Streptomycetes: A Storehouse of Bioactive Compounds and ...in Bergey’s Manual of Determinative Bacteriology [1984]. The Class Actinobacteria falls in Phylum 14 in Domain II, Bacteria

Research Journal of Recent Sciences _________________________________________________ ISSN 2277-2502

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

Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502



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




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




Figure–2

Varied Colonial Appearance of Actinomycetal Isolates

a b

c

e f

g h

d




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




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.




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




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.




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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