58 J SCI IND RES VOL 67 JANUARY 2008Journal of Scientific & Industrial Research

Vol. 67, January 2008, pp.58-64

*Author for correspondence

E-mail: rc_ray@rediffmail.com

Partial characterization and optimization of extracellular thermostable Ca2+

inhibited α-amylase production by Streptomyces erumpens MTCC 7317

S Kar and R C Ray*

Regional Centre of Central Tuber Crops Research Institute, PO, Dumduma Housing Board, Bhubaneswar 751 019

Received 12 March 2007; revised 20 September 2007; accepted 27 September 2007

Culture parameters for optimum production of extracellular α-amylase by moderately thermostable Streptomyces

erumpens MTCC 7317 isolated from a brick-kiln soil were found to be pH (6.0), temperature (50°C) and incubation period

(36 h). Enzyme production was more in soluble starch (1%), beef extract (1%) and glycerol (0.02%) as compared to other

carbon (1%: wheat flour, cassava starch, potato starch, cassava flour, etc.), nitrogen (1%: yeast extract, peptone, casein,

ammonium chloride, etc.) and surfactant [0.02%: Cween (Tween) 20, Cween 40, Cween 60, Cween 80, sodium lauryl

sulphate (SLS) and triton x 100] sources tested, respectively. Glycerol concentration (0.02 %) was most stimulatory in

enzyme production. Addition of Ca2+ (even at 10 mM) inhibited α-amylase production. S. erumpens cells immobilized on

sodium alginate beads produced more (9.0-23.4%) amylase as compared with free cells. Partially purified enzyme (by

ammonium sulphate precipitation) had a molecular mass of 54,500 Da in native SDS-PAGE.

Keywords: Actinomycete, α-Amylase, Streptomyces erumpens MTCC 7317, Thermostable

Introduction

Thermostable enzymes1-3 are most widely exploited

and are of considerable commercial interest in starch

processing4,5, leather, food, paper, textile, detergents,

drugs and toxic waste removal industries4,6. Among

starch hydrolyzing enzymes, thermostable α-amylase (EC

3.2.1.1) is of utmost significance4,6. Most thermostable

amylases are of bacterial origin7, especially from Bacillus

spp. (B. licheniformis, B. amyloliquefaciens,

B. amylovorans, etc). Among actinomycetes, about

three-fourths of Streptomyces spp. produce antibiotics8,

and also produce several extracellular enzymes such as

chitinase, lipase and β-xylosidase9-11.

This study presents production and characterization

of thermostable Ca2+ independent α−amylase by

S. erumpens MTCC 7317 isolated from a brick kiln soil.

Material and MethodsIsolation and Identification of Thermostable Microorganism

Producing α-Amylase

Soil samples from brick-kilns (1000°C) were collected

from outskirts of Bhubaneswar, Orissa, India. Soil

samples (1 g each) were serially diluted with sterile

distilled water to 10-6 dilution and one ml of soil suspension

(10-4-10-6) was pour-plated on Medium A12 (peptone 2.0,

yeast extract 1.0, NaCl 1.0 and agar 2.0%; pH 7.0) and

incubated at 50°C for 48 h. Three replicates were

maintained for each dilution. Colonies (140) that appeared

on agar surface were considered as thermostable

microorganisms, which were inoculated on starch

containing Medium B12 (soluble starch 1.0, yeast extract

0.2, peptone 0.5, MgSO4 0.05, NaCl 0.05, CaCl

2 0.015

and agar 2.0%; pH 7.0) at 50°C for 48 h. After incubation,

iodine solution (KI/I 0.1 N) was spread on agar surface

(culture) plate (containing Medium B) to isolate amylase

producers by measuring halo-zones12. Strain producing

higher amount of amylase halo-zones (>3.6 mm) than

others was selected and maintained on agar slants made

up of Medium B at 4°C. Selected isolate was identified

as S. erumpens Calot & Cercos (Institute of Microbial

Technology, Chandigarh, India) based on microscopic and

biochemical tests and was coded as MTCC 7317. It was

further confirmed by 16S rRNA sequence analysis.

Effect of Various Parameters on α−α−α−α−α−Amylase Production

In all experiments, three replicates were maintained

and expressed as mean of three replicates.

Incubation Period

Inoculum (1x105 CFU/ ml) was prepared in soluble

starch-peptone broth (soluble starch 1.0, peptone 0.5,

KAR & RAY: α-AMYLASE PRODUCTION BY STREPTOMYCES ERUMPENS MTCC 7317 59

yeast extract 0.25, K2HPO

4 0.2 and MgSO

4 0.1%;

pH 7.0) by transferring a loop full of organism from a

fresh culture and incubating at 50°C and 120 rpm for 24

h in an orbital incubator shaker (Remi Pvt Ltd, Bombay,

India).

Amylase production was carried in a starch–beef

extract (SB) liquid medium (soluble starch 1.0, beef

extract 1.0, yeast extract 0.2 and MgSO4 0.01%;

pH 7.0). Sterile SB medium (50 ml) taken in an

Erlenmeyer flask (250 ml) was incubated with inoculum

(2%) and agitated at 120 rpm for 60 h in an incubator

shaker at 50°C. At an interval of 12 h, growth of

microorganism was monitored by recording absorbancy

[optical density (OD)] at 600 nm. Then culture broth

was centrifuged at 8000 rpm in a refrigerated centrifuge

(Model C-24, Remi Pvt Ltd, Bombay, India) for 20 min

at 4°C. The clear cell free supernatant was used for

amylase assay.

pH

SB medium of different pH (4.0-9.0) was incubated

at 50°C for 36 h at 120 rpm in an incubator shaker.

pH stability of amylase was also studied after incubating

enzyme at various pHs of assay buffer (4.0-9.0) for

10 min. pH were measured with a pH meter (Systronics,

Ahamadabad, India) using glass electrode. pHs (4.0-

6.0) were maintained with acetate buffer (0.2 M) while

higher pH (6.0-9.0) were achieved with phosphate buffer

(0.1 M).

Temperature

Culture of S. erumpens was incubated at 30-90°C in

SB medium for 36 h in an incubator shaker at 120 rpm.

After 36 h of incubation, cell free supernatants were

prepared and analyzed for amylase activity. Temperature

stability of enzyme was determined by incubating cell

free supernatants between 40-90°C for 30 min, using

0.1 M phosphate buffer (pH 6.0).

Carbon and Nitrogen Sources, and Surfactants

S. erumpens was grown in several variants of SB

medium prepared by: (i) substituting starch with various

carbon sources (1%); (ii) replacing beef extract with

different organic and inorganic nitrogen sources (1%);

and (iii) supplementing with different surfactants

(0.02%). Medium was incubated at 50°C for 36 h at

120 rpm. After 36 h of incubation, cell free supernatants

were used for amylase assay. In continuation of this

experiment, concentrations of soluble starch (1-5%),

beef or yeast extract (0.25-2.00%) and glycerol (0.01-

0.05%) were studied for amylase production.

Ca2+ Ions

Ca2+ (10-40 mM) was introduced into production

medium (SB) at 50°C and incubated for 36 h in an

incubator shaker at 120 rpm. Amylase was assayed in

cell free supernatant.

Cell Immobilization

S. erumpens cell suspension (1 x 105 CFU/ ml) was

added to 4% (w/w) sodium alginate solutions (1:1 vol)

and mixed thoroughly. Cell alginate mixture was then

casted into beads by dropping from a hypodermic syringe

into 0.1 M CaCl2 solution. These beads (diam, 3.0 mm)

were hardened by keeping in dilute (0.1 M) CaCl2

solution for 24 h and washed with sterile distilled water.

For high cell density, cells were grown on beads for 24 h

before using for enzyme production. Gel beads

containing immobilized S. erumpens cells were immersed

in soluble starch-beef extract broth for 24 h at 50°C and

cell population in immobilization increased from 104 to

108 cells/ g gel.

TLC (Thin Layer Chromatography)

The end products liberated by the action of amylase

on starch were identified by spotting starch digest and

standard sugars (glucose and maltose) on a silica gel plate

activated at 80°C for 30 min. The plates were developed

in butanol: ethanol: water (50: 30: 20) and dried overnight

at room temperature (32 ± 2°C). Individual sugars were

visualized by spraying with acetone-silver nitrate solution

(0.1 ml saturated solution of AgNO3 in 20 ml of acetone).

Amylase Assay

Reaction mixture consisted of 0.2 ml enzyme (cell free

supernatant), 0.25 ml of 0.1% starch solution and 0.5 ml

of phosphate buffer (0.1 M, pH 6.0) incubated at 50°C

for 10 min. The reaction was stopped by adding 0.25 ml

of 0.1 N HCl and colour was developed by adding 0.25

ml of I/KI solution (2% KI in 0.2% I). Optical density of

blue colour solution was determined using a UV-Vis

Spectrophotometer (Cecil Instrument, UK) at

690 nm. One unit of enzyme activity is defined as the

quantity of enzyme that causes 0.01% reduction of blue

colour intensity of starch iodine solution at 50°C in one

minute per ml13.Partial Purification of Enzyme

α-Amylase was partially purified by ammonium

sulphate fractionation followed by dialysis and gel filtration

60 J SCI IND RES VOL 67 JANUARY 2008

chromatography. A total of 100 ml of culture filtrate was

centrifuged at 8000 rpm for 20 min at 4°C to remove

cells. Supernatant was brought to 50% ammonium

sulphate saturation at 4°C in an ice bath. Precipitated

protein was collected by centrifugation at 8000 rpm at

4ºC and dissolved in a minimum volume of phosphate

buffer (0.1M; pH 6.0). Enzyme solution was dialyzed at

4°C against the same buffer for 24 h at 4°C with

continuous stirring and three changes of same buffer.

DEAE cellulose-ion exchange column was pre-

equilibrated with same buffer. Dialysate was

concentrated through a rotary evaporator at 50°C and

applied to DEAE cellulose column (flow rate

0.6 ml/min) with 50 ml linear NaCl gradient (0-0.5 M).

Each fraction (10 ml) was analyzed for protein

concentration and α- amylase activity. Active fractions

were pooled and concentrated through a rotary evaporator

at 50°C. Final concentrated enzyme solution was taken

for sodium dodecyl sulphate-polyacrylamide gel

electrophoresis (SDS-PAGE). Optimal production and

stability of partially purified enzyme at various pH (4.0-

9.0) and temperature (30-90°C) were studied.

Electrophoresis and Molecular Mass Determination

SDS-PAGE14 was performed with 12%

polyacrylamide gel using a Mini GEL electrophoresis

system (Model No 0502, Bangalore Genei Pvt Ltd,

Bangalore, India). Bacterial proteins were stained with

0.2% Coomassie Brilliant Blue. Molecular mass of

partially purified amylase was estimated using a standard

‘protein marker’ (PMW-M) of known molecular mass

(14300-97400 Da) (Bangalore Genei Pvt Ltd, Bangalore,

India).

Results and Discussion

Although amylase is mainly a eubacterial product but

the possibility of using Streptomyces spp. for enzyme

production has been recently investigated15.

Isolation of Thermostable Amylase Producing Streptomyces sp.

Isolated strain possessed the ability to hydrolyze starch

and could grow on a variety of sugars (glucose,

arabinose, mannitol, meso-inositol, raffinose, rhamnose,

galactose and fructose). Strain could also grow on

nutrient broth at 25-60°C (optimum 50°C) and

pH 5.0-9.0 (optimum 6.0).

Effect of Various Parameters on Amylase Production

Incubation Time

Highest amylase production (3700 units) was obtained

at 36 h of incubation (Fig. 1). Amylase production

gradually progressed with growth of S. erumpens;

enzyme production was highest when cell entered

stationary phase (36 h). Similar results were obtained

for many Bacillus spp. (B. subtilis, B. licheniformis and

B. coagulans)16,17. Another species of actinomycetes

(S. rimosus) was reported to produce amylase after 48

h of incubation in the stationary phase of growth18.

Similarly, incubation period by several Bacillus spp.

(B. coagulans and B. licheniformis) reported to require

a batch time of 48-72 h for optimum amylase

production17.

Chromatogram indicates formation of two major

sugars (maltose and glucose) from soluble starch; this

suggest that enzyme was essentially an α-amylase15,17.

Major end products of starch hydrolysis by

S. megasporus were maltose, maltotriose and glucose15.

pH

pH (6.0-6.5) for optimal (Fig. 2A) amylase production

and stability were similar to that reported (6.0-7.0) for

most bacteria (B. thermooleovorans19 and B. subtilis13),

yeast (Filobasidium capsuligenum20 and Candida

antarcticai21) and actinomycetes (Streptomyces

megasporus15, S. rimosus18 and Thermoactinomyces

vulgaris22).

Temperature

Optimum α-amylase production (maximum activity,

3480 units) at 50°C (Fig. 2B) is characteristic of a

moderately thermostable microorganism4,5. Beyond

50°C, there was sudden decrease in enzyme production.

Optimum α−amylase production for actinomycetes,

Thermoactinomyces vulgaris and S. rimosus, were

found at 62.5°C and 45°C, respectively18,22.

0

500

1000

1500

2000

2500

3000

3500

4000

12 24 36 48 60

Incubation period, h

En

zym

e p

rod

uctio

n,

Un

its

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Gro

wth

OD

at

600

nm

Enzyme Growth

Fig. 1 — Growth of S. erumpens MTCC 7317 and α -amylase

production during 60 h incubation period

KAR & RAY: α-AMYLASE PRODUCTION BY STREPTOMYCES ERUMPENS MTCC 7317 61

Carbon and Nitrogen Sources

Maximum production of α-amylase was achieved

when soluble starch (1%) as carbon (3500± 103.4 units)

and beef extract (1%) as nitrogen source (3680± 106.2

units) were used in SB (production) medium. Other

carbon sources (1%) resulted in enzyme production (in

units) were as follows: wheat flour, 3090± 98.0; cassava

starch, 2788± 110.4; potato starch, 2690± 103.4; cassava

flour, 2338± 121.3; sweet potato flour, 2155± 87.0; and

sweet potato starch, 1990± 121.1. Similarly, other

nitrogen sources (1%) resulted in enzyme production

(in units) were as follows: yeast extract, 3547± 93.23;

peptone, 3480± 65.2; casein, 3377± 121.0; ammonium

chloride, 1936.5± 95.0; asparagines, 1173.5± 123.0; urea,

1396.5± 115.0; ammonium sulphate, 1396.5± 105.5;

potassium nitrate, 1948.5± 121.0; ammonium molybdate,

1408.5± 103.0; and ammonium nitrate, 1173± 110.0.

Many species of Streptomyces18,23, Bacillus and other

microorganisms4,5 gave similar results.

Surfactants

Surfactants often increase enzyme secretion and

production24. Addition of glycerol had a marginal

stimulating effect [9% more than the control (no

surfactant added)] on α-amylase production whereas

Cween 40 had innocuous effect and all others [(Cween

20, Cween 60, Cween 80, sodium lauryl sulphate (SLS),

etc)] had inhibitory effect (Fig. 3). Glycerol (0.02%)

showed highest amylase production (3680 units),

whereas at 0.01, 0.03, 0.04 and 0.05% the enzyme yield

was 3425, 3550, 3475 and 3400 units, respectively.

Increased production of α-amylase from Thermomyces

lanuginosus has been reported by addition of Tween 8025.

It appears that same surfactant may stimulate enzyme

production in one species and inhibit in another species.

It is reported that growth and biochemical production of

actinomycetes are stimulated by glycerol8,26, which

solubilize membrane proteins that lead to an increase in

cell membrane permeability, thereby enhancing secretion

of biomolecules27.

Ca2+ Ion Concentration

For production and stability of α-amylase by Bacillus

spp., addition of Ca2+ ion is often necessary28. However,

in this study, with the increase in Ca2+ concentration (10-

40 mM) in medium, α-amylase production was

proportionally inhibited (Fig. 4). Thus, α-amylase

production by S. erumpens was Ca2+ independent.

Similar results were obtained for Bacillus

thermooleovorans, B. coagulans, B. licheniformis,

Bacillus sp. WN11, etc.4,5,17,19. There are reports that

Ca2+ inhibit glucose isomerase29. Calcium-independent

0

500

1000

1500

2000

2500

3000

3500

4000

No

surfa

ctan

t

Glyce

rol

Cwee

n 20

Cwee

n40

Cwee

n 60

Cwee

n 80

SLS

Triton

x-1

00

Different surfactants

Enzym

e p

roduction, U

nits

0

500

1000

1500

2000

2500

3000

3500

4000

4 5 6 7 8 9

Different medium/ assay buffer pH

Enzym

e p

roduction, U

nits

Production Stability

0

500

1000

1500

2000

2500

3000

3500

4000

40 50 60 70 80 90

Incubation temperature, 0C

En

zym

e p

rod

uctio

n,

Un

its

Production Stability

Fig. 2 — Effect of medium pH (A) and temperature (B) on α –amylase production and stability by S. erumpens MTCC 7317

Fig. 3— Effect of surfactants (0.02%) on α -amylase production

by S.erumpens MTCC 7317

(B)

(A)

62 J SCI IND RES VOL 67 JANUARY 2008

amylases merit consideration for starch liquefaction,

especially in manufacture of fructose syrup, where

calcium is a known inhibitor of glucose isomerase5.

Cell Immobilization

Amylase production using immobilized S. erumpens

cells was higher (9-23%) than free cells during 24-60 h

of enzyme production (Fig. 5). Cells survived and were

physiologically active on the support used for

immobilization even after three cycles of operations,

which could save considerable time and energy. Similar

results were obtained on amylase production using

B. licheniformis30,31.

Partial Purification and Molecular Mass Estimation

Crude extract of α-amylase, partially purified with

ammonium sulphate precipitation, contained

2.55 mg/ml protein and showed a specific activity of

1492.06 units/mg protein. After partial purification,

specific activity increased to 3912.92 units/mg proteins

with a yield of 20.7 % and 2.6 fold purification (Table 1).

Electrophoresis studies showed that molecular mass of

partially purified enzyme was approx 54,500 Da

(Fig. 6). There were wide variations in molecular mass

of different Actinomycetes spp., Thermoactinomyces

vulgaris (53,000 Da)22; S. megasporus strain SD12

(97 kDa)15 and S. rimosus (43,000 Da)32. Partially

0

500

1000

1500

2000

2500

3000

3500

4000

4500

12 24 36 48 60

Incubation period, h

Enzym

e p

roduction, U

nits

Normal cell Immobilized cell

9 7 ,0 0 0 D a

6 6 ,0 0 0

4 3 ,0 0 0 D a

A B

Fig. 5. — Amylase production using normal and immobilized cell

of S. erumpens MTCC 7317

Fig. 6 — Determination of molecular weight by SDS-PAGE: (A)

molecular weight markers; (B) α-amylase from S. erumpens

MTCC 7317

0

500

1000

1500

2000

2500

3000

3500

4000

0 10 20 30 40

Concentration of Ca2+

, mM

Enzym

e p

roduction, U

nits

Fig. 4 — Effect of Ca2+ concentration on α -amylase production

by S .erumpens MTCC 7317

Table 1— Purification of α-amylase from S. erumpens MTCC 7317

Purification Total Total Total Yield Specific Fold of

Steps volume enzyme protein mg % activity purification

ml activity Units/mg

Units protein

Culture 100 380700 255.2 100 1492.1 0

filtrate(crude)

Ammonium 15 72390 24.8 19.0 2921.3 2.0

sulphate

precipitation

After dialysis 16 78884.48 20.2 20.7 3912.9 2.6

KAR & RAY: α-AMYLASE PRODUCTION BY STREPTOMYCES ERUMPENS MTCC 7317 63

purified enzyme of S. erumpens showed similar

pH (6.0-6.5) and temperature (50°C) optima and stability

as in crude enzyme.

Conclusions

This study shows the production of thermostable

α-amylase by free and immobilized cells of S. erumpens

MTCC 7317 in submerged culture. α-amylase production

from this actinomycete strain is significant as it is Ca2+

independent, which is useful in confectionary industries

particularly in making of fructose syrups.

Acknowledgement

Authors thank Director, CTCRI, Thiruvanathapuram

for providing facilities.

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