Maximum Protease Production Research Articles

Bioprocess optimization for production of thermoalkali-stable protease from Bacillus subtilis K-1 under solid-state fermentation

ABSTRACTCost-effective production of proteases, which are robust enough to function under harsh process conditions, is always sought after due to their wide industrial application spectra. Solid-state production of enzymes using agro-industrial wastes as substrates is an environment-friendly approach, and it has several advantages such as high productivity, cost-effectiveness, being less labor-intensive, and less effluent production, among others. In the current study, different agro-wastes were employed for thermoalkali-stable protease production from Bacillus subtilis K-1 under solid-state fermentation. Agricultural residues such as cotton seed cake supported maximum protease production (728 U ml−1), which was followed by gram husk (714 U ml−1), mustard cake (680 U ml−1), and soybean meal (653 U ml−1). Plackett–Burman design of experiment showed that peptone, moisture content, temperature, phosphates, and inoculum size were the significant variables that influenced the protease production. Furthermore, statistical optimization of three variables, namely peptone, moisture content, and incubation temperature, by response surface methodology resulted in 40% enhanced protease production as compared to that under unoptimized conditions (from initial 728 to 1020 U ml−1). Thus, solid-state fermentation coupled with design of experiment tools represents a cost-effective strategy for production of industrial enzymes.

Catalytic Role of Thermostable Metalloproteases from Bacillus subtilis KT004404 as Dehairing and Destaining Agent.

Proteases with characteristic stabilities are considered attractive candidates for industrial catalysis. In the present study, a potent bacterial strain KT004404, an inhabitant of hydrothermal vents, was isolated and characterized for protease production. Initial screening indicated that this strain produced a hydrolytic zone of 30mm 16S rRNA-based identification revealed that our isolate was a strain of Bacillus subtilis. Optimum reaction condition for maximum protease production was determined as 55°C, pH 6, 1% inoculum size and malt extract as primary growth substrate supplemented with 1% dextrose. Yield of the enzyme was increased up to 7.53 folds with a specific activity of 55.125U/mg after gel filtration chromatography. SDS-PAGE analysis confirmed the size of protease as 28.24kDa. Purified enzyme retained its catalytic activity over a broad range of temperature (5 to 65°C) and pH [5-8]. Addition of metal ions shown to have a stimulatory effect on catalytic properties while EDTA inhibited the efficiency of the enzyme confirming it as a metalloprotease. Protease exhibited excellent stability and activity in the presence of anionic surfactants, solvents, and detergents. The results of dehairing and destaining experiments suggested that the protease produced by B. subtilis KT004404 could be used in leather and textile industries with ecological benefits.

Improvement of shelf life of soymilk using immobilized protease of Oerskovia xanthineolytica NCIM 2839.

Protease enzyme has lot of commercial applications, so the cost-effective production of protease using sunflower oil seed waste was carried out from Oerskovia xanthineolyitca NCIM 2839. The maximum protease production was after 24 h of incubation with 2.5 % oil seed waste concentration. O. xanthineolytica was found to produce two proteases—P1 and P2. The proteases were purified using 60 % cold acetone precipitation and DEAE-cellulose ion exchange chromatography. SDS-PAGE revealed molecular weight of P1 and P2 was 36 and 24 kDa, respectively. P1 and P2 were optimally active at pH 7.0 and pH 7.5 at temperature 35 and 40 °C, respectively. Analysis of hydrolyzed product of P1 and P2 by HPLC reveals that the P1 has endoprotease and P2 has exoprotease activity. The treated soy milk with immobilized proteases showed increased shelf life and removal of off flavor.

Optimization of thermotolerant alkaline protease production from Brevibacillus brevis strain BT2 using surface response methodology

Abstract An efficient thermotolerant alkaline protease producer, strain Brevibacillus brevis BT2 was isolated from waste water effluents from textile dyeing industry. Effect of variables, i.e. soybean meal, soluble starch, inoculums, temperature and pH and their mutual interaction were analyzed by Box-Behnken design (BBD) for optimizing their levels in the medium and effect of their mutual interaction on thermotolerant alkaline protease production. All the above variables were found significantly influencing protease activity individually and also in interaction with other variables. The model predicted maximal protease production of 121.1 (U ml−1) at the soybean meal 7.46 (gL−1), soluble starch 10.00 (gL−1), inoculums 1.76%, temperature 49.3 °C, and pH 9.1. The maximum protease production obtained experimentally was found to be 128.5 U/ml. The result was coincident with the predicted value and the model was proven to be adequate.

Agroindustrial/Forestry Residues as Substrates for Production of Thermoactive Alkaline Protease from Bacillus licheniformis K-3 Having Multifaceted Hydrolytic Potential

Microbial proteases due to their enormous application potential in numerous biotechnological processes have gathered considerable research impetus during recent years. However, lack of process suitable characteristics viz. thermostability, wide range pH stability, resistance to inhibitors, among others, in most of the available proteases motivated the search for better and efficient proteases. High production cost of microbial proteases mainly due to expensive substrates (carbon/nitrogen sources used for cultivation of microorganisms) is another limitation for wide range industrial application of these enzymes. Current investigation reports production of a thermostable and pH stable protease from a bacterial isolate Bacillus licheniformis K-3 using agroindustrial/forestry residues as inexpensive substrates for cost-effective enzyme production. Pine needle biomass is being reported for the first time as a proficient carbon source that supported maximum protease production (1321 U/ml), and was followed by wheat bran (1303 U/ml), potato peel (1167 U/ml) and cane bagasse (1122 U/ml). Gram husk as nitrogen source enhanced enzyme production maximally (932 U/ml) as compared to control (389 U/ml), and was followed by soybean meal (823 U/ml), malt extract (742 U/ml), mustard cake (724 U/ml) and fish scales (670 U/ml). Optimum pH and temperature for enzyme was 9.0 and 60 °C, however, protease showed considerable activity over high temperatures and broad pH range. Protease from B. licheniformis K-3 showed remarkable tolerance against detergents like CTAB, SDS and Tween-20. The protease exhibited excellent potential for industrial applications like degelatinization of X-ray films and dehairing of animal hide.

Molecular characterization and growth optimization of halo-tolerant protease producing Bacillus Subtilis Strain BLK-1.5 isolated from salt mines of Karak, Pakistan.

Microbial proteolytic enzyme is one of the most important industrial enzymes that hydrolyze proteins. The applications of proteases under harsh industrial conditions like alkalinity, salinity, and temperature make them inactive and unstable. This suggests need for search for novel microbial sources for protease production having diverse properties. For this purpose, 54 bacterial strains were isolated from different salt mines of Karak, Pakistan and were investigated for their proteolytic activity on skim milk agar plates. The strain which showed maximum protease activity was characterized by 16S rRNA gene sequence analysis. Furthermore, growth and protease production was optimized for the characterized bacteria under different physical factors, i.e., pH, temperature and salinity. The isolate BLK-1.5 exhibited strong protease production and was identified as Bacillus subtilis based on biochemical characteristics and 16S rRNA gene sequence analysis. Maximum production of protease was recorded at pH 10, 37°C and 7% (w/v) NaCl. Molecular weight of proteases was estimated 38kDa and its optimum activity was observed at pH 10, 50°C and 2% (w/v) NaCl. In conclusion, the protease produced by halo-tolerant Bacillus subtilis strain BLK-1.5 has diverse characteristics and could be useful in various industrial applications.

Characterization of halo-alkaline and thermostable protease from Halorubrum ezzemoulense strain ETR14 isolated from Sfax solar saltern in Tunisia.

A total of 54 halophilic strains were isolated from crystallizer TS18 (26 strains) and non-crystallizer M1 (28 strains) ponds and screened for their ability to produce protease, amylase, and lipase activities. Enzymatic assays allowed the selection of thirty-two active strains, among them, the ETR14 strain from TS18 showed maximum protease production yields and therefore, selected for further analysis. The results from 16S rRNA gene sequence analysis revealed that the strain belonged to Halorubrum ezzemoulense (Hrr. ezzemoulense) species. Further results indicated that optimum growth and protease production yields were obtained with 10-15% NaCl concentrations in the DSC-97 medium. The enzyme was also able to maintain high levels of protease activity at salt concentrations of up to 25%. While readily available carbon sources were noted to significantly reduce protease production, the combination between yeast extract and peptone enhanced protease excretion, which reached a maximum of 284 U ml(-1) at the end of the exponential growth phase. The enzyme exhibited optimum activity at pH 9 and 60 °C. The halophilic protease retained 87% of its initial activity after 1 h incubation at 70 °C and showed high stability over a wide range of pH, ranging from 7 to 10. This protease exhibited good temperature, pH, and salinity tolerance, which distinguishes it from other proteases previously described from other members of the holoarchaea genera and makes it a promising candidate for application in various industries.

Production, Purification, and Biochemical Characterization of Thermostable Metallo-Protease from Novel Bacillus alkalitelluris TWI3 Isolated from Tannery Waste.

Protease enzymes in tannery industries have enormous applications. Seeking a potential candidate for efficient protease production has emerged in recent years. In our study, we sought to isolate proteolytic bacteria from tannery waste dumping site in Tamilnadu, India. Novel proteolytic Bacillus alkalitelluris TWI3 was isolated and tested for protease production. Maximum protease production was achieved using lactose and skim milk as a carbon and nitrogen source, respectively, and optimum growth temperature was found to be 40°C at pH 8. Protease enzyme was purified using ammonium sulfate precipitation method and anion exchange chromatography. Diethylaminoethanol (DEAE) column chromatography and Sephadex G-100 chromatography yielded an overall 4.92-fold and 7.19-fold purification, respectively. The 42.6-kDa TWI3 protease was characterized as alkaline metallo-protease and stable up to 60°C and pH 10. Ca(2+), Mn(2+), and Mg(2+) ions activated the protease, while Hg(2+), Cu(2+), Zn(2+), and Fe(2+) greatly inhibited it. Ethylenediaminetetraacetic acid (EDTA) inhibited TWI3 protease and was activated by Ca(2+), which confirmed that TWI3 protease is a metallo-protease. Moreover, this protease is capable of dehairing goat skin and also removed several cloth stains, which makes it more suitable for various biotechnological applications.

Studies on the effect of agitation and aeration for the improved protease production by bacillus licheniformis NCIM-2042

The present research work deals with the effect of agitation and aeration for protease production using Bacillus licheniformis NCIM-2042. The experiments were carried out in a lab scale 2.2 l bioreactor with 2 l working volume at 37 °C for 84 hour. Determination of kLa was estimated by dynamic gassing out method. The value of kLa was found to be 41, 54 and 56 1/h at 1, 2 and 3 vvm of airflow rate respectively. The maximum protease production was found to be 382.46 U at optimum flow rate of 2 vvm and agitation speed of 180 rpm.

Medium Formulation and its optimization for increased protease production by Penicillium sp. LCJ228 and its potential in blood stain removal

The production of protease by Penicillium sp. LCJ228 was optimized under submerged fermentation. Nutritional and physical factors that influence protease production were optimized by one factor at a time (OFAT) method in order to achieve high yield of protease. Maximum protease production was obtained on the 4th day in a liquid medium containing glucose (15 g/L), yeast extract (15 g/L), black gram husk (10 g/L) with an initial pH of 10 and an inoculum size of 2 g/L of fungal mycelium. About 2.4 fold increase in protease production was observed in the optimized medium. The maximally yielded crude protease was then precipitated and characterized by SDS-PAGE and native-PAGE. The molecular weight of the ammonium sulphate precipitated protease was ~66 to 70 kDa and native-PAGE showed three isoenzymes. The crude and precipitated protease had the ability to completely remove blood stains on cotton fabric indicating its potential to be used as a stain remover in detergents.

Alkaline protease from a new halotolerant alkaliphilic Bacillus agaradhaerens strain ak-r isolated from egyptian soda lakes

Alkaline proteases are hydrolytic enzymes that cleave peptide bonds in proteins and peptides in alkaline conditions, which occupy a pivotal importance with respect to their industrial applications. This study aimed to isolate new alkaline protease producing alkaliphilic bacteria from Egyptian soda lakes and optimize the fermentation process to enhance the enzyme production. The extensive screening process of the samples collected from Egyptian soda lakes resulted in isolation of a potent alkaline protease producing alkaliphilic strain AK-R. The isolate was identified as Bacillus agaradhaerens strain AK-R based on 16S rRNA gene analysis (99%). Wheat bran and gelatin supported maximum alkaline protease production as carbon and nitrogen sources, respectively. Strain AK-R is halo-tolerant thermo-tolerant alkaliphilic bacterium in nature, as it can grow over a wide range of NaCl concentrations (up to 25%) and up to 55 °C, with maximal growth and enzyme production at 2.5-5%, and pH 11 at 35 °C. Among the tested cations, only Mg 2+ and Ca 2+ ions significantly enhanced the enzyme production by about 1.2, and 1.3 fold compared to control, respectively. Alkaline protease secretion was coherent with the growth pattern, reaching maximal yield after about 32 h (mid stationary phase). In conclusion a new halo-tolerant thermo-tolerant alkaliphilic alkaline protease producing Bacillus agaradhaerens strain AK-R was isolated from Egyptian soda lakes. Optimization of the nutritional and cultivation conditions resulted in increase of enzyme yield by 20 fold. Strain AK-R and its extracellular alkaline protease with salt, pH and temperature, tolerance signify their potential application in laundry and pharmaceuticals industries.

Optimization of Alkaline Protease Production by Alkaliphilic Bacillus sp. KW2 in Low Cost Medium using Statistical Approaches

Objective: Isolation of alkaline protease producing alkaliphilic bacteria and optimization of protease production in low cost medium. Methods: Optimization of protease production was performed by one-variable-at-a-time (OVAAT) approach and statistical approaches using Design Expert 6 software. Results: Nine (9) morphologically distinct alkaliphilic bacterial strains were isolated. Of these isolates 6 strains were found to be positive for protease production. Among these proteolytic strains, Bacillus sp. KW2 was selected for protease production optimization studies. The optimal protease production was observed at 30°C and pH 10.7. Among the various carbon and nitrogen sources studied rice bran and soybean meal were found to be the best Carbon and Nitrogen sources respectively. Plackett-Burman design (PBD) was used to screen signal factors that influenced the protease production. Rice bran, soybean meal, inoculum age, CaCl2 and inoculum size gave positive effects whereas KH2PO4 and MgSO4 gave negative effects on protease production. The four most critical factors viz. rice bran, soybean meal, calcium chloride and potassium dihydrogen phosphate were selected for optimization studies by Response Surface Methodology (RSM). The response surface graphs showed significant interactions among the factors and a final optimized medium (FOM) was obtained after PBD and RSM experiments. Overall, a 4.8 fold increase in protease production was observed after optimization. Studies of the time course of protease production by Bacillus sp. KW2 revealed that maximal protease production occurred at the stationary phase (84 h) of growth. Conclusions: The present study shows that protease production is greatly influenced by cultural conditions and media constituents. This study also established that the strain KW2 could produce alkaline protease in a low cost medium containing rice bran and soybean meal as major carbon and nitrogen sources. KW2 and its protease may be promising agents for biotechnological applications.

A versatile system based on substrate formulation using agroindustrial wastes for protease production by Aspergillus niger under solid state fermentation

Abstract This study aimed to evaluate the substrate formulation using different agroindustrial wastes (wheat bran, soybean meal, cottonseed meal and orange peel) as a tool to purpose a versatile system for protease production by Aspergillus niger LBA 02 under solid state fermentation. The results showed interesting synergistic and antagonistic effects between the agroindustrial wastes during the protease production. The highest protease activity was found using the medium containing wheat bran (0.50) and soybean meal (0.50), reaching 262.78 U g−1 after 48 h fermentation. The most prominent synergetic effects were observed on fermentations performed using the medium composed by the four agroindustrial wastes in equal proportions at 48 and 72 h fermentation, reaching increases of 33.7%, 7.6%, 30.8% and 581.7% and 11.6%, 131.4%, 69.5% and 547.0%, respectively, in protease production as compared to the individual substrates. From these results was also possible to establish the use of at least three different culture media formulations with a maximum protease production above 260 U g−1, making it a versatile and efficient system for protease production and that can be easily applicable to other groups of enzymes.

OPTIMIZATION OF NUTRITIONAL AND CULTURE CONDITIONS FOR IMPROVED PROTEASE PRODUCTION BY ASPERGILLUS NIDULANS AND ASPERGILLUS FLAVUS

Aspergillus sp. is considered to be a good source of protease. Medium optimization is essential for each isolate in order to maximise the protease production. In the present study, different media were evaluated for maximising protease biosynthesis under submerged fermentation (SmF) using A. nidulans LCJ249 and A. flavus LCJ253. Among the six media tested, Medium 6 was found to be the best medium for maximum protease production in both A. nidulans LCJ249 (1178.0 U/mL) and A. flavus LCJ253 (950.6 U/mL). Effect of essential nutrient parameters like carbon, nitrogen and casein and other culture conditions such as incubation time, inoculum size, pH and shaking and static conditions were evaluated by the conventional one factor at a time approach. In the case of A. nidulans LCJ249, a medium containing 20 g/L glucose, 15 g/L malt extract, 5 g/L casein, initial medium pH of 7 and inoculum size of 30 mg/L favoured maximum protease production. Similarly, in A. flavus LCJ253, a medium containing 20 g/L starch, 20 g/L peptone, 15 g/L casein, initial medium pH of 7 and inoculum size of 10 mg/L favoured maximum protease production. In both the cultures, protease production was higher under shaking condition than in static conditions. The study showed that the optimised medium with optimal conditions enhanced protease production when compared to the original medium.

Production of extracellular alkaline protease by new halotolerant alkaliphilic Bacillus sp. NPST-AK15 isolated from hyper saline soda lakes

Alkaline proteases are among the most important classes of industrial hydrolytic enzymes. The industrial demand for alkaline proteases with favorable properties continues to enhance the search for new enzymes. The present study focused on isolation of new alkaline producing alkaliphilic bacteria from hyper saline soda lakes and optimization of the enzyme production. A new potent alkaline protease producing halotolerant alkaliphilic isolate NPST-AK15 was isolated from hyper saline soda lakes, which affiliated to Bacillus sp. based on 16S rRNA gene analysis. Organic nitrogen supported enzyme production showing maximum yield using yeast extract, and as a carbon source, fructose gave maximum protease production. NPST-AK15 can grow over a broad range of NaCl concentrations (0–20%), showing maximal growth and enzyme production at 0–5%, indicated the halotolerant nature of this bacterium. Ba and Ca enhanced enzyme production by 1.6 and 1.3 fold respectively. The optimum temperature and pH for both enzyme production and cell growth were at 40°C and pH 11, respectively. Alkaline protease secretion was coherent with the growth pattern, started at beginning of the exponential phase and reached maximal in mid stationary phase (36 h). A new halotolerant alkaliphilic alkaline protease producing Bacillus sp. NPST-AK15 was isolated from soda lakes. Optimization of various fermentation parameters resulted in an increase of enzyme yield by 22.8 fold, indicating the significance of optimization of the fermentation parameters to obtain commercial yield of the enzyme. NPST-AK15 and its extracellular alkaline protease with salt tolerance signify their potential applicability in the laundry industry and other applications.

Hide Dehairing and Laundry Detergent Compatibility Testing of Thermostable and Solvents Tolerant Alkaline Protease from Hot Spring Isolate <i>Bacillus cohnii</i>U3

In the present study, alkaline protease producing thermophilic Bacillus cohniiU3 strain was isolation from Unnai hot spring, India. The maximum production of protease (344 U/mL) was reported after 72 h of inoculation at 50°C in shake flask culture using the gelatin casein medium. The protease was found to remain active up to 96 h and production was growth dependent. The activity of parital purified protease was reported in abroad range of pH ranging from 4.0 to 11.0 with an optimum of 9.0 pH; Enzymes was indicated the highest activity at 50°C temperature. Salt independent catalysis and activity with a broad range of substrate concentration is the key feature of the protease. Thermos table nature, the stability in alkaline pH and stability in high salt concentration for 45 min were outstanding features of protease. The enzyme was stable in the presence of various organic solvents like Dimethyl Sulphoxide (DMSO), Methanol, Butanol, n-Hexane, Benzene at 25% (v/v) concentration. A good compatibility of the enzyme with most commercial detergents indicated its application in detergent industry. The remarkable dehairing in goat hide and destaining of blood spot in 2 h using 10 U/mL of protease assure that it could be a potential candidate for leather and detergent industries.

Deproteinization of Distillery Yeast Biomass Waste by Protease-producing Bacillus megaterium PB 4

The distillery yeast biomass (DYB), a waste by-product from distilleries is one of the under-utilized protein sources to be exploited for the production of protein hydrolysates. In this study, a neutral protease produced from Bacillus megaterium PB4 isolated from agro wastes enriched soil exhibited significant deproteinization of distillery yeast biomass to yield protein hydrolysates. Among three protease producing strains isolated, the maximum protease production of 120.3 ± 1.4 U mL-1 was exhibited by PB4 grown in Luria-Bertani (LB) broth supplemented with casein at pH 7.0 and 30°C. The minimal media supplemented with various carbon and nitrogen sources, glucose and peptone significantly improved the protease production in PB4. The effect of metal ions such as Zn2+, Mg2+, Mn2+, Cu2+ at 0.5 and 1.0 mM final concentration in the media on protease production indicated that Zn2+ (1.0 mM) was favorable in enhancing the protease production. The Km and Vmax of PB4 protease for casein substrate was 0.6 U ml-1 and 217.3 μM min-1 mg-1 protein, respectively. The deproteinization rate for distillery yeast biomass was 84 and 76.4% using culture and crude enzyme extracted from PB4, respectively. To the best of our knowledge, deproteinization of distillery yeast biomass using proteolytic bacterial isolate has never been demonstrated before. The experimental results in the present study suggest that Bacillus megaterium PB4, can be utilized for the eco-friendly, economical production of protein hydrolysates and may be considered as a potential candidate in various biotechnological applications.

Production and characterization of haloalkaline protease from ascidian-associated Virgibacillus halodenitrificans RSK CAS1 using marine wastes

A marine ascidian-associated bacterium, Virgibacillus halodenitrificans RSK CAS1, was optimized for protease production by response surface methodology using marine waste as substrate. The central composite design was employed, and the optimal medium constituents for maximum protease production (1461.11 U/ml) were determined to be shrimp shell powder (15.32 g/l), casein (5.37 g/l), MgSO4 (3.0 g/l) and NaCl (55.31 g/l). The protease was purified from the culture supernatant to homogeneity in a three-step procedure consisting of ammonium sulfate precipitation, ion exchange chromatography (DEAE-cellulose column) and gel-filtration chromatography (Sephadex G-75 column), resulting in a 8.7-fold-change in purified protein. This protein had a specific activity of 1,086.78 U/mg and a molecular weight of 21 kDa. It exhibited optimal activity at 50 °C, pH 9 and 25 % NaCl. The significant stability of this protein at higher levels of salt, metal ions, organic solvents and commercial detergents and at higher, temperature, as well as its application as a cleaning additive in blood stain removal, suggests its possible use the laundry detergent industry.

Optimization of fibrinolytic protease production from Bacillus subtilis I-2 using agro-residues

The aim of this work was to study the production of fibrinolytic protease by Bacillus subtilis I-2 on agricultural residues. Molasses substantially enhanced (63%) protease production (652.32 U/mL) than control (398.64 U/mL). Soybean meal supported maximum protease production (797.28 U/mL), followed by malt extract (770.1 U/mL), cotton cake (761.04 U/mL), gelatin (742.92 U/mL) and beef extract (724.8 U/mL). Based on the Plackett-Burman designed experiments, incubation time, soybean meal, mustard cake and molasses were identified as the significant fermentation parameters. Ammonium sulfate precipitation and DEAE sephadex chromatography resulted 4.8-fold purification of protease. Zymography showed the presence of three iso-forms in the partially purified protease preparation, which was confirmed by the SDS-PAGE analysis (42, 48, 60 kDa). Protease exhibited maximum activity at 50oC and at pH 8.0. Significant stability was observed at 30-50oC and at pH 7.0-10.0. Mg2+, Zn2+, Co2+, Ca2+, Mn2+ and Cu2+,EGTA, EDTA and aprotinin severely decreased the enzyme activity.

Optimization and partial purification of a protease produced by selected bacterial strains grown on trash fish meal substrate and its antagonistic property against bacterial pathogens

Abstract In the present study protease producing bacterial strains such as Bacillus cereus, Proteus mirabilis, Proteus vulgaris and Enterobacter aerogenes were isolated from spoiled fish and shrimp tissues. The isolated strains were individually inoculated into the formulated culture media supplied with various concentrations (2.5–20%) of trash fish (Odonus niger) meat powder as major substrate. Among the tested substrate concentrations, 5% substrate supplied medium favored maximum protease (3.82–5.13 IU ml−1) production. After optimization of substrate concentration, the organisms were tested individually for their protease production capacity at various pHs and temperatures. The results showed that all the tested strains were capable of producing high protease at pH 6 and 50 °C. The crude proteases produced by the tested organisms were individually purified by MLFTPP and ASBP methods. The partially purified enzymes were screened against few pathogenic bacterial strains, and it was observed that the enzyme produced by P. vulgaris exhibited the highest antibacterial activity with the inhibition zone ranged between 168.0±0.82 and 204.3±1.25 AU/ml. Further, the partially purified enzyme of P. vulgaris alone characterized through SDS-PAGE analysis and determined its molecular mass as 65.0 KDa.