Optimization Conditions of Extracellular Proteases Production...

ISSN: 0973-4945; CODEN ECJHAO

E-Journal of Chemistry

http://www.ejchem.net 2012, 9(2), 949-961

Optimization Conditions of Extracellular

Proteases Production from a Newly Isolated

Streptomyces Pseudogrisiolus NRC-15

El-SAYED E. MOSTAFA §, MOATAZA M. SAAD

§, HASSAN M. AWAD

*,

#MOHSEN H. SELIM and

§HELMY M. HASSAN

§Microbial Chemistry Department, National Research Centre (NRC)

El-Bohouth Street, Dokki, P.O. Box 12622, Cairo, Egypt *Chemistry of Natural and Microbial Products Dept. National Research Centre (NRC)

El-Bohouth Street, Dokki, P.O. Box 12622, Cairo, Egypt #Institute of Bioproduct Development, Universiti Teknologi Malaysia (UTM), 81310

Skudai, Johor, Malaysia

[email protected]

Received 23 August 2011; Accepted 4 October 2011

Abstract: Microbial protease represents the most important industrial enzymes,

which have an active role in biotechnological processes. The objective of this

study was to isolate new strain of Streptomyces that produce proteolytic

enzymes with novel properties and the development of the low-cost medium.

An alkaline protease producer strain NRC-15 was isolated from Egyptian soil

sample. The cultural, morphological, physiological characters and chemotaxonomic

evidence strongly indicated that the NRC-15 strain represents a novel species of

the genus Streptomyces, hence the name Strptomyces pseudogrisiolus NRC-15.

The culture conditions for higher protease production by NRC-15 were

optimized with respect to carbon and nitrogen sources, metal ions, pH and

temperature. Maximum protease production was obtained in the medium

supplemented with 1% glucose, 1% yeast extract, 6% NaCl and 100 µmol/L of

Tween 20, initial pH 9.0 at 50 ºC for 96 h. The current results confirm that for

this strain, a great ability to produce alkaline proteases, which supports the use

of applications in industry.

Keywords: Streptomyces NRC-15, Phenotypic identification, Alkaline protease, Optimum

culture conditions.

Introduction

Proteases are important industrial enzymes accounting for 60% of total global enzyme sales1.

Bacterial proteases show more promise than animal and fungal proteases, accounting for

20% of the world market2 with the predominant use in detergents, especially for alkaline

bacterial proteases3. Extremophilic especially alkaliphilic, halophilic and thermophilic

proteases are preferred due to ease of operation, higher activity, enhanced stability, faster

mailto:[email protected]

HASSAN M. AWAD et al.

950

reaction and less proneness to contamination. Microbial alkaline proteases for industrial uses

are produced and studied mainly from Bacillus and Streptomyces4. Little is known about

proteases from other actinomycetes, much less from Nocardiopsis sp.5. Proteases are studied

in protein chemistry and protein engineering as well as for applications as cleaning agents,

food additives and dehairing (depilating) agents6.

The possibility of using Streptomyces for protease production has been investigated because of

their capacity to secrete the proteins into extracellular media, which is generally regarded as safe

with food and drug administration. Streptomyces sps that produce proteases include Str.

Clavuligerus, Str. griseus, Str.rimouses, Str. thermoviolaceus, Str. thermovulgaris7,8.

Actinomycetes, in addition to antibiotics, elaborate extracellular enzymes, e.g. proteases, chitinases,

amylases, etc. Compared to Bacillus sp., actinomycetes have been less explored for proteases. The

species belonging to the genus Streptomyces constitute 50% of the total population of soil

actinomycetes and 75-80% of the commercially and medicinally useful antibiotics have been

derived from this genus9.

The aim of this study was to screen local actinomycete isolate for protease production,

the taxonomy of the protease producer strain as well as detailed enzyme production

optimization.

Experimental

Soil sample, Streptomyces isolation and screening for protease activity

Ten farming soil samples, from 5-20 cm depth, were collected from different locations in

Egypt and diluted in sterile saline solution. The diluted samples (up to 10-7

) were plated onto

a medium composed of (g/L): casein, 20.0; glucose, 1.0; KH2PO4, 1.5 and Na2HPO4, 1.5 at

pH 8.0 supplemented with Cycloheximide (50 mg/mL). Plates were incubated at 50 °C for

7-10 days. A clear zone of casein hydrolysis gave an indication of protease producing

organisms. Depending upon the zone of clearance, strain NRC-15 was selected, maintained

on ISP-2 slants at 4 ºC and identified for further experimental work.

Taxonomic grouping of streptomyces isolate

Actinomycete colonies were characterized morphologically and physiologically following the

directions given by the International Streptomyces project (ISP) according to Shriling and

Gottlieb10

and Bergey's Manual of Systematic Bacteriology11

. Cultural characteristics of pure

isolate in various media were recorded after incubation for 7 to 14 days at 28 oC. Morphological

observations were carried out with a light microscope Model SE (Nicon, NY, USA) and

Transmission electron microscope (TEM) a Ziess EM 10 (Carl Zeiss, Oberkocben, West

Germany) using the method of Shriling and Gottlieb10

. Colors of spores (aerial and substrate

mycelia) were visually estimated by using a Stamp Color Key based on the computer color

wheels of Tresner and Backus12

.

Carbon utilization was determined on plates containing ISP basal medium 913

. Carbon

sources separately-sterilized were added to a final concentration of 1.0%. The plates were

incubated at 28 οC and the growth was noticed after 7, 14 and 21 days using glucose as positive

control. Cell wall analysis of DAP isomers in the cell wall composition was analyzed using paper

chromatography by Lechevalier and Lechevalier14

.

Measurement of protease activity

Protease activity was assayed by a modified method of Tsuchida et al.15

with some

modification by using casein as the substrate. Hundred μL of enzyme solution was added to

900 μL of substrate solution (2 mg/mL (w/v) casein in 10 mM Tris-HCl buffer, pH 8.0). The

Optimization Conditions of Extracellular Proteases Production 951

mixture was incubated at 37 °C for 30 min. Reaction was terminated by the addition of an

equal volume of 10% (w/v) trichloroacetic acid then the reaction mixture was allowed to

stand in ice for 15 min to precipitate the insoluble proteins. The supernatant was separated

by centrifugation at 12,000 rpm for 10 min at 4 °C; the acid soluble product in the

supernatant was neutralized with 5 mL of 0.5 M Na2CO3 solution. The color developed after

adding 0.5 mL of 3-fold diluted Folin Ciocalteau reagent was measured at 660 nm. All

assays were done in triplicate. One protease unit is defined as the amount of enzyme that

releases 1 µg of a tyrosine per mL per minute under the above assay conditions. The specific

activity is expressed in the units of enzyme activity per milligram of protein.

Protein concentration

Protein concentration was determined by the method of Lowry et al.16

with bovine serum

albumin as standard.

Cell dry weight (CDW) determination

The biomass concentration was determined as cell dry weight after centrifugation (5 000 g

for 5 min) of 10 mL of culture broth in duplicate and dried at 105 ºC overnight until constant

weight.

Optimization of cultural and environmental conditions

Protease production media and cultivation conditions

Five different types of broth media were used in this study for primary evaluation of

medium optimization process. All these media were reported before for their high

support of protease production. The compositions of these media were as follows: a

medium 1: 800 mL of solution (A): KH2PO4 3 g; Na 2HPO4 3 g; NH4Cl 2 g and NaCl 50 mg

and 200 mL of solution (B): 8 g glucose and 1 g MgSO4. The medium 2: (g/L): 20

starch; 3 yeast; 1 K2HP4; 3 CaCO3 and 0.01 g FeSO4. The medium 3: (g/L): 30 sucrose;

0.5 KCl; 0.01 Fe SO4; 0.5 Mg SO4; 1 K2HPO4 and 3 KNO3. A medium 4 (%): 2 starch;

1.2 K2HPO4; 0.05 Mg SO4. A medium 5: (%) 1 glucose; peptone 0.5; yeast extract 0.5;

NaCl 0.5 and CaCl 0.2. For all media used, the pH was adjusted to 9.0 before

sterilization. The carbon source was sterilized separately and added to the fermentation

medium before inoculation. Fifty ml of these liquid media was dispensed into each 250 mL

Erlenmeyer flasks and autoclaved at 121 °C for 20 min. The flasks were inoculated in

duplicates with 5% of vegetative cell from seven-day-old culture grown on ISP-2

medium. The inoculated flasks were kept at 50 °C on a rotary shaker (New Brunswick

Scientific Co., NJ, USA) at 200 rpm for 96 h. The contents of each flask were harvested

by centrifugation at 5.000 rpm for 10 min and the supernatant was analyzed for enzyme

activity and cell growth.

Time course of protease production

Strain NRC-15 cells were incubated at the optimum conditions to select the optimal

growth phase and biomass for enzyme activity. Samples were withdrawn at 24 h intervals and the supernatant was analyzed for enzyme activity and biomass.

Optimization of the culture medium

The effect of different carbon sources on protease production was studied by supplementing

the optimized medium with different sugars. These are glucose, galactose, lactose, xylose,

sucrose, maltose, cellobiose, cellulose and starch at 1% (w/v). The carbon source was

separately sterilized and added to the medium before inoculation.


952

To test the effect of different nitrogen source on protease production, an optimum liquid

medium was supplemented with various organic nitrogen sources such as casein, peptone,

yeast extract and several inorganic nitrogen sources such as NaNO3, KNO3, NH4 NO3,

NH4Cl, NH4H2PO4, (NH4)2HPO4 and (NH4)2SO4 at 1% (w/v). The culture media were

incubated at 50 °C for 96 h on 200 rpm and the supernatant was analyzed for cell growth and

protease activity.

To determine the effect of metal ions on the protease production, different metal salts

were individually added to the optimized medium. The metal ions are CaCl2, MgCl2.7H2O,

MnCl2 and KCl at 0.6% w/v concentration was added. Sodium chloride at 6 % concentration

was also added. The flasks were inoculated and incubated on 200 rpm at 50 °C for 96 h. Cell

dry weight was determined and cell free supernatant was analyzed for protease activity.

Effect of initial medium pH on protease production

To study the effect of pH on cell growth and protease production, the optimized medium pH

was adjusted in the range from 7-11 with 6% NaCl. The cultures were incubated at 50 °C on

200 rpm for 96 h and analyzed for cell growth and protease activity.

Effect of surfactant on protease production

The effect of surfactants such as Tween 20, Tween 60 and Tween 80 on Streptomyces

proteases production was investigated. Tween types each separately, were added to the

optimized medium and cultivated by strain NRC-15 at optimum conditions. Cell free

supernatants were analyzed for enzyme activity.

Results and Discussion

Soil sampling, streptomyces isolation and screening for protease activity This study was undertaken with an aim of highlighting the selecting of the strains with

protease activity. Using the selective media and cultivation conditions described

previously, fifty Streptomyces were obtained from ten soil samples that were collected

from various areas in Egypt and were screened for protease activity on casein agar

medium. Twenty isolates produced a significant extracellular proteases activity as

noticed by large halos. The most potent isolate, NRC-15, a salt-tolerant actinomycete

strain, was selected and subjected to phenotypic identification for further studies . The

addition of antifungal agents to the isolation medium suppresses the growth of fungal

species on the plates. For this purpose either Cycloheximide (50-100 µg/mL), or

nystatin (10-50 µg/mL) was used17

.

Identification of an actinomycete isolated strain

Cultural and Morphological characteristics

The morphology of the spore chains of aerial mycelium and the individual spores of the

strain were classified in the spiral section and spiny surface as shown in (Figure 1a, b).

Laboratories having access to an electron microscope should include electron micrographs

of the spore surface as one of the descriptive characterization for each type culture18

.

Cultural characteristics are presented in (Table 1) indicated that the aerial mycelium was

gray. Therefore, the culture is assigned to the Gray (Gy) series. The characterization of

Streptomyces species is mainly based on the color of aerial, substrate mycelia and

soluble pigment, the shape and ornamentation of spore surface because of its stability.

Other additional tests are also considered to ascertain species' classification of new

isolate strains as recommended by Holt et al.19

.


Figure 1a. Light microscope of aerial

mycelium (Spiral) for strain NRC-15 (800X)

for 14 days at 28 ºC using Bennett’s medium.

Figure 1b. Transmission electron

micrograph (spores spiny) for strain NRC-

15 at 20.000X for 21 days at 28 º C using

Bennett’s medium.

Table 1. Cultural characteristics of Str. pseudogrisiolus NRC-15 at 7-14 days.

Medium No. Growth Color of Soluble

Pigment AM SM

ISP2 abundant Gray pale yellow brown

ISP3 fair Gray beige no

ISP4 abundant Gray beige no

ISP5 abundant Gray brown no

ISP7 abundant Gray brownish no

Bennett’s abundant Gray pale yellow brown

Czapeks moderate Gray Light brown Light brown

Nutrient agar moderate White yellowish no

Glucose asparagine moderate white beige no

Physiological and biochemical properties

The results in (Table 2) show that the strain NRC-15 exhibits the ability to reduce

nitrate to nitrite and liquefies gelatin. On the other hand, it is characterized by its

inability for melanin pigment production and coagulation of milk. Additionally,

Streptomyces NRC-15 utilized all tested sugars as C-sources (Table 2) except raffinose.

With further identification of a new isolate strains, some physiological characters such

as degradation of starch, gelatin, reduction of nitrates and the use of arabinose, glycerol,

inositol, rhamnose, galactose and mannitol as a sole C-source, were recommended by

Shriling and Gottlieb10

.

Chemotaxonomic analysis showed that the cell wall of strain NRC-15 contained

chemo type, I LL diaminopimelic acid (LL-DAP) (Table 2). The presence of this type in

the cell wall indicates that this isolate is Streptomyces as identified by Lechevalier and

Lechevalier14

who established that cell wall composition analysis is one of the main

chemotaxonomic characters of Streptomyces identification.

Based on the taxonomic properties described above, strain NRC-15 belongs to the

genus Streptomyces. Comparison of the characteristics of strain NRC-15 and related


954

members of genus Streptomyces with published descriptions of various Streptomyces

species 20

showed that the strain represents a novel species of the genus. The NRC-15

strain was most similar to Str. pseudogriseolus as shown in (Table 2). Therefore, the

proposed name for this strain is Str. pseudogriseolus NRC-15.

Table2. Identification of strain NRC-15 by physiological and biochemical tests

comparing with Str. Pseudogrisiolus.

Properties Results

Strain NRC-15 Str. pseudogrisiolus

Morphological and physiological properties Sporulated aerial mycelium color Gray (Gy) Gray (Gy)

Spore chain morphology Spiral (S) Spiral (S) Spore wall ornamentation Spiny (SPY) Spiny (SPY)

Action of milk No coagulation in 14 days ND

Nitrate reduction Positive ND

Gelatin liquefaction Positive ND

Melanin production None None

Chemotaxonomic analysis Diaminopimelic acid (DAP) LL-DAP LL-DAP

Carbon sources Degree of utilization

D-glucose + a +

D-xylose + +

L-arabinose + +

L-rhamnose + +

D-fructose + +

D-galactose + +

Raffinose -b -

D-mainitol + +

Meso-inositol + +

Salicin + +

Sucrose + - a = positive growth, b = no growth, ND = not detected.

Optimization of cultural and environmental conditions

Media screening for protease production

Different production media were used in this experiment to evaluate their capacity to

support cell growth and protease production. These media have a different composition and

were used by previous authors for protease production. The results in (Figure. 2) show that

the maximum proteases yield of, 760.3 U/mL, with a specific activity of 165 U/mg protein

were recorded using a medium No.1. The medium No.4 was the poorest for protease

production. This yield may be due to induction of enzyme secretion by glucose and

ammonium chloride. In Streptomyces, the enzyme production varied greatly with the culture

media used3.

The medium No. 5 and medium No.2 were the next best ones for enzyme production.

This indicated that this organism can produce protease on different substrates. There was

minimum yield of protease in a medium No.4. It could be attributed to the inability of the


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test organism to utilize starch as a carbon source for initial growth of inoculum. Presence of

more quantity of sugar in the medium was reported by Chaloupka21

to enhance the protease

production. The medium No.1 proved to be the best for protease production, which might be

due to the presence of readily available sugar (8 g/L glucose) and inorganic phosphates.

Shirato and Nagatsu22

also reported that 0.8 g/L of KH2PO4 was optimum for protease

production using Str. griseus. For the next best media, No 5 and No. 2 which might be due

to the presence of yeast extract, which supplied most of the essential growth factors. The

medium No.1 was used in the present study keeping in view the protease production and

cost of the constituents. The maximum CDW was recorded with a medium No.3, but the

poorest CDW was noticed using a medium. No.4. The total protein ranged between 2.9

mg/mL from a culture using a medium No.1 to 4.4 mg/mL by using medium No. 5.

Figure 2. Media screening for proteases production by Str. pseudogrisiolus NRC-15 at

shake flask on 200 rpm at 96 h.

Time course of protease production

The effect of incubation time on cell growth and protease production by strain NRC-15 are

shown in (Figure 3). The proteases production starts at 24 h of 200 U/mL, then gradually

increased and reached its maximal of 798 U/mL with specific activity of 270 U/mg protein

at 96 h. After this stage it gradually decreased to 450 U/mL at 144 h. The total protein record

2.73 mg/mL at 24 h and reached its maximal of 4.75 mg/mL at 96h. These results are in

agreement with those obtained by several authors such as Moreira et al.2, Jignasha and

Satya3, Hadeer et al.

24 who found that the maximal proteases production starts in early

stationary phase of growth. However, the maximum protease production was observed at 72 h

using Str. gulbargensis25

and 120 h using Str. albidoflavus26

. There was a gradual increase in

biomass during stationary phase. This indicates that high level of protease production is

observed during active biomass production.

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Carbon source and protease production

The results presented in (Figure 4) show that the maximum protease yield of 920 U/mLwith

specific activity of 221.35 U/mg protein was obtained from glucose. The next ones are

galactose and xylose with yield of 798 and 756 U/mL and specific activity of 185 and

184 U/mg protein, respectively. Both of maltose and starch showed the same yield of

protease, were 630 U/mL with specific activity of 150 U/mg protein. Furthermore, sucrose

and cellobiose gave the same yield of, 546 U/mL, with the specific activity of 140 U/mg

protein. The alkaline protease yield of 588 and 504 U/mL was obtained from the culture

using lactose and cellulose with specific activity of 147 and 126 U/mg protein, respectively.

These results were in agreement with El-Shafei et al.26

who found that glucose (1.25%) was

finally the best carbon source for protease production by Str. albidoflavus.

Figure 3. Effect of incubation period on proteases production by Str. pseudogrisiolus NRC-

15 on 200 rpm at 96 h.

These results contrast with Dastager et al.25

, Jignasha and Satya3 who reported that

starch and sucrose as the best C-source for Str. gulbargensis and Str. clavuligerus MIT-1

respectively. The total protein of the carbon sources yielded between 3.9-4.3 mg/mL.

Nitrogen source effect on protease production

The nitrogen sources of the fermentation medium under study were investigated as shown in

(Figure 5). A maximum protease yield of 998 U/mL with specific activity of 256 U/mg

protein was obtained from the culture using yeast extract. The culture containing peptone

and ammonium phosphate cultures were the next with yield of 996 and 983 U/mL and the

specific activity of 249 and 240 U/mg protein, respectively. The lowest protease yield was

recorded using KNO3 in the production medium. These results contrast with Ahmad et al.27

who found that soybean meal was reported as the best N source for Str. avermectinus.

Incubation of period, h


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Figure 4 Effect of different carbon source on the production of alkaline protease produced

by Str. pseudogrisiolus NRC-15 on 200 rpm at 96 h.

By studying the effect of different carbon and nitrogen sources, it was found that the

optimum enzyme yield had been established in case of glucose, and yeast extract. These

results are in agreement with Kathiresan and Manivannan28

, Narayana and Vijayalakshi29

that used the same constituents in addition to peptone for alkaline protease production from

the coastal mangrove Streptomyces sp. isolate and Str. albidoflavus, respectively.

Optimal initial pH for enzyme production

The effect of medium pH on cell growth and enzyme production was investigated as shown in

(Figure. 6). The maximal protease yield of 1018 U/mL with a specific activity of 254.44 U/mg

protein was obtained from the culture at initial medium pH 9.0. On the other hand, the lowest

yield was obtained at initial medium pH 5.0. The next best yield was 1016 U/mL with a

specific activity of 247.70 U/mg protein using a culture at initial medium pH 10.0 followed by

initial medium pH 8. The cell growth varied and ranged between 0.0106 g/mL at pH 5.0 to

0.0126 g/mL using initial pH 7.0. These results are in accordance with Jignasha and Satya3

who found Str. clavuligerus grows optimally at pH 9.0. Furthermore, Li et al.30

mentioned that

alkaliphilic Streptomyces sp. grows at an optimum pH 8-9 with scant growth at pH 7.0.

Although the growth was almost similar to the culture at pH 9.0, adversely affected at lower

pH compared to that culture at pH 10, confirming the alkali nature of enzyme. These results

contrast with Seong et al.31

, Yeoman and Edwards8 who indicated that Streptomyces have

optimum pH at an acid or neutral range: Str. tendae, pH 6.0 and Str. microflavus, pH 7.0.

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Figure 5. Effect of different nitrogen sources on the production of alkaline protease

produced by Str. pseudogrisiolus NRC-15 on 200 rpm at 96 h.

Figure 6. Effect of different pH on the production of alkaline protease produced by Str.

pseudogrisiolus NRC-15 on 200 rpm at 96 h.

Different pH

Different nitrogein sources


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The metal ions' requirements for protease production

The results in (Table 3) show that the effect of different divalent ions on the cell growth and

enzymatic activity. The maximum protease yield of 1068 U/mL with a specific activity of

290.20 U/mg protein was obtained using the culture containing NaCl as compared to all the

other tested elements and the control. These results contrast with Mizusawa32

who found that

Mn++

was stimulated the production of protease when he studied the effect of Mg++

and

Mn++

on protease production of thermophilic Streptomyces. In general, cations are known to

induce enzyme secretion and increase the thermo stability of the enzyme33

. The cell growth

differed in the culture supplement from one element to the other. However, CDW of all

elements' culture was more than the control. The total protein of media used from different

metal ions ranged between 3.6 to 4.2 mg/mL.

Figure 7. Effect of surfactants on the production of alkaline protease produced by Str.

pseudogrisiolus NRC-15 on 200 rpm at 96 h.

Table 3. Effect of metal ions on the production of alkaline protease produced by Str.

pseudogrisiolus NRC-15.

Total protein,

mg/mL

Specific activity,

U/mg protein

Enzyme activity,

U/mL

CDW,

g dry wt/mL Metal ion

3.60 263.44 948.40 0.0106 CaCl2 4.20 185.10 777.45 0.0139 MgCl2 3.98 166.30 661.95 0.0090 MnCl2 3.99 138.34 552.00 0.0136 KCl 3.68 290.24 1068.10 0.0126 NaCl 3.63 261.27 948.42 0.0106 Control

Surfactants


960

Effect of surfactants on protease activity

There is no available information concerning the effect of surfactants (Tween 20, Tween 60

and Tween 80) on Streptomyces proteases production. Therefore, the aim of the present

experiment was to study the effect of different tween types on alkaline protease production

by strain NRC-15. The results in (Figure 7) show that, the addition of different types of

Tween at 100 µmol/L to the optimized production medium based on spent grains, increased

alkaline protease production. The highest protease yield of 1093 U/mL with a specific

activity of 291.5 U/mg protein was obtained from the culture containing Tween 20 in

comparing with control. It may be due to the adsorbed surfactant film around the cell which

decreased or increased permeability or enhanced the availability of important ions, which

has a favorable effect. Results are in a good agreement with that obtained by Orpin34

. The

total protein of media used from different types of tween ranged between 2.6 to 3.75 mg/mL.

Conclusion

A new species of Str. pseudogrisiolus isolated and identified by phenotypic evidence. This

strain proposed the name, Str. pseudogrisiolus NRC-15. It was able to produce alkaline

proteases. The optimization of culture conditions required for the maximal extracellular

protease production was investigated. Thus, the optimization studies resulted in the

following findings: the most suitable nutrient medium are 1% glucose, 1% yeast extract, 6%

NaCl and 100 µmol/L of Tween 20 at initial pH 9.0 on 50 ºC for 96 h. Under these

conditions, the maximal alkaline proteases of 1093 U/mL were achieved. The present work

shows potential of industrial protease production from a Streptomyces.

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