellurite (0.001 and 0.01).

15 ml of sterile molten Bennet’s agar medium was cooled to 450C and each of the

above inhibitory compounds was added to petri plate. The isolate was streaked onto the surface

of the medium, incubated at 280C for 7 days and the presence or absence of growth was

recorded.

2.2.A.4.7. Test for carbon utilization medium: (Pridham and Gottlieb, 1948)

50

ISP 9: Basal medium

Carbon source 10.0 g

(NH4)2SO4 2.64 g

KH2PO4 2.38 g

K2HPO4 5.65 g

MgSO4.7H2O 1.0 g

CuSO4.5H2O 0.1 g

FeSO4.7H2O 0.1 g

MnCl2.4H2O 0.1 g

ZnSO4.7H2O 0.0015 g

Agar 15.0 g


pH 6.8 – 7.0


The pH of the medium was adjusted to 7.0 and 5 ml of media was dispensed into test

tubes and autoclaved. After cooling to about 450C, sterile aqueous solutions of the carbon

compounds were added at desired concentration. The carbohydrates, polyhydric alcohols, DL-

inositol and salicin were added such that the final concentration was 1 % (w/v), the phenols at

0.1% (w/v) and the sodium salts of organic acids at 0.15% (w/v). Those materials sufficiently

soluble in water were sterilized by filtration through Sietz EK filter pads. Some compounds

(dextrin, starch, DL-inositol and salicin) that were relatively insoluble or did not filter well

were added directly to the basal medium in the proper concentration prior to tubing and

sterilization. After addition of the carbon sources, the tubes were slanted, allowed to solidify

and incubated to determine sterility.

2.2.A.4.8. Test for nitrogen utilization medium: (Williams et al, 1983)

Nitrogen source 1.0 g

D-glucose 10.0 g

MgSO4. 7H2O 0.5 g

51

NaCl 0.5 g

FeSO4. 7H2O 0.01 g

K2HPO4 1.0 g

Agar 20.0 g


pH 7.0


52

2.2.A.4.9. Test for production of acid and gas: (Hugh and Lieifson, 1953)

Media composition:

Bacto peptone 2.0 g

Bacto meat extract 1.0 g

Bacto agar 3.0 g

Bromothymol blue solution (0.2% w/v) 15 ml

pH 7.2


The ability of isolate to produce acid and gas was tested on the different carbohydrates

by adding them to a final concentration of 1% (w/v) to the acid gas medium. The carbohydrates

tested were L-arabinose, cellulose, sucrose, starch, D-xylose, meso-inositol, D-fructose, D-

glucose, L-rhamnose, maltose, D-mannose, D-lactose, inulin, D-melibiose, D-galactose,

melibiose, mannitol, inulin and xylitol (1.0 % w/v) and sodium acetate, sodium citrate (0.1%

w/v). Durham’s tubes were kept in the inverted position for testing gas production. Both the

tests were carried out in the same media. Presence of acid was determined by change in

medium colour to green.

2.2.A.4.10. Media for cultivation of cells for determination of cell wall composition:

Yeast extract glucose broth:

Yeast extract 10.0 g

Glucose 10.0 g


pH 7.0


Cells used for chemotaxonomic analysis were obtained after incubation at 30ºC for 3 days in

yeast extract glucose broth.

2.2.A.5. Primary screening medium:

53

For development of inoculum, well sporulated, 7 to 10 days old glycerol aspargine slant

was used for the production of streptolipin. 5 ml of 2% v/v tween 20 was added to the slant and

spore suspension was prepared. This spore suspension was transferred to 95 ml of sterile

production medium (composition of the medium given below) in 500 ml Erlenmeyer flask. The

culture flasks were incubated at 220 rpm, 300C for 7 days.

Media components:

Soybean meal 1.0 g

Corn steep liquor 0.5 g

Soluble starch 1.0 g

Dextrose 0.5 g

Calcium carbonate 0.7 g

The media components were added to distilled water and pH was adjusted to 7.2 with 0.1 N

HCl or 0.1 N NaOH and final volume made up to 95 mL with distilled water. 95 mL of this

medium was taken in 500 mL Erlenmeyer flasks and autoclaved for 15 minutes at 1210C.

2.2.A.6. Secondary screening media:

Production Medium 1:

Yeast extract 0.5 g

Dextrose 1.0 g

Starch 2.0


The media components were added to distilled water and pH was adjusted to 7.2 with 0.1 N

HCl or 0.1 N NaOH and final volume made up to 100 mL with distilled water. 100 mL of this

medium was taken in 500 mL Erlenmeyer flasks and autoclaved for 15 minutes at 1210C.

Production Medium II:

Soybean meal 1.0 g



54

Dextrose 0.5 g


The above media components were added to the distilled water and pH was adjusted to 7.2

with 0.1 N HCl or 0.1 N NaOH and final volume was made up to 100 mL with distilled water.

100 mL of this medium was taken in 500 mL Erlenmeyer flasks and autoclaved for 15 minutes

at 1210C.

2.2.A.7. Selection of inoculum media for production of streptolipin

In order to minimize the time lag in fermentation process, inocula are raised in media

with a composition similar to that of fermentation medium.

Composition of the two inoculum media are given below:

Inoculum medium I (g/L):

Soyabean meal 10.0

Corn steep liquor 10.0

Glucose 10.0


pH 7.0

Inoculum medium II:

Soyabean meal 15.0

Glycerol 5.0

NaCl 5.0


pH 7.0

The media was sterilized at 1210C for 15 minutes.

Spore suspension from maintenance slant was transferred to the above mentioned

inoculum media and incubated on rotary shaker (220 rpm) at 280C for 48 hrs. After 48 hrs

inoculum level (10% v/v) was transferred to the selected production medium (ISP VIII) and

incubated on rotary shaker (220 rpm) at 280C. Samples were withdrawn at the end of the

fermentation cycle (168 hrs) and HPLC analysis for the streptolipin estimation was carried out.

55

2.2.A.8. Standard media for screening streptolipin production:

Streptolipin production of the isolate was carried out in twenty three different

production media. The composition of media are given below (g/L).

M.1. Lindenberg synthetic media M.2. Hobb’s medium

Glycerol 30.0 Glucose 20.0

NaNO3 2.0 NaCl 5.0

K2HPO4 1.0 Na2SO4 5.0

MgSO4.7H2O 0.5 NaNO3 4.5

FeSO4.7H2O 0.4 K2HPO4 1.2

pH 7.0 Trisbase 1.2

MgSO4.7H2O 1.0

ZnSO4 0.01

pH 7.0

M.3. Czepek-Dox broth M.4. O-Brien synthetic media

Sucrose 30.0 Glucose 20.0

NaNO3 3.0 Glycine 2.6

K2HPO4 1.0 Sodium acetate 1.36

MgSO4.7H2O 0.5 (NH4)2SO4 0.54

KCl 0.5 K2HPO4.3H2O 0.05

FeSO4.7H2O 0.01 ZnSO4.7H2O 0.03

pH 7.0 FeSO4.7H2O 0.025

CuSO4.5H2O 0.016

MnSO4.4H2O 0.012

CaCl2. 2H2O 0.05

MgSO4.7H2O 0.5

pH 6.8-7.0

M.5. Dulaney’s medium M.6. Thornberry’s medium

Glucose 10.0 Glucose 10.0

NaCl 5.0 KH2PO4 2.38

56

K2HPO4 2.0 K2HPO4 5.65

MgSO4.7H2O 0.4 NH4NO3 4.0

CaCl2 0.4 MgSO4.7H2O 0.25

FeSO4.7H2O 0.02 Sodium lactate 11.2

ZnSO4.7H2O 0.01 ZnSO4.7H2O 0.14

(NH4)2HPO4 4.0 FeSO4.7H2O 0.014

pH 7.0 MnSO4.4H2O 0.084

CuSO4.5H2O 0.0016

pH 8.4

M.7. Baron’smedium M.8. Numerof’s medium


NH4NO3 4.0 Glycine 2.6

MgSO4.7H2O 0.25 Sodium acetate 1.36

NaCl 5.0 (NH4)2SO4 0.54

Sodium citrate 1.0 FeSO4.7H2O 0.03

KH2PO4 0.1 CuSO4.5H2O 0.5

K2HPO4 0.1 K2HPO4 0.5

CaCO3 3.0 CaCl2 0.05

pH 8.2 pH 5.5

M.9. Complex organic media M.10. Lumb’s medium


Soya bean flour 25.0 MgSO4.7H2O 10.0

Yeast extract 3.0 Sodium citrate 1.0

(NH4)2SO4 2.0 NaCl 2.5

CaCO3 2.0 CaCl2 0.87

NaCl 2.0 KH2PO4 0.5

KH2PO4 0.15 Glycine 5.0

pH 8.4 FeSO4.7H2O 0.0075

MnSO4.4H2O 0.008

CuSO4.5H2O 0.001

57

ZnSO4.7H2O 0.0014

(NH4)2MO4.4H2O 0.0018

pH 8.8

M.11. Corn meal salt medium M.12. Carbohydrate carcode medium

Cornmeal 50.0 Potato starch 5.0

Na2HPO4 1.15 Glucose 5.0

KH2PO4 0.25 Ribose 5.0

KCl 0.2 Glycerol 5.0

MgSO4.7H2O 0.2 Soyaflour 20.0

pH 7.0 (NH4)2SO4 0.2

Yeast extract 2.0

Bactopeptone 2.0

pH 7.0

M.13. ISP Production media I: M.14. ISP Production media II:

Soyabean meal 25.0 Soluble starch 25.0

Glucose 25.0 Corn steep liquor 10.0

NaNO3 4.0 (NH4)2SO4 5.0

K2HPO4 0.05 CaCO3 5.0

NaCl 2.5 ZnSO4 0.04

CaCO3 0.4

pH 7.0

pH 7.0

M.15. ISP Production media III: M.16. ISP Production media IV:

Glycerol 20.0 Glucose 10.0

Peptone 5.0 Soluble starch 10.0

Yeast extract 3.0 Peptone 7.5

Meat extract 3.0 Meat extract 7.5

CaCO3 2.5 NaCl 3.0

pH 7.0 pH 7.0

58

M.17. ISP Production media V: M.18. ISP Production media VI:

Soyabean meal 15.0 Glucose 33.0

Glucose 15.0 Soluble starch 33.0

Glycerol 2.5 Soyabean meal 34.0

Sodium Chloride 5.0 (NH4)2SO4 13.0

CaCO3 3.0 K2HPO4 13.0

pH 7.0 NaCl 2.5

CaCO3 12.5

pH 7.0

M.19. ISP Production media VII: M.20. ISP Production media VIII:

Soyabean meal 20.0 Soyabean meal 10.0

(NH4)2SO4 5.0 Dextrose 5.0

Meat extract 4.0 Corn steep liquor 5.0

Yeast extract 2.5 Soluble starch 1.0

Glucose 6.0 CaCO3 7.0

KCl 4.0

CaCO3 0.1 pH 7.2

K2HPO4 0.1

pH 7.0

2.2.B. Fungi

2.2.B.1. Culture maintenance medium for fungi:

24 g of the potato dextrose medium (containing potatoes infusion from 200 g/L and 20

g dextrose/L) obtained from Himedia, India, was dissolved in one liter of distilled water and

pH was adjusted to 5.5 with 0.1 N HCl. For preparation of agar slants (PDA), 2% w/v bacto-

agar was used. Autoclaved at 1210C for 15 minutes.

59

2.2.B.2. Screening of fungal cultures for the production of lipase inhibitor:

Fungal cultures available at CFTRI Culture Collection Center were used for selective

screening studies for lipase inhibitor. All the cultures were sub cultured on potato dextrose agar

slants. A three day old slant of each fungal culture was used in the experiments. The following

strains were screened for the production of lipase inhibitor.

1. Aspergillus awamori – 1042

2. Aspergillus carbonarius – 1047

3. Aspergillus flavus – 1058

4. Aspergillus niger – 1038


6. Aspergillus niger – CFR-W-105


8. Aspergillus oryzae – 1120

9. Aspergillus sp. CFR-H-105

10. Aspergillus sp. CFR-J-105

11. Fusarium sp – 1128

12. Penicillium sp. 1062

13. Polyporus squamosus – 1134

2.2.B.3. Medium for submerged fermentation (SmF) of fungi:

1000 mL distilled water containing 200 g of peeled potato slices was boiled for 30

minutes and filtered. To the cooled filtrate 20 g of dextrose was added and pH was adjusted to

5.5 with 0.1 N HCl and final volume was made up to 1000 mL with distilled water. 100 mL of

this medium was taken in 500 mL Erlenmeyer flasks and autoclaved for 15 minutes at 1210C.

2.2.B.4. Medium for solid state fermentation (SSF) of fungi:

The medium used for basal wheat bran: Each 250 mL Erlenmeyer flask contained, 10 g

dry wheat bran, moistened with 10 mL of 0.2 N HCl comprising 2.1 mg each of ferrous

60

sulphate, zinc sulphate and copper sulphate and 5 mL of distilled water. This solid medium was

autoclaved at 1210C for 60 minutes and cooled to room temperature before inoculation.

2.3. General fermentation conditions

2.3.1. Inoculum development for screening

The spores were subcultured on glycerol aspargine agar media, incubated at 300C for 7

to 10 days. To the well sporulated slant, sterile 5 ml of 2% v/v tween 20 was added to the slant

and spore suspension was prepared for each media. This spore suspension was transferred to a

sterile 95 ml of the production medium in 500 ml Erlenmeyer flask, which were previously

sterilized at 1210C for 15 minutes and incubated at 280C on a rotary shaker at 220 rpm.

2.3.2. Submerged fermentation for actinomycetes cultures

Media components:

Soybean meal 1.0 g



Dextrose 0.5 g


The above media components were added to 95 ml of distilled water and pH was

adjusted to 7.2 with 0.1 N HCl or 0.1 N NaOH and final volume was made up to 100 mL with

distilled water. 100 mL of this medium was taken in 500 mL Erlenmeyer flasks and autoclaved

for 15 minutes at 1210C.

2.3.3. Solid state fermentation for fungal cultures (SSF)

For development of inoculum, a loopful of spores from a three day old PDA slant was

transferred to a sterile 100 ml PDB in 500 ml Erlenmeyer flask. The culture flasks were

incubated at 220 rpm, 300C for 3 days. 2-3 pellets from a day old broth of each culture were

transferred to a sterile wheat bran medium.

61

After inoculation, the bran was spread as a thin layer and left in slanting position in an

incubator maintained at 300C for one day. After a one day growth, the bed was disturbed in

each flask by mixing thoroughly for a few min and was left for 7 days without any further

disturbance.

2.3.4. Submerged fermentation for fungal cultures (SmF)

A loopful of spores from a three day old PDA slant was transferred to a sterile 100 ml

PDB in 500 ml Erlenmeyer flask. The culture flask were incubated on a rotary shaker operating

at 220 rpm at 300C.

62

2.4. General extraction conditions

2.4.1. Extraction of inhibitor from SmF broth:

After fermentation, each flask containing 100 mL fermented medium was extracted into

50mL ethyl acetate and kept on shaker for 60 minutes at 220 rpm. The organic layer was

separated, dried using anhydrous sodium sulphate and distilled under reduced pressure. The

crude extract was redissolved in DMSO to give a stock of 50mg/mL for fungal cultures and 7.5

mg/mL for actinomycetes cultures. 10 µL of this solution was tested for pancreatic lipase

inhibition assay.

2.4.2. Extraction of inhibitor from SSF bran:

After 8 days fermentation, each culture was treated with 100 mL ethyl acetate and kept

on shaker for 1 hour at 220 rpm. The bran was filtered on a muslin cloth, ethyl acetate layer

was separated, the moisture was removed by using anhydrous sodium sulphate and distilled

under reduced pressure. For screening the crude extract was dissolved in DMSO at a

concentration 50 mg/mL and 10 µL of this solution was tested for pancreatic lipase inhibition

assay.

2.5. Analytical methods

2.5.1. Lipase assays:

Method I: Spectrophotometric assay (For primary screening):

Buffer preparation:

The buffer was prepared by adding 100 mM sodium phosphate monobasic, 150 mM of

anhydrous sodium chloride and 0.5 % (v/v) triton x-100 in distilled water and the final pH was

adjusted to pH 7.4.

63

Assay of pancreatic lipase for preliminary studies:

The assay was carried out by monitoring the appearance of paranitro phenol at 400 nm

with a micro molar extinction coefficient of 0.0148. The assay mixture contained 50 mM

paranitrophenyl butyrate (30 µL stock) and 10 µL of inhibitor sample dissolved in DMSO,

sodium phosphate buffer (pH 7.4). The reaction was initiated by addition of enzyme solution

(<0.15 unit). One unit of the enzyme activity is defined as the enzyme required forming 1 nM

of the product per minute at 370C under assay conditions. The fraction containing the inhibitor

was dissolved in a minimum quantity of DMSO, which was found to have no effect on enzyme

activity at less than 0.1 % concentration. The assay was initiated by adding substrate to the

reaction mixture (Shirai and Jackson, 1982).

Inhibition was expressed as a percentage relative to solvent control. All activities were

carried out in triplicates and the average has been reported. The relative activity was expressed

as percentage ratio of enzyme activity in the presence of inhibitors to the enzyme activity in the

absence of enzyme inhibitors at the end 3 minutes of the enzyme reaction time.

Method I1: pH stat assay (For secondary screening):

The crude extract that remained after distillation of solvent was taken as a source of

inhibitor and dissolved in DMSO to achieve a concentration 75 µg/ml. The determination of

lipase activity and the hydrolysis of fatty acids from trioleate was followed at pH 8 for 10

minutes at room temperature, using a recording pH-stat. The substrate emulsion was prepared

by ultrasonication of trioleate (30 mg/ml) in a solution containing taurodeoxycholate 1mM,

taurocholate 9 mM, cholestrol 0.1 mM, lecithin 1 mM, bovine serum albumin 15 mg/ml, Tris-

HCl 2 mM, NaCl 100 mM and CaCl2 1 mM. This composition of the test emulsion was chosen

to mimic as closely as possible the in vivo conditions. After addition of test compound, or

vehicle solution alone in to 8 mM Tris-HCl, the pH was set to 8.0 and the reaction was started

by the addition of porcine pancreatic lipase. The release of fatty acid was estimated by 0.1 N

NaOH (Weibel et al, 1987). Secondary screening was carried out by this method.

Normality of NaOH: To potassium hydrogen thalate (0.1N), 3 drops of phenolphthalein was

added as an indicator and titrated against 0.1N NaOH.

64

N1V1=N2V2

N1= Normality of potassium hydrogen thalate (0.1N)

V1= Volume of potassium hydrogen thalate consumed

N2= Normality of NaOH

V1= Volume of NaOH

Specific activity = (Test-Blank) x 1000 x N NaOH / A x Incubation time

N NaOH=Normality of NaOH

A=Protein concentration

2.5.2. Tests for cell wall composition of isolate N2:

2.5.2.1. Amino acid analysis: (Becker et al, 1965 and Hasegawa et al, 1983)

3 mg of dried cells are hydrolyzed with 1 ml of 6 N HCl in a screw capped vial at

1000C for 12 hours. After cooling, the hydrolysate is filtered and 1 ml of water was added. It

was concentrated under reduced pressure. The residue was dissolved in 1 ml of water, dried

again and redissolved in 0.3 ml of water.

2 µl of the sample along with the standards at a concentration of 0.1M (meso-

diaminopimelic acid and levo- diaminopimelic acid) was applied at the base line of the

cellulose TLC sheet. Ascending TLC was performed with the solvent system containing

methanol, water, 6 N HCl and pyridine in the ratio of 80: 26: 4: 10 (v/v). After the

chromatogram was air dried, spots were visualized by spraying 0.2% (w/v) ninhydrin in

acetone and heating at 1000C for 3 min.

2.5.2.2. Sugar analysis: (Becker et al, 1965)

50 mg of dried biomass in a reaction tube with a polytetrafluoroethylene cap and 1 ml

of 1 N H2SO4 was added. It was heated at 1000C for 2 hours, cooled and 0.5 mg of barium

hydroxide to neutralize the solution. The cell debris was separated by centrifugation and the

65

supernatant was evaporated on a rotary evaporator under reduced pressure. The residue was

dissolved in 400 µl of water.

1µl of the sample along with the standard solutions (galactose, arabinose and xylose,

rhamnose, mannose and ribose each at 1% concentration) was applied at the base line of the

cellulose TLC sheet. Ascending TLC was performed with the solvent system containing n-

butanol: water: pyridine: toluene (10: 6: 6: 1, v/v). After the chromatogram was air dried, spots

were visualized by spraying the chromatogram with aniline acid phthalate reagent (3.3 g of

phthalic acid dissolved in 100 ml of water saturated n-butanol with 2.0 ml of aniline) and

heated at 1000C for 4 min. Hexoses appear as yellowish, brown spots and pentoses appear as

maroon spots.

2.5.2.3. Analysis of menaquinones: (Collins et al, 1977)

100 mg dried biomass was mixed with 20 ml chloroform/methanol (2:1, v/v) and the

suspension was stirred continuously over night. The biomass was then removed by filtration

and the extract was evaporated to dryness under reduced pressure 370C. Analytical thin layer

chromatography of quinones was performed using 0.5 mm layers of Merck Kiesegel-gel HF254

and developing solvent system consisting of petroleum ether and acetone (85: 15, v/v).

Menaquinones were detected on thin-layer chromatography with short wave ultraviolet light

(254 nm). Chloroform was used to elute the quinone from the silica.

Ultraviolet spectra of menaquinones were recorded in hexane solution. The GC/EIMS

data was generated using VG Auto Spec M mass spectrometer equipped with HP 5890 series II

gas chromatograph under GC/EIMS conditions. For GC, an HP-5 capillary column was used

with the following temperature program: 1000C for 5 min; 100C/min; 2300C, 15 min. injection

temperature was at 2100C. For MS conditions, the source temperature was 2500 C at an

electrical energy of 70 eV.

2.5.2.4. Analysis of fatty acids: (Tamaoka et al, 1983)

50 mg of dried biomass was taken in an 8.5 ml tube fitted with a

polytetrafluoroethylene seal with 3 ml methanol: toluene: concentrated H2SO4 (30:15:1, v/v)

66

and heated for 12 hours. It was then cooled to room temperature and 2 ml of hexane was added

and centrifuged at 100 rpm. A small column of prewashed ammonium hydrogen carbonate in a

Pasteur pipette plugged with cotton wool was prepared and washed with methanol: chloroform

(1:2, v/v). The supernatant hexane was chromatographed to the column and eluted with

methanol: chloroform (1:2, v/v).

The residue was dissolved in hexane and chromatographed on silica gel sheet (Merck

5735) and developed by using hexane: diethyl ether (85:15 , v/v). the sheet was air dried in the

fume hood and sprayed with 0.01% (w/v) rhodamine 6G in 95 % ethanol and visualized under

366 nm. The purple colour band was cut and extracted with 1ml of diethyl ether. This was

further chromatographed on neutral aluminium oxide column and eluted with diethyl ether.

The extraction was repeated and dried by flushing with nitrogen.

The resultant methyl esters were separated on a gas chromatograph (model 5890;

Hewlett-Packard) fitted with a model of HP-5 capillary column (0.53 mm by 30 m; Hewlett-

Packard). The column was conditioned at 1500C for 2 min and then programmed from 150 to

2500C at the rate of 100C/min, with helium as a carrier gas.

2.5.2.5. Analysis of phospholipids: (Minnikin et al, 1977)

The phospholipids were extracted from frozen cells with a solution containing

chloroform: methanol: 0.3% aqueous sodium chloride (9: 10: 3, v/v). The polar lipid extract

was dissolved in 100 µl of chloroform: methanol (2:1, v/v). 5 µl of the sample was spotted on

silica-gel plates (Merck 5554) along with the standard samples (phosphatidylglycerol,

phosphatidylethanolamine, phosphatidylmethylethanolamine, phospholipids containing

glucosamine) and the plate was developed by ascending TLC with a mobile phase containing

chloroform: methanol: water (65:25:4, v/v). The plate was air dried to remove the residual

traces of the solvent and further developed with a solvent system containing chloroform: acetic

acid: methanol: water in the ratio of 40:7.5:6:2 in the second direction. The plates were dried

and the different components were detected by spraying with reagents. One plate was sprayed

with molybdophosphoric acid (5%, w/v) and heated at 1800C for 15 minutes. All polar lipids

appeared as dark spots on a light green ground. Ninhydrin (2% w/v) in water saturated butanol

67

was sprayed lightly and heated at 1000C for 5 minutes. Lipids containing amino groups

appeared as pink-red spots. The spots were marked and sprayed with Zinzadze reagent.

Phosphorus containing lipids appeared as blue spots on a white back ground. The third plate

was sprayed with metaperiodate and left aside for 10 minutes for oxidation. Decolourized with

sulphurdioxide gas and sprayed with schiff reagent for the detection of lipids containing vicinal

diol groups. The fourth plate was lightly sprayed with α-naphthol reagent heated at 1000C for

10 minutes for the detection of glycolipids. Glycolipids appear as brown spots.

2.5.2.6. Analysis of mycolic acids: (Tomiyasu, 1982)

The extraction and purification of mycolic acids was the same as carried out for fatty

acids. Methyl esters of mycolic acids were detected and purified using chromatographic system

described for menaquinones above. Conditions for the analysis of mycolic esters by mass

spectroscopy were the same as for the menaquinone samples.

2.5.2.7. Phylogenetic analysis of isolate N2:

Genomic DNA was prepared as described by Shivaji et al (1992). The 16S rDNA was

amplified (Shivaji et al, 2000; 2004), the PCR product purified using QIA quick PCR

purification kit (Qiagen) and sequenced using an ABI PRISM model 3700 automatic DNA

sequencer using the Big Dye Terminator cycle sequencing kit (Applied Biosystems). The

almost complete sequence of 16S rDNA containing 1464 bp was aligned with closely related

sequences that were deposited in EMBL using CLUSTAL W (Thompson et al, 1994). The

pair-wise evolutionary distances were computed using the DNADIST program with the

Kimura 2-parameter model (Kimura, 1980). Phylogenetic trees were constructed using

NEIGHBOR JOINING and DNAPARS of the PHYLIP package (Felsenstein, 1993). The

stability among the clades of a phylogenetic tree was assessed by taking 1000 replicates of the

data set and was analyzed using the programs SEQBOOT, DNADIST, NEIGHBOR and

CONSENSE of the PHYLIP package.

2.5.3.1. Column chromatography:

68

Silica gel (60-120 mesh) was dried in an oven for one hour at 1000C. 30 gm of this was

packed on to a glass column (15 x 3 cm) fitted with a G0 filter, in hexane with a flow rate of 1

mL/min. Elution of the crude extract was carried out using hexane, chloroform, ethyl acetate,

methanol and various combinations of these solvents. Two bed volumes were taken as a

fraction. Each fraction was analyzed by qualitative TLC and enzyme assay.

2.5.3.2. Thin layer chromatography (TLC):

Qualitative TLC plates preparation:

Qualitative TLC plates were prepared by making slurry of 2 gm of silica gel-G with 5

mL of water and spread over the plate manually on a 5 X 20 cm glass plate followed by air-

drying. The plates were then activated in oven for one hour at 1000C. After activation, the TLC

plates spotted with crude extracts or purified compounds and run using a benzene : methanol (9

: 1) mobile phase.

Preparative TLC plates preparation:

For preparative TLC plates, the slurry was prepared by mixing 8 gm of silica gel-G

with 20 mL of water and spread over the plate manually on a 20 X 80 cm glass plate followed

by air-drying. The plates were then activated in oven for one hour at 1000C. After activation,

the TLC plates spotted with partially purified compound and run using a suitable mobile phase.

2.5.3.3. High pressure liquid chromatography (HPLC) method:

The HPLC method for streptolipin was developed on RP-C18 column [5µm, 250 mm x

4.6 mm, Shimpack CLC-ODS (L1) column (Shimadzu, Japan)] using LC-10A (Shimadzu,

Japan) with a gradient mobile phase of acetonitrile and water. The programme was as follows:

69

0.01 min acetonitrile 10%, 20.0 min acetonitrile 70%, 40.0 min acetonitrile 100% operating at

a flow rate of 1 ml/ minute and the UV detector fixed at 210 nm.

2.5.3.4. Melting point determination:

2 mg of compound was packed into a capillary tube sealed at one end. This tube was

placed on SELACO-650 hot stage apparatus and the melting point were determined and are

uncorrected in a 230V PEW-thermal block and temperature was read manually using a

mercury bulb thermometer.

2.5.3.5. Elemental analysis:

Elemental analysis was carried out on Elementar Vario EL III. Oxygen was used for

combustion and Helium as the mobile phase. The combustion chamber temperature was 1150 0C and the reduction chamber temperature was at 8500C. Detector used for thermal

conductivity and the liberated sulphur dioxide was detected at 1400C. The carbon dioxide,

nitrogen and water were detected at room temperature. The oxygen and helium flow were 1.25

and 2–3 Kg per square cm respectively. Desiccants used were sodium hydroxide and

phosphorus pentaoxide to remove the moisture content.

2.5.3.6. Infrared spectroscopy:

IR absorption spectra were obtained with a Perkin Elmer model 2000 Infrared Fourier-

transform spectrophotometer using an attenuated total reflectance cell on a thin layer of the

sample (1 mg/mL) dissolved in nujol.

2.5.3.7. Nuclear magnetic resonance spectroscopy (NMR):

NMR spectra were recorded at 500 MHz on a Bruker DRX-500 MHZ spectrometer

(500.13 MHz proton and 125 MHz carbon frequencies) at 27 0C. Proton and carbon 900 pulse

widths were 11.2 and 8.8 µs respectively. About 20 mg of the sample-dissolved in CDCl3 was

used for recording spectra.

70

Two-dimensional heteronuclear multiple quantum coherence transfer spectra (2D-

HMQCT) were recorded in magnitude mode with sinusoidal shaped z gradients of strength

25.7, 15.42 and 20.56 G/cm in the ratio of 5:3:4 were applied for duration of 1 ms each with a

gradient recovery delay of 100 µs to defocus unwanted coherence. The t1 was incremented in

256 steps. The size of the computer memory used to accumulate the 2D data was 4K. The

spectra were processed using unshifted and π/4 shifted sine bell window function in F1 and F2.

2.5.3.8. Liquid chromatography mass spectroscopy (LC-MS):

Mass spectral data were achieved by LCMS (waters 2690, HPLC system connected to

micro mass LCZ mass spectrometer) with electro spray ionization in positive mode [ESP+].

The following ion optics were used: capillary 3 Kv conc 30 volt and 60 volt and extra per 7

volts. The source block temperature was 1200C and the desolvation temperature 2500C. The

electrospray probe flow was adjusted to 100 µl/min. continuous mass spectra were recorded

over the range M/z 110 to 800 with scan time 1 second and inter scan delay 0.1 sec. For LC

conditions used were same as HPLC.

2.5.3.9. Detection of elements by chemical method:

A small quantity of test sample with a piece of sodium in a sodium fusion tube was

heated gradually at first and then strongly till the evolution of vapours and smoke ceased. The

red-hot tube was plunged in to 10 ml of water taken in a mortar. The glass tube was crushed

and transferred in to a 100 mL beaker, boiled and filtered and the filterate was collected. The

filterate was used for both nitrogen and sulphur test.

Nitrogen test:

To the filterate, ferrous sulphate was added and boiled. After cooling dil. H2SO4 was added till

a blue precipitate was formed, which confirmed the presence of nitrogen.

Sulphur test:

71

i. To the filterate, sodium nitroprusside and sodium hydroxide were added. The

appearance of purple colour confirmed the presence of sulphur.

ii. To the filterate, excess of acetic acid and lead acetate were added. Black precipitate

confirmed the presence of sulphur.

Test for ester: This was carried out by two methods.

i. Phenolphthalein test:

The compound was dissolved in alcohol, added 2 to 3 drops of alcoholic KOH solution

and one drop of phenolphthalein. The solution was heated gently. Pink colour

discharged showed the presence of ester group.

ii. Ferric hydroxymate test:

To the test compound, 0.5 ml 1N hydroxylamine chloride in rectified spirit was added

along with few drops of NaOH solution. The solution was heated to boiling, gradually

cooled, acidified and two drops of FeCl3 was added. The appearance of purple colour

confirms the presence of Ester group in the compound.

2.6. Microorganisms used for comparative studies:

Reference actinomycete strains used in this study are Streptomyces erumpens (NRRL

B-3163), Streptomyces yatensis (NRRL B-24116), Streptomyces hygroscopicus (NRRL 2387),

Streptomyces malaysiensis (DSM 41697), Streptomyces rimosus (NRRL 2234), Streptomyces

violaceusniger (NRRL-B 1476), Streptomyces sparsogenes (ATCC 25498), Streptomyces

melanosporofaciens (ATCC 25473), Streptomyces platensis (NRRL 8035), Streptomyces

thermodiastaticus (ATCC 27472), Streptomyces rutgersensis (NRRL B-1256), Streptomyces

olivaceiscleroticus (ATCC 15722) and Streptomyces kasugaensis (ATCC 1574).

2.7. In vivo efficacy of streptolipin on experimental animal models: 2.7.1. Effect of single and multiple doses of streptolipin on fat absorption:

72

Mice were used to assess the effect of single and multiple administration of streptolipin

on intestinal fat absorption. Female Swiss albino mice weighing 30-35 g were maintained on

mouse diet. The diet contained (w/w) casein 20%, sucrose 69.8%, refined groundnut oil 5%,

salt mixture 4%, vitamin mixture 1%, choline chloride 0.2%, fat- vitamin mix per kg diet

(retinyl acetate) 600 International Unit (I.U), calciferol 6000 I.U, α-tocopheryl acetate 100 mg.

Two sets of experiments were carried to evaluate the single and multiple dose effect of

streptolipin on faecal triglyceride excretion. One set for single dose was observed for three

days and the other set for multiple dose for seven days. In the multiple dose study, three doses

were administered with each dose on every alternate day. Each set contained five groups, 3

groups for streptolipin, one group for commercially available compound (Orlistat) and one

group for the vehicle control, where each group consisted of ten mice. Streptolipin was

administered orally at 5, 10 and 20 mg/kg body weight and Orlistat was administered at 10

mg/kg body weight. Overnight fasted mice were given streptolipin and Orlistat in a suspension

of 5% gum arabic and 5% defatted milk powder or vehicle solution alone (0.2 ml/animal) and

return to ad-libitum eating. Faeces were collected every day and triglyceride was estimated.

At the end of the feeding period, the animals were starved overnight and sacrificed by

heart puncture under ether anaesthesia. Blood was collected by heart puncture, allowed to clot

and centrifuged for 10 minutes at 2500 rpm to obtain serum. The liver was quickly excised,

washed with ice cold saline, wiped with filter paper, cleaned and weighed. Serum and liver

samples were stored at – 800 C until use for analysis. Small intestine (20-25 cm long segment

between jejunum and caecum leaving about 5 cm on either side) was immediately excised and

flushed with ice cold 0.9% saline. The intestinal segments were then cut open longitudinally

and mucosa was scraped with microscopic slide. The mucosal scrapings were homogenized in

0.9% saline used for lipase assay (Platel and Srinivasan, 1996), washed with ice cold saline and

stored at 40C with saline until use. The intestinal mucosa was scrapped with 2 mM Tris HCl

buffer (pH 8.0) and further analysis was carried out.

2.7.2. Faecal triglycerides:

73

Faeces samples (1 g) were mixed with 1 ml of ethanol and 2 ml of aqueous solution of

gum arabic (20 % w/v) and were homogenized for 5 min and homogenates were used for

estimation of triglyceride.

For the determination of total faecal triglyceride, a 5g aliquot of the homogenate was

boiled with 22 ml of acidified sodium chloride (4.28 mole/litre) for 1 min. after cooling, 40 ml

of amyl alcohol and ethanol (v/v= 1/250) was added and fat extracted with 50 ml of petroleum

ether. A 25 ml aliquot of the petroleum ether layer was evaporated and the residue was

redissolved in 2 ml of ethanol. After adding alcoholic potassium hydroxide, 8 ml (0.11

mol/litre) and 5 drops of thymol blue (15 mmol/ litre), the solution was boiled at 1100C for 20

min. Immediately afterwards, 10 ml of ethanol was added and the amount of potassium

hydroxide not used for saponification was titrated with 0.1 mole/litre hydrochloric acid

(Hartmann et al, 1993).

2.7.3. Lipid Peroxides:

Plasma lipid peroxides were estimated by the fluorimetric measurement of Thiobarbituric

acid complex by the method of Yagi (1984). The fluorimetric measurement was carried out at

an excitation wavelength of 515 nm and emission wavelength of 553 nm and compared with

the standards prepared by reacting 0.5 n mole 1,1,3,3-tetraethoxypropane with TBA reagent.

Lipid peroxides in liver were determined by the method described by Ohkawa et al (1979)

involving photometric measurement of Thiobarbituric acid complex extracted into butanol.

Absorbance of butanol extract was measured at 532 nm. Values were compared with the

tetraethoxypropane, which is used as standard treated similarly.

2.7.4. Antioxidant enzymes:

Five percent liver homogenate was prepared with 0.15 M KCl and centrifuged at 500 X

g for 10 min. The cell-free supernatant was used for the assay of activities of glutathione

peroxidase, catalase and Superoxide dismutase (SOD).

2.7.4.1. Catalase Assay:

74

The catalase assay was carried out according to the method of Aebi (1984). One

milliliter of liver homogenate was added to 1.9 ml of phosphate buffer (50mM, pH 7.4). The

reaction was initiated by the addition of 1 ml of hydrogen peroxide (30mM). Blank without

liver homogenate was prepared with 2.9 ml of phosphate buffer and 1 ml of hydrogen

peroxide. The decrease in optical density due to decomposition of hydrogen peroxide was

measured at the end of 1 min against the blank at 240 nm. Units of catalase were expressed as

the amount of enzyme that decomposes 1µM H2O2 per milligram of protein.

2.7.4.2. Estimation of Superoxide dimutase:

The assay of SOD was based on the reduction of nitro blue tetrazolium (NBT) to water

insoluble blue formazan measured by the method of Beauchamp and Fridovish (1971). Liver

homogenate (0.5 ml) was mixed with 1 ml of 50 mM sodium carbonate, 0.4 ml of 24 µM NBT,

and 0.2 ml of 0.1 mM EDTA. The reaction was initiated by adding 0.4 ml of 1 mM

hydroxylamine hydrochloride. Zero time absorbance was recorded at 560 nm followed by

recording the absorbance after 5 min at 250 C. The control was simultaneously run without

liver homogenate. Units of SOD activity were expressed as the amount of enzyme required to

inhibit the reduction of N—50%. The specific activity was expressed in terms of units per

milligram of protein.

2.7.4.3. Estimation of Glutathione peroxidase:

Glutathione peroxidase assay was carried out according to the method of Nicholos

(1962). Liver homogenate (0.5 ml) was mixed with 1 ml of 10 mM KI solution and 1 ml of

40mM sodium acetate solution. The absorbance of potassium periodide was read at 353 nm,

which indicates the amount of peroxidase. Twenty microliters of hydrogen peroxide (15mM)

was added and the change in the absorbance in 5 min was recorded. Units of peroxidase

activity were expressed as the amount of enzyme required to change the OD by 1 unit per

minute. The specific activity was expressed in terms of units per milligram of protein.

2.7.5. Plasma non-specific enzymes:

75

Alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase

activities were determined in serum by using commercially available test kits (Span Biotech,

India).

2.7.6. Lipid analysis:

Cholesterol in the lipid extracts from plasma and liver was estimated as described by

Searcy and Bergquist (1960). Plasma cholesterol associated with HDL fraction was

determined after precipitation of apolipoprotein-B containing lipoproteins with heparin-

manganese reagent according to the method of Warnick and Albers (1978). Triglycerides in

plasma and liver were determined by the method described by Fletcher (1968) using

triglyceride purifier (Sigma Chemical Co., USA) to remove phospholipids. Phospholipids

were estimated by the ammonium ferrothiocyanate method of Charles and Stewart (1980).

76

3.A.1. Screening of different terrestrial substrates for selective isolation of actinomycetes:

Identification of the area for the collection of soil samples was done randomly.

However, while choosing the terrestrial samples sufficient care was taken to see that the points

of collection had, as widely varying characteristics as possible, with regard to the organic

matter content, moisture content, particle size, color of soil and geographical distribution. Six

samples from Karnataka state, two samples from Andhra Pradesh state, five samples from

Tamilnadu state from India and two soil samples from the desert of Muscat Saudi Arabia, were

collected for the selective isolation of actinomycetes. All the samples were collected into

sterile containers. A brief account of the different samples collected is given in Table 3.1.

3.A.2. Techniques for the isolation of actinomycetes:

Isolation and screening of actinomycetes cultures is being intensively pursued for the

discovery and commercialization of a number of novel antibiotics as they have the capability to

synthesize secondary metabolites (Jong-Gwan et al, 2002). Several methods have been

suggested in literature for the preferential growth and isolation of actinomycetes, of which the

soil dilution plate and soil plate technique (Warcup, 1950; Corke and Chase, 1956; Corbaz et

al, 1963; Porter, 1960; Williams, 1965) are the most widely used methods. Many differential

media for suppressing the growth of fungi and bacteria have been described. By following

these methods, it may be possible to miss some important strains as the bacteriostatic and

fungistatic agents incorporated in the media may also suppress some actinomycetes. However,

the simplest, traditional and the best method of isolation of actinomycetes is the crowded plate

technique and some of the best antibiotic producers have been isolated in this way. Hence, this

method was employed in the present study.

Table 3.1: Different soil samples for screening of actinomycetes:

Sample Nature of the Type of the sample Place of collection

77

code sample

B Grayish free

flowing

Forest soil Coonoor, Tamilnadu

C Shiny, moist

reddish brown

Lake front Ooty, Tamilnadu

V Reddish free

flowing

Tea plantations Ooty, Tamilnadu

S Greenish muddy Sugarcane field soil Manali, Tamilnadu

E Reddish granular Tea plantations Ooty, Tamilnadu

M Brownish

granular

Coconut field soil Nanjangud, Karnataka

T Reddish brown

granular

Sugarcane Mandya, Karnataka

F Reddish, free

flowing

Lalbagh botanical

garden

Bangalore, Karnataka

N Black dry, lumpy Cow barn yard Mysore, Karnataka

K Grayish, muddy Kukkarahalli lake Mysore, Karnataka

G Black, sticky Rice field Naganahalli, Karnataka

A Blackish brown

granular

Hill station Tirumala, Andhra Pradesh

P Black dry, lumpy Sugarcane Tirupathi, Andhra Pradesh

D Light brown

sandy

Desert Muscat, Saudi Arabia

H Light brown

sandy

Desert Muscat, Saudi Arabia

Tsao et al (1960) and Lawrence (1956) have reported that bacterial and fungal

contamination could be reduced by physical treatment (centrifugation and heat treatment) of

the soil sample. Reduction in contamination of bacteria and fungi through chemical treatment

78

(phenol and calcium carbonate) of the soil sample was reported by Nonomura and Ohara

(1969) and Rehacek (1959).

In the present study, both physical and chemical pretreatments were employed for the

isolation of actinomycetes, to decrease the bacterial and fungal contaminants. It was observed

that the soil sediments treated with phenol resulted in complete elimination of fungal growth

but was ineffective against bacterial contamination. The control plates, which were not

pretreated, exhibited overgrowth by bacteria and fungi. The use of heat treatment reduced the

number of bacteria and fungi in isolation plates and number of actinomycetes colonies (Table

3.2).

These results were in concurrence with the results reported by Nonomura and Ohara

(1969), where the heat treatment effectively reduced the outgrowth of the soil sediments.

Pretreatment with calcium carbonate decreased the contamination of bacteria and fungi and the

number of actinomycetes colonies significantly increased, whereas in centrifugation method

the bacterial and fungal contamination was more with significantly less actinomycetes. Also,

there was not much difference between the centrifugation method and control, as the

actinomycetes colonies and contaminants were similar (Table 3.2).

Actinomycetes can be isolated from soil samples and other natural substrates by plating

them on suitable agar media after diluting them appropriately. Actinomycetes colonies, when

grown on suitable media are usually compact and have leathery and dry surface which makes

them to be distinguished from those of fungi and bacteria on dilution plates. Many media,

which preferentially encourage the growth of actinomycetes, have been suggested such as

czapeck dox agar (Lawrence, 1956), egg albumin agar, glucose aspargine agar, half strength

nutrient agar (Lacey and Goodfellow,

Table 3.2: Different pretreatments of various soil samples.

79

Soil Pretreatment Actinomycetes (% of

total microbial

population)

Actinomycetes per

gram of soil

(x 103)

Control 37 27.5

Phenol 56 22

Heat treatment 20 10

Calcium carbonate 96 200

Cow barn yard

Centrifugation 38 32.5

Control 33 32

Phenol 58 24

Heat treatment 25 11


Forest

Centrifugation 34 36

Control 34 39

Phenol 55 28

Heat treatment 26 8.6


Tea plantation

Centrifugation 36 42.5

Control 32 34

Phenol 60 30

Heat treatment 27 9.2


Lake

Centrifugation 38 35

1975), starch casein agar (Kuster and Williams, 1964), nutrient agar, raffinose-histidine

agar, czapeck dox yeast extract casamino acid agar (CYC), Gauze agarized medium No

1(Kuznetsova et al, 1988), Benedict’s modification of Lindenbein’s medium, Humic

acid vitamin agar (Hayakawa and Nonomura, 1987; Hayakawa et al 1991), Potassium

80

tellurite, AV agar (Nonomura and Ohara, 1969) and oatmeal agar (Williams and Cross,

1971).

In the present work four media were employed for selective isolation of actinomycetes.

1. Starch casein agar medium.

2. Oatmeal-agar medium.

3. Potassium tellurite agar medium.

4. Half strength nutrient agar medium.

Out of four media, starch casein agar medium was found to be the best media for isolation

of actinomycetes (Table 3.3) and further work was carried out using the same medium.

Table 3.3: Isolation of actinomycetes on different media:

Medium Actinomycete colonies (x 104)/g soil

Starch casein agar medium 3.2

Oatmeal-agar medium. 2.4

Potassium tellurite agar medium 2.1

Half strength nutrient agar medium. 1.8

The soil samples, following calcium carbonate treatment were plated on starch casein agar

medium. At the end of incubation, the plates were observed for actinomycetes and the selected

colonies were subcultured on the glycerol aspargine agar slants depending up on the colour of

aerial mycelium, reverse colour, soluble pigment and colony texture to the naked eye. Doubtful

colonies were confirmed by microscopic examination. The distribution of actinomycetes in

different samples is shown in Table3.4. Screening of

Figure 3.1: Screening of actinomycetes on starch casein agar:

81

Figure 3.2: Isolated culture slants of actinomycetes:

82

Table 3.4: Distribution of actinomycete isolates in various soil samples:

Samples code Total number of isolated actinomycetes

83

B 20

C 18

V 16

S 2

E 14

M 4

T 3

F 17

N 30

K 39

G 3

A 25

P 7

D 17

H 15

Total isolated: 230

actinomycetes on starch casein agar was shown in Figure 3.1 and some of the isolated culture

slants actinomycetes were shown in Figure 3.2.

84

Table 3.5: Distribution of actinomycete isolates from various samples.

Number of isolates Colour of aerial mycelium

Source

Gray White Red Blue Green Yellow Cream Polymorphic (Pink or orange )

No aerial mycelium

Total Soluble pigment

Desert 15 9 1 3 0 2 0 1 2 33 8 Hill station 13 3 1 0 2 1 0 2 3 25 10 Cow barn yard

3 19 2 1 0 4 0 0 1 30 9

Forest 7 2 3 3 0 2 3 0 0 20 12 Garden 8 7 1 1 17 Lake 30 16 4 2 0 2 0 1 2 57 16 Fields 14 5 0 0 0 0 0 0 0 19 Tea plantations

16 8 0 0 0 2 0 2 1 29 13

Total 106 69 11 9 2 14 4 6 9 230 68 Percentage of total

46.0 30.0 4.78 3.91 0.86 6.08 1.7 2.6 3.9 100 29.56

85

230 isolates recovered from terrestrial samples were distributed in to series

according to color of the mature sporulated aerial mycelium (Table 3.5). Members of the

gray series were found to represent 46 percent of the total number of isolates and the

lowest occurrence

was noted for the green series (0.86%). The highest occurrence of isolates of the gray

series is in agreement with that reported by Ndonde and Semu (2000).

Masmeh (1992) in his study on distribution of Streptomyces flora in Jordan

reported that the white color class dominated the soil samples (43.6%). Table 3.5 shows

that, out of 230 isolates, 68 produced soluble pigment, representing 29.56%. The

differences in color of aerial mycelia of the isolates as well as those of the pigments

produced, may be an indication of the diversity of actinomycetes in the sites investigated.

Almost all the isolates showed vegetative mycelium and aerial hyphae, with a few (4%)

with only the substrate mycelium, which were red and cream colored.

3.A.3. Screening of microorganisms for pancreatic lipase inhibitor production:

Fermentation of the cultures was done by solid state and submerged methods

using wheat bran and potato dextrose broth as media, respectively for fungi. These media

were selected, as earlier reports in our laboratory showed fungal cultures had the ability

to produce novel and new enzyme inhibitors (Rao et al, 2001; 2002a; 2002b; 2002c; 2002d;

2003). The quantity of crude extract from solid state fermentation was more as compared

to submerged fermentation. This might be due the solubility of the components from

wheat bran to ethyl acetate during extraction. However, the concentration of the inhibitor

in solid state fermentation was less, when compared to submerged fermentation. Taking

this into consideration, the crude extract from solid state fermentation was used at higher

concentration for enzyme inhibition studies.

15 fungal cultures from CFTRI culture collection center were screened for lipase

inhibition. Among the 15 fungal isolates screened, the highest activity was shown by

Penicillium sp-1062 followed by Polyporus squamosus-1134 in submerged fermentation,

86

Table 3.6: Primary screening of fungal cultures from CFTRI Culture Collection

Center

for pancreatic lipase inhibition:

%Inhibition of pancreatic lipase

S.No

Culture SmF (at 0.5 mg

crude)

SSF (at 1.0 mg

crude)

1. Aspergillus niger-1038 0.15 0.31




5. Aspergillus niger-CFR-W-

105

2.34 2.16

6. Aspergillus flavus-1058 1.23 1.85

7. Aspergillus carbonarius-1047 5.21 4.55

8. Aspergillus oryzae-1120 1.11 2.35

9. Aspergillus awamori-1042 2.34 5.67


11. Aspergillus sp-CFR-H-105 3.21 2.98

12. Aspergillus sp-CFR-J-105 4.22 7.96

13. Penicillium sp-1062 9.82 8.76

14. Polyporus squamosus-1134 7.97 9.03

15. Fusarium sp-1128 5.34 7.67

87

where as in solid state fermentation, the highest activity was shown by Polyporus

squamosus-1134 followed by Penicillium sp-1062. However, the culture extracts

obtained from both solid state and submerged fermentation showed less than 10%

inhibition (Table 3.6). The results obtained were not convincing. Hence further studies

using these cultures were discontinued.

Primary screening allows the detection and isolation of new microorganisms

exhibiting desired property and remove the unrequired microorganisms on the basis of

relatively simple and fundamental criteria. Secondary screening is strictly essential in

systematic screening programmes intended to select highly potent isolate of the desired

activity. It is very useful in sorting out microorganisms that have real commercial value

from many isolates obtained during primary screening. At the same time, microorganisms

that have poor applicability in a fermentation process are discarded. It provides

information pertaining to the effect of different components of a medium and product

yield potentials of different isolates. It detects gross genetic stability in microbial

cultures. This type of information is very important, since microorganisms tending to

undergo mutation or alteration in some way may lose their capability of maximum

accumulation.

Two hundred and thirty actinomycetes cultures, which were isolated were grown

for seven days and at the end of fermentation each culture was extracted with ethyl

acetate. Ethyl acetate was chosen as extraction solvent because of its immiscible nature

with aqueous phase and its partitioning ability of hydrophobic and hydrophilic

compounds. For sake of brevity, a flow chart is given below for the process of isolation

and screening of actinomycetes (Figure 3.3).

In submerged fermentation, as ethyl acetate extraction alone does not remove all

the metabolites from the fermented broth, the aqueous broth filtrates were also tested for

lipase inhibition. As none of the culture filtrates showed inhibition, further studies were

focused only on inhibitions shown by the ethyl acetate extracts. The biomass was

separated by cheese cloth, the aqueous layer and solvent layer were separated by

separating funnel. The solvent

88

Figure3.3: Isolation and screening of actinomycetes isolates

Soil samples Pretreatment with CaCO3 for 10 days

1g soil sample with 50ml of sterile water Serial dilution

Plating with Starch Casein Agar Medium

Incubated for 3 weeks

Isolation of actinomycetes colonies

Sub-culturing

Fermentation for 8 days in ISP medium

Solvent extraction (Ethyl acetate)

Crude extract dissolved in DMSO 75µg

Inhibition of the target pancreatic lipase enzyme

89

was distilled under reduced pressure and the residue remained after complete distillation

was used as a source of inhibitor. The crude residue was dissolved in DMSO to give an

extract of 7.5 mg/mL and 75µg each of these crude extracts were then tested for lipase

inhibition. The control experiment contained the ethyl acetate extract of the unfermented

broth at the same concentration as the sample.

From a total of 230 strains of actinomycetes isolated from various soil samples,

84 cultures did not exhibit any inhibition against pancreatic lipase and 146 isolates were

found to produce pancreatic lipase enzyme inhibitors. The results indicate that K12, V22,

N62, B80, V41, A56, N2, N18, A37, K44, B18, A21, D4, D7, N21, H5, N46, K10, N32, C4, C18, N35

strains are effective against pancreatic lipase enzyme (Table 3.7). The isolate N2 was

found to be the most potent with 91.8% inhibition followed by A37. Of the 146 active

isolates, 124 cultures exhibited inhibition below 20% and 22 isolates have shown activity

against pancreatic lipase above 20%. Among the 22 strains, 7 cultures were found to have

high potency to inhibit pancreatic lipase (Table 3.8). Isolates showing more than 50%

inhibition were further selected and subjected to secondary screening.

From a total of 230 isolates, 22 isolates were found to produce pancreatic lipase

inhibitors. Among these, 7 were recovered from the soil sediment collected from cow

barnyard, 5 from lakes, 2 from desert, 2 from tea plantations, 3 from hill station, 2 from

forest and 1 from coconut field (Table 3.9). Table 3.10 shows that, only one active

culture was obtained from heat treated sediments, three from sediments pretreated with

phenol and most of the cultures were from calcium carbonate pretreatment. Further, the

greatest number of active strains were isolated from samples, which were pretreated with

calcium carbonate followed by phenol.

The percentage of active isolates also varied within each colour series (Table

3.11). 9.5% of the isolates were active against the target enzyme. The highest number of

isolates exhibiting activity against the target enzyme was from gray series (12) followed

by white series (7) and the percentage inhibitions were 11.3 and 10.1 respectively.

Saadoun et al (1998), studying the antimicrobial activity of isolates from northern Jordan

90

Table 3.7: Primary screening of actinomycetes isolates for pancreatic lipase

inhibition:

S.No Culture number Percent of pancreatic

lipase inhibition at 75 µg

of crude extract

1 B2 Nil 2 B4 12.1 3 B8 Nil 4 B9 18.25 5 B10 12.34 6 B11 Nil 7 B13 17.5 8 B14 18.53 9 B17 16.12 10 B18 25.7 11 B20 10.5 12 B22 Nil 13 B24 Nil 14 B25 5.8 15 B28 Nil 16 B29 19.5 17 B32 Nil 18 B35 Nil 19 B37 7.8 20 B80 52.6 21 C2 18.4 22 C3 Nil 23 C4 35.7 24 C6 Nil 25 C7 10.7 26 C8 16.0 27 C11 Nil 28 C12 19.4 29 C15 8.7

Continued….

91

30 C18 25.8 31 C19 15.7 32 C22 17.8 33 C24 Nil 34 C26 10.4 35 C27 13.6 36 C28 15.8 37 C31 Nil 38 C32 Nil 39 V2 Nil 40 V4 10.46 41 V7 13.93 42 V8 2.83 43 V11 3.36 44 V12 12.55 45 V15 12.39 46 V16 12.35 47 V18 13.93 48 V19 Nil 49 V20 Nil 50 V22 66.6 51 V24 Nil 52 V27 Nil 53 V28 Nil 54 V41 35.9 55 S4 Nil 56 S6 Nil 57 E2 15.8 58 E6 15.2 59 E7 13.6 60 E9 Nil 61 E11 7.9 62 E12 1.5 63 E14 Nil 64 E15 Nil

Continued….

92

65 E16 Nil 66 E18 18.9 67 E21 Nil 68 E22 9.7 69 E24 Nil 70 E27 11.77

71 M9 Nil 72 M11 4.7 73 M23 Nil 74 M30 5.85 75 T3 8.56 76 T7 Nil 77 T14 Nil 78 F3 12.5 79 F5 15.8 80 F6 Nil 81 F7 11.6 82 F9 Nil 83 F11 6.8 84 F12 13.2 85 F15 12.2 86 F16 13.7 87 F19 Nil 88 F21 19.2 89 F23 13.4 90 F24 Nil 91 F26 Nil 92 F29 7.9 93 F30 Nil 94 F35 3.4 95 N1 14.32 96 N2 91.8 97 N8 4.5 98 N7 Nil

Continued….

93

99 N10 Nil 100 N12 Nil 101 N18 26.11 102 N20 14.2 103 N21 26.15 104 N24 Nil 105 N26 Nil 106 N27 Nil 107 N32 30.2 108 N35 25.1 109 N38 10.2 110 N39 7.0 111 N41 15.1 112 N46 25.38 113 N55 10.5 114 N59 Nil 115 N60 6.4 116 N62 61.4 117 N67 Nil 118 N69 9.5 119 N71 13.4 120 N73 Nil 121 N75 17.5 122 N76 7.5 123 N79 18.5 124 N82 33.7 125 K5 9.4 126 K4 Nil 127 K8 Nil 128 K10 33.08 129 K12 72.8 130 K16 Nil 131 K18 11.5 132 K22 10.1

Continued….

94

133 K25 13.07 134 K26 8.8 135 K28 8.88 136 K32 6.2 137 K34 Nil 138 K35 14.62 139 K36 4 140 K39 19.61 141 K40 Nil 142 K41 Nil 143 K44 58.2 144 K45 4.5 145 K46 15.96 146 K47 19.23 147 K49 12.31 148 K50 15.54 149 K51 6.66 150 K53 16.81 151 K55 12.41 152 K56 17.89 153 K57 13.84 154 K59 14.12 155 K60 20.0 156 K61 9.33 157 K64 19.33 158 K67 15.6 159 K72 Nil 160 K77 Nil 161 K81 13.08 162 K82 15.38 163 K83 16.53 164 G21 Nil 165 G36 8.9 166 G38 Nil 167 A2 16.15

Continued….

95

168 A4 11.16 169 A5 13.5 170 A6 13.4 171 A7 Nil 172 A9 Nil 173 A11 16.77 174 A12 18.53 175 A13 15.67 176 A15 5.8 177 A17 Nil 178 A18 14.6 179 A19 13.7 180 A21 26.7 181 A24 26.7 182 A26 Nil 183 A27 9.4 184 A29 Nil 185 A30 11.6 186 A32 Nil 187 A33 18.6 188 A34 25.7 189 A36 Nil 190 A37 80.2 191 A56 45.9 192 P2 12.7 193 P7 Nil 194 P8 Nil 195 P9 17.1 196 P10 11.7 197 P15 Nil 198 P24 Nil 199 D3 7.4 200 D4 25.67 201 D6 Nil 202 D7 31.6

Continued….

96

203 D9 Nil 204 D11 18.2 205 D14 Nil

206 D15 15.9 207 D16 Nil 208 D17 9.4 209 D21 Nil 210 D23 12.7 211 D24 Nil 212 D25 17.5 213 D28 Nil 214 D29 Nil 215 D30 Nil 216 H4 Nil 217 H5 26.53 218 H8 Nil 219 H9 13.7 220 H11 Nil 221 H12 Nil 222 H13 12.5

223 H18 14.9 224 H19 Nil 225 H21 13.6 226 H23 Nil 227 H26 18.7 228 H28 Nil 229 H29 14.8 230 H33 11.7

97

Table 3.8: Classification of actinomycete isolates based on inhibition (%) of

pancreatic lipase:

Inhibition (%) Sample code

Nil 1-10 11-20 21-50 >50

B 8 2 8 1 1

C 6 1 9 2 0

V 6 2 6 1 1

S 2 0 0 0 0

E 6 3 5 0 0

M 2 2 0 1 0

T 2 1 0 0 0

F 6 3 8 0 0

N 9 5 8 5 2

K 8 8 20 1 2

G 2 1 0 0 0

A 7 2 13 2 1

P 4 0 3 0 0

D 9 2 4 2 0

H 7 0 8 0 0

Total 84 32 92 15 7

98

Table 3.9: Distribution of actinomycetes isolates showing inhibition greater than

20% on soil sample type:

Sample code Sample type Positive cultures

B Forest 2

C Lake 2

V Tea plantation 2

M Coconut field 1

N Cow barn yard 7

K Lake 3

A Hill station 3

D Desert 2

Total: 22

Table 3.10: Distribution of actinomycetes isolates showing inhibition greater than

20% on soil pretreatment basis:

Sample code Pretreatment Positive cultures

B Calcium carbonate 2

C Calcium carbonate 2

V Calcium carbonate 2

M Calcium carbonate 1

N Calcium carbonate 5

N Phenol 2

K Phenol 1

K Calcium carbonate 2

A Calcium carbonate 2

A Heat 1

D Calcium carbonate 2

99

Table 3.11: Distribution of active actinomycetes isolates on the basis of colour:

Gray White Red Blue Green Yellow Cream Polymorhica

(pink or orange) No aerial mycelium

Total

Number of isolates 106 69 11 9 2 14 4 6 9 230

Number of active isolates 12 7 0 2 0 1 0 0 0 22

Percentage of active isolate 11.30 10.10 0 22.20 0 7.10 0 0 0 9.50

Results and Discussion

100

soils, found that the higher percentage of active isolates was found in red and gray series and

the lower percentage ones in green and white series. Arai (1976), however reported that most

active species of Streptomyces were found in the gray and yellow series of non chromogenic

type and no antibiotic producing species were described in the white and green series of

chromogenic type.

The comparison of the enzymatic inhibition between all colour classes against the

targeted enzymes showed that the isolate N2 and A37 in the gray series displayed the highest

enzyme inhibition against pancreatic lipase enzyme and no activity was noted in the green, red,

yellow, cream, polymorphic and no aerial mycelium series. The highest percentage of isolates

was found in blue series followed by gray and white. Most of the actinomycetes from gray

series inhibited lipase enzyme followed by white. Isolates in gray series were found to be the

most active against target enzymes. These differences in percentage of enzyme inhibition may

imply that the investigated actinomycetes isolates may produce different bioactive compounds.

In fact, it was observed that in the same colour class, most of the isolates showed a range

enzyme inhibition.

From the preliminary screening, seven actinomycete isolates were selected for

secondary screening, which showed inhibition greater than 50 % and were designated as K12,

V22, B80, N2, N80, A37 and K44 (Table 3.7). Production of pancreatic lipase inhibitor by selected

isolates and their consistency and reproducibility was checked on two different media,

production medium I and production medium II. This approach was useful to detect the effect

of various media components, genetic stability of the microbial culture and the yield.

The cultures were grown in four replicates in submerged fermentation. The inhibition

shown by the four replicates of each culture is represented in Table 3.12. Results indicated that

the isolate N2 not only produced the maximum amount of inhibitor but also showed greater

reproducibility. The remaining cultures K12, V22, B80, N62, A37 and K44 were not as potent as N2.

Production media (PM-II) was found to be the best (Table 3.12). As the inhibition shown by

actinomycete N2 in submerged fermentation were reproducible and more potent than the other

cultures, further studies were carried out using this culture.


101

Table 3.12: Secondary screening of actinomycetes cultures for reproducibility and

consistent production of lipase inhibitor:

Inhibition concentration (µg) to obtain 50% inhibition of pancreatic lipase

Run-I Run-II Run-III Run-IV

Culture

Number

PM-I PM-II PM-I PM-II PM-I PM-II PM-I PM-II

B80 77.01 71.29 76.42 73.03 78.21 72.80 78.84 72.38

V22 61.20 56.30 61.36 55.89 62.40 54.90 62.60 56.89

N2 39.42 33.00 40.01 33.80 40.26 34.20 41.23 32.40

N62 67.12 61.07 67.81 62.12 69.20 61.78 68.60 62.86

K12 58.89 51.51 57.62 53.36 57.98 52.90 59.20 53.42

K44 69.89 64.43 71.02 65.20 71.62 65.60 70.89 66.10

A37 51.28 46.75 53.21 48.20 54.62 47.45 55.16 47.98


102

.

3.B.1. Morphological characters of isolate N2:

The isolate N2 was a gram-positive, non-acid fast, non-motile actinomycete with

extensively branched substrate hyphae (0.3-0.5 µm in diameter). Aerial mycelia form complete

spiral chains of spores with more than 20 spores per chain. The colour of the spores was gray.

The size of the spores ranges from 1.3-1.0 x 1.5 µm, spore surface was warty (Figure 3.4) and

the spores were non motile. No synnemata, sclerotia or sporangia were observed. There were

no distinctive substrate mycelial pigments, diffusible pigments and no sensitivity of substrate

pigment and diffusible pigment to pH was observed. There were no spores on substrate

mycelium (Table 3.13).

3.B.2. Cultural characteristics of isolate N2 on different media:

The growth characteristics of isolate N2 in twelve different media are described in

Table 3.14. The growth was good on all the tested media except on peptone yeast extract iron

agar, nutrient agar and Bennets agar. Gray aerial mycelium was observed on oat meal, starch

casein agar and czapek dox agar, white coloured substrate on starch casein and czapek dox

agar, and dark brown on oat meal agar. White aerial mycelium and colourless substrate

mycelium was observed on nutrient agar. The aerial mycelium was grayish black on peptone

yeast extract iron, tyrosine and glycerol arginine agar, gray substrate mycelium on peptone

yeast extract iron and tyrosine, but colourless on glycerol arginine agar. The growth of isolate

N2 on glycerol aspargine agar was shown in Figure 3.5. Only on Bennet’s agar colourless aerial

and substrate mycelium was observed. The aerial mycelium was grayish white on inorganic

salts and starch, glycerol aspargine agar and potato dextrose agar, but reddish brown substrate

mycelium was observed on inorganic salts and starch, blackish brown substrate on glycerol

aspargine agar and dirty


103

Figure 3.4: Scanning electron micrograph showing N2 warty spores and spiral spore chains.


104

Table 3.13: Morphological characters of isolate N2:

S.No Morphological character

and pigmentation

Observation

1 Spore chain morphology Spirales

2 Spore chain ornamentation Warty

3 Colour of aerial spore mass Gray

4 No distinctive substrate

mycelial pigments

Nil

5 Pigmentation of substrate

mycelium

Nil

6 Production of diffusible

pigments

Nil

7 Sensitivity of substrate

pigment to pH

Nil

8 Sensitivity of diffusible

pigment to pH

Nil

9 Melanin production on

peptone yeast extract iron

agar

Nil

10 Melanin production on

tyrosine agar

Nil

11 Fragmentation of mycelium Nil

12 Sclerotia formation Nil

13 Sporulation on substrate

mycelium

Nil


105

Table 3.14: Cultural characteristics of isolate N2 on different media:

S.No Media Growth Aerial

mycelium

Substrate

mycelium

1 Yeast extract Malt

extract agar

Good Blackish gray Black

2 Oat meal agar Good Gray Dark brown

3 Inorganic salt and

starch agar

Good Grayish white Reddish brown

4 Glycerol aspargine

agar

Good Grayish white Blackish brown

5 Peptone yeast extract

iron agar

Moderate Grayish black Gray

6 Tyrosine agar Good Grayish black Gray

7 Starch casein agar Good Gray White

8 Potato dextrose agar Good Grayish white Dirty green

9 Nutrient agar Moderate White Colorless

10 Glycerol-arginine agar Good Grayish black Colorless

11 Bennets agar Moderate Colorless Colorless

12 Czapek dox agar Good Gray White


106

Figure 3.5: Culture growth on glycerol aspargine agar.


107

green colour on potato dextrose agar. Blackish gray aerial mycelium and black substrate

mycelium was observed on yeast extract malt extract. Soluble pigment was not produced

on any of the tested media. Melanin was also not produced.

3.B.3. Antimicrobial activity of isolate N2:

The culture and its aqueous extract was tested against various microorganisms

(Table 3.15). The test organisms were gram positive and gram negative bacteria,

Streptomyces and fungi. However, the culture did not show any antimicrobial activity

against the tested microorganisms (Table 3.15).

Table 3.15: Antimicrobial activity of isolate N2:

S.No Antimicrobial activity Observation

1 Bacillus subtilis NCIB 3610 Negative

2 Pseudomonas fluorescens

NCIB 9046

Negative

3 Escherichia coli NCIB 9132 Negative

4 Micrococcus luteus NCIB 196 Negative

5 Candida albicans CBS 562 Negative

6 Saccharomyces cerevisiaae

CBS 1171

Negative

7 Streptomyces murinus ISP

5091

Negative

8 Aspergillus niger LIV 131 Negative


108

3.B.4. Enzyme activity tests for isolate N2: Lecithinase, proteolysis, pectin, chitinolysis, hippurate hydrolysis and lipolysis tests:

Chitinolytic activity, proteolysis, lecithinase was detected by the appearance of

zones of clearing around the growth (Table 3.16). There was no zone of hydrolysis for

pectinolytic, hippurate and lipolysis.

Table 3.16: Enzyme activity of isolate N2:

S.No Enzyme activity Observation

1. Lecithinase Positive 2. Pectin Negative 3. Lipolysis Negative 4. Proteolysis Positive 5. Chitin Positive 6. Nitrate reduction Positive 7. Hydrogen sulphide

production Negative

8. Hippurate hydrolysis Negative 9. Milk coagulation Doubtful 10. Milk peptonization Negative

H2S production test:

No characteristic greenish brown, brown, blackish brown, bluish black or black

color of the substrate was observed indicating the absence of H2S production.

Nitrate reduction test:

Pink, red or orange colour was not observed, indicating the test to be negative.

Further, the presence of nitrate was confirmed by adding a pinch of zinc dust after

addition of reagents, wherein pink colour was observed, indicating the presence of nitrate

in the broth.


109


110

3.B.5. Degradation tests of isolate N2:

Clearance of the insoluble compounds around the growth area was scored as

positive for the degradation of adenine, hypoxanthine, xanthine and guanine. There was

no clearance of zone for aesculin, elastin and xylan indicating a negative test (Table

3.17). Blackening of the media was not observed, which indicated a negative test for

arbutin. Tween 20, 40, 60 and 80 plates were observed for opacity along with control.

There was no change in opacity of the plates and the test was negative. Absence of

orange yellow to pink or purple indicated negative result for allantoin. Bluish black or

black diffusible pigment was not observed and the test was negative for tyrosine.

Hydrolyzed zone was observed for both gelatin and starch which indicates the test to be

positive. The organism hydrolyzed urea producing a characteristic red- violet colour on

the plate. As incubation proceeded, the colour extended towards the bottom of the tube

indicating complete hydrolysis.

3.B.6. Antibiotics resistance of isolate N2:

The organism was found to be susceptible to ampicillin (10µg), chloramphenicol

(30µg), erythromycin (15µg), neomycin (30µg), oxytetracycline (30µg), penicillin G (10

IU), rifampicin (10µg), gentamycin (10µg) and streptomycin (10µg), but not susceptible

to kanamycin (30µg) (Table 3.18).

3.B.7. Effect of temperature and pH on growth of isolate N2:

Good growth was observed between 10 to 370C, no growth was observed at 40C

and poor growth was observed at 450C (Table 3.19). There was no growth at pH 2.0 and

the growth was good between pH 5 to 11 (Table 3.20).

3.B.8. Growth of isolate N2 in the presence of inhibitory compounds:

The growth was good in the presence of sodium chloride upto a concentration of

5% (w/v) after which there was no growth, on further increase in sodium chloride


111

concentration upto 13% (w/v). The growth was good on sodium azide, poor on phenol

and there was no growth in the presence of potassium tellurite, thallous acetate and

crystal violet (Table 3.21).

The chromogenic Streptomycetes (melanin producers) tended to be less tolerant to

sodium chloride than the non chromogenic species. Similarly, smooth spored species

were less tolerant than spiny spored forms. Gray coloured species of Streptomycetes were

more tolerable when compared with other colour series (Tresner et al, 1968). 50% of all

Streptomycetes fall in to low or high tolerance group, which is of considerable taxonomic

significance.

3.B.9. Test for carbon source utilization by isolate N2:

The ability of N2 to grow on the test media varied considerably. The growth in

control tube showed very little or no growth and those tubes in which isolates could

effectively utilize the particular carbon source had very profuse growth. Very slight

growth was observed with some carbon sources, indicating that the particular compound

was not adequate source of carbon in that concentration or that the materials used

contained traces of other compounds. The utilization of carbon sources by the isolate N2

is shown in Table 3.22.

The results recorded as follows:

Utilization positive: when growth on tested carbon source was significantly better than

on the basal medium without carbon source but some what less than on glucose.

Utilization doubtful: when growth on tested carbon source was only slightly better than

on the basal medium without carbon source and significantly less than with glucose.

Utilization negative: when growth was similar or less than growth on basal medium

without carbon source.

The isolate utilizes L-arabinose, starch, sorbitol, D-glucose, D-fructose, D-xylose,

meso-inositol, D-mannitol, L-rhamnose, maltose, D-mannose, D-lactose, trehalose, D-

melibiose, dextran, D-sorbitol, adonitol, D-galactose, cellobiose for growth, but not

sucrose, inulin, sodium malonate, sodium benzoate and sodium tartarate. Little or poor

growth was


112

Table 3.17: Degradation of various compounds by isolate N2:

S.No Degradation of

Observation

1 Hypoxanthine Degraded

2 Guanine Degraded

3 L- Tyrosine No degradation

4 Elastin No degradation

5 Adenine Degraded

6 Xanthine Degraded

7 Tween 20 No degradation




11 Starch Degraded

12 Xylan Degraded

14 Urea Degraded

15 Allantoin No degradation

16 Gelatin Degraded

17 Aesculin No degradation

18 Arbutin No degradation


113

Table 3.18: Resistance of isolate N2 to antibiotics:

S.No Antibiotic Observation

1. Ampicillin (10µg) Not resistant

2. Chloramphenicol (30µg) Not resistant

3. Erythromycin (15µg) Not resistant

4. Neomycin (30µg) Not resistant

5. Oxytetracycline (30µg) Not resistant

6. Penicillin G (10 IU) Not resistant

7. Rifampicin (10µg) Not resistant

8. Gentamycin (10µg) Not resistant

9. Streptomycin (10µg) Not resistant

10. Kanamycin (30µg) Resistant


114

Table 3.19: Growth of isolate N2 at different temperatures:

S.No Temperature (0C) Observation

1 4 No growth

2 10 Good growth

3 20 Good growth

4 28 Good growth

5 37 Good growth

6 45 Doubtful growth

Table 3.20: Growth of isolate N2 at different pH

S.No PH Observation

1 2 No growth

2 5 Good growth

3 7 Good growth

4 9 Good growth

5 11 Good growth


115

Table 3.21: Growth of isolate N2 in the presence of inhibitory compounds:

S.No Inhibitory compound

(% w/v)

Observation

1 Sodium chloride (1) Good growth



4 Sodium chloride (7) No growth




8 Sodium azide (0.01) Good growth

9 Sodium azide (0.02) Good growth

10 Phenol (0.1) Doubtful growth

11 Potassium tellurite (0.001) No growth

12 Potassium tellurite (0.01) No growth

13 Thallous acetate (0.001) No growth

14 Thallous acetate (0.01) No growth

15 Crystal violet (0.0001) No growth


116

Table 3.22: Growth of isolate N2 on different carbon sources:

S.No Carbon source (1.0% w/v) Observation

1 L-Arabinose Positive

2 Cellulose Negative

3 Sucrose Negative

4 Starch Positive

5 Sorbitol Positive

6 D-Xylose Positive

7 Meso-inositol Positive

8 Mannitol Positive

9 D-Fructose Positive

10 D-Glucose Positive

11 L-Rhamnose Positive

12 Raffinose Negative

13 Maltose Positive

14 D-Mannose Positive

15 D-Lactose Positive

16 Inulin Negative

17 Trehalose Positive

18 D-Melibiose Positive

19 Dextran Positive

20 D-Galactose Positive

21 D-sorbitol Positive

22 Adonitol Positive

23 Cellobiose Positive

24 Xylitol Doubtful

25 Sodium acetate (0.1% w/v) Doubtful

26 Sodium citrate (0.1% w/v) Doubtful

27 Sodium malonate (0/1% w/v) Negative

28 Sodium propionate (0.1% w/v) Doubtful

29 Sodium pyruvate (0.1% w/v) Doubtful

30 Sodium benzoate (0.1% w/v) Negative

31 Sodium tartarate (0.1% w/v) Negative


117

observed with cellulose, xylitol, sodium acetate, sodium citrate, sodium propionate and

sodium pyruvate.

3.B.10. Test for nitrogen utilization by isolate N2:

Only slight growth was observed with some compounds, indicating that the

particular compound was not adequate source of nitrogen in that concentration or that the

compounds used contained traces of other compounds and results are shown in Table

3.23. The isolate was found to utilize alanine, L-dopa, L-leucine, glycine, ornithine

mono HCl, tyrosine, glutamic acid, tryptophan, L-hydroxy proline, L-histidine, L-

methionine, L-lysine, L-serine, L-threonine, L-cysteine, potassium nitrate and DL-

amino-n-butyric acid for growth, but not aspartic acid. Little or poor growth was

observed with L-valine, L-arginine and L-phenylalanine.

3.B.11. Test for production of acid and gas by isolate N2:

Production of acid was observed on starch, melibiose, inositol, arabinose, lactose,

rhamnose, fructose, glucose, galactose, maltose, inulin and xylitol. Production of acid

was not observed on mannose, xylose, cellulose, sucrose, sodium acetate and sodium

citrate. However, on none of the carbohydrates gas was produced (Table 3.24).

3.B.12. Chemotaxonomic characteristics of isolate N2:

3.B.12.1.Test for sugars and amino acids:

The extract of isolate N2 contains levo- diaminopimelic acid, which was

confirmed by comparison with the sigma standard samples of meso-diaminopimelic acid

and levo- diaminopimelic acid on the TLC plate.

The carbohydrates migrated in the following sequence from the origin (slowest to

fastest) galactose, glucose, arabinose, mannose, xylose, ribose and rhamnose. Yellowish,


118

brown and maroon coloured spots were not observed indicating the absence of hexoses

and pentoses in the cell wall composition of isolate N2. It was concluded that strain N2

has levo- diaminopimelic acid, no characteristic sugar was detectable in the cell wall

(wall chemotype I) (Lechevalier and Lechevalier, 1970) which is a characteristic feature

of Streptomyces.

3.B.12.2. Test for menaquinones:

Peaks corresponding to molecular ions (790, 792 and 788) were found in the mass

spectra of menaquinones isolated from strain (Figure 3.6). From the mass spectra, major

menaquinones detected were MK-9(H8), MK-9(H6) and MK-9(H4) which is a

characteristic feature of Streptomyces.

3.B.12.3. Test for mycolic acid:

The sample was compared with the standard sigma sample of mycolic acid. The

results indicated it to be absent, which is a characteristic feature of Streptomyces.

3.B.12.4. Test for fatty acids:

The resultant peaks were identified with a mixture of standard methyl esters with

gas chromatography. The cellular fatty acids consisted of 12-methyltetradeconic acid (ai-

15:0), hexadecanoic acid (16:0), 14-methylhexadecanoic acid (ai-17:0) and 14-

methylpentadecanoic acid (I-16:0) (Figure 3.7), which is a characteristic feature of

Streptomyces (Kroppenstedt, 1985).

3.B.12.5. Test for phospholipids:

All polar lipids appeared as dark spots on a light green ground

molybdophosphoric acid. Lipids containing amino groups appeared as pink-red spots

with Ninhydrin. Phosphorus containing lipids appeared as blue spots on a white back

ground with Zinzadze reagent.


119

Table 3.23: Growth of isolate N2 on sole nitrogen source:

S.No Nitrogen source

(0.1% w/v)

Observation

1 DL-α-Amino-n-butyric acid Good growth

2 Potassium nitrate Good growth

3 L-Cysteine Good growth

4 L-Valine Doubtful growth

5 L-Threonine Good growth

6 L-Serine Good growth

7 L-Phenylalanine Doubtful growth

8 L-Lysine Good growth

9 L-Methiionine Good growth

10 L-Histidine Good growth

11 L-Arginine Doubtful growth

12 L-Hydroxy proline Good growth

13 Tryptophan Good growth

14 Glutamic acid Good growth

15 Tyrosine Good growth

16 Ornithine mono HCl Good growth

17 Glycine Good growth

18 L-Leucine Good growth

19 Aspartic acid Good growth

20 Dopa Good growth

21 Alanine Good growth


120

Table 3.24: Production of acid and gas by isolate N2:

S.No Carbon source

(1% w/v)

Acid Gas

1 Starch Positive Negative

2 Melibiose Positive Negative

3 Meso-inositol Positive Negative

4 Arabinose Positive Negative

5 Mannose Negative Negative

6 Xylose Negative Negative

7 Mannitol Negative Negative

8 Lactose Positive Negative

9 Tyrosine Positive Negative

10 Rhamnose Positive Negative

11 Fructose Positive Negative

12 Glucose Positive Negative

13 Galactose Positive Negative

14 Maltose Positive Negative

15 Inulin Positive Negative

16 Xylitol Positive Negative

17 Sodium acetate Negative Negative

18 Sucrose Negative Negative

19 Sodium citrate Negative Negative

20 Cellulose Negative Negative


121

Glycolipids appeared as brown spots with α-naphthol. By comparing with the standard it

was observed that the sample contains the type II phospholipids, namely

phosphatidylinositol and phosphatidylmethy-lethanolamine (Lechevalier et al, 1977),

which is a characteristic feature of Streptomyces.

3.B.13. Phylogenetic analysis of isolate N2:

The assignment of N2 to the genus Streptomyces was also supported by the 16S

rRNA gene sequence analysis of N2. The almost complete sequence of 16S rRNA gene

(1464 nt) of N2 following BLAST analysis indicated that N2 is related to species of

Streptomyces ranging from a minimum of 95% [as in S. venezuelae (AB045890), S.

subrutilus (X80825), S. bikiniensis (X79851) and S. bottropensis (D63868)] to a

maximum of 99% [as in S. violceusniger (AJ391822)] (Table 3.25). There are many

other species to which N2 has a similarity ranging between 97 to 98% at the rRNA gene

level (Table 3.25), To further analyse the phylogenetic relationship of N2 a phylogenetic

tree was constructed using NEIGHBOR JOINING and DNAPARS and the results clearly

indicated that N2 falls within the genus Streptomyces (Figure 3.8). It is obvious from

Figure 3.8 that N2 forms a sub clade along with S. violaceusniger (AJ391822), with

which it is closely related (99%) and four other species namely S. malayensis

(AFF117304), S. yatensis (AFF336800), S. melanosporofaciens (AJ391837) and S.

rutgersensis (AY508511). These four species are related to N2 by 98% at the 16S rRNA

gene level and as anticipated form one clade with high bootstrap values (64 to 96%). Five

other species namely S. hygroscopicus (AB045864), S. kasugaensis (AB02442), S.

cebimarensis (AJ560629), S. erumpens (AJ621603) and S. rimosus (AB045883) despite

having 98% similarity with N2 were distributed in different clades and appeared to be

distanced away from the N2 clade and their grouping was not supported by high bootstrap

values. N2 could be differentiated from all the species having more than 97% similarity at

rRNA gene level based on a number of phenotypic and chemotaxonomic characteristics

as listed in Table 3.26. Table 3.27 lists the phenotypic differences between N2 and S.

violaceusniger with which it exhibits 99% similarity. Thus based on the overwhelming

differences in morphological and chemotaxonomic features and


122

Figure 3.6: Mass spectra of menaquinones isolated from N2


123

Figure 3.7: Cellular fatty acids of the isolate N2


124

16S ribosomal RNA gene partial sequence Working GenBank flatfiles:

LOCUS AY950450 1464 bp DNA linear BCT 21-MAR-2005

DEFINITION Streptomyces vayuensis 16S ribosomal RNA gene, partial sequence.

ACCESSION AY950450 VERSION AY950450

KEYWORDS .

SOURCE Streptomyces vayuensis

ORGANISM Streptomyces vayuensis

Bacteria; Actinobacteria; Actinobacteridae; Actinomycetales; Streptomycineae;

Streptomycetaceae; Streptomyces.

REFERENCE 1 (bases 1 to 1464)

AUTHORS Shivaji,S., Prabagaran,S.R., Naveen Babu,K. and Sattur,A.P.

TITLE Streptomyces vayuensis sp. nov.: a new species of the genus Streptomyces

JOURNAL Unpublished

REFERENCE 2 (bases 1 to 1464)

AUTHORS Shivaji,S., Prabagaran,S.R., Naveen Babu,K. and Sattur,A.P.

TITLE Direct Submission

JOURNAL Submitted (02-MAR-2005) Microbiology, Centre for Cellular and

Molecular Biology, Uppal Road, Hyderabad, AP 500007, India

FEATURES Location/Qualifiers

Source 1..1464

/organism="Streptomyces vayuensis"

/mol_type="genomic DNA"

/db_xref="taxon:319468"

/note="taxonomic information"

rRNA <1..>1464

/product="16S ribosomal RNA"


125

1 tttgagtttt gatcctggct caggacgaac gctggcggcg tgcttaacac atgcaagtcg

61 aacgatgaac cggtttcggc cggggattag tggcgaacgg gtgagtaaca cgtgggcaat

121 ctgccctgca ctctgggaca agccctggaa acggggtcta ataccggata cgactgccga

181 ccgcatggtc tggtggtgga aagctccggc ggtgcaggat gagcccgcgg cctatcagct

241 tgttggtggg gtgatggcct accaaggcga cgacgggtag ccggcctgag agggcgaccg

301 gccacactgg gactgagaca cggcccagac tcctacggga ggcagcagtg gggaatattg

361 cacaatgggc gcaagcctga tgcagcgacg ccgcgtgagg gatgacggcc ttcgggttgt

421 aaacctcttt cagcagggaa gaagcgcaag tgacggtacc tgcagaagaa gcgccggcta

481 actacgtgcc agcagccgcg gtaatacgta gggcgcaagc gttgtccgga attattgggc

541 gtaaagagct cgtaggcggc ttgtcgcgtc ggatgtgaaa gcccggggct taactcccgg

601 gtctgcattc gatacgggca ggctagagtt cggtagggga gatcggaatt cctggtgtag

661 cggtgaaatg cgcagatatc aggaggaaca ccggtggcga aggcggatct ctgggccgat

721 actgacgctg aggagcgaaa gcgtggggag cgaacaggat tagataccct ggtagtccac

781 gccgtaaacg ttgggaacta ggtgtgggcg acattccacg ttgtccgtgc cgcagctaac

841 gcattaagtt ccccgcctgg ggagtacggc cgcaaggcta aaactcaaag gaattgacgg

901 gggcccgcac aagcggcgga gcatgtggct taattcgacg caacgcgaag aaccttacca

961 aggcttgaca tacaccggaa acatccagag atgggtgccc ccttgtggtc ggtgtacagg

1021 tggtgcatgg ctgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg

1081 caacccttgt tctgtgttgc cagcatgcct ttcggggtga tggggactca caggagactg

1141 ccggggtcaa ctcggaggaa ggtggggacg acgtcaagtc atcatgcccc ttatgtcttg

1201 ggctgcacac gtgctacaat ggccggtaca atgagctgcg aagccgtgag gtggagcgaa

1261 tctcaaaaag ccggtctcag ttcggattgg ggtctgcaac tcgaccccat gaagtcggag

1321 tcgctagtaa tcgcagatca gcattgctgc ggtgaatacg ttcccgggcc ttgtacacac

1381 cgccccgtca cgtcacgaaa gtcggtaaca cccgaagccg gtggcccaac ccttgtggag

1441 ggagccgtcg aatgtgggac tggc


126

Table 3.25: rRNA gene sequence similarity of Streptomyces vayuensis sp. nov.

(strain

N2) with species of Streptomyces exhibiting > 95% similarity at the

rRNA gene level as determined by BLAST

Species RNA gene similarity homology (%)

Strain Accession no.

S. violaceusniger 99 NRRL B-1476 AJ391822 S. hygroscopicus 98 IFO 13598 AB045864 S. yatensis 98 DSM 41771 AF336800 S. kasugaensis 98 MB273-C4 AB024442 S. rutgersensis 98 DSM 40830 AY508511 S. cebimarensis 98 DSM 41798 AJ560629 S. erumpens 98 DSM40941 AJ621603 S. rimosus 98 JCM 4667 AB045883 S. malaysiensis 98 ATB-11 Af117304 S. melanosporofaciens 97.8 NRRL B-12234 AJ391837 S. platensis 97.7 SAFN-030 AY167807 S. sclerotialus 97.6 DSM 43032 AJ621608 S. mashuensis 97.5 DSM40221 X79323 S. griseocarneus 97.5 DSM40004 X99943 S. catenulae 97.5 DSM 40258 AJ621613 S. tubercidicus 97.5 DSM40261 AJ621612

S. niger 97.4 DSM 43049 AJ621607 S. olivaceiscleroticus 97.4 DSM 40595 AJ621606 S. sparsogenes 97.3 NRRL 2940 AJ391817 S. peucetius 97.1 JCM 9920 AB045887 S. thermodiastaticus 97.0 DSM40573 Z68101 S. yunnanensis 97.0 YIM 41004 AF346818 S. cattleya 97.0 JCM 4925 AB045870 S. thermocoprophilus 97.0 B19 AJ007402 S. lydicus 96 ATCC 25470 Y15507 S .sampsonii 96 DSM 40394 Z76680 S. thermoviolaceus 96 DSM 41392 Z68095 S. griseus 96 IFO 13550 AB045867 S. yeochonensis 96 CN 732 AF101415 S. somaliensis 96 DSM 40738 AJ007403 S. venezuelae 95 NRRL2277 AB045890 S. subrutilus 95 ATCC27467 X80825 S. bikiniensis 95 ATCC11062 X79851 S. bottropensis 95 ATCC25435 D63868


127

S t r a i n N 2

Figure 3.8: Neighbour-joining tree based on 16S rRNA gene sequences showing the

phylogenetic relationship between Streptomyces vayuensis sp. nov. and other species of

the genus Streptomyces and related reference microorganisms. Bootstrap values

(expressed as percentages of 1000 replications) greater than 50% are given at the nodes.


128

the phylogenetic analysis it is proposed to assign N2 the status of a new species for which

the name Streptomyces vayuensis sp. nov. is proposed.

3.B.14. Comparative studies of isolate N2:

Isolate N2 was compared with 19 similar strains and the comparative studies were

shown in Table 3.26. Although the N2 isolate was closely related to Streptomyces

violaceusniger at species level, however, it differed with the isolate on counts of, the

spore surface of N2 isolate was warty, where it was rugose in case S. violaceusniger. The

isolate N2 cannot utilize both raffinose and sucrose, but S. violaceusniger utilizes. S.

violaceusniger degrades both arbutin and tyrosine, whereas N2 isolate did not degrade. S.

violaceusniger hydrolyzes esculin, whereas N2 isolate did not hydrolyze. S.

violaceusniger shows antifungal activity against Aspergillus niger whereas N2 isolate did

not show the antifungal activity. Growth of the isolate N2 was good at 10 and 450C and

there was no growth in case of S. violaceusniger at both these temperatures (Table 3.27).

It is evident from the chemical, molecular, systematic and phenotypic data that the isolate

N2 should be given species status in the genus Streptomyces (Waksman and Henrici,

1943). Therefore, the name Streptomyces vayuensis is proposed for the isolate N2.

3.B.15. Final and brief description of isolate N2 as Streptomyces vayuensis sp.nov:

Streptomyces vayuensis sp. nov, gram-positive, non-acid fast, non motile

actinomycete with extensively branched substrate hyphae (0.3-0.5 µm in diameter).

Aerial mycelia form complete spiral chains of spores with more than 20 spores per chain.

The size of the spores ranges from 1.3-1.0 x 1.5 µm, spore surface was warty and the

spores are non motile. No symnemata, sclerotia or sporangia are observed. The reverse

sides of colonies are colorless/gray/reddish brown. The colour of the aerial and substrate

mycelia varied depending on the growth medium.

Melanin and soluble pigments were not produced on any media tested. Grows

between 10-37°C and pH 5-13. Tolerates upto 5% NaCl. No hydrolysis of starch, no


129

production of hydrogen sulphide, liquefies gelatin, coagulates milk and is positive for

urease but negative for catalase and is not reduced to nitrite. Degrades casein, xanthine,

adenine, hypoxanthine and guanine but not tyrosine, tryptophan, Tween 20, 40, 60, 80,

xylan, allantoin, aesculin and arbutin. Produces acid from L-arabinose, galactose,

cellulose, starch, D-melibiose, meso-inositol, D-lactose, tyrosine, L-rhamnose, D-

fructose, D-glucose, maltose, inulin, Xylitol and no acid production from mannose,

xylose, mannitol and sucrose. Cells are susceptible to ampicillin, chloramphenicol,

erythromycin, neomycin, oxytetracycline, penicillin G, rifamycin, gentamycin and

streptomycin; but negative to kanamycin. Utilizes L-arabinose, starch, sorbitol, D-

glucose, D-fructose, D-xylose, meso-inositol, D-mannitol, L-rhamnose, maltose, D-

mannose, D-lactose, trehalose, D-melibiose, dextran, D-galactose, cellobiose, alanine, L-

dopa, L-leucine, glycine, ornithine mono HCl, tyrosine, glutamic acid, tryptophan, L-

hydroxy proline, L-histidine, L-methionine, L-lysine, L-serine, L-threonine, L-cysteine,

potassium nitrate and DL- amino-n-butyric acid for growth, but not sucrose, inulin,

sodium malonate, aspartic acid and adonitol. Little or poor growth is observed with

cellulose, xylitol, sodium acetate, sodium citrate, sodium propionate, sodium pyruvate,

raffinose, L-valine, L-arginine and L-phenylalanine. Mycolic acids were absent. Major

menaquinones were MK-9(H6), MK-9(H8) and MK-9(H4). Cellular fatty acids consisted

of 12-methyltetradeconic acid, hexadecanoic acid, 14-methylhexadecanoic acid, 14-

methylpentadecanoic acid. The strain Streptomyces vayuensis has been deposited in

Microbial Type Culture Collection MTCC at IMTECH, Chandigarh with accession

number MTCC 5219.


130

Table 3.26: Phenotypic and chemotaxonomic characteristics that differentiate Streptomyces vayuensis sp. nov. (strain N2) from the closely related species of the genus Streptomyces

Test 1 2* 3* 4* 5* 6* 7* 8* 9* 10* 11* 12* 13* 14* 15 16* 17 18 19 20 Carbon utilization L-arabinose + + + + + - - + + + d - d + d d - + - d D-fructose + + + + + + d + + + d + d + d d + + d d D-galactose + + + + + + + + + + d - d + d d d + d d Meso-inositol + + + + + + + + + + - + + + - + + + - D-mannitol + + + + + - + + + + + + + + + + + - + + Raffinose + + + - + + + + - + d + d + - + + + - - L-Rhamnose + + + + + - + + + + + + - - - + - - - - Sucrose - + - + + - - - - + d + + - + d + - - +

D-xylose - + + + + - + + + + d + d - d d + - d d Enzymatic activity Hippurate - - + - - - - - - - d - - + d d d - d d Esculin + + + + + + + + - - - - d + d d d - d d Allantoin - - + - - - - - - - - - d + d d d - d d Hydrogen sulphide production

- - + - - - - - - - + - - - + + d - d +

Nitrate reduction - - - + - + + - - - - - - + - - d - d - Urea utilization + - - + + + - + - d - d + d d d - d d Starch hydrolysis + - + - + - + + + d + + d d d + d d Degradation Adenine + + - + + + + + + - d - d + d d d d d d Arbutin + + + + + - + - - - + - + + + + d d d + Elastin + + - - + + - - - - + + + + + + + + Xylan + + + + - - - - - - d - d + d d d d d d


131

Tween-80 + + + + - - - - - - d - d + d d d d d d Xanthine - - - + - - - - + - + - - + + + + + + L-tyrosine - + + + + + + + - - - + d d d d d d d Cellulose hydrolysis - - - - - - - - - - d - - d d d d d d d Gelatin liquefaction + + + + + + + + + - d - + d d d d d d d Milk coagulation - - - - - + - - - - - - - d d d d d d Milk peptonization + - - + - + - - - - - - - d d d d d d Melanin - - - - - - - + - - - - - - - - - - - - Melanin - - - - - - - + - - - - - - - - - - - - Antimicrobial activity

Bacillus subtilis - - - - - - - + - - - - + + - - d - - - Candida albicans - - + - - - - + - - - - - + + - d d - + Aspergillus niger + + + + + + + + - - - + - + + - d d - + Growth at (°C) 10 - - - - - - - - + + - + - + + d d d d d 45 - - - - - - - - + + - + - + - + - - - Sodium chloride 7% w/v

- - - - - - - - + + + + + + + + d d d d

+: good growth, -: no growth, ± : doubtful, d: data not available * Present study The numbers in the top row of the table represent characteristics of the following species: 1, S. yunnanensis (Qi Zhang et al., 2003); 2, S.hygroscopicus; 3, S. melanosporofaciens; 4, S. sparsogenes; 5, S. violaceusniger; 6, S. kasugaensis; 7, S. yatensis; 8, S. malaysiensis; 9, Strain N2; 10, S. erumpens; 11, S. thermodiastaticus; 12, S. olivaceiscleroticus; 13, S. platensis; 14, S. rimosus; 15, S. niger (Goodfellow et al, 1986c); 16, S. rutgersensis; 17, S. peucetius (Grein et al, 1963); 18, S. tubercidicus (Nakamura, 1961); 19, S. catenulae (Williams et al, 1989); 20, S. selerotialus (Williams et al, 1989).


132

Table 3.27: Differences in the phenotypic and chemotaxonomic features of

isolate N2 and S. violaceusniger, the phylogenetically nearest neighbour.

Characteristics Isolate N2 S. violaceusniger Spore surface Warty Rugose Utilization of raffinose Negative Positive Utilization of sucrose Negative Positive Esculin hydrolysis Negative Positive Degradation of arbutin Negative Positive Degradation of L-tyrosine Negative Positive Antimicrobial activity on Aspergillus niger Negative Positive Growth at 10°C Positive Negative Growth at 45°C Positive Negative


133

3.C.1. Isolation and purification of pancreatic lipase inhibitor:

The isolation of streptolipin was carried out by using of porcine pancreatic lipase assay

(Figure 3.9). At the end of fermentation, the biomass was separated from 6.5 litres cultured

broth by centrifugation and dried at 300C. The biomass (40 g) was then ground to small

granules to which ethyl acetate (1.6 litres) was added and kept on rotary shaker for two hours.

The organic layer was separated by centrifugation and concentrated under reduced pressure to

obtain crude extract (6.02 g). The crude extract was loaded on to silica gel column

chromatography and eluted successively with hexane, chloroform and methanol and their

combinations. The active fraction in hexane: chloroform (1:1) eluted as an yellow eluent

which upon concentration under reduced pressure (4.3 g) and was further chromatographed on

silica gel column chromatography with hexane and ethyl acetate at different ratios of

increasing polarity. The active fraction eluted as colourless fraction (2.6 g) and was further

chromatographed on Sephadex LH-20 with methanol. The active fractions were pooled (1.68

g) and further purified by preparative TLC using benzene: ethyl acetate (7:3) and benzene:

methanol (9:1) and benzene. The inhibitor was extracted from silica with diethyl ether to obtain

an off white solid (26.2 mg). The purified compound moved as a single spot on silica gel F254

with an Rf 0.34 in benzene: methanol (9:1). The structure was accomplished with this

compound.

3.C.2. Adaptation of the PNPB spectrophotometric assay to TLC system:

During the purification of the compound the spectrophotometric assay was adapted to

the TLC system. The adaptation to the TLC method was as follows: The principle of the assay

is that the substrate, p-nitrophenyl butyrate is hydrolyzed by lipase to give a p-nitrophenol

(yellow colour). The presence of the inhibitor is indicated by the non action of the enzyme on

the substrate and thereby appearing as a colourless spot. The inhibitor was run on a TLC plate

with a mobile phase Benzene: Methanol (9:1). The plate was then air dried to remove the traces

of solvent. After complete evaporation of the solvent, pancreatic lipase enzyme was sprayed

on the TLC plate at a concentration of 20 mg / ml with sprayer and incubated at 300C for 3

minutes, after which the substrate para-


134

Figure 3.9: Purification protocol of lipase inhibitor

Fermented broth (6.5 L) Centrifugation

Mycelium (40 g)

Ethyl acetate extraction

Crude extract (6.0 g)

Silica gel(#60-120) (4.3 g) Hexane: CHCl3 (1:1)

Silica gel(#60-120) (2.6 g)

Hexane: ethyl acetate (9:1)

Sephadex LH-20 (1.689 g) Methanol 40 fractions

12 active fractions checked on TLC Preparative TLC (1.2 g) Benzene

Benzene: methanol (9:1) Benzene: ethyl acetate (7:3)

Pure compound (26.2 mg)


135

nitro phenyl butyrate (100 mM) was sprayed and incubated again at 300C for 3 minutes. The

background of the plate was yellow and the inhibitor appears as a white spot. The procedure in

the form of a flow sheet is given in Figure 3.10.

Lipase Spectrophotometer Assay:

Lipase +

PNPB PNP Butyric acid

3.C.3. Physico-chemical properties of streptolipin

Streptolipin is an off white solid. It is freely soluble in chloroform, dimethyl sulphoxide

and acetone (Table 3.29). It is sparingly soluble in methanol, acetonitrile, diethyl ether and

ethyl acetate and insoluble in hexane, various buffers [phosphate buffer (pH 7.4 to 8.0), acetate

buffer (pH 5.0 to 6.0), trisbuffer (pH 8.0 to 9.0)], aqueous solutions [5% NaOH, 5% acetic

acid, 5% sodium bicarbonate] and water. The inhibitor decomposes at 184.4oC. The λ max nm

(ξ) in methanol 210 (21,752), 260 (11,400). HPLC analysis shows retention of 19 minutes on a

RP-C18 column with a gradient mobile phase of acetonitrile and water at 210 nm. Elemental

analysis: theoretical C, 67.86; H, 9.67; N, 3.68; P, 4.07; S, 4.21 found C, 67.02; H, 9.86; N,

3.94; P, 3.89; S, 4.36. IR (ν values in cm-1): 1434, 1312, 1046, 954, 761 for quinoxaline, 3436,

2913, 1771,1714, 1659, 1236, 667 (Figure 3.11). The molecular formula of the compound was

calculated as C43H73N2O5SP, based on the mass spectra and 1H and 13C NMR spectra. 1H and 13C NMR data (Figure 3.12,3.13, 3.14 and Table 3.26A). EI-MS m/z: 761 (M+),

Figure 3.10: Adaptation of the PNPB (p-nitrophenylbutyrate) spectrophotometric assay

to TLC system

O

NO2

C

O

(CH2)2 CH3

NO2

OH

CH3 (CH2)2 C

O

OH


136

TLC Plate with Inhibitor

Benzene: Methanol (9:1)

Sprayed with pancreatic lipase enzyme (20 mg/ ml)

Incubated for 3 min at 300C

Sprayed with Substrate (PNPB, 100 mM)

Incubated for 3 min at 300C

Inhibitor and Background

(white) (yellow)


137

Figure 3.11 IR spectrum of inhibitor, isolated from Streptomyces vayuensis.


138

Figure 3.12: 1H NMR spectrum of inhibitor from Streptomyces vayuensis


139

Figure 3.13: 13C NMR spectrum of inhibitor from Streptomyces vayuensis


140

Figure 3.14: LCMS of inhibitor from Streptomyces vayuensis


141

763 [M+2]+, [M-C5H11]+, 579, 295, 337, 225, 128, 113, 112,111 (Figure 3.14 and Table

3.28B). The lassaigne’s sodium test indicated the presence of nitrogen, phosphorous and

sulphur.

3.C.4. Structure elucidation of the inhibitor

The structure of the inhibitor was established with the help of extensive spectroscopic

studies such as UV, IR, NMR (1H and 13C; 1 and 2-D) and LC-MS along with the support from

elemental analysis and colour reactions. The elemental analysis showed the presence of P, N

and S and indicated the molecular formula as C43H73N2O5SP. The UV absorption spectra

showed the maxima at 210 and 260 nm, these corresponds to the aromatic -C=N (π- π*

transition) group in a quinoxaline nucleus. IR spectra of the inhibitor revealed the absorption

bands attributable to quinoxaline (1434, 1312, 1046, 954, 761, imine (2249 and 2123 cm-1),

OH or NH (3,452 cm-1) groups and a characteristic P–O and P–O–C stretching (1046 cm-1). 1H

NMR spectrum showed signals in the range of δ 7.05-7.15 as multiplet for the aromatic

protons. The 13C NMR spectrum showed six signals between δ 130-140 along with two more

signals at δ 155.3 and 171.1, which are characteristic of quinoxaline nucleus. The IR

absorption at 954 cm-1 and the doublet of doublet signal at δ 5.86 (16 Hz) for two protons in

proton NMR spectrum indicated the presence of a trans olefinic double bond in alkyl chain,

which is confirmed by the presence of two carbon signals at δ 122.4 and 147.8 ppm in Carbon

NMR spectrum. This is also supported by the presence of two multiplets δ 1.6-1.7 and 1.92-

1.96 in proton NMR spectrum and two carbon signals at δ 30.1 and 39.6 indicated the protons

and carbons adjacent to double bond. The signal at δ 179.7 shows the presence of carbonyl

carbon of an ester group. Mass spectral analysis disclosed the structure of the inhibitor further

(Figure 3.14). LC-EI-MS showed the M+ ion at m/z 761 along with a low abundance ion at

m/z 763 (M+2)+, which are in agreement with the molecular formula proposed from elemental

analysis. The ion at m/z 295 suggested the presence of nonadecenoyl moiety, while the ion at

m/z 579 with loss of 182 units (C13H26) moiety is due to the presence of double bond on C-6,7

in nonadecenoyl moiety. The presence of double bond in nonadeca-6-enoyl moiety was also

confirmed by the NMR signals as described earlier. The LC/MS data also substantiated the

presence of quinoxaline moiety [at m/z 128], which was further confirmed by the chemical


142

tests for the presence of both nitrogen and phosphorous. The ions at m/z 111, 112 and 113

confirmed the presence of thiophosphoryl group, that is suggested from the IR spectrum. The

triplet at δ 2.36-2.39 in proton NMR spectrum and signal at δ 24.3 indicated the OCH2 group

of hexadecyl moiety attached to thiophosphoryl group.

HMBC spectrum showed the connectivities between signals δ 147.8 and 122.4 ppm (C-

6’ and 7’ respectively) and the proton signals at δ 1.92-1.96 ppm (H-5’) related to the double

bond. The connectivities between the carbon signals δ 14.5 ppm (C-19’ and 16’’) and the

proton signals at δ 1.25-1.35 ppm (H-17’, 18’ and H-14’’, 15’’) were also observed. The

δ 171.1 ppm (C-2) signal showed weak connectivities with the proton signals at δ 7.05-7.15

ppm (H-8) and 2.04-2.12 ppm (H-2’) indicating the link between nonadeca-6-enoicacid and

quinoxaline nucleus. The carbon signal at δ 27.8 ppm (C-1’’) showed peak connectivity with

the proton signal at δ 5.37 ppm (hydroxyl proton of thiophoshphoryl group). The quaternary

carbon signals at δ 138.8 and 140.2 ppm (C-9 and 10 respectively) were not observed in the

HMBC spectrum due to the low concentration of the compound.

The ions at m/z 337 and 225 suggested the second alkyl chain as –O-P=S (OH) -O-

(CH2)15-CH3. Further conformation of the structure was obtained from 2DHMQCT and1H-1H

COSY. 2DHMQCT spectra also gave corresponding carbon signals wherever protons were

attached. The molecular formula C43H73N2O5SP was confirmed on the basis of high resolution

LC/MS [M+ 761, M+2 763] in combination with proton and carbon NMR data and finally

confirmed by its elemental analysis. Two dimensional NMR analysis including COSY, HMQC

and HMBC spectra led to the assignment of this compound as nonadeca-6-enoicacid-3-

(hexadecyloxy- hydroxy thiophosphoryloxy)-quinoxalin-2-yl ester.


143

Based on the above data, the most probable structure of the compound was found to be

as follows:

N

N O

OP

OS

OH

O

1'

2'3'

4'5'

6' 7'8'

9'10'

11'12'

13'14'

15'

12''

13''

14''

16'

16''

18'17'

15''

1''2''

3''

4''

5''

6''

7''

8''

9''

10''

11''

19'

1

2

3

45

6

7

89

10

Figure 3.15: Chemical structure of streptolipin

Based on this data, proposed structure of the inhibitor was chemically named as nonadeca-6-

enoic acid-3-(hexadecyloxy-hydroxy-thiophosphoryloxy)-quinoxalin-2-yl ester and is given as

Figure 3.15. A literature search revealed that this compound did not match with any reported

lipase inhibitors or of any Streptomyces metabolites. The inhibitor is henceforth designated as

streptolipin [Streptomyces, lipase inhibitor]. The isolated inhibitor exhibited a dose dependent

inhibition against pancreatic lipase inhibition at an IC50 of 349 nM (Figure 3.16).

3.C.5. Kinetic studies on the inhibition of streptolipin against pancreatic lipase: 3.C.5.1. Lineweaver-Burk (LB) plot of streptolipin inhibition by streptolipin:

In order to determine the nature of inhibition, pancreatic lipase was exposed at different

concentrations of streptolipin. The results illustrated in Figure 3.18 shows that increasing the

concentration of streptolipin resulted in a family of lines with a common intercept in the

second quadrant resulting in increase in Vmax with different slope values. However, as

streptolipin concentration was increased from 0.125 to 0.625 µM, Km value was same which

denoted that it is non competitive inhibition. The equilibrium B plot versus the inhibitor

concentration, which is linear as shown in the Figure 3.19 and the Ki value was found to be

0.714 µM. These results indicate that streptolipin is a non-competitive inhibitor.

Table 3.28.A: 1H and 13C NMR chemical shifts of compound


144

H/C No δ (Η) Multiplicity J (Hz) δ ©

2 171.1

3 155.3

5 130.4

6 128.4

7 128.2

8

7.05-7.15

m

7.0

130.1

9 138.8

10 140.2

1′ 179.7

2′ 2.04-2.12 t 8.0 25.1

5′ 1.92-1.96 m 39.5

6′

(CH=CH)

5.86-5.89 m 147.8

7′(CH=C

H)

5.86-5.89 m 16 122.4

8′ 1.6-1.7 m 10 30.3

19′ (CH3) 0.88 t 6.5 14.5

1″

(OCH2)

2.36-2.39 t 7.5 27.8

16″(CH3) 0.89 t 6.0 14.5

OH 5.37 s

-(CH2)n- 1.25-1.35 m 29.5-30.1

* :500 MHz ** :125MHz


145

Table 3.28.B: Mass spectrometry; fragmentation of streptolipin in EI-MS (m/z)

(m/z) Fragment

761.1 (M+)

763.4 [M+2]+

468.4 [M- X]+

427.4 [M- Y]+

579.4 [M-Z]+

514.4 [M-C18H33]

337 . PO3S-C16H34[Y]

295.2 O2C19H35 [X]

181 C13H26 [Z]

255.2 C15H15O2N2

112 . PO3HS

225.2 . CH2-(CH2)14-CH3

128 C8H4N2


146

Table 3.29: Physico-chemical properties of the inhibitor

Characteristics Compound data

Appearance Off white solid

Solubility Chloroform, dimethyl sulphoxide, acetone

U.V 210, 260

HPLC (Rt) (minute) 19

Melting point 184.40C

Theoretical value C: 67.86; H: 9.67; N: 3.68

Found C: 67.02; H: 9.86; N: 3.94.

Molecular formula C43H73N2O5S

Molecular weight 761.09

Exact mass 760.5

IR (cm-1) 1434,1312, 1046, 954, 761 for quinoxaline,

3436,2913, 1771,1714, 1659, 1236, 667

NMR See Table 3.28.A

LC/MS (EI-MS m/z)

Fragmentation

See Table 3.28.B

IUPAC name nonadeca-6,10-dienoic acid-3-

(hexadecylloxy-hydroxy-

thiophosphoryloxy)-quinoxalin-2-yl ester.

Designated as Streptolipin


147

3.C.5.2. Determination of irreversibility of the pancreatic lipase enzyme inhibition:

Streptolipin was checked for reversibility/irreversibility of specific activity of the

enzyme exposure to the inhibitor. Enzyme fraction eluting in the void volume was used for

reversibility/irreversibility studies. The inhibitor was not bound to Sephadex G-25 column

chromatography indicating irreversible nature.

3.C.6. Other biological activities of streptolipin:

Streptolipin did not show any inhibition against soybean 15-lipoxygenase, rat lens

aldose reductase, rat brain acetyl cholinesterase and other pancreatic enzymes such as

phospholipase A2, amylase, trypsin and chymotrypsin upto 200 µM. Streptolipin did not

show any activity against lipases like Mucor javanicus, Rhizopus oryzae, Candida rugosa,

Penicillium roqueforti, Pseudomonas sp, Candida rugosa, Candida antarctica and Humicola

sp upto 200 µM. Streptolipin revealed no antimicrobial activity upto a concentration of 200 µg

by disc plate method against Bacillus subtilis, B. pumilis, E. coli, Pseudomonas aeruginosa,

Penicillium notatum, Aspergillus niger, Saccharomyces cerevisiae and Candida utilis.


148

Figure 3.16: Concentration dependent inhibition of streptolipin on pancreatic lipase

0

20

40

60

80

100

120

0 100 200 300 400 500 600 700

Inhibitor concentration (nM)

Rem

aini

ng a

ctiv

ity (%

)


149

Figure 3.17:[V] versus [S] plot in the presence of different fixed concentrations of streptolipin

0

0.2

0.4

0.6

0.8

1

1.2

0 0.05 0.1 0.15 0.2 0.25 0.3

[S]

[V]

I=0(control) I=0.125 µM I=0.250µM I=0.375µMI=0.5 µM I=0.625 µM


150

Figure 3.18: L-B plot of 1/[V] vs 1/[S] in the presence of different fixed concentraions of streptolipin

-10

-5

0

5

10

15

20

25

30

35

40

-20 -10 0 10 20 30 40 50 60

1/[S]

1/[V

]

I=0(control) I=0.125 µM I=0.250µM I=0.375µM I=0.5 µM I=0.625 µM


151

Figure 3.19: The slope (km/Vmax) of the lines described from the double reciprocal

plot are plotted against the streptolipin concentration in order to derive

the Ki value for the inhibitor:

y = 0.1341x + 0.0408R2 = 0.8017

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

-2 0 2 4 6

Inhibitor Concentration

Slop

e of

rec

ipro

cal p

lot


152

3.D. Studies on media optimization for streptolipin production:

Large-scale fermentation processes are frequently developed with complex media that

contain a large variety of nutrients and growth factors. These media are generally economical

and able to support high product yields. However, many of these processes experience

unacceptable variability in the productivity and product profiles (especially trace

contaminants). This variability is usually attributed to changes in the composition of one or

more of the complex ingredients. One approach at addressing this problem is to develop

chemically defined media that use pure compounds in known proportions and have

traditionally been employed only in the laboratory to support biosynthetic studies. Defined

medium can offer other advantages such as low foaming, translucency and the relative ease of

product recovery and purification.

3.D.1. Standard HPLC curve for streptolipin: Different concentrations of pure streptolipin ranging from 10 to 50 µg, were injected

into HPLC and the peak area was determined. A standard curve was constructed by plotting the

peak area versus the streptolipin concentration (Figure 3.20). Based on this standard curve

streptolipin concentration in crude samples was determined.

3.D.2. Time course fermentation for streptolipin production:

A typical time course of fermentation is shown in Figure 3.21. The production medium

as used in screening studies (ISP medium I) was used for the experiment. It was seen that

biomass increased steadily till it reached the maximum of 8.35 g/L at 168 h after which it

reached stationary phase. The death phase was not reached till 240 h of fermentation time. The

initial pH of the medium was 7.2 and gradually increased to 8.8 and maintained till the end of

the fermentation. The course of extracellular and intracellular streptolipin production was quite

characteristic. The production of the inhibitor both extracellularly and intracellularly started

after 48 h and increased till it reached a maximum at 168 h. The maximum amount of


153

streptolipin produced extracellularly was 15 mg/L and intracellularly 42 mg/L after which it

decreased.

Figure 3.20: Standard curve for streptolipin

0100000002000000030000000400000005000000060000000700000008000000090000000

0 10 20 30 40 50 60

Streptolipin (µg)

Peak

are

a

As the intracellular inhibitor concentration was higher than extracellular, further studies

on this organism were carried out only on intracellular inhibitor. The optimum time of 168 h

for the production of streptolipin was taken for the termination of fermentation.

3.D.3. Optimization of physical parameters for production of streptolipin:

3.D.3.1. Selection of inoculum media for production of streptolipin:

Two inoculum media were tried in this study. The difference in the medium was the

presence of glycerol in the inoculum medium II and corn steep liquor and glucose in inoculum

medium I(Section 2.2.A.7). The inoculum medium I showed highest production of streptolipin,

than inoculum medium II (Table 3.30). The maximum biomass was 8.38 and 6.98 g/L and the

productivity of streptolipin was found to be 42.6 and 34.98 mg/L in the inoculum media I and

II, respectively. Inoculum medium I was used for further studies.


154

Figure 3.21: Time course fermentation for streptolipin production

0

5

10

15

20

25

30

35

40

45

0 24 48 72 96 120 144 168 192 216 240

Time (hrs)

Stre

ptol

ipin

(mg/

L)

0

1

2

3

4

5

6

7

8

9

10

pH, D

ry b

iom

ass

(g/L

)

Intracellular (mg/L) Extracellular (mg/L) pH Dry biomass(g/L)

Table 3.30: Selection of inoculum media for production of streptolipin:


155

Inoculum media Dry biomass (g/L) Streptolipin (mg/L)

I 8.38 42.60 II 6.98 34.98

3.D.3.2. Effect of temperature on production of streptolipin:

The effect of different temperature on streptolipin production was tested using the ISP

medium I and the results are shown in Figure 3.22. There was marked increase in the

production of streptolipin from 10 to 300C reach the highest 48.81 mg/L and biomass to 12.52

g/L. The productivity of Streptolipin increased from 0.05 to 0.29 mg/L/h from 10 to 300C and

further increase of the temperature decreased the productivity. Further increase of temperature

till 450C, decreased the production of streptolipin as well as biomass. The optimal cultivation

temperature on cell growth and streptolipin production was 300C and used for further studies.

3.D.3.3. Effect of initial pH of the medium on production of streptolipin:

The effect of initial pH from pH 2.0 to 9.0 was studied on the ISP medium I. The

results indicate that the production of streptolipin to be strongly dependent on the pH of culture

broth. The cell growth and streptolipin production increased from pH 2.0 to 7.0. Further

increase in pH from 7.0 to 9.0 decreased the dry biomass and streptolipin production (Figure

3.23). The streptolipin productivity increased from pH 2.0 to 7.0 (0.05 to 0.29 mg/L/h) and

further decreased. The highest level of biomass (12.29 g/L), streptolipin (48.99 mg/L) and the

maximum productivity (0.29 mg/L/h) was observed at pH 7.0.


156

Figure 3.22: Effect of temperature on production of streptolipin

0

10

20

30

40

50

60

10 20 25 30 35 40 45

Temperature 0C

Stre

ptol

ipin

(mg/

L),

Dry

bio

mas

s (g/

L)

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Stre

ptol

ipin

pro

duct

ivity

(mg/

L/h

)

Streptolipin (mg/L) Dry biomass (g/L) Streptolipin Productivity (mg/L/h)


157

Figure 3.23: Effect of initial pH on production of streptolipin

0

10

20

30

40

50

60

2 5 7 8 9 10 11

pH

Stre

ptol

ipin

(mg/

L)

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Dry

bio

mas

s (g/

L),

Stre

ptol

ipin

Pro

duct

ivity

(mg/

L/h

)

Streptolipin (mg/L) Dry biomass (g/L) Streptolipin Productivity mg/L/h

3.D.3.4. Effect of aeration on production of streptolipin:


158

The oxygen demand of a fermentation process is generally met by aeration. One

important aspect of aeration in fermentation is the resistance to the transfer of dissolved

oxygen through the medium to the microbial cell, which could be overcome by increased

agitation. The transfer of oxygen from air to solutions forms the second important factor in

aeration. It is usually was solved in shake flasks by modeling working volume (medium to

flask volume) ratios. The effect of aeration on streptolipin production was carried out with a

flask volume: flask ratio (medium to flask volume) from 0.05 to 0.4.

Biomass production increased gradually as the volume: flask ratio increased (Figure

3.24). The highest biomass was reached at 17.66 g/L at a volume: flask ratio of 0.4. On the

other hand, streptolipin production increased dramatically from 0.05 to reach the highest of

48.93 mg/L at 0.2. At ratios above this, the inhibitor yield fell to a low of 9.01 mg/L at a ratio

of 0.4.

3.D.4. Optimization of nutritional parameters for the production of streptolipin: 3.D.4.1. Screening of standard media for the production of streptolipin

The composition of the growth medium has an important influence on metabolite

production (Porter, 1975). There is no generalized medium applicable to all organisms,

standard media reported in literature for production of various secondary metabolites (Table

3.31) were screened for the production of streptolipin and the results shown in Table 3.32. The

poor growth of the strain on Czapek-Dox medium was evidently due to the inability of the

organism to utilize sucrose. Among the International Streptomyces Project (ISP) production

media, accumulation of biomass and streptolipin production was highest on ISP media I

(biomass 12.766 g/L, streptolipin 48.81 mg/L) followed by ISP V (biomass 12.57 g/L,

streptolipin 45.78 mg/L) and ISP VIII (biomass 11.96 g/L, streptolipin 44.92 mg/L) and

among other media tried, accumulation of biomass and streptolipin production was highest on

carbohydrate carcode medium (biomass 15.61 g/L,


159

Figure 3.24: Effect of aeration on production of streptolipin

0

10

20

30

40

50

60

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45

Volume: Flask ratio

Stre

ptol

ipin

(mg/

L),

Dry

bio

mas

s (g

/L)

Dry biomass weight (g/L) Streptolipin (mg/L)

streptolipin 47.79 mg/L) followed by Baron’s media (biomass 10.04 g/L, streptolipin 25.20

mg/L) and Numerof’s medium (biomass 10.60 g/L, streptolipin 20.32 mg/L). However ISP


160

medium I showed almost the same highest productivity of streptolipin as carbohydrate carcode

medium (0.29 mg/L/h and 0.28 mg/L/h respectively). This could be due to the presence of Zinc

a micronutrient in the ISP production medium I that could have played role in the maximum

production of the compound. The organism when grown on complex organic medium

containing higher amounts of carbon sources and the micronutrients Ca++, Na+, K+ did not

show the production of the compound although it accumulated maximum amount of biomass.

Highest yield 35.21mg/L of streptolipin and a low biomass of 2.925 g/L on Lindenberg

synthetic medium suggested a role of Mg++ and Fe++ in the production of compound. However

reduced biomass in this medium may be due to the glycerol, which is not a good carbon source

for this culture. Out of 20 media, ISP production media I showed the highest productivity of

streptolipin and was taken as the basal medium for further experiments since the organism

produced the highest amount of inhibitor on this medium.

3.D.4.2. Effect of different carbon sources on production of streptolipin:

Different carbon sources such as polysaccharides, oligosaccharides (starch, dextrin,

cellulose), trisaccharides (raffinose and rhamnose), disaccharides (melibiose, sucrose, maltose,

xylose and lactose), monosaccharides (glucose, galactose, arabinose, fructose, mannose and

inositol), sugar alcohols (mannitol, xylitol), sodium salts of organic acids (sodium acetate,

citrate and pyruvate) and a control (without carbon source) were screened for optimizing media

components for the production of streptolipin. Each carbon source was incorporated at 15 g/L

level in to the basal medium in place of glucose.

Removal of glucose from the ISP medium I was taken as control. The biomass and

streptolipin production was very low (Table 3.33). These results showed poor production of

streptolipin on the polysaccharides > trisaccharides > disaccharides > sugar


161

Table 3.31: A list of standard media for the production of antibiotics:

Media Metabolite Reference

Czapek – Dox medium Inulinase Gill, 2003 O – Brien synthetic medium Streptomycin O’Brien et al, 1956 Complex organic medium Streptomycin Shirato and Nagatsu, 1965

Dulaney medium Streptomycin Dulaney, 1948 Thornberry medium Streptomycin Shirato and Motoyama, 1966 Baron medium Streptomycin Shirato and Motoyama, 1966 Numerof medium Streptomycin Numerof et al, 1954 Lumb medium Streptomycin Shirato and Motoyama, 1966 Cornmeal salt medium Streptomycin Shunzo et al, 1982 Carbohydrate carcode medium

Nystatin Jonsbu et al, 2002

Hobb medium Streptomycin Hobb et al, 1989 Lindenberg synthetic medium

Actinorhodin Elibol et al, 1995


162

Table 3.32: Screening of standard media for the production of streptolipin

Media Dry Biomass (g/L)

Streptolipin (mg/L) Streptolipin productivity (mg/L/h)

Czapek – Dox medium 1.92 8.00 0.04 O – Brien synthetic medium 3.27 8.96 0.05 Complex organic medium 0.32 8.10 0.04

Dulaney medium 1.80 12.80 0.07 Thornberry medium 2.20 6.63 0.03 Baron medium 4.05 25.20 0.15 Numerof medium 4.61 20.32 0.12 Lumb medium 2.72 4.82 0.02 Cornmeal salt medium 0.91 11.17 0.06 Carbohydrate carcode medium

15.62 47.79 0.28

Hobb medium 3.64 9.54 0.05 Lindenberg synthetic medium

2.93 35.21 0.20

ISP Production Medium I 12.77 48.82 0.29 ISP Production Medium II 10.37 37.75 0.22 ISP Production Medium III 9.53 30.20 0.17 ISP Production Medium IV 4.29 15.45 0.09 ISP Production Medium V 12.57 45.78 0.27 ISP Production Medium VI 10.58 26.95 0.16 ISP Production Medium VII 11.65 39.99 0.23 ISP Production Medium VIII 11.97 44.92 0.26


163

Table 3.33: Effect of different carbon sources on production of streptolipin:

Supplement (15 g/L) Dry biomass (g/L) Streptolipin (mg/L) None (control) 0.82 1.20 Carbon sources Monosaccharides

D-glucose 12.70 48.05 D-Galactose 6.87 47.76 Arabinose 6.48 44.74 Fructose 9.05 43.23 Mannose 6.98 38.23 Inositol 8.57 13.39 Disaccharides

Melibiose 6.49 13.33 Maltose 7.79 23.26 Sucrose 7.80 15.90 Lactose 6.90 29.22 Xylose 5.90 31.82 Trisaccharides

Raffinose 6.13 10.70 Rhamnose 4.81 15.89 Polysaccharides

Starch 10.56 19.29 Cellulose 18.51 12.90 Dextrin 9.08 19.69 Sugar alcohols

Mannitol 8.46 25.72 Xylitol 7.95 11.16 Sodium salts

Sodium acetate 5.73 29.27 Sodium citrate 4.43 12.48 Sodium pyruvate 5.79 46.05


164

alcohols and higher productivity on monosaccharides and sodium salts of acids. Poor

production with polysaccharides may be due to poor metabolic turn over of the compound.

Among monosaccharides tried, the maximum biomass was accumulated from glucose

(12.70 g/L) followed by inositol and fructose and the maximum streptolipin yield (48.05 mg/L)

was from glucose followed by galactose and arabinose. Among disaccharides studied, the

maximum biomass was accumulated from sucrose (12.70 g/L) followed by maltose and lactose

and the maximum streptolipin yield (48.05 mg/L) was from xylose followed by lactose and

maltose. The trisaccharides tried were rhamnose and raffinose. The maximum biomass was

accumulated from raffinose (6.13 g/L), but the maximum streptolipin yield (15.89 mg/L) was

from rhamnose. Among polysaccharides tried, the maximum biomass was accumulated from

cellulose (15.51 g/L) followed by starch and dextrin and the maximum streptolipin yield (19.69

mg/L) was from dextrin followed by starch and cellulose. Sugar alcohols tried were mannitol

and Xylitol. The maximum biomass and streptolipin was from mannitol (8.46 g/L of biomass

and 25.72 mg/L streptolipin). Among sodium salts tried, the maximum biomass was

accumulated from sodium pyruvate (5.79 g/L) followed by sodium acetate and sodium citrate

and the maximum streptolipin yield (46.05 mg/L) was from sodium pyruvate followed by

sodium acetate and sodium citrate.

Among all carbon sources sodium pyruvate and galactose showed highest yield (46.046

and 47.7 mg/L respectively) followed by arabinose and fructose. Significant rise on the yield of

the compound on sodium pyruvate suggests that pyruvate could be one of the intermediate

compounds in the biosynthetic pathway of the inhibitor. However significant reduction in the

biomass on sodium pyruvate and galactose suggests higher metabolic turnover of the

compound by the organism on the carbon sources. But on glucose, not only highest production

of the metabolite but also higher accumulation of biomass was observed. Hence it was a good

carbon source for the production of streptolipin and glucose was used as a carbon source for

screening of nitrogen sources.


165

3.D.4.3. Effect of inorganic nitrogen sources on production of streptolipin:

Seven nitrogenous compounds i.e., (ammonium acetate, urea, sodium nitrate,

ammonium nitrate, ammonium hypophosphate, ammonium sulphate and potassium nitrate)

were used as single sources in the basal medium at a level of 15 g/L in place of soyabean meal.

In addition calcium carbonate was added at a level of 3.5 g/L, to keep the pH relatively neutral

and allow the organism to grow and produce the inhibitor.

It was generally observed that ammonium salts gave low biomass and relatively low

streptolipin values (Table 3.34). Sodium nitrate and potassium nitrate, however was readily

utilized. The results indicate that a medium containing sodium nitrate as the sole nitrogen

source was more suitable for streptolipin production than in a medium with ammonium

nitrogen. When sodium nitrate served as the sole nitrogen source, the pH remained relatively

neutral and yields of approximately 41.92 mg per liter were obtained. However, when the

sodium nitrate was tried at a higher level (20 g/L), it resulted in little or no inhibitor formation,

indicating the critical nature of sodium nitrate in the production of streptolipin. Such critical

nutritional studies on inorganic salts was generally poor nitrogen sources (Martin and Daniel,

1977) have been reported for polyene production.

3.D.4.4. Effect of organic nitrogen sources on production of streptolipin:

A number of complex organic nitrogen sources were tested at 15 g/L for their effect on

the production of streptolipin. The results are shown in Table 3.35. It was observed that the

organic nitrogen sources had a marked effect on the fermentation. Growth occurred more

readily and significantly higher streptolipin levels were reached. The most remarkable increase

in the streptolipin yield was obtained by the addition of yeast extract (54.248 mg/L). A number

of authors have suggested that stimulation of higher yields of secondary metabolites might

been possible due to free amino acids and short peptide (two to three amino acids long) and

also more growth factors than other protein hydrolysates in yeast extract and soyabean meal,


166

perhaps a single amino acid or a combination of amino acids (Aasen et al, 2000; De Vuyst,

1995; De Vuyst and combinat-

Table 3.34: Effect of inorganic nitrogen sources on production of streptolipin:

Inorganic nitrogen sources (15 g/L)

Dry biomass (g/L) Streptolipin (mg/L)

Potassium nitrate 11.65 36.94 Sodium nitrate 12.01 41.20 Ammonium nitrate 9.12 26.60 Ammonium acetate 7.62 17.82 Ammonium sulphate 6.92 13.38 Ammonium hypophosphate 8.92 24.42 Urea 5.74 18.80

Table 3.35: Effect of organic nitrogen sources on production of streptolipin:

Complex nitrogen sources (15 g/L)


Peptone 9.12 24.13 Casein 11.86 41.85 Soyabean meal 11.94 48.79 Yeast extract 12.62 54.25 Malt extract 8.92 23.27 Corn meal 7.89 18.00 Corn steep liquor 5.92 16.22 Skim milk 11.75 40.87

Table 3.36:Effect of different amino acids on production of streptolipin: Amino acids ( 15 g/L) Dry biomass (g/L) Streptolipin (mg/L) Glycine 1.25 8.64 Histidine 5.56 18.31 Isoleucine 6.00 27.10 Aspargine 5.23 32.94 Tryptophan 6.07 16.37


167

ion of amino acids (Aasen et al, 2000; De Vuyst, 1995; De Vuyst and Vandamme 1992;1993;

Egorov et al, 1971; Kozlova et al, 1972).

This possibility was investigated by adding 5 amino acids, glycine, tryptophan,

histidine, isoleucine and aspargine separately at a level of 15 g/L instead of soya bean meal.

The results are given in the Table 3.36. Asparagine showed the highest yield of streptolipin.

This could be due to the presence of two amino groups in aspargine. Higher yield of inhibitor

on aspargine may be due to the long carbon chain (6 carbons) of the amino acid, which would

have contributed to the biomass accumulation. Growth was little on medium supplemented

with glycine.

3.D.4.5.Effect of trace elements on production of streptolipin: ISP medium I, used as basal medium contains 5 trace elements. The effect of

individual trace element from the medium was studied by removing one trace element at a

time.

Results showed that removing the trace elements of potassium and zinc reduced the

production of streptolipin from 48.75 to 38.24 and 41.91 respectively (Table 3.37). The results

indicate both dipotassium hydrogen phosphate and zinc sulphate play a role in

Table 3.37: Effect of trace elements on production of streptolipin:

Basal medium without Dry biomass weight (g/L) Streptolipin (mg/L)

NaNO3 12.06 47.81 K2HPO4 11.92 38.24

NaCl 12.77 47.02 ZnSO4 12.35 41.91 CaCO3 12.66 46.99 Control 12.82 48.75


168

Figure 3.25:Effect of dipotassium hydrogen sulphate on production of streptolipin

12.2

12.4

12.6

12.8

13

13.2

13.4

13.6

13.8

0.01 0.025 0.05 0.1 0.5 1

Dipotassium hydrogen sulphate (g/L)

Dry

bio

mas

s (g/

L)

44

46

48

50

52

54

56

58

Stre

ptol

ipin

(mg/

L)

Dry biomass weight (g/L) Streptolipin(mg/L)


169

Figure 3.26: Effect of zinc sulphate on production of streptolipin

11.8

12

12.2

12.4

12.6

12.8

13

13.2

13.4

0.01 0.025 0.05 0.1 0.5 1 Zinc sulphate (g/L)

Dry

bio

mas

s (g

/L)

46

47

48

49

50

51

52

53

54

55

56

Stre

ptol

ipin

(mg/

L)


the production of streptolipin. So, the next experiments were carried out on different

concentrations (0.01g/L to 1g/L) of dipotassium hydrogen phosphate and zinc sulphate. The


170

optimum concentration of dipotassium hydrogen phosphate and zinc sulphate wasfound to be

0.5 g/L and 0.1 g/L respectively (Figure 3.25 and 3.26). These gave the highest Streptolipin

values 56.76 mg/L and 54.80 mg/L of dipotassium hydrogen phosphate and zinc sulphate

respectively.

3.D.4.6. Effect of lipids on production of streptolipin:

Preliminary experiments revealed that this organism grew well on a medium

containing soya bean meal and glucose, the soya bean meal was replaced by lipids including

cottonseed oil, coconut oil, groundnut oil, olive oil, stearic acid and lauric acid. The biomass

was very less when compared with the control. The growth was sparse in groundnut oil (Table

3.38). This suggests the interference of lipids with primary metabolism of the organism which

in turn would have lead to reduction in biomass. The maximum biomass was observed in the

medium supplemented with olive oil (5.02 g/L) and the highest streptolipin was observed in

coconut oil (18.95 mg/L). However, total production of the biomass and is very less compared

to control indicating that lipids cannot be used as inhibitory source of raw material for

production of Streptolipin.

Table 3.38: Effect of lipids on production of streptolipin:

Lipids (10 g/L) Dry biomass weight (g/L) Streptolipin (mg/L) Olive oil 5.02 12.75 Cotton seed oil 2.54 4.63 Ground nut oil 0.37 3.03 Coconut oil 4.02 18.95 Stearic acid 1.15 9.36 Lauric acid 0.32 9.06 Control 12.36 48.99


171

Figure 3.27: Effect of molasses on production of streptolipin

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

10 20 40 60 80 100

Molasses (g/L)

Dry

bio

mas

s (g

/L)

0

2

4

6

8

10

12

14

16

18

20

Stre

ptol

ipin

(mg/

L)



172

3.D.4.7. Effect of molasses on production of streptolipin: Earlier reports (Lan et al, 2002) suggested increased productivity of antibiotics on

molasses when used as a commercial source. Different concentrations of molasses, varying

from 1 to 10% were used for the production of streptolipin. There was gradual increase in the

compound with increase in molasses concentration to reach a highest of

17.81 mg/L at 8% after which that inhibitor concentration declined (Figure 3.27). As,

production of Streptolipin was significantly low when compared with the basal medium,

further optimization on molasses was not done.

3.D.4.8. Formulation of medium for the production of streptolipin:

From results shown in Table 3.33, it was observed that glucose, galactose and sodium

pyruvate were the best carbon sources for the production of streptolipin and from the Table

3.34 and 3.35, casein, soyabean meal, yeast extract and sodium nitrate to be the best nitrogen

sources.

Effect of different nitrogen sources on the production of streptolipin in the presence of

either galactose or sodium pyruvate was carried out, since there was highest productivity of the

compound on galactose and sodium pyruvate. Results (Table 3.39 and Table 3.40) showed the

yields on sodium nitrate in the presence of either galactose or sodium pyruvate to be high.

However, the streptolipin production was highest with glucose as the carbon source and yeast

extract as nitrogen source (Table 3.33 and Table 3.34).

Table 3.39: Production of streptolipin on different nitrogen sources in presence of 15 g/L Galactose:

Nitrogen source, (15 g/L )


NaNO3 5.54 14.55 Casein 8.30 20.54 Yeast extract 9.45 32.71 Soya bean meal 7.06 31.35 Table 3.40: Production of streptolipin on different nitrogen sources in presence of

15 g/L sodium pyruvate:


173

Nitrogen source, (15 g/L) Dry biomass weight (g/L) Streptolipin (mg/L) NaNO3 4.79 16.53 Casein 7.40 23.73 Yeast extract 7.39 28.80 Soya bean meal 8.36 31.35

3.D.4.9. Effect of different glucose concentrations on the kinetics of streptolipin

production:

The results of nutritional studies on carbon sources indicated glucose to be the best for

achieving the highest streptolipin production (Section 3.D.4.2.). Hence, various concentrations

of glucose from 10 to 80 g/L were used to study the kinetics of streptolipin production. In these

experiments, the yeast extract concentration was fixed at 15 g/L along with the other salts of

basal medium.

Kinetics of Biomass formation:

Figure 3.28 shows the time course of biomass growth with initial glucose concentration

(So) as the parameter. The biomass increased with increasing glucose concentrations. Table

3.41 shows that the maximum biomass accumulation increased with increasing glucose

concentration up to 50 g/L, above which there was a decrease in the biomass accumulation. It

was also noted that the time taken to reach the stationary phase increased with increasing

glucose concentration (Table 3.41). Beyond a concentration of So=40 g/L, however, stationary

phase was not reached and biomass continued to increase, albeit, slowly.

In case of So = 10 g/L, the stationary phase was reached very early during the

fermentation (96 h) to reach a total biomass of about 6.04 g/L. Further increase in the initial

glucose concentration increased the biomass, till 168 h to 23.22 g/L (Table 3.41) and stationary

phase.

The biomass growth rate is described by the equation

dx/dt=µX---- (1)


174

Where µ is the specific growth rate.

This is the most frequently applied model for biomass growth. The specific growth rate related

to the substrate concentration as given by Monods equation

µ=µmax.So/(Ks + So)----- (2)

Where µmax was the maximum specific growth rate

When specific growth rates are calculated, it is important to take only the initial specific

growth rates (i.e µi) as in the early hours of fermentation, the cells are in active (logarithmic)

growth phase.

Table 3.41: Effect of initial glucose concentration on biomass synthesis:

Glucose g/L 10 20 30 40 50 80

Maximum

biomass g/L

6.04 12.02 15.83 18.48 21.85 22.92

Time to reach

stationary phase

96 132 144 168 Not

reached

Not

reached

For our experiments, through the plots of ln X vs time (Figure 3.29) at different initial

glucose concentrations, corresponding specific growth rates were obtained from the slopes of

these plots.

Specific growth rates thus calculated were plotted against the initial glucose

concentration (So) in Figure 3.30. The effect of initial glucose concentration on the specific

growth rate can be seen by a double reciprocal plot of initial specific growth rate against the

initial substrate concentration (Figure 3.31). This gives a straight line with an intercept 1/Vmax

on Y-axis. The Monods constants obtained from Figure 3.31 are: Ks value was 3.85 and the

µmax was 1.1 h-1.


175

Kinetics of Streptolipin production:

Figure 3.32 shows the effect of initial glucose concentration on the streptolipin

accumulation pattern. For all glucose concentrations tested, streptolipin accumulation started

after 24 hours of growth except So=20g/L. The accumulation of streptolipin increased with

increase of initial glucose concentration till 50 g/L after which it decreases (Figure 3.32). At

the lower glucose concentration (So=10 g/L) the accumulation of streptolipin started after 44h.

The time taken to reach the maximum streptolipin concentration increased with initial glucose

concentration.

The maximum streptolipin concentration was observed for So=50 g/L at 168 h and

further increase was not observed (Table 3.42). It may be noted that the maximum streptolipin

concentration reached at different initial glucose concentrations occurred during the secondary

metabolism at 132 h for So=10 and 20 g/L, and 168 h above So=20 g/L.

A plot of the rate of streptolipin synthesis (dp/dt) against time indicated that, generally

in all glucose tested, the rate of product synthesized increased drastically in the early hours of

fermentation after which it slowed and then dropped drastically (Figure 3.33). The rate of

streptolipin production increased with increase of glucose concentration till So=40 g/L and

further increase decreased. The overall rate of streptolipin increased from So=10 to 20 g/L, but

on further increase above So=20 g/L the rate decreased. Here, the production of streptolipin

was dependent on the biomass, which might be the reason for the decrease of time of

streptolipin production, when the initial glucose concentration increased.

Kinetics of sugar utilization:


176

Figure 3.34 shows the substrate utilization pattern at various initial glucose

concentrations. It was observed that, as the glucose concentration increased, the

Figure 3.28: Variation of total biomass with initial glucose concentration

0

5

10

15

20

25

0 50 100 150 200

Time (h)

Bio

mas

s g/L

Glucose (g/L)10 20 30 40 50 80


177

Figure 3.29: Semilogarithmic plot of biomass with time

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

0 50 100 150 200

Time (h)

Inx,

g/L

Glucose (g/L) 10 20 30 40 50 80


178

Figure 3.30: Monod's plot of initial specific growth rates vs substrate concentration

0

0.5

1

1.5

2

2.5

3

3.5

0 10 20 30 40 50 60 70 80 90

Glucose, So, g/L

ul, h


179

Figure 3.31: Double reciprocal plot of specific growth rate against the initial glucose concentration

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

-1 -0.5 0 0.5 1 1.5

1/So, lit/mol

1/u1

, h-1


180

Figure 3.32: Variation of total streptolipin with initial glucose concentration

-20

0

20

40

60

80

100

120

0 50 100 150 200

Time (h)

Stre

ptol

ipin

(mg/

L)

Glucose (g/L)10 20 30 40 50 80


181

Figure 3.33: Rates of streptolipin formation at different initial glucose concentration

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 20 40 60 80 100 120 140 160 180

Time (h)

dp/d

t mg/

L/h

Glucose (g/L) 10 20 30 40 50 80


182

Table 3.42: Effect of initial glucose concentrations on streptolipin synthesis:

Glucose g/L 10 20 30 40 50 80

Maximum streptolipin

concentration mg/L

23.32 58.42 72.96 80.12 92.42 90.27

Time of starting

streptolipin production

(h)

60 48 36 36 36 36

Time to reach

maximum streptolipin

concentration (h)

132 132 168 168 168 168

Overall rate of

streptolipin

synthesized (mg/L/h)

0.28 0.35 0.29 0.24 0.22 0.14

Table 3.43: Effect of initial glucose concentration on glucose utilization pattern:

Glucose g/L 10 20 30 40 50 80

Percent of glucose

utilization( g/L)

100 100 100 100 100 75.4

Glucose exhausted (h) 144 144 144 144 168 >168

Overall rate of glucose

utilization (g/L/h)

0.0069 0.013 0.02 0.027 0.029 0.028


183

Figure 3.34: Time course of initial glucose consumption during fermentation

-10

0

10

20

30

40

50

60

70

80

90

0 50 100 150 200

Time (h)

Glu

cose

g/L

Glucose (g/L) 10 20 30 40 50 80


184

Figure 3.35: Rates of glucose utilization at different initial glucose concentration

-0.01

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0 50 100 150 200

Time (h)

ds/d

t g/L

/h

Glucose (g/L) 10 20 30 40 50 80


185

time taken to exhaust the glucose also increased. Since all the fermentation runs were

terminated at 168 h for all the experiments, glucose utilization was incomplete for So=80 g/L

(Table 3.43). The Table also reveals that overall rate of glucose utilization increased with

increasing glucose concentrations till So= 50 g/l, then stayed just about constant.

From the Figure 3.35, the rate of glucose utilization (ds/dt) increased steeply to reach

its maximum value of 0.012 g/l/h to 0.069 g/L/h from 10 to 80 g/L. The overall rate of

streptolipin increased from 10 to 80 g/L of initial glucose concentration. The results of the

glucose kinetics indicates that the initial sugar, glucose, or 50g/L to be optimum for the best

production of streptolipin.

3.D.4.10. Effect of different concentrations of yeast extract on streptolipin production:

Studies on the optimization of nitrogen sources (both organic and inorganic

nitrogen sources) showed that yeast extract at a concentration of 15 g/L resulted in the highest

concentration of streptolipin (54.25 mg/L) (section 3.D.4.4). Since maximum streptolipin

production corresponded to the maximum concentration of cell mass, achieving a high biomass

indicated as a prerequisite for a successful fermentation. In order to improve the streptolipin

yields by further generating higher cell densities, cell growth and streptolipin production were

determined over a period of time as a function of yeast extract concentration in the range of 10

to 80 g/L. The glucose concentration was fixed at 15 g/L along with the other salts of basal

medium.

Kinetics of Biomass formation:

The time course of biomass growth with respect to initial yeast extract concentration as

the parameter was followed. The maximum dry biomass accumulation increased from 5.08 to

19.05 g/L with increase of initial yeast extract (YEo) concentration (YEo= 10 to 80 g/L)

(Figure 3.36). It was also observed that the time taken to reach the stationary phase increased

with increasing YEo=10 to 80 g/L (Table 3.44).


186

Kinetics of Streptolipin production:

When the YEo concentrations increased from 10 to 50 g/L, the maximum streptolipin

production increased concomitantly. Streptolipin production started after 44 h of fermentation

(Figure 3.37). Interestingly, at all the yeast extract concentrations tried, the inhibitor was

detected when the biomass was around 35% of the final biomass (Xmax) had reached.

Table 3.44: Effect of initial yeast concentrations on biomass formation:

Yeast extract concentration (g/L)

Dry biomass (g/L) Xmax

Time taken to reach stationary phase (h)

10 5.08 108 20 10.05 120 30 12.23 120 40 15.84 120 50 18.44 144 80 19.05 144

Table 3.45: Effect of initial yeast extract concentrations on streptolipin synthesis:

Yeast extract concentration (g/L)

Maximum streptolipin concentration reached (mg/L)

Time taken to reach highest streptolipin concentration (h)

Rate of streptolipin production (mg/L/h)

10 23.40 108 0.14 20 55.00 132 0.33 30 66.90 132 0.40 40 80.64 132 0.48 50 92.71 144 0.55 80 90.01 144 0.54


187

The streptolipin production increased with increase YEo, the streptolipin yield was 23.4

mg/L where YEo=10 g/L and it increased to 92.62 mg/L when YEo=50 g/L, but on further

increase (YEo =50 g/L) streptolipin production declined. Table 3.45. The time taken to reach

stationary phase, increased from 108 h for YEo=10 g/L to 144 h for YEo= 80 g/L for

streptolipin production. Rate for streptolipin production increased with increase of YEo, from

YEo=10 g/L (0.13 mg/L/h) to YEo=50g/L (0.55 mg/L/h) and further increase of initial yeast

extract decreased the rate of streptolipin production to 0.53 mg/L/h (Table 3.45).

Increasing YEo concentration not only led to an increase in the biomass and streptolipin

production, but also led to an increase in the rate of streptolipin production from 0.139 mg/L/h,

for YEo=10g/L to 0.55 mg/L/h for a YEo=50g/L. Further increase in the YEo led to a decline

in the rate of streptolipin production and the time taken to reach highest streptolipin

concentration increased from 108 h to 144 h with increase of initial yeast extract concentration

(Table 3.45).

Achieving maximum cell density at the end of the fermentation seems to be a very

important parameter as the inhibitor is intracellular and biomass concentration at 144 h is the

highest. In the current studies above 50 g/L of yeast extract, streptolipin production was

decreased. Figures 3.36 and 3.37 indicate that, dry cell weight and streptolipin yield increased

with increasing concentration of yeast extract reaching an optimum at 50.0 g/L.

3.D.4.11. Production of streptolipin with optimum conditions on shake flask:

Based on the above observations, fermentation studies were carried out by using

optimized production medium, which contained glucose as carbon source and yeast extract as

nitrogen source. The trace elements were added as indicated in the optimization studies.


188

Figure 3.36: Influence of initial yeast extract concentrations on cell growth

0

5

10

15

20

25

0 50 100 150 200

Time (h)

Dry

bio

mas

s (g/

L)

Yeast extract (g/L) 10 10 g 20 g 30 g 50 g 80 g


189

Figure 3.37: Influence of initial yeast extract concentrations on streptolipin production

-20

0

20

40

60

80

100

0 50 100 150 200

Time (h)

Stre

ptol

ipin

(mg/

L)

10 g 20 g 30 g 40 g 50 g 80 g

Composition of the final optimized production media (g/L):


190

Glucose 50.0

Yeast extract 50.0

NaNO3 4.0

K2HPO4 0.5

NaCl 2.5

ZnSO4 0.1

CaCO3 0.4

Table 3.46. Comparison of medium studies before and after optimization in shake

flask:

Shake flask fermentation

(before media optimization)

Shake flask fermentation

(after media optimization)

Maximum biomass (g/L)

(Xmax)

8.35 28.01

Time taken to reach

highest biomass (h)

168 84


concentration (mg/L)

42 212.60

Time taken to reach

highest streptolipin (h)

168 84

Spore suspension was added from a well-sporulated glycerol aspargine agar slant to the above

production media and incubated at 300C at an initial pH 7.0, on a rotary shaker (220 rpm).

Medium flask volume ratio was maintained at 0.2 level and fermentation was carried out for

168 h. The maximum streptolipin and biomass was reached at 72 h. The pH decreased from 7.0

to 6.4 till 84 h, where glucose was exhausted, at this point, the pH increased to pH 8.0 till 168h

(Figure 3.38). The specific growth rate (µ) during early exponential growth was phase 0.42,

maximum biomass (Xmax) 28.01 (g/L) and final


191

Figure 3.38: Production of streptolipin with optimum conditions on shake flask

0

5

10

15

20

25

30

0 12 24 36 48 60 72 84 96 108 120 144 168

Time (h)

Bio

mas

s (g/

L),

pH

0

50

100

150

200

250

Stre

ptol

ipin

(mg/

L)

pH Biomass weight (mg/L) Streptolipin (mg/L)


192

streptolipin concentration 212.6 mg/L. The maximum biomass increased from 8.35 to 28.01

g/L from initial unoptimized studies to the optimum shake flask conditions and the time taken

to reach the highest biomass was decreased from 168 to 84h. The maximum streptolipin

concentration increased from 42 to 212.6 mg/L and the time taken to reach the streptolipin

decreased from 168 to 84 h (Table 3.46). Therefore, there was significant improvement in the

production of streptolipin was observed with the modified medium with decrease of

fermentation time from 168 to 84 hours (Table 3.46).

3.D.4.12. 10 L Fermentor study:

Batch culture for production of streptolipin was carried with all the optimum conditions

of the shake flask optimization studies. pH was controlled at 7.0 and the initial DO was set at

100% in a stirred tank fermentor (Figure 3.39.A). The maximum streptolipin (199.1 mg/L) and

biomass (28.4 g/L) was found at 84 hrs, although there was some linear growth till 108 hours,

where the glucose was completely exhausted. When the biomass finally reached 30.47g/L

(Figure 3.39.B).

Table 3.47: Comparison between shake flask and fermentor studies after

optimization of media:

Shake flask fermentation Fermentor studies

Maximum biomass (g/L)

(Xmax)

28.01 30.47

Time taken to reach

highest biomass (h)

84 84

Specific growth rate (µ) 0.42 0.39


concentration (mg/L)

212.60 199.10

Time taken to reach

highest streptolipin (h)

84 84


193

Figure 3.39.A. Production of streptolipin in 10 L laboratory fermentor


194

Figure 3.39.B: 10 L Fermentor study

0

50

100

150

200

250

0 12 24 36 48 60 72 84 96 108 120 144 168

Time in Hours

Stre

ptol

ipin

(mg/

L)/G

luco

se p

erce

nt

0

5

10

15

20

25

30

35

Bio

mas

s wei

ght (

g /L

)

Streptolipin (mg/L) Percent of glucose in the broth

Biomass weight (g/L)


195

The specific growth rate during early exponential growth phase (µmax) was 0.39. The

DO% had decreased from 100 to 63.5% along with increase of biomass. Therefore, significant

improvement in the production of streptolipin was observed with the modified medium.

The maximum biomass obtained from fermentor studies and shake flask fermentation

and the time taken to reach highest biomass was same in the both shake flask and fermentor

studies (84 h) (Table 3.47). The specific growth rate (µ) was 0.42 g/L in the shake flask, where

as in fermentor studies it was 0.39 g/L. The maximum streptolipin concentration decreased

212.6 to 199.1 g/L from shake flask to the scale up studies, whereas, the time taken to reach the

highest streptolipin production was 84 h for both shake flask and scale-up studies (Table 3.47).

The result clearly demonstrated that streptolipin production was predominantly growth

associated. The optimized medium developed was fairly simple containing the basic nutrients

and succeeded in quickly developing a reliable and very productive defined medium process,

which yielded to rapid large-scale cultivation study for streptolipin production.

3.D.5. Optimization of down stream processing conditions for streptolipin extraction:

3.D.5.1. Choice of the extraction solvent:

The effect of organic solvents was studied on the extraction of streptolipin from the dry

biomass. Both polar and non polar Organic solvents, were tried for efficient extraction of

streptolipin. Among the different organic solvents tried, ethyl acetate extraction gave

maximum recovery of streptolipin from the biomass, followed by chloroform and acetone

(Table 3.48).


196

Table 3.48: Choice of the extraction solvent:

Solvent Streptolipin (mg/10 g of dry biomass weight)

Ethyl acetate 50.26

Chloroform 46.94

Hexane 8.6

Methanol 4.3

Ether 13.26

Ethanol 12.76

Acetone 46.35

3.D.5.2. Biomass to solvent ratio:

The effect of biomass to solvent ratio on extraction of streptolipin was followed by

varying the amount of ethyl acetate to the 10 g (Figure 3.40). When the biomass to solvent

ratio increased from 1:5 to 1: 40, extraction of streptolipin, increased from 9.48-to 50.6-mg/10

g of dry biomass weight. Biomass to solvent ratio above 1:40 did not further increase

streptolipin yields. These results indicate that biomass to ethyl acetate ratio 1:40 to be ideal for

efficient Streptolipin extraction.

3.D.5.3. Effect of static and agitated conditions during extraction:

The extent of recovery was studied using the optimized conditions of biomass to ethyl

acetate ratio of 1:40 (w/v) in conical flasks under static and agitated conditions at 300C.

Agitation of the fermented biomass with the solvent was carried out by incubating the flasks on

a rotary shaker at 220 rpm. Under static conditions, a maximum of 9.12mg/ 10 g of dry

biomass could be extracted after 60 min of extraction. Beyond this time, streptolipin

concentration in the extract remained constant (Figure 3.41). Further it was observed that under

agitation conditions, the rate of extraction of streptolipin increased gradually till 60 min

(49.91mg). As compared to static extraction, the agitated mode of extraction gave much higher

yields. This was particularly true with extraction times of less than 60 minutes. For both modes

of extraction, the optimum time of extraction was 60 minutes.


197

3.D.5.4. Repeated extraction:

Repeated extractions were carried out by incubating the biomass with ethyl acetate

(1:40 w/v ratio) on a rotary shaker at 300C for 30 minutes each. At the end of incubation, the

solvent was removed and fresh solvent was added to the same biomass. Six such cycles were

carried out to study the efficiency of repeated extraction, which was a function of the number

of cycles needed to achieve maximum streptolipin yield from the biomass. It was observed

that, the first extract had maximum streptolipin concentration was 49.89 mg/10 g dry biomass,

while the second extract had 15.6 mg/g dry biomass, while the third extract 8.4 mg/g dry

biomass, while the fourth 4.6 mg/g dry biomass. There was no streptolipin extracted in the next

subsequent two extractions. This indicated that the biomass be extracted for four times to

extract all the streptolipin.

3.D.5.5. Sequential extraction:

This mode of extraction was studied in three flasks in which the solvent containing

streptolipin extracted from biomass was added to the fresh biomass, taking are to maintain the

solvent to biomass ratio (1:40). This was done for three flasks (Figure 3.42). The extract

obtained from the third flask was thus in contact with biomass for a total of 180 minutes. The

concentration of streptolipin was checked at the end of each step in the process. The

concentration of streptolipin in the extract increased from 48.23 mg in the first extraction to

63.84 mg/g of dry biomass in the final extraction. This would mean that there was substantial

increase in the overall yield of streptolipin extraction from the biomass. One advantage, using

this method was ethyl acetate could be reused for at least three cycles of extraction. This would

mean significant savings in terms of overall downstream processing costs.


198

Figure 3.40: Effect of biomass to ethyl acetate ratio on extraction of streptolipin

0

1

2

3

4

5

6

0 10 20 30 40 50 60

Ratio of biomass : ethyl acetate

Stre

ptol

ipin

(mg/

g dr

y bi

omas

s)

3.E. In vivo efficacy of streptolipin on experimental animal models:


199

Obesity and hyperlipidemia are to a relevant degree related to a high dietary fat intake.

The major ingredient (Over 95%) of dietary fat is triglyceride. The key enzyme that hydrolyses

dietary triglyceride during digestive process is lipase secreted by pancreas. Pancreatic lipase

(poured through pancreatic juice) exerts its action in the intestinal lumen at the water-lipid

interphase, in conjunction with bile juice and co-lipase (also present in pancreatic juice). A

clear demonstration that such an inhibition of dietary fat absorption results from inhibition of

the lipase activity has been made in the case of Orlistat isolated from Streptomyces toxytricini.

Orlistat has been observed to be a very potent, selective and irreversible inhibitor of pancreatic

lipase.

Investigations were made on laboratory mice to examine the health beneficial anti-

obesity effects of streptolipin by inhibition of lipase and hence interference with digestion and

absorption of dietary fat. This was done in both single dose as well as multiple dose

streptolipin administered animals.

3.E.1. Influence on lipase activity and dietary triglyceride absorption In this investigation, streptolipin has been administered at 3 different doses by gavage

(as 0.2 ml suspension in 5% gum arabic in 5% defatted milk) namely 5, 10 and 20 mg/kg body

weight. The middle dose (Weibel et al, 1987) i.e. 10 mg/Kg body weight corresponds to the

dose of reference drug Orlistat.

In the single dose study, the fat intake and faecal excretion of triglycerides were

monitored for 48 hours following the drug administration. In the multiple dose study, the

animals were administered with the compound on three alternate days (Day-1, Day-3 and Day-

5). Fat intake and faecal excretion of dietary triglycerides were followed until 48 hours after

the last dose. In single dose study, the animals were sacrificed on the third day and in multiple

dose, on the seventh day.

Faecal excretion of triglycerides in streptolipin administered animals is presented in

Table 3.49 and 3.50. Excretion of triglyceride in faeces was 164, 297 and 334% higher in three

Streptolipin groups compared to the control animals during first 24 hour of the drug dosage.

Thus, there was a dose dependent higher excretion of triglyceride as a result of streptolipin


200

administration. Further, the effect of Streptolipin was higher than that of Orlistat given at the

same dose level. Higher faecal excretion of triglyceride continued on the second day, but to a

much lesser degree compared to day 1. Faecal excretion of triglycerides on day 3 after

streptolipin administration was compared to controls.

Significantly, higher excretion faecal triglyceride was evidenced in the multiple dose

study throughout the duration of the experiment. As expected, the extent of higher excretion of

triglyceride was much higher on days of drug administration Day-1, Day-3 and Day-5).

Compared to the second day after drug administration.

Figure 3.43 and 3.44 present amounts of dietary triglyceride ingestion, faecal

triglyceride excretion and net triglyceride absorbed in mice administered streptolipin. It is very

clear that triglyceride absorption was dose -dependently lower in streptolipin-treated mice, and

was concomitant with higher faecal excretion of triglycerides.

Activity of lipase was examined in the mucosal scrapings of small intestine in

streptolipin administered animals (Figure 3.45). Lipase activity resident in intestinal mucosa

was significantly lowered (29 to 42%) in animals administered streptolipin, at the end of two

days following single oral administration. The activity was concurrable to controls, in Orlistat

administered mice. Similarly, lower activity of lipase in intestinal mucosa was seen in the

multiple dose two days after the third dose of administration of streptolipin. The decrease in

lipase activity was 19 to 41 %. Multiple dose of Orlistat does not have any inhibitory effect on

lipase resident in intestinal mucosa.


201

Table 3.49: Effect of streptolipin on faecal excretion of triglycerides (Single dose study)

Mouse Group Day 1 Day 2

Day 3

Control 20.9 + 4.85 19.2 + 3.25 18.2 + 3.25

Orlistat (10 mg/kg b.w.) 77.5 + 6.2 42.7 + 7.35 19.2 + 3.5

Streptolipin (5 mg/Kg b.w.) 54.5 + 5.25 36.4 + 7.9 18.3 + 2.6



Values expressed as mg triglyceride/ mice are mean ± SEM of 10 number of animals per group.


202

Table 3.50: Effect of streptolipin on faecal excretion of triglycerides (Multiple dose study) Mouse Group Day 1 Day 2 Day 3 Day 4 Day 5 Day 6

Control 17.2 + 3.6 18.3 + 2.9 19.2 + 4.5 38.3 + 9.0 19.9 + 2.8 20.1 + 4.05

Orlistat (10 mg/Kg b.w.) 66.6 + 4.7 37.5 + 5.8 77.6 + 8.4 83.5 + 17.6 76.3 + 6.25 39.3 + 7.95

Streptolipin (5mg/Kg b.w.) 58.7 + 4.65 36.0 + 9.3 53.5 + 9.05 61.5 + 8.9 54.2 + 7.3 33.2 + 6.2

Streptolipin (10 mg/Kg b.w.) 74.7 + 5.25 42.0 + 4.8 81.9 + 8.05 80.3 + 9.6 78.9 + 5.95 44.4 + 2.75

Streptolipin (20 mg/Kg b.w.) 69.6 + 4.5 42.7 + 6.15 78.8 + 6.0 45.3 + 5.65 82.3 + 6.05 46.6 + 4.7

Values expressed as mg triglyceride/ mice are mean ± SEM of 10 number of animals per group.


203

��

��

��

��

��

��

��

��

��

��

Figure 3.43: Effect of streptolipin on absorption of dietary triglyceride (Single dose)

0

50

100

150

200

250

Control Orlistat 10mg/kg b.w

Streptolipin 5mg/kg b.w



�� Total triglyceride intake Faecal triglyceride

�� Absorbed triglyceride


204

��

��

��

��

��

��

��

��

��

��

Figure 3.44: Effect of streptolipin on absorption of dietary triglyceride (Multiple dose)

0

100

200

300

400

500

600

700

800

Control Orlistat 10mg/kg b.w




�� Total triglyceride intakes Faecal triglyceride

�� Absorbed triglyceride


205

��

��

��

��

��

Figure 3.45: Effect of streptolipin on activity of intestinal lipase

66.2 67

46.8

39.4 38.1

54.6 53.8

44.5

32.3

41.1

0

10

20

30

40

50

60

70

80

Control Orlistat (10mg/kg b.w)

Streptolipin (5mg/kg b.w)

Streptolipin(10 mg/kg b.w)

Streptolipin(20 mg/kg b.w)

Spec

ific

activ

ity (U

nits

/mg

prot

ien)

�� 3 days 7 days

Values expressed as TBARS represent mean ± SEM of 10 animals in each group.


206

Thus, the higher excretion of fecal triglycerides is attributal to the strong lipase

inhibitory potential of streptolipin. It is probable that streptolipin has similar inhibitory

potential on pancreatic lipase which in the key enzyme in the digestive hydrolysis of

dietary triglycerides. In the absence of inhibitory effect of Orlistat on lipase activity of

intestinal mucosa, the higher excretion of fecal triglycerides, as a result of Orlistat

administration should be attributable to previously reported inhibitory influence of

Orlistat on pancreatic lipase.

3.E.2. Influence on blood and liver lipid profile: Serum lipid profile of streptolipin administered mice are presented in Table 3.51

and 3.52. Concomitant with decreased dietary triglyceride absorption. Serum triglyceride

levels were significantly lower in streptolipin treated mice. The decrease in serum

triglyceride was in the range 17–29% as a result of single dose (Table 3.51) and in the

range of 19-36% as a result of multiple dose (Table 3.52). Although, total cholesterol

concentration in serum remained unchanged, as a result of single of administration of

streptolipin, the proportion of cholesterol associated with LDL fraction was somewhat

lower in these test groups. Such a decrease in LDL cholesterol was accompanied by a

proportionate increase in HDL cholesterol (Table 3.51). Similar trend serum cholesterol

was also evidenced in streptolipin administered animals in the multiple dose study (Table

3.52).

Liver lipid profile in streptolipin administered mice, as a result of single as well as

multiple dose is prevented in Table 3.53 and 3.54. Triglyceride content of liver was

significantly lower, as a result of multiple dose streptolipin administration (Table 3.54).

As a result, the total lipid content of the liver was also proportionally lower.

Perirenal fat (depot fat) was significantly lowering in experimental mice, as a

result of streptolipin administration (Table 3.55 and 3.56). The decrease in perirenal fat

content and liver triglyceride content are in agreement to interferance of streptolipin with

dietary triglyceride absorption. These effects of streptolipin appear to exceed the effect of

Orlistat (Reference drug) administered with an equal dose. In otherwise, streptolipin is


207

Table 3.51: Effect of streptolipin on serum lipid profile (Single dose study)

Cholesterol Groups/parameters Total triglycerides Total HDL LDL

Total phospholipids

Control 178.2 ± 18.6 228.3±8.32 51.8±1.46 176.5±7.98 160.5±2.13

Orlistat (10 mg/kg b.w.) 207.5 ± 11.2 220.5±4.26 64.8±1.05 155.8±5.09 129.6±10.0

Streptolipin (5 mg/Kg b.w.) 134.2 ± 9.37 204.7±3.67 64.3±0.57 140.4±4.10 171.0±10.2



Values expressed as mg/dl are mean ± SEM of 10 number of animals in each group


208

Table 3.52: Effect of streptolipin on serum lipid profile (Multiple dose study)

Cholesterol Groups/parameters Total triglycerides Total HDL LDL

Total phospholipids

Control 168.2 ± 7.73 205.2 ± 10.9 48.5 ± 0.44 156.7 ± 10.4 95.3 ± 1.38

Orlistat (10 mg/kg b.w.) 136.8 ± 3.37 181.9 ± 7.81 65.3 ± 0.62 116.7 ± 7.80 125.6 ± 9.95

Streptolipin (5 mg/Kg b.w.) 137.0 ± 2.74 188.9 ± 7.94 66.9 ± 0.88 122.0 ± 7.92 128.3 ± 9.30



Values expressed as mg/dl are mean ± SEM of 10 number of animals in each group


209

Table 3.53: Effect of streptolipin on liver lipid profile (Single dose study)

Groups/parameters Total lipids Triglycerides Cholesterol Phospholipids Control 75.2 ± 2.88 38.9 ± 1.37 8.11 ± 0.12 20.6 ± 0.95

Orlistat (10 mg/kg b.w.) 71.9 ± 1.87 41.4±2.44 7.34±0.22 24.5±0.88

Streptolipin (5 mg/Kg b.w.) 96.6 ± 3.04 46.9±1.28 7.72±0.23 24.5±0.88

Streptolipin (10 mg/Kg b.w.) 94.2 ± 6.33 45.0±1.93

9.23±0.33 32.4±1.02

Streptolipin (20 mg/Kg b.w.) 79.7 ± 2.19 40.5±1.08 7.43±0.26 26.4±0.99

Values expressed as mg/gm are mean ± SEM of 10 number of animals in each group.


210

Table 3.54: Effect of streptolipin on liver lipid profile (Multiple dose study)

Groups/parameters Total lipid Triglyceride Cholesterol Phospholipids

Control 98.7 ± 3.98 31.9 ± 0.67 8.97 ± 0.25 22.9 ± 1.29

Orlistat (10 mg/kg b.w.) 67.2 ± 3.88 18.4 ± 1.67 6.28 ± 1.67 21.0 ± 1.57

Streptolipin (5 mg/Kg b.w.) 85.8 ± 4.12 25.5 ± 0.52 7.49 ± 0.49 18.3 ± 0.32



Values expressed as mg/gm are mean ± SEM of 10 number of animals in each group.


211

Table 3.55: Effect of streptolipin on liver and adipose tissue weight (Single dose study)

Perirenal fat Liver Groups/parameters Perirenal fat

(mg) mg% Perirenal

fat Total liver (g) % Liver weight

Control 130.0 ± 1.20 479.7 ± 4.48 1.11 ± 0.031 4.09 ± 0.114

Orlistat (10 mg/kg b.w.) 103.1 ± 3.00 389.1 ± 11.32 1.05 ± 0.05 3.96 ± 0.188





212

Table 3.56: Effect of streptolipin on liver and adipose tissue weight (Multiple dose study)

Perirenal fat Liver Groups/parameters Perirenal fat (g) mg% Perirenal fat Total liver (g) % Liver weight

Control 167.0 ± 6.02 592.2 ± 21.3 1.24 ± 0.04 4.39 ± 0.131

Orlistat (10 mg/kg b.w.) 122.6 ± 9.06 433.2 ± 32.0 1.26 ± 0.04 4.45 ± 0.141




Values represent mean ± SEM of 10 animals in each group.


213

evidenced in this animal study to be a more potent inhibitor of lipase activity and hence

of dietary triglyceride absorption, when compared to Orlistat.

3.E.3. Influence on antioxidant status: Lipid peroxide values in serum and liver streptolipin administered mice is

presented in Figure 3.46. Significantly decreased serum (or) circulatory lipid peroxide

content was evidenced in streptolipin administered mice. This decrease was as much as

50% in the highest dose. Similarly, hepatic lipid peroxide content was lower as a result

of streptolipin administration. The extent of decrease in lipid peroxide content of both

circulation and hepatic tissue was higher in streptolipin administration as compared to the

effect produced by an equal dose of Orlistat.

Activities of three antioxidant enzymes in the catalase, GSH peroxidase, superoxide

dismutase in the liver of streptolipin administered mice are presented in Table 3.57.

Streptolipin administration produced a significant increase in the activities of hepatic

catalase, both as a result of single dose as well as multiple dose. There was no much

difference in activities of plasma non-specific enzymes in streptolipin administered mice

in multiple dose study (Table 3.58).

Similarly, the activities of hepatic glutathione peroxidase and of superoxide

dismutase were significantly higher in streptolipin administered mice especially as a

result of multiple dose. This effect was higher compared to the effect produced by an

equal dose of Orlistat. Thus, concomitant with lowered lipid peroxides, the endogenous

antioxidant enzymes were beneficially modulated in hepatic tissue of streptolipin

administered mice.

Thus, in addition to its lipase inhibitory activity, streptolipin is evidenced in this

animal study to exert beneficial influence on the antioxidant status of the animal.


214

Figure 3.46: Effect of streptolipin on lipid peroxides in serum (Circulatory)

and liver (Multiple dose study)

Se

rum

µM/dl

0

10

20

30

40

Liver

nM/m

g pro

tein

0

1

2

3

4

5

Serum µM/dl Liver nM/mg protein

Values expressed as TBARS represent mean ± SEM of 10 animals in each group.

Control Lipstatin Streptolipin Streptolipin Streptolipin (10 mg/kg b.w) (5 mg/kg b.w) (10 mg/kg b.w) (10 mg/kg b.w)


215


216

Table 3.57: Effect of streptolipin on activities of hepatic antioxidant enzymes (Multiple

dose study) :

Mouse Group Streptolipin

Liver

antioxidant Enzymes

Control

Orlistat (10 mg/kg b. w) 5 mg/ Kg

b.w 10 mg/ Kg b.w

20 mg/ kg b. w

Catalase 3 days 214.4 ± 4.5 290.6 ± 8.7 243.3 ± 5.6 304.3 ± 3.7 326.9 ± 5.6 7 days 155.0 ± 11.9 297.5 ± 26.7 286.2 ±

19.2 389.0 ± 27.9

428.1 ± 15.6

Peroxidase 3 days 27.1 + 2.9 27.8+ 2.8 26.2 + 2.2 30.6+ 4.2 31.9 + 1.8 7 days 16.8 + 2.0 23.5 + 0.6 20.0 + 3.2 27.5 + 7.7 29.1 + 5.5 Super Oxide Dismutase 3 days 7.0+2.9 7.7 + 0.1 7.5 + 0.2 10.0 + 1.2 14.7 + 1.2 7 days 6.4 + 1.3 8.1 + 0.9 8.1 + 1.2 10.1+2.4 15.4+1.1


217

Table 3.58: Effect of streptolipin on the activities of plasma non-specific enzymes (Multiple dose study).

Mouse Group Streptolipin

Serum Enzyme

Control Orlistat

10 mg/Kg b.w. 5 mg/ Kg b.w. 10 mg/ Kg b.w. 20 mg/ Kg b.w. Alanine

aminotransferase

137.7 + 2.90 132.7 + 3.33 127.4 + 1.19 124.6 + 1.51

129.0 + 2.31

Aspartate

aminotransferase

293.2 + 1.59 271.2 + 5.40 269.5 + 1.98 268.6 + 4.13

279.0 + 3.89


218

Figure 3.42: Effect of sequential extraction on streptolipin recovery from the biomass

0

10

20

30

40

50

60

70

80

1 2 3

Sequential extraction number

Stre

ptol

ipin

(mg/

10 g

dry

bio

mas

s wei

ght)


219

Summary and highlights of the investigation:

15 fungal cultures were screened for lipase inhibition from CFTRI culture collection

center and none of the fungal cultures have shown potent inhibition against pancreatic lipase.

Several soil samples were collected from randomly chosen locations, taking sufficient care to

see that the points of collection had, as widely varying characteristics as possible with regard to

physical and physiological characters. Several methods for pretreatments of soil sample and

media were tried for isolation of actinomycetes, out of which calcium carbonate treatment was

found to be the best for isolation of actinomycetes and starch casein media was the best among

the media tried.

230 isolates were isolated from terrestrial samples. According to color of the mature

sporulated aerial mycelium, isolates of the gray series were found to represent 46 percent of the

total number of isolates and the lowest occurrence was noted for the green series (0.86%).

22 isolates were found to produce pancreatic lipase inhibitors. Among these, 7 were

recovered from the soil sediment collected from cow barnyard, 5 from lakes, 2 from desert, 2

from tea plantations, 3 from hill station, 2 from forest and 1 from coconut field, the greatest

number of active strains were isolated from samples, which were pretreated with calcium

carbonate followed by phenol.

From these 22 isolates, 7 actinomycete isolates which showed inhibition greater than 50

% were selected for secondary screening. Results indicated that only one isolate N2 not only

produced the maximum amount of inhibitor but also showed greater reproducibility. Two

different types of media were employed for the production of lipase inhibitor for secondary

screening. This approach was useful to detect the effect of various media components, genetic

stability of the microbial culture and the yield. As the inhibition shown by actinomycete N2 in

submerged fermentation were reproducible and more potent than the other cultures, further

studies were carried out using this culture.


220

Identification of the actinomycete N2 was carried out. It is gram-positive, non-acid fast,

non motile with extensively branched substrate hyphae was assigned to Streptomyces genus.

The assignment of N2 to the genus Streptomyces was also supported by the 16S rRNA

gene sequence analysis of N2. The almost complete sequence of 16S rRNA gene (1464 nt) of

N2 following BLAST analysis indicated that N2 is related to species of Streptomyces and

further analyse the phylogenetic relationship of N2 a phylogenetic tree was constructed using

NEIGHBOR JOINING and DNAPARS. A detailed study on morphological and

chemotaxonomic features complemented by phylogenetic analysis showed overwhelming

differences with other closely related Streptomyces species, which led to a proposal to assign

N2 the status of a new species for which the name Streptomyces vayuensis sp. nov. was given.

The extraction and purification of the lipase inhibitor carried out. Techniques such as 1H, 13C NMR, 2DHMQCT, 1H-1H COSY, HMQC and HMBC spectra along with LC/MS and

elemental analysis were used which led to the identification of a novel metabolite as nonadeca-

6-enoicacid-3-(hexadecyloxy- hydroxy thiophosphoryloxy)-quinoxalin-2-yl ester with a

molecular formula C43H73N2O5PS with a molecular weight of 761.

A literature search revealed that this compound did not match with any reported lipase

inhibitors or of any Streptomyces metabolites. The inhibitor is henceforth designated as

STREPTOLIPIN [Streptomyces lipase inhibitor]. This has an IC50 of 349 nM and did not

show inhibition against plant and fungal lipases. Streptolipin revealed no antimicrobial activity

upto a concentration of 200 µg by disc plate method. Studies on the mode of inhibition of

streptolipin against pancreatic lipase revealed it to be irreversible with a non competitive type

of inhibition with a Ki value of 0.714 µM.

The optimisation of the fermentation and nutritional parameters and downstream

processing of production of streptolipin through submerged fermentation was studied. An

HPLC method was developed to determine the streptolipin concentration in crude samples

from the standard graph. Streptolipin production was influenced by carbon, nitrogen and trace

salt supplements. Streptolipin production was highest when glucose and yeast extract were

used at 50 g/L and incubation at 300C for 84 h. The maximum streptolipin obtained at

optimized physico-chemical parameters was 212 mg/L.


221

Extraction of the biomass using ethyl acetate ratio 1:10 at pH under agitated conditions

resulted in maximum inhibitory recovery. Sequential extraction showed substantial increase in

the overall yield of inhibitor extraction. At optimum levels of physico-chemical and down

stream process parameters streptolipin yield was found to be 75.9 mg/10 g of dry biomass.

There was a dose dependent higher excretion of triglyceride as a result of streptolipin

administration. It is very clear that triglyceride absorption was dose -dependently lower in

streptolipin-treated mice, and was concomitant with higher faecal excretion of triglycerides.

The higher excretion of fecal triglycerides is attributal to the strong lipase inhibitory potential

of streptolipin. Total cholesterol concentration in serum remained unchanged, the proportion of

cholesterol associated with LDL fraction was somewhat lower in these test groups. The

decrease in perirenal fat content and liver triglyceride content are in agreement to interferance

of streptolipin with dietary triglyceride absorption.

The activities of hepatic glutathione peroxidase, catalase and superoxide dismutase

were significantly higher in streptolipin administered mice especially as a result of multiple

doses. Thus concomitant with lowered lipid peroxides, the endogenous antioxidant enzymes

were beneficially modulated in hepatic tissue of streptolipin administered mice. Streptolipin is

evidenced in this animal study to exert beneficial influence on the antioxidant status of the

animal along with a more potent inhibition of lipase activity and hence of dietary triglyceride

absorption, when compared to Orlistat.

Recommendations for further work:

• Studies on strain improvement for further increase of streptolipin production is also

another area which is needed for the commercialization of the process.

• Spectroscopic studies such as circular dichroism and fluorescence studies are to be

carried out to get a detailed insight into the mode of action of the inhibitor at the

molecular level.


222

• Biochemical and physiological parameters such as interactions with serum proteins to

determine bioavailability and dosage development would be valuable in determining

the efficiency of the inhibitor in vivo.

• Fermentation techniques such as fed-batch fermentation to enhance the streptolipin

production are also recommended.


223

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Clin. Pharm. Ther: 56, 82-85.


240

List of publications and patents:

Papers communicated:

1. Naveen Babu, K., Shivaji, S. and Sattur, A.P. Streptomyces Vayuensis sp. nov: A new

species of Genus Streptomyces. International Journal of Systematic and Evolutionary

Microbiology (Communicated).

2. Naveen Babu, K., Jagan Mohan Rao, L. and Sattur, A.P. Streptolipin, a novel

pancreatic lipase inhibitor. Journal of Antibiotics (Communicated).

3. K.Naveen Babu and A.P.Sattur. Streptolipin production by Streptomyces vayuensis:

kinetics and the influence of nutrients. Prcocess Biochemistry (Communicated).

US product patent:

Sattur, Avinash Prahalad; Babu, Kilaru Naveen; Rao Lingamallu Jagan Mohan; Karanth,

Naikanakatte Ganesh; An inhibitor compound and its isolation and method of treatment

against pancreatic lipase for use as an antiobesity agent (Filed).

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