Optimization Conditions of Extracellular Proteases Production from a Newly Isolated Streptomyces Pseudogrisiolus NRC-15

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.


Introduction
Proteases are important industrial enzymes accounting for 60% of total global enzyme sales 1 .Bacterial proteases show more promise than animal and fungal proteases, accounting for 20% of the world market 2 with the predominant use in detergents, especially for alkaline bacterial proteases 3 .Extremophilic especially alkaliphilic, halophilic and thermophilic proteases are preferred due to ease of operation, higher activity, enhanced stability, faster reaction and less proneness to contamination.Microbial alkaline proteases for industrial uses are produced and studied mainly from Bacillus and Streptomyces 4 .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) agents 6 .
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.thermovulgaris 7 , 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 genus 9 .
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.

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; KH 2 PO 4 , 1.5 and Na 2 HPO 4 , 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 Gottlieb 10 and Bergey's Manual of Systematic Bacteriology 11 .Cultural characteristics of pure isolate in various media were recorded after incubation for 7 to 14 days at 28 o C. 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 Gottlieb 10 .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 Backus 12 .
Carbon utilization was determined on plates containing ISP basal medium 9 13 .Carbon sources separately-sterilized were added to a final concentration of 1.0%.The plates were incubated at 28 ο C 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 Lechevalier 14 .

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 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 Na 2 CO 3 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.

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): KH 2 PO 4 3 g; Na 2 HPO 4 3 g; NH 4 Cl 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 K 2 HP 4 ; 3 CaCO 3 and 0.01 g FeSO 4 .The medium 3: (g/L): 30 sucrose; 0.5 KCl; 0.01 Fe SO 4 ; 0.5 Mg SO 4 ; 1 K 2 HPO 4 and 3 KNO 3 .A medium 4 (%): 2 starch; 1.2 K 2 HPO 4 ; 0.05 Mg SO 4 .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.
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 NaNO 3, KNO 3, NH 4 NO 3, NH 4 Cl, NH 4 H 2 PO 4 , (NH 4 ) 2 HPO 4 and (NH 4 ) 2 SO 4 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 CaCl 2 , MgCl 2 .7H 2 O, MnCl 2 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.

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 used 17 .

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 culture 18 .
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 .

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 Gottlieb 10 .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 Lechevalier 14 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 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.

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 used 3 .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 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 Chaloupka 21 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 Nagatsu 22 also reported that 0.8 g/L of KH 2 PO 4 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.

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 Satya 3 , 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.gulbargensis 25 and 120 h using Str.albidoflavus 26 .There was a gradual increase in biomass during stationary phase.This indicates that high level of protease production is observed during active biomass production.

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.These results contrast with Dastager et al. 25 , Jignasha and Satya 3 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 3.9-4.3mg/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 KNO 3 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.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 Manivannan 28 , Narayana and Vijayalakshi 29 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 Satya 3 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 Edwards 8 who indicated that Streptomyces have optimum pH at an acid or neutral range: Str.tendae, pH 6.0 and Str.microflavus, pH 7.0.
Different corben sources  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 Mizusawa 32 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 enzyme 33 .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.

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 Orpin 34 .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.

Figure 2 .
Figure 2. Media screening for proteases production by Str.pseudogrisiolus NRC-15 at shake flask on 200 rpm at 96 h.

Figure 3 .
Figure 3.Effect of incubation period on proteases production by Str.pseudogrisiolus NRC-15 on 200 rpm at 96 h.

Figure 4
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.

Figure 5 .
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 .
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

Figure 7 .
Figure 7. Effect of surfactants on the production of alkaline protease produced by Str.pseudogrisiolus NRC-15 on 200 rpm at 96 h.

.
Identification of strain NRC-15 by physiological and biochemical tests comparing with Str.Pseudogrisiolus.
a = positive growth, b = no growth, ND = not detected.

Table 3 .
Effect of metal ions on the production of alkaline protease produced by Str.pseudogrisiolus NRC-15.