08 Chapter 2

Review of Literature

Murali Krishna. CH Page 23

2.0 REVIEW OF LITERATURE

Amylases are starch degrading enzymes. They are widely distributed

in microbial, plant and animal kingdoms (Banks et al, 1975). They degrade

starch and related polymers to yield products characteristic of individual

amylolytic enzymes. Initially the term amylase was used originally to

designate enzymes capable of hydrolysing -1, 4- glycosidic bonds of

amylose, amylopectin, glycogen and their degradation products (Damien et

al, 2010). They act by hydrolysing bonds between adjacent glucose units,

yielding products characteristic of the particular enzyme involved (Dhanya

et al, 2009).

In recent years a number of new enzymes associated with degradation

of starch and related polysaccharides structures have been detected and

studied (Mohammad et al, 2010). The enzymes having potential commercial

importance of microbial origin that split -1,4 or -1,4 and/or -1,6 bonds

in these structures, may be divided in the following six classes.

1. Enzymes that hydrolyse alpha-1,4 bonds and bypass alpha -1,6

linkages e.g. -amylase (endo acting amylases).

2. Enzymes that hydrolyse -1,4 and cannot bypass -1,6 linkages

e.g. -amylase (exoacting amylases producing maltose as a major

end product).

3. Enzymes that hydrolyse -1,4 and -1,6 linkages e.g. amylo

glucosidase (gluco amylase) and exo acting amylase.

4. Enzymes that hydrolyse only -1,6 linkages e.g. pullulanase and

other debranching enzymes.

5. Enzymes that hydrolyse preferentially -1,4 linkages in short chain

oligosaccharides produced by the action of other enzymes on

amylose and amylopectin e.g. -glucosidases.



6. Enzymes that hydrolyse starch to a series of non reducing cyclic

D-glucosyl polymers called cyclodextrins or sachardinger dextrins

e.g. Bacillus macerans amylase (cyclodextrin producing enzyme)

(Archana et al, 2011)

2.1 STARCH

Before describing the action pattern and properties of amylolytic

enzymes it is essential to discuss the features of the natural substrate,

starch is a major reserve carbohydrate of all higher plants (Encarnacion et

al, 2011). In some cases it accounts for as high as 70% of the undried plant

material. It occurs in the form of water insoluble granules (Guadalupe et al,

2011). The size and shape of the granules is often characteristic of the plant

species from which they are extracted. When heated in water the hydrogen

bonds holding the granules together begin to weaken and this permits them

to swell and gelatinize (Tomasz et al , 2011).Ultimately they form paste or

dispersion, depending on the concentration of polysaccharide (Xuerong et al,

2008). Starches are produced commercially from the seeds of plants, such

as corn, wheat, sorghum or rice, from the tubers and roots of the plants

such as cassava, potato, arrowroot and the pith of sago palm (Cynthia et al,

2011).The major commercial source of starch is corn from which it is

extracted by a wet milling process (Dhanya et al, 2009).

Starch is a heterogeneous polysaccharide composed of two high

molecular weight entities called amylose and amylopectin (Damien et al,

2010). These two polymers have different structures and physical properties.



TABLE 2.1.1 COMPARISON OF AMYLOSE AND AMYLOPECTIN

Properties Amylose Amylopectin

Basic structure Linear Branched

Stability in aqueous solution Retrogrades Stable

Degree of Polymerization C.103 C.104 - C.105

Average chain length C.103 C.20-25

Beta amylase hydrolysis 87% 54%

Beta amylase & Debranching enzyme

hydrolysis

98% 79%

Maximum Iodine complex 650 nm 550 nm

Starch may be separated into two components by addition of a polar

solvent, e.g. n-butanol, to a dispersion of starch (Natasa et al, 2011). The

insoluble amylose complex can then be separated from soluble amylopectin

fraction. Amylose is composed of linear chains of -1,4 linked D-glucose

residues(Chi-Wenlin et al, 2011). Hence it is extensively degraded by -

amylase. Some amylose is not totally degraded to maltose by this enzyme.

Amylose has a degree of polymerization of several thousands of glucose

units (Bank et al 1975; Maryam et al, 2010). Because of the molecular

shape and structure of amylase, it is not stable in aqueous solution and

retrogrades (precipitates spontaneously), (Ahmad et al, 2010). This is

because linear chains align themselves by hydrogen bonding and thus forms

aggregates. This process is irreversible retrograded amylase will only

dissolve in alkaline solution (Burhan et al, 2008). Amylose has considerable

viscosity in alkaline solutions due to its molecular shape. Amylase this

forms complex with iodine to form intense blue colour and this forms the

basis of a method for quantitative determination of amylase (Aw et al, 1969).



Amylopectin may account for 75 to 80% of most starches. It has

molecular weight in excess on 10-10 and has a branched structure

composed of chains about 20-25 -1,4 linked D-glucose residues.

Amylopectin which is branched by -1,6 D-glucosidic bonds (Henrissat et al,

1991).In aqueous solutions, amylopectins are relatively stable due to

branched molecules and are not able to form compact aggregates. There is

no apparent relationship between the limiting viscosity number and the

degree of polymerization. Due to the nature of branched structure, the

iodine binding power is reduced (Aw et al, 1969). The branched components

of starch are amylopectin which has different types of chains referred to as

A, B and C chains (Mohammad et al, 2010).

The hydrolysis of starch may be carried out using either acid or

enzyme as catalyst (Diana et al, 2007). Enzyme hydrolysis has several

advantages; it is more specific, therefore fewer by products are formed, and

hence yields are higher. Conditions for enzyme hydrolysis are milder

therefore refining stages to remove ash and colour is minimized. The

enzymatic hydrolysis of starchy has been practiced on an industrial scale for

many years and is gradually replacing the traditional acid hydrolysis

process (Kazunari et al, 2011).

2.2 SOURCES OF ALPHA AMYLASE

Alpha amylases are ubiquitous enzymes produced by plants, animals

and microbes, where they play a dominant role in carbohydrate metabolism

(Varel et al, 1994). Amylases from plant and microbial sources are employed

for centuries as food additives (Mabel et al, 2008). Barley amylases are used

in Brewing industry. Fungal amylases are widely used in preparation of

oriental foods (Popovic et al, 2009). Fungal and bacterial amylases are

mainly used for industrial applications due to their cost effectiveness,

consistency, less time and space requirement for production and ease of

process optimization and modification (Ellaiah et al, 2002).



Among bacteria Bacillus.sp is widely used for the production of

amylases. Species like B.subtilis, B.stearothermophilus, B.licheniformis,

and B.amyloliquefaciens are known to be good producers of alpha amylase

(Harshemi et al, 2011). Similarly filamentous fungi have been widely used

for the production of amylases for centuries (Juliana et al, 2011). As these

moulds are known to be prolific producers of extracellular proteins, they are

widely exploited for the production of different enzymes including alpha

amylases (Kozunari et al, 2011). Fungi belonging to the genus Aspergillus

have been most commonly employed for the production of alpha amylase.

Production of enzymes by solid state fermentation using these moulds

turned a cost effective production technique (Parveen et al, 2011).

2.3 FERMENTATIVE PRODUCTION OF AMYLASE

To meet the demand of industries, low cost medium is required for

the production of alpha-amylase (Aliyu et al, 2011). Both SSF and

submerged fermentation (SmF) could be used for the production of amylase,

although traditionally these have been obtained from submerged cultures

because of easy handling and greater control of environmental factors such

as temperature and pH (Xusheng et al, 2011).Mostly synthetic media have

been used for the production of bacterial amylase through SmF (Ajay et al,

2010). The contents of synthetic media such as nutrient broth, soluble

starch, as well as other components are very expensive and these could be

replaced with cheaper agricultural by products for the reduction of the cost

and the medium(Solange et al, 2010). The solid substrate may provide only

support and nutrition (Hashemi et al, 2011). SSF is considered as an

interesting alternative since the metabolites so produced are concentrated

and purification is of less quality (Nasrin et al, 2010). SSF is preferred to

SmF because of simple technique, low capital investment, lower levels of

catabolite repression and end product inhibition, low waste water out put

better product recovery and high quality has been reported to produce

promising results (Ajay et al, 2010) other substrates such as sunflower

meal, rice husk, cotton seed meal, soybean meal, rice husk, cotton seed



meal, soy bean meal, and pearl millet and rice bran have been tried for SSF

(Maryam et al, 2010).

SSF technique is generally confined to the process involving fungi

(Kiran et al, 2010). However, successful bacterial growth in SSF is known

much in natural fermentation (Lonsane et al, 1990). The production of alpha

amylase by SSF is limited to the genus Bacillus like B.subtilis, B.polymaxa,

B.mesentiricus, B.vulgarus, B.coagulans, B.megaterium and B.licheniformis

have been used for alpha amylase production in SSF (Natasa et al,

2011).The production of bacterial amylase using alpha amylase technique

requires less fermentation time which leads to considerable reduction in the

capital and recurring expenditure (Li Zhuang et al, 2011). Research on the

selection of suitable substrates for SSF has mainly been centred around

agro industrial residues due to their potential advantages for filamentous

fungi which are capable of penetrating into the hardest of these solid

substrates, aided by the presence of turgor pressure at the tip of the

mycelium (Maryam et al, 2010). In addition, the utilization of these agro

industrial wastes, not only provides alternative substrates but also on the

other hand helps in solving pollution problems (Priya et al, 2011). Table 1

summarizes various agro residues reported for microbial alpha amylase

production.

TABLE 2.3.1 VARIOUS SUBSTRATES USED FOR ALPHA AMYLASE

PRODUCTION

Substrate Organism Activity (U/g)

Wheat bran Bacillus sp.PS-7 464,000

Spent brewing grain A.oryzae NRRL 6270 6583

Maize bran B.coagulans 22956

Rice bran Bacillus sp.PS-7 145,000

Coconut oil cake A.oryzae 3388

Amaranthus grains A.flavus 1920



2.4 PROCESS OPTIMIZATION

Optimization of the various parameters and manipulations of media

are one of the most important techniques used for the over production of

amylase in large quantities (Balasubramanien et al, 2011). To meet

industrial demands production of alpha amylase in fungi is known to

depend on both morphological and metabolic state of the culture (Juliana et

al, 2011). Growth of mycelium is crucial for extracellular enzyme like alpha

amylase (Sangeeta et al, 2009). Various physical and chemical factors have

been known to effect the production of alpha amylase such as temperature,

pH, Incubation period, carbon, nitrogen sources, surfactants, phosphate,

different metal ions, moisture and agitation with respect to SSF and SmF

(Ellaiah et al, 2002).

2.4.1 TEMPERATURE:

The influence of temperature on amylase production is related to the

growth of the organism (Pandey et al, 1990). Hence the optimum

temperature depends on whether the culture is mesophilic or thermophilic.

Among the fungi most amylase production studies have been done with

mesophilic fungi within the temperature range of 25-37oC (Takahiro et al,

2011). A raw starch degrading amylase was produced by Aspergillus ficum

at 30oC by Hayashida et al in 1986. Yeast such as Saccharomyces kluyveri

and S.cerevisiae was reported to produce alpha amylase at 30oC (Moller et al

2004). Amylase production at optimum level has been reported between 50-

55oC for the thermophilic fungal cultures such as Talaromyces emersonni,

Thermomonospora fusca etc (Ahmad et al, 2010).

2.4.2 pH:

pH is one of the important factors that determine the growth and

morphology of microorganisms as they are sensitive to the concentration of

hydrogen ions present in the medium(Ellaiah et al, 2002). Earlier studies

have revealed that fungi required slightly acidic pH and bacteria required

neutral pH for optimum growth. pH is known to effect the synthesis and



secretion of alpha amylase just like its stability (Yakup et al, 2010). Fungi of

Aspergillus sp. such as A.oryzae, A.ficuum and A.niger were found to give

significant yields of alpha amylase at pH equal to 5.0 to 6.0 in SmF (Parveen

et al, 2011). Alpha amylase producing yeast strains such as S.cerevisiae and

S.kluyveri exhibited maximum enzyme production at pH 5.0 (Samrat et al,

2011).

2.4.3 CARBON SOURCES:

Carbon sources such as galactose, glycogen and Inulin have been

reported as suitable substrates for the production of amylases by

B.licheniformis and Bacillus.sp.1-3 (Xusheng et al, 2011). Starch and

glycerol were known to increase enzyme production in B.subtilis IMG22,

Bacillus sp, PS-7 and Bacillus sp.1-3(Maryam et al, 2010). Soluble starch

has been found as the best substrate for the production of alpha amylase by

B.stearothermophilus (Srivastava et al 1986). Bacillus sp. was noted to give

a maximum raw starch digesting amylase in a medium containing lactose

(1%) and yeast extract (15%). Thermomyces lanuginosus was reported to

give maximum alpha amylase yield when maltodextrin was supplemented to

the medium (Nguyen et al, 2000). Agricultural wastes are being used for

both liquid and solid fermentation to reduce the cost of fermentation media.

The waste consists of carbon and nitrogen sources necessary for the growth

and metabolism of organism. These nutrients sources include orange waste,

peer millet starch, potato, corn, tapioca, wheat and rice as flours (Lin Hui et

al, 2011).

2.4.4 NITROGEN SOURCES:

Soybean meal was found as the best nitrogen source for alpha

amylase by Bacillus sp. 1-3. Tanyildizi et al reported that peptone increased

enzyme activity while yeast extract exhibited no effect on alpha amylase

production (Arpana et al, 2011). Strains of Bacillus stearothermophilus and

B.amylolyticus secreted maximum alpha amylase in a medium

supplemented with 1% peptone, 0.5% yeast extract and 0.5% maltose under



vigorous shaking conditions (Elif et al, 2005), compared the influence of

organic and inorganic nitrogen sources and reported peptone to be a better

nitrogen source for enzyme production by B.licheniformis SPT 278 than

ammonium phosphate, the best among inorganic nitrogen sources. L-

asparagine was reported to be one of the most promising nitrogen sources

for alpha amylase production by Thermomyces lanuginosus (Adinarayana et

al, 2005). Yeast extract also resulted in a significant alpha amylase yield.

Supplementation of Casein hydrolysate to the medium resulted in 143%

increase in alpha amylase productivity by A.oryzae 1560 compared to

ammonia.

2.4.5 SURFACTANTS:

Surfactants in the fermentation medium are known to increase the

secretion of proteins by increasing cell membrane permeability. Therefore

addition of these surfactants is used for the production of extracellular

enzymes (Samrat et al, 2011). Addition of tween 80 (1.3%) to the

fermentation medium increased alpha amylase production by 2 fold in

Thermomyces lanuginosus (Sivaramakrishnan et al, 2006). A study on the

effect of supplementation of Poly ethylene glycols (PEG) (molecular mass of

600,3000,4000,8000 and 20,000) in fermentation medium for alpha

amylase production by two bacillus sp. Researchers indicated that 5% PEGs

600 and PEG 3000 yielded 31% increase in enzyme production by

B.amyloliquefaciens and 21% increase by B.subtilis (Goes et al, 1999).

2.4.6 METAL IONS:

Supplementation of salts of certain metal ions provided good growth

of microorganisms and thereby better enzyme production as most alpha

amylases are known to be metalloenzymes (Zoe et al, 2006). Ca+2 are

reported to be present in majority of these enzymes. Addition of Calcium

chloride to the fermentation media increased the enzyme production (Arthur

et al, 1996). Positive results of the influence of CaCl2 (0.1%) and NaCl (0.1%)

on alpha amylase production in SSF using Amaranthus grains as substrates



were recorded (Vishwanathan et al, 2001). LiSo4 (25 mM) and MgSo4 (1mM)

increased alpha amylase production by Bacillus sp.1-3 (Reeta et al, 2009)

but FeCl3 and MgSo4 exhibited negative influence on alpha amylase

production ( Vishwanathan et al 2001).

2.4.7 MOISTURE CONTENT:

Moisture is one of the most important parameters in SSF that

influences the growth of the organism and thereby enzyme production

(Pandey et al, 2000). Low and high moisture levels of the substrate effect the

growth of the microorganisms resulting in lower enzyme production (Ellaiah

et al, 2002). High moisture content leads to reduction in substrate porosity,

changes in the structure of substrate particles and reduction of gas volume.

Bacteria are generally known to require initial moisture of 70-80%. Alpha

amylase production by Bacillus licheniformis M27 was highest with 65%

initial moisture content in an SSF system (Namita et al, 2007). Significant

decrease in enzyme production was observed with high increase in moisture

content which was due to the decrease in the rate of oxygen transfer.

Studies indicated that enzymes titres could be increased significantly by

agitation of the medium with high moisture content (Lonsane et al, 1990).A

thermotolerant B.subtilis requires initial moisture of 30% for its growth and

maximum enzyme production (Ahmad et al, 2010).

2.4.8 PARTICLE SIZE OF THE SUBSTRATE:

In SSF, particle size of the substrate effects growth of the organism

and thereby influences the enzyme production (Ellaiah et al, 2002). The

adherence and penetration of micro organisms as well as the enzyme action

on the substrate clearly depend upon the physical properties of the

substrate such as the crystalline or amorphous nature, the accessible area,

surface area, porosity, particle size etc (Chen et al, 2011). In all the above

parameters, particle size plays a major role because all these factors depend

on it (Pandey et al, 1991). Smaller substrate particles have greater substrate

surface area for growth but inter particle porosity is lower (Pandey et al,



1991).For larger particle sizes, the porosity is greater but the saturated

surface area is smaller hence determination of particle size corresponding to

optimum growth and enzyme production is necessary (Reeta et al, 2009).

2.5 PURIFICATION:

Down stream processing for the production of pure enzymes can

generally constitute a major percentage of overall production cost especially

if end purity requirements are stringent (Lalit et al, 2010). Purification

process in down stream processing after fermentation strongly depend on

the market, processing cost, final quality and available technology. Most

enzymes are purified by Chromatographic techniques after crude isolation

by precipitation and membrane separations (Prakash et al, 2009). The need

for large scale cost effective purification of proteins has resulted in evolution

of techniques that provide fast, efficient and economical protocols in fewer

processing steps (San-Lang et al, 2011). Purification techniques that

produce homogenous preparation of amylase in a single step are given in

below table (Anni linden et al, 2000).



2.5.1 METHODS OF ONE STEP PURIFICATION OF ALPHA AMYLASES

Method Adsorbent Yield

(%)

Purification

fold

Reference

Affinity Adsorption

Chromatography

Beta

cyclodextrin-

iminodiacetic

acid- Cu+2

95 - Liao et al

Expanded bed

chromatography

Alginic acid

cellulose cell

beads

69 51 Amritkar

et al

High speed counter

current

chromatography

PEG 4000

aqueous two

phase system

73.1 - Zhi et al

Magnetic affinity

adsorption

Magnetic

alginate

microparticles

88 9 Safari kova

et al

Substitute affinity

method

Insoluble corn

starch at 4oC

78 163 Najafi et al

2.6 IMPROVEMENT OF THE STRAIN

The mutant strains of Bacillus have better ability to produce alpha

amylase, which can be derived by mutagenesis and extra screening. Both

chemical mutagenic agents as well as UV irradiations can be used to

improve the Bacillus strains for the production of alpha amylase. The UV

irradiations produce mutants by the photolysis of pyramidines to from

dimers. These dimers can cause errors in the replication, which result in

mutation (Guadalupe et al, 2011). During the course of ultra violet studies

with the bacillus subtilis (ATCC6051), a mutant was obtained which was



stimulated by a factor in yeast extract with glucose inorganic salts medium

containing monoacids, vitamins, purines and a pyramidine. This mutant

was a stable one and can be stored at 5oC for several months.

Markkanen and suihko (1947) found that UV irradiation was suitable

mutagen of alpha amylase and proyteolytic enzyme production by Bacillus

subtilis. Cells exposed to UV radiation produced large number of

permutations, mostly as a result of dimerization of thiamine. Those

premutants usually under go partial or complete repair on return to visible

light, when a specific enzyme acts to separate the dimerized thiamine

molecules. Irradiation causing death rate of 90% was found to be most

effective in the production of mutants with improved amylase yields. Among

mutants, the best frequencies of positive mutations were 1:50 and 1:20 for

amylase and proteolytic enzyme, respectively.

Bailey et al, (1979) employed various mutagenic agents in

succession to produce mutant strains of Bacillus subtilis with improved

yield of amylase. The parent strain had previously been selected as a good

producer of amylase. Highly productive mutant strains were selected

through out the work as the basis for further treatment. In shake flask

cultures, the yields of amylase of the best strains were double that of parent

strain and in Fermentor cultivation the improvement was even greater.

Yu et al (1986) have used NTG as mutagen for the mutation of

Bacillus subtilis. Two mutant strains K1 and K5 were isolated from 2500

isolates. These strains gave increase in the yield of enzyme than the parental

strain BF7658. The average yield of alpha amylase of K1 and K5 strains

were 320 and 381 U/ml. respectively.

Shah et al (1989) have isolated a high yielding mutant of B.subtilis

by subjecting its parental strain and subsequent highest yielding mutant,

after each exposure, to successive to N-Methyl-N-Nitro-N-Nitrosoguanidine

(NTG) at various concentrations and finally to a single UV irradiation. This

mutant secretes 5-fold more alpha amylase activity than the parental strain.



In screening higher producer of thermostable alpha amylase, Bacillus

licheniformis B198 was chosen as an original strain, which was treated

repeatedly with various mutagens. The mutant A.4041 was selected after

repeated natural selection. The thermostable alpha amylase activity of this

mutant was in 100 fold of the original one, reaching 200U/ml in the shake

flask. The mutant was resistant to catabolite repression by glucose (Xuezhi

et al, 1991).

Qirang and Zhao (1994) investigated the selection and breeding of a

high productivity of a amylase from multi resistant mutant of Bacillus. Jin

et al ,(1998) have developed a hyper producing alpha amylase mutant of

Bacillus licheniformis. The mutant shows 50 times higher enzyme than the

parental strain.

Bin et al (1999) screened out alpha amylase high producing strains

from Bacillus subtilis. In screening high producer of alpha amylase, Bacillus

subtilis 14140 was chosen as an original strain. The strain was treated

repeatedly with N-Methyl-N-Nitro-N-Nitrosoguanidine (NTG). After screening,

the mutant B.subtilis GS was selected. The enzymatic activity of alpha

amylase was raised from 3000U/ml. Effect of carbon sources and nitrogen

sources on the formation of alpha amylase were also studied in the shake

flask. Niziolek, (1998) investigated the production of extracellular, amylolytic

enzymes in 41 strains of the genus Bacillus representing 13 species using

different liquid media and cultivation temperature of 30oC and 38oC. It was

found that 8 strains were amylase negative, 19 strains were low productive

and 12 were medium productive strains (10-25 U/ml). B.subtilis AS-1-108,

B.subtilis NCIB 8159 and B.licheniformis NCIB 7198 strains were included

among the higher producers as they produced about 370, 170 and 40 U/ml

of alpha amylase. The amylase production by B.subtilis was variously

affected by medium composition and temperature of cultivation. The

enzymes from B.subtilis AS-1-108 and NCIB 8159 strains were more thermo

sensitive than those of the medium productive strains of B.subtilis. The



action pattern of alpha amylase from B.subtilis strains was affected by pH

and temperature.

Alkaline amylase producing mutants were successfully induced from

this strain by mutagenesis with two different agents like ultra violet light

and NTG (1-methyl 1-3-nitro-nitroguanidine).In the first mutation step (UV

mutagenesis) they selected a mutant strain, Bacillus sp. ICCF 276/18 which

was induced with UV mutant strain. The amylolytic activity of Bacillus sp.

ICCF 276/18 in the optimized medium was 17.5 U/ml, being approximately

2.5 higher fold than that of the wild strain (Dinu et al, 2001).

Haq et al (2002) investigated the biosynthesis of alpha amylase by

chemically treated mutant of B.subtilis GCBUCM-25.The strain of B.subtilis

was treated with NTG for different intervals of time (5-60 min). One hundred

mutant strains were isolated and tested for the production of alpha amylase

and Bacillus subtilis GCBUCM-25 gave maximum production of enzyme

(2210 U/ml). The optimum conditions for the production of alpha amylase

were sodium nitrate as nitrogen source, pH 7.5 phosphate buffer and 4mM

CaCl2 as diluents.

Table 2.6.1 Alpha Amylases Exhibiting Different Temperature Stability

Organism Temperature Residual

activity

Optimum

Temperature

Reference

Lactobacillus

manihotivarans

50-60 70 (50oC for

1.0h)

55 Aguilar et al

Bacillus sp 1-3 65-100 50 (80oC for

2.5h)

70 Goyal et al

Pyrococcus

furiosus

80-100 50 (98oC for

13h)

100 Viellie et al

Aspergillus

tamarii

50-60 90 (65oC for

3h)

55 Moreria et al

Cryptococcus

flavus

50-60 60 (60oC for

60 min)

50 Wanderley et

al



2.6.2 CONCENTRATION EFFECT OF DENATURING AGENTS ON ALPHA

AMYLASE ACTIVITY

Inhibitor Concentration effect Organism Reference

EDTA 10mM Resistant

10mM Inhibitory

Bacillus sp. L1711

Bacillus sp.1-3

Bernharsboteer

Goyal et al

SDS 1% slight inhibition B.halodurmas

LBK 34

Hagihara et al

Urea 8M Inhibitory Bacillus sp. ANT-6 Burhan et al

Hydrogen

Peroxide

1.8M Resistant Bacillus KSM-K38 Hagihara et al

2.6.3 Applications Of Amylases In Various Industrial Sectors

Sector Applications References

Food Industry Production of glucose syrup, crystalline glucose

Production of HFCS

Production of Maltose syrups

Reduction of viscosity of sugar syrups

Reduction of haze formation in juices

Solubilization & Saccharification of starch for alcohol fermentation in

brewing industries

Retardation of staling in baking industry

Hans et al,

2009.

Detergent

Industry

Used as an additive to remove starch based

dirts

Gupta et al,

2005



Paper Industry Reduction of viscosity of starch for

appropriate coating of paper

Ellaiah et al,

2002

Textile

Industry

Warp sizing of textile fibres Reeta et al,

2009

Pharmaceutical

industry

Used as a digestive aid Guler et al,

2010

2.7 APPLICATIONS OF AMYLASE

The history of the industrial production of enzymes dates back to the

time when Dr. Jhokichi Takamine began the production of digestive enzyme

preparation by wheat brankoji culture of Aspergillus oryzae in 1894.

Industrial production of dextrose powder and dextrose crystals from starch

using -amylase and glucoamylase began in 1959 (Pandey et al, 2000).

Since then, amylases are being used for various purposes. Conversion of

starch into sugar, syrups and dextrins forms the major part of the starch

processing industry (Noda et al, 2001). The hydrolysates are used as carbon

sources in fermentation as well as sources of sweetness in a range of

manufactured food products and beverages. Hydrolysis of starch to products

containing glucose, maltose etc, is brought about by controlled degradation

(Hans et al, 2009; Uma et al, 2007).Some of the applications of amylase are

as follows

2.7.1 LIQUEFACTION

Liquefaction is a process of dispersion of insoluble starch granules

in aqueous solution followed by partial hydrolysis using thermostable

amylases. In industrial processes, the starch suspension for liquefaction is

generally in excess of 35% (w/v) (Damien et al, 2010). Therefore the viscosity

is extremely high following gelatinization (Vander et al, 2002). Thermostable

-amylase is used as a thinning agent, which brings about reduction in

viscosity and partial hydrolysis of starch. Retrogradation of starch is thus

avoided during subsequent cooling (Sang-Lang et al, 2000).



The traditional thinning agent used in starch technology was acid

(hydrochloric or oxalic acids). The introduction of thermostable -amylases

has meant milder processing conditions. The formation of by products is

reduced and refining and recovery costs are lowed (Dhanya et al, 2009).

In the enzymatic process the hydrolytic action is terminated when

the average degree of polymerization is about 10-12. Two distinct types of

thermostable -amylases are commercially available and used extensively in

starch processing technology (Tomasz et al, 2011). The amylase of Bacillus

amyloliquefaciens was the first liquefying -amylase used on a large scale.

Later a more heat stable enzyme from Bacillus licheniformis was introduced

commercially (Madsen et al, 1973). Liquefaction can be done by two

methods like:

Single stage enzyme liquefaction: In 1973, Novo industry at

Copenhagen developed and patented the process. In this process, starch

slurry containing 30-40% dry solids is prepared in the feed tank. The PH is

adjusted to about 6-6.5 with sodium hydroxide. Calcium salts may be added

if the level of the free calcium ions is below 50 ppm. The liquefying enzyme

is then added. The slurry is then pumped continuously through a jet cooker

where the temperature is raised to 105o C by direct injection of live stream.

Tremendous shearing forces are exerted on the slurry as it is pumped

through the jet cooker. So in addition to the viscosity reduction action of the

enzyme, some mechanical thinning also occurs. The slurry is maintained at

this high temperature in the pressurized holding cell for about 5 min after

which it is discharged via a spring located release valve into a reaction,

where enzyme action is allowed to continue for about 2 hours at 95o c after

this treatment the liquefied starch will have dextrose equivalent (DE) of

10-20 depending on amount of enzyme used .DE is defined as a reducing

sugars expressed as dextrose and calculated as a percentage of dry

substance. This process is simple energy consumption is relatively low

because the maximum operating temperature is only 105oc as compared to

140-150oc normally used (Borge et al, 1995).



2.7.1.1 ACID ENZYME LIQUEFACTION:

This is another process which takes advantage of the

thermostability B.licheniformis amylase. The amylase is added after the

starch has been cooked and cooled to 100-95oC. Starch slurry containing

30-40% dry solids is cooked at a high temperature for about 5 mins. A jet

cooker is used so that sufficient mechanical thinning due to shearing takes

place (Madsen et al, 1973).

Liquefaction is the first and most important step in starch

processing. The purpose is to provide a partially hydrolysed starch

suspension of relatively low viscosity which is free from by products (Sameh

et al, 2011). Stable to retro gradation and suitable for further processing i.e.,

saccarification with the liquefaction process doesnt go well, problems like

poor filtration and turbidity of the processed solution occurs (Archana et al,

2011). The most important factor for ideal liquefaction of starch is that the

starch slurry which contains suitable amount of alpha amylase is treated at

105- 107oC as quickly and uniformly as possible (Fuentes et al, 2010).

Thermostable amylase is not sufficiently heat stable to be used during

liquefaction process, but they can be used as saccharifying enzymes. The

most widely used enzymes in this group are the maltogenic enzymes (Noda

et al, 2001).

2.7.2 MANUFACTURING OF MALTOSE

Maltose is a naturally occurring disaccharide. Its chemical structure

has 4-0--D-glucopyronosil-D-glucopyranose. It is the main component of

maltose sugar syrup (Yakup et al, 2010). Maltose is widely used as

sweetener and also as intravenous sugar supplement. It is used in food

industry because of low tendency to be crystallized and is relatively non

hygroscopic (Sameh et al, 2011).

Corn, potato, sweet potato and cassava starches are used for maltose

manufacture (Uma et al, 2007). The concentration of starch slurry is

adjusted to be 10-20% for production of medical grade maltose and 20-40%



for food grade. Thermostable alpha amylase from B.licheniformis and

B.amyloliquefaciens are used (Archana et al, 2011).

2.7.3 MANUFACTURE OF HIGH FRUCTOSE CONTAINING SYRUP

High fructose containing syrups (HFCS) 42 F (Fructose content

equal to 42%) is prepared by enzymic isomerization of glucose with glucose

isomerase. The starch is first converted to glucose by enzyme liquefaction

and saccharification (Shekufeh et al, 2010).

2.7.4 MANUFACTURE OF OLIGOSACCHARIDES MIXTURE

Oligosaccharides mixture (Maltooligomer mixture) is obtained by

digestion of corn starch with alpha amylase, beta amylase and pullulanase.

Maltooligomer mix is a new commercial product. Its composition is usually

as follows: Glucose, 2.2%; maltose, 37.5%; maltotriose, 46.4%; and

maltotetrose and larger malto oligosaccharides, 14% (Marc et al, 2002).

Maltooligomer mix powder obtained by spray drying is highly

hygroscopic. Therefore it serves as a moisture regulator of the food with

which it is mixed (Takata et al, 1992). Maltooligomer mix tastes less sweet

than sucrose. It has lower viscosity than corn syrup because of its low

content of glucose (Shekufeh et al, 2010). Maltooligomer mix is mainly used

as a substitute for sucrose and other saccharides. It is also used for

preventing crystallization of sucrose in foods (Vander et al, 2002).

2.7.5 MANUFACTURE OF MALTOTETROSE SYRUP

Maltotetrose syrup (G4 syrup) is produced by subjecting starch to the

action of maltotetrose forming amylase. The sweetness of the syrup is as low

as 20% of sucrose. Therefore a partial replacement of sucrose with G4 syrup

reduces the sweetness of food without affecting their taste and flavour

(Gulay et al, 2004). It has high moisture retention power which serves to

prevent retrogradation of starch ingredient and retains suitable moisture in

foods (Uma et al, 2007). It has high viscosity than sucrose thus improving

the food texture. G4 syrup can be used to control the freezing points of



frozen foods. It can be used in industries such as paper sizer (Damien et al,

2010).

2.7.6 MANUFACTURE OF HIGH MOLECULAR WEIGHT BRANCHED

DEXTRINS

Branched dextrins of high molecular weight are prepared by

hydrolysis of corn starch with alpha amylase. The extent of starch

degradation depends on the type of the starch and physical properties

desired (Dhanya et al, 2009). They are obtained as powder after

chromatography and spray drying. These are used as extender and a glozing

agent for production of powdery foods and rice cakes respectively (Harmeet

et al, 2005).

2.7.7 REMOVAL OF STARCH SIZER FROM TEXTILES (DESIZING)

In textiles weaving, starch paste is applied for warping. This gives

strength to textiles at weaving. It also prevents the loss of string by friction,

cutting and generation of static electricity on the string by giving softness to

the surface of the string due to laid down wrap. After weaving the cloth, the

starch is removed and the cloth goes to scouring and dyeing. The starch on

cloth is usually removed by application of alpha amylase (Querong et al,

2008)

2.7.8 DIRECT STARCH FERMENTATION TO ETHANOL

The amylolytic activity rate and amount of starch utilization and

ethanol yields increase in several folds in co cultures (Reeta et al, 2009)

.Moulds amylases are used in alcohol production and brewing industries.

The advantages of such systems are uniform enzyme action in mashes,

increase rate of saccharification, alcohol yield and yeast growth (Maria et al,

2011)



2.7.9 TREATMENT OF STARCH PROCESSING WASTE WATER (SPW)

Starch is also present in waste produced from food processing plants

(Jamuna et al, 1989).Starch waste causes pollution problems.

Biotechnological treatment of food processing waste water can produce

valuable products such as microbial biomass proteins and also purifies the

effluent (Ashis et al, 2009).

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