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Vol. 2(ISC-2012), 330-339 (2013) Res. J. Recent. Sci.
International Science Congress Association 330
Streptomycetes: A Storehouse of Bioactive Compounds and Enzymes
A: Production of Glucose Isomerase
Bhasin Sheetal1 and Modi H.A.
2
1Dept. of Biosciences, Maharaja Ranjit Singh College of Professional Sciences, Hemkunt Campus, Khandwa Road, Indore, MP, INDIA 2Dept. of Life Sciences, University School of Sciences, Gujarat University, Ahmedabad, Gujarat, INDIA
Available online at: www.isca.in Received 2012, revised 2012, accepted 2013
Abstract
Streptomycetes, the filamentous prokaryotic microbes, possessing exceptionally large size genome, carrying around 8-Mb
DNA sequence, are of immense commercial importance. They exhibit extensive primary as well as secondary metabolic
activity which accounts for their indispensible role in commercial and environmental scenario. They are exhaustively
investigated for the production of bioactive compounds and enzymes. We have explored the capacity of Streptomycetes to
produce glucose isomerase. Glucose isomerase converts glucose into fructose and xylose to xylulose. This reaction has
immense commercial potential as it is used for the production of High Fructose Corn Syrup. It is used as a sweetening agent
in pharmaceutical and food industry. Varieties of different strains of Actinomycetes were isolated from compost pit and
categorised on the basis of cultural and morphological characters according to Bergey’s Manual. Production of glucose
isomerase was qualitatively screened by plate assay method. The high yielding extracellular glucose isomerase producer
Streptomyces sp. SB-P1was studied by submerged fermentation process and the fermentation period was optimised.
Keywords: Streptomycetes, glucose isomerase, extracellular, HFCS, xylose.
Introduction
Streptomycetes are common inhabitants of soil. They are a
subdivision of Actinomycetes which are a heterogeneous group of
microbes consisting of unicellular as well as filamentous bacteria.
They are the producers of more than 75% of the antibiotics
available in the market. They produce geosmin which is
responsible for the earthy fragrance after the rains1. Streptomycetes
produce a wide variety of secondary metabolites possessing
antibacterial, antifungal, antiviral, antitumor, immunosuppressive,
antihypertensive and antihypercholesterolemic properties. They
have indispensible role in mineralization of all complex organic as
well as inorganic matter. Streptomycetes are important
decomposers of plant and animal remains and recalcitrant
compounds in the soil. They produce a large repertoire of enzymes
for performing degradation activities1-4
. They have been also
explored for the production of pigments. These pigments are in
great demand due to their biological origin and non-carcinogenic
nature as compared to chemical pigments5.
Streptomycetes are placed in Group 23 of class Actinobacteria
in Bergey’s Manual of Determinative Bacteriology [1984]. The
Class Actinobacteria falls in Phylum 14 in Domain II, Bacteria
in Volume 4 of Bergey’s Manual of Systematic Bacteriology
[2001]6.
We have developed a collection of different types of
Actinomycete isolates and worked on their glucose isomerase,
amylase, protease, cellulase, lipase, keratinase, antibacterial,
antifungal and pigment production. The screening and
production of glucose isomerase is elaborated here.
Glucose isomerase (GI) reversibly converts glucose into
fructose. GI from Streptomyces is a tetramer composed of four
identical polypeptide chains of 43,000 Daltons each. It is a heat
stable enzyme possessing wide range of optimum temperature
between 60 – 70°C and pH for enzyme activity 7–8 depending
on its source. GI requires magnesium for optimum activity and
cobalt for thermostability7,8
. GI has tremendous commercial
scope because of the increasing demand of High Fructose Corn
Syrup (HFCS) in domestic as well as International market9. It is
an equilibrium mixture of glucose and fructose. HFCS is
produced by enzymatic conversion of corn starch into glucose
by amylase, which is eventually isomerized by glucose
isomerase to fructose. Fructose has higher solubility and 1.7
times more sweetening capacity than glucose. It is also 1.3 times
sweeter than sucrose, therefore it has higher sweetening index.
Applications of GI lie in medicated syrups, beverage, baking,
canning, and confectionary items10
. Isomerisation can be
effective if GI is immobilised by a suitable method11-14
. GI is
used at commercial scale in immobilized form in fermenters for
the production of HFCS12
.
Glucose Isomerase was first reported to be produced by
Pseudomonas hydrophila by Marshall and Kooi in 1957 since then
many microbes have been screened for the production of the
enzyme. GI is reported to be extracellular as well as intracellular10
.
This enzyme can be produced by a variety of microorganisms
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Vol. 2(ISC-2012), 330-339 (2013) Res. J. Recent. Sci.
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Actinomyces olivocinereus, A. phaeochromogenes, Actinoplanes
missouriensis, Streptomyces olivochromogenes, Bacillus
stearothermophilus, B. megabacterium, B. coagulans,
Bifidobacterium sp. Brevibacterium incertum, B.
pentosoaminoacidicum, Lactobacillus brevis, L. buchneri, L.
fermenti, L. bifermentans and many more. Streptomycetes are the
preferred producers at commercial as well as research level10,15-17
.
We have investigated a large number of Actinomycete isolates for
the production of GI and worked on its extracellular production.
Materials and Methods
Isolation of Actinomycetes: Compost pit soil samples were
collected from Western region of Madhya Pradesh. Samples
were sun dried and treated with calcium carbonate. The samples
were diluted 10 times in sterile distilled water. Bennett’s Agar
and Actinomycete isolation agar were used for the isolation of
Actinomycetes. The purified cultures were preserved on
Actinomycete isolation agar slants. The characterization of
isolates was done by morphological, cultural, biochemical and
molecular analysis.
Screening for GI producers: All the isolates were screened
qualitatively for GI by plate assay method. The screening
strategy was designed according to the method described by
Manhas and Bala18
with some modifications. Production of
glucose isomerase was checked on media containing xylose as a
sole source of carbon and wheat bran medium9. We used three
different media combinations for primary screening, X+P+
medium containing (g/L) Xylose (10), Peptone (0.3), KNO3 (2),
K2HPO4 (2), NaCl (2), MgSO4.7H2O (0.5), FeSO4 (0.1), CaCO3
(0.2), Agar (20), pH (7). Another medium named X+P-
contained all above ingredients excluding peptone. Third one
was wheat bran medium, it contained all above ingredients
present in X+P+ but xylose was replaced with wheat bran. The
organisms producing glucose isomerase can isomerise xylose to
xylulose besides glucose to fructose. Xylose has to be first
converted to xylulose which is further channelized into pentose
phosphate pathway for generation of energy. The organisms
possessing very low or negligible GI activity might not grow on
such a media. The organisms growing on medium containing
xylose as a sole source of carbon will be utilizing xylose as a
source of carbon. The cultures were spot inoculated on all the
media combinations and incubated at 30°C. The plates were
observed daily. The isolates developing early on the plates and
giving luxurious growth were picked up as GI producers.
Submerged Fermentative Production of GI: The isolates
which were found to be good producers by plate assay method
were picked up for further studies. They were subjected to
submerged fermentation in liquid medium to check the amount
of extracellular GI produced. Conical flasks containing 20 mL
of Bennett’s broth containing (g/L): Glucose (10), Tryptone (1),
Meat extract (1), Yeast extract (1). The pH of the medium was
adjusted to 7 and sterilized by autoclaving. The selected isolates
were inoculated in Bennett’s broth and incubated in orbital
shaker. The fermentation was terminated on the fourth day and
broth was harvested in sterile centrifuge tubes. The fermented
broth was centrifuged at 5000 RPM for 10 minutes19
. The
supernatant was used as crude extracellular enzyme extract. The
centrifuged supernatant of fermented broth was used as crude
enzyme extract for determination of GI activity by assay method
described by Chen et al.20
. The reaction mixture for GI assay
contained 500 µL of 0.2 Molar sodium phosphate buffer, 200
µL of 1 M glucose, 100 µL of 0.1 M magnesium sulphate, 100
µL of 0.01 M cobalt chloride and 200 µL of crude enzyme
extract. The final volume of assay mixture was made up to 2
mL. This reaction mixture was incubated in water bath at 70°C
for 60 minutes. The reaction was stopped by adding 2 mL of 0.5
M perchloric acid. To 0.05 mL aliquot of above 0.95 mL of
distilled water was added. To this 200 µL of 1.5% cysteine
hydrochloride, 6mL of 70% sulphuric acid and 200 µL of 0.12%
alcoholic Carbazole is added. The intensity of purple colour so
developed was estimated spectrophotometrically at 560 nm21
.
One unit of glucose isomerase activity was defined as the
amount of enzyme that produced 1µmol of fructose per minute
under the assay conditions described9.
Optimization of growth and fermentation period for
Streptomyces sp. SB – P1: The best extracellular producer of GI
was selected and picked for further studies. The isolate was
inoculated in Bennett’s broth for determination of optimum
fermentation period. A set of ten 100 mL conical flasks
containing 20 mL of Bennett’s broth were inoculated with
Streptomyces sp. SB – P1. All the flasks were incubated at 30°C
and 120 RPM on orbital shaker. One flask was harvested after
every 24 h. The biomass was separated from the nutrient
medium by centrifugation and washed with distilled water. The
biomass was transferred on a filter paper and kept for drying at
55°C. The dry weight was determined after 24 h9. The optimum
fermentation period was determined by analysing the amount of
GI produced at 24 h intervals. The increase in GI accumulation
was determined by performing enzyme assay described above.
The centrifuged crude supernatant was used to measure
extracellular glucose isomerase.
Results and Discussion
The investigation revealed the presence of abundant
Actinomycetal strains in soil samples which can be harnessed in
various directions for the development of eco-friendly and
economic bioprocesses.
Isolation of Actinomycetes: The compost pit samples were rich
in diverse kinds of Actinomycetes. We developed a collection of
75 isolates. The isolates were picked from soil inoculated plates
after four to six days of incubation. The Actinomycetal colonies
were picked up on the basis of their cultural characters. These
microorganisms exhibit chalky, powdery or velvety
appearance22
. The purified cultures were further studied
microscopically by slide culture technique23
. On the basis of
spore arrangement pattern observed under the microscope the
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International Science Congress Association 332
isolates were grouped in different categories like Streptomyces
Saccharomonospora, Streptoverticillium, Nocardioides,
Streptoverticillium, Streptomyces, Thermomonospora,
Saccharomonospora, Dermatophillus etc. The colonial details
of the isolates are presented in figure-1. Streptomycetes being
fast growing Actinomycetes dominated on the soil isolation
plates. The colonies appear leathery till the development of
substrate mycelium but turn to velvety or chalky on sporulation.
Actinomycetes are peculiar in their appearance on agar medium;
they possess different aerial spore mass colour (figure-1 and
figure-2), colony reverse colour (figure-4) and soluble
pigmentation (figure-5). Actinomycetes are known to be well
distributed in different habitats of nature. Researchers have
reported the presence of Actinomycetes in abundance in garden
soil24
, desert area, marine soil25,26
and polar regions27
also. They
are also found to inhabit certain animals like termite28
and
earthworms. Varied aerial spore mass colour of some isolates is
depicted in figure-1.
The colonies appear velvety and exhibit varied appearance like
colony with a bulge in the center (figure-2a), colony with a
depression in the center (figure-2b), wrinkled (figure-2c and
2d), membranous (figure-2e), powdery (figure-2f) and glossy in
absence of sporulation, radiating fibers. Actinomycetes also
show concentric rings around their colonies on aging (figure-1e,
1c, 2a, 2f, 2g and 2h). These filamentous microbes grow as
beads in the liquid medium (figure-3a and 3b). The colonies
appearing similar by aerial spore mass could be differentiated
from their different colony reverse (figure-4). Many exhibited
melanin pigmentation (figure-5).
Figure-1
Varied Spore Mass Colour of Isolates; a: Isolate V6; Ivory spore mass colour, b: P1; Light Gray, c: P2; Ash Gray, d: V1;
Brownish Gray, e:V2; Pinkish White, f: AII5; Bluish Gray.
a b
d e f
c
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Figure–2
Varied Colonial Appearance of Actinomycetal Isolates
a b
c
e f
g h
d
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Figure-3
Actinomycetes growing in the form of beads in liquid medium
Figure–4
Varied colony reverse exhibited by the Actinomycetal isolates
Figure–5
A few representatives of the collection of isolates
b a
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Screening for GI producers: The isolates exhibited varying
degrees of GI production on xylose agar plates. The early
responders were considered as probable GI producers. A few
isolates appeared within 24 hours of inoculation, many grew
after 48 hours and a few did not grow till 96 hours. The growth
was best on wheat bran agar medium which must be due to
elaborate nutrients present in it. The presence of wheat bran
enriches the medium with growth promoters. This medium also
promoted heavy and early sporulation as also observed by
Manhas and Bala18
and Srinivasan et al.29
. X+P- supported
growth of isolates to a lesser extent as compared to X+P+
because of the absence of peptone. The isolates still growing on
such a medium are utilizing xylose as a sole source of carbon.
This is possible only if the isolate can produce GI which also
converts xylose into xylulose which is further channelized for
carbohydrate metabolism. Varied 40 isolates were positive for
GI production but 18 among them responded very well. Our
results indicated that more than half of the isolates were positive
for GI production which supports the enormous scope of
exploring Actinomycetes as GI producers. GI production by
Streptomycetes was initially reported by Tsumura, N., and
Sato30
. Actinomycetes are known to produce GI from numerous
sources like marine water, marine soil, garden soil, compost pit
soil etc. The difference in response of the isolates on screening
medium is depicted in figure-6.
Figure–6
Screening for Glucose Isomerase (GI) producers
Table-1
Screening for GI
Sr. No. Isolate Code Growth
Response Sr. No.
Isolate
Code
Growth
Response Sr. No.
Isolate
Code
Growth
Response
1 P1 ++++ 26 KV - 51 NPII2 +++
2 P2 ++ 27 KC1 - 52 NPII4 ++
3 V1 ++ 28 KC2 +++ 53 NPII5 ++
4 V2 - 29 KC3 - 54 NPII6 -
5 V3 ++ 30 KC4 - 55 K -
6 V4 - 31 KC5 +++ 56 MR1 -
7 V5 ++++ 32 KC6 ++ 57 S1 -
8 V6 ++ 33 KC7 ++ 58 S2 ++
9 V7 ++ 34 KC8 ++ 59 S4 -
10 Ab ++ 35 KB1 ++++ 60 VJ1 ++++
11 Ga1 - 36 KB2 - 61 VJ2 +++
12 Ga2 - 37 KB3 - 62 AI1 ++
13 Ga3 - 38 KB4 ++++ 63 AI2 -
14 Ga4 - 39 M2 ++ 64 AII1 ++
15 Gu1 - 40 M3 - 65 AII2 ++
16 Gu2 - 41 M4 - 66 AII3 +
17 Gu3 - 42 MJ1 ++++ 67 AII4 ++++
18 Gu4 - 43 MJ2 +++ 68 AII5 ++++
19 Gu5 ++ 44 BII1 ++ 69 KNI1 -
20 Gy1 +++ 45 NPI1 ++ 70 KNI2 -
21 Gy2 - 46 NPI2 ++++ 71 KNII1 ++
22 N1 - 47 NPI4 - 72 KNII2 ++
23 N2 - 48 NPI5 - 73 KNII3 -
24 R1 +++ 49 NPI6 - 74 KNII4 ++
25 R2 - 50 NPII1 +++ 75 LP -
Note: GI production was noted by the presence of luxurious growth on the media and denoted by (+) sign.
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Table-2
Details of the symbols used for GI production in Table-1.
Symbol Response Inference ++++ Very Good Growth High GI production
+++ Good Growth Moderate GI production ++ Fair Growth Less GI production
+ Very Less Growth Negligible GI production
- No Growth No GI production
Submerged Fermentative Production of GI: The top 18 GI
producers were subjected to submerged fermentation process for
determination of best extracellular producer of GI. The medium
used for primary screening did not contain any inducer as used
by some investigators10,31
. Our investigation aims at developing
GI production technology from an extracellular producer
without the addition of any inducer. An extracellular GI
producer in place of intracellular one shall reduce the
production cost by bypassing the cell disintegration steps9. GI is
reported to be induced by the presence of xylose. Xylose is
expensive therefore an organism producing high amount of GI
in absence of xylose shall be of great commercial importance.
The process can also be made feasible by incorporating an agro-
residue rich in xylose. Researchers have also worked on agro-
residues rich in xylan, which can be converted to xylose for the
use as an inducer. Gong et al.32
reported Actinoplanes
missouriensis, produces intracellular glucose isomerase which
does not need xylose as an inducer. Many isolates in our
collection exhibited the isomerisation ability extracellularly.
The enzyme yield among the collection ranged from 0.97
Units/mL to 2.8 Units/mL. Isolate named P1 yielded highest
extracellular GI. Chen et al.20
reported 1.5U/mL of extracellular
GI produced by Streptomyces flavogreiseus. Teeradakorn et al.33
reported 1.1U/mL of GI using a fusant of Streptomyces cyaneus
and Streptomyces greiseoruber. Most of the researchers have
reported the production of GI from intracellular sources; there
are very few reports on extracellular producers. Deshmukh et
al.34
reported 12U/mL of intracellular GI production by
Streptomyces thermonitrificans. Chen et al.20
reported 3.5U/mL
whereas Givry and Duchiron15
reported intracellular production
of GI to be 7.24U/mL. The results are depicted in figure - 7.
Optimisation of growth and fermentation period for
Streptomyces sp. SB – P1: The technology for production of
glucose isomerase can be designed by considering a few points
in detail i.e. the growth requirements of the organism and the
growth stage for the production of enzyme. The enzyme is used
in the primary metabolism of the organism’s growth therefore
it’s production may also coincide with the exponential growth
phase. The maximum accumulation of enzyme was observed by
different investigators between 36 to 96 h6,10
.
The growth for Streptomyces sp. SB – P1 was determined by
measuring the dry weight of the biomass. The biomass increased
till the sixth day of incubation progressively but there was no
significant increase in the dry weight beyond this. The increase
in biomass is depicted in figure - 8.
Production was terminated in a set of flasks after every 24 h and
the broth was harvested to determine the amount of GI
produced. The accumulation of enzyme increased till 96 h of
incubation. Further incubation did not support the increase in
productivity of glucose isomerase. This enzyme is produced by
the organisms for utilization of available sources such as
glucose or xylose as nutrients therefore the production start as
the growth starts. As the number of bacterial cells goes on
increasing the number of enzyme molecules shall increase,
therefore high biomass accumulation will result in good enzyme
yield35
. The production of glucose isomerase is reported by
Hasal et al.31
in 24 h, Manhas and Bala18
in 48 h whereas Chen
et al.20
observed the production in 72 h and Chou et al.36
in 96h
by Streptomycetes.
Figure-7
GI activity of different isolates
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Figure–8
Growth curve of Streptomyces sp. SB – P1
Figure–9
Production of Extracellular GI at different intervals by Streptomyces sp. SB - P1
Maximum accumulation of enzyme in our case was observed in
96 h of incubation as also reported by Srinivasan et al.29
for
extracellular production by Chainia species. Further incubation
resulted in loss of enzyme activity. Gong et al.32
reported
maximum production in 120 hours. The decrease of the enzyme
activity during the stationary phase may be explained by the
detrimental effects of acidic pH or some by-products formed in
the medium. Presence of proteases may also breakdown the
enzyme present in the medium. The GI activity during seven
days period is graphically depicted in figure - 9.
Conclusion
The compost pit samples are rich in Actinomycetes. The
enrichment of Actinomycetes was observed to a great extent by
addition of calcium carbonate to soil. Streptomycetes present in
soil samples dominated other species during isolation, as also
reported by other researchers. The primary screening studies
revealed good growth response of the isolates on wheat bran
medium which opens a way for utilising agro residues at
commercial level also; this will be helpful in reducing the
production cost.
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Around fifty per cent of the isolates were positive for GI and
nearly twenty five percent were good producers. This is
encouraging for investigation of an isolate with the properties
required by the market.
The biomass increase was observed continuously for 10 days
but there was a progressive increase till 4th
day. The maximum
GI was also recovered on 4th
day which signifies the association
between GI production and growth. The slower increase in
biomass after 5 days also indicates that the organism’s shift to
stationary phase where the increase in enzyme accumulation
ceases.
Production of GI is reported to be intracellular by majority of
researchers. GI is extracted out by rupturing the cells or it can
also be released in the medium by autolysis. The lowering yield
of GI coinciding with the isolate reaching the stationary phase
indicates the release of GI from the Streptomycetal cells by
autolysis. The programmed release of GI by isolate
Streptomyces sp. SB – P1 helps us to avoid cell disruption steps
making the downstream processing economically effective.
The isolates were checked for the production of GI in absence
of xylose. The extracellular production of GI was observed in
absence of the inducer xylose which establishes the isolate as
non-inducer requiring strain. This is the need of the hour as
incorporation of xylose increases the cost of the production
process.
Acknowledgement
The work presented here is due to the gratifying support by the
management of Maharaja Ranjit Singh College of Professional
Sciences, Indore (MP) India for which I am highly thankful
towards the institute. I deeply express gratitude to the
Department of Life Sciences, Gujarat University, Ahmedabad,
(Gujarat), India also for their support.
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