International Journal of Pharma and Bio Sciences

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1 Research Article Biotechnology International Journal of Pharma and Bio Sciences ISSN ISOLATION, IDENTIFICATION, PARTIAL PURIFICATION, OPTIMIZATION AND CHARACTERIZATION OF ALKALINE PROTEASE FROM E. coli SHRUTI SHUKLA* AND O. P. VERMA *1 Department of Molecular & Cellular Engineering, JSBB, SHIATS, Allahabad, India. ABSTRACT In the present study E. coli was used for the production of alkaline protease in eight different fermentation media. This bacterium was isolated from soil sample and screened for protease production. The enzyme production from E. coli was estimated using casein as substrate and its molecular weight was determined by SDS-PAGE. The enzyme activity was also studied at different parameters like ph, temperature, carbon source, nitrogen source, metal ions concentration and effect of inhibitors. KEYWORDS: E.coli, alkaline protease, SDS-PSGE, submerged fermentation, optimization. SHRUTI SHUKLA Department of Molecular & Cellular Engineering, JSBB, SHIATS, Allahabad, India. *Corresponding author B

2 INTRODUCTION The alkaline proteases are produced by a range of bacteria, yeast and moulds. Among them the bacterial alkaline proteases are produced in large amount for various applications in leather processing, Medical diagnostics, and recovery of silver from X-ray films 1-2. It is also used in baking, brewing, meat tenderization, peptide synthesis, medical diagnosis, cheese making, certain medical treatments of inflammation and virulent wounds and in deharing of sheepskins. It also has wide application in Bioremediation process 3-4. Alkaline protease added to laundry detergents plays a specific catalytic role in the hydrolysis of protein strains such as blood, milk, human sweat, etc. The increased usage of the protease as a detergent additive is mainly due to its cleaning capabilities in environmentally acceptable, non-phosphate detergents 5. Although microbes from terrestrial sources are employed for industrial production of enzymes, the potential for the synthesis of several novel enzymes by marine microorganisms has been recognized 6. Diverse techniques have been used for the screening of novel enzymes with new bio-catalytic capabilities and great potential for several industrial processes and other applications 7. Large number of microorganisms produces proteases, but Bacillus strains are recognized as important sources of commercial alkaline protease because of their ability to secrete large amounts of enzymes with high activity 8-9. In the present study the alkaline protease was extracted from E. coli then the enzyme was purified and optimized at different parameters. MATERIALS AND METHODS Isolation and Screening of Bacteria for Protease production Bacterial species were isolated from soil sample using serial dilution methods. Identification of selected isolate was studied based on different morphological and biochemical characteristics. This data was compared with a standard description given in Bergy s Manual of Systematic Bacteriology. The isolated bacterial species were screened for the protease production viz. skim milk agar media. Production and estimation of alkaline protease Production of alkaline protease was done in eight different fermentation media inoculated with isolated bacterial species for three days. After alkaline protease production the estimation of enzyme was done using casein as substrate and carbonate-bicarbonate buffer (sodium carbonate - sodium bicarbonate buffer solution). Partial purification and SDS-PAGE The isolated enzyme was partially purified by gel filtration chromatography viz. sephadex G- 75 and the molecular weight was determined through SDS-PAGE technique. Optimization of Alkaline protease production The production of alkaline protease enzyme in all the eight different fermentation media was optimized at different parameters like ph (3, 5, 7, 9 & 11), temperature (40 o C, 50 o C, 60 o C, 70 o C & 80 o C), carbon source (lactose, sucrose, fructose, maltose and dextrose), nitrogen source (gelatin, ammonium chloride, urea, ammonium nitrate & sodium nitrate) and metal ions (sodium chloride, potassium chloride, calcium chloride, magnesium chloride & zinc chloride). RESULTS AND DISCUSSION The biochemical property of isolated bacterial species was done by various biochemical tests and identified through Bergy s manual, the bacterial species were found to be E.coli. The E. coli was further screened for protease production using skim milk agar media (viz. steak plate method); formation of clear zone around the bacterial colony indicates the presence of protease production as shown in Figure 1(a). B

3 Figure 1 Positive result of Casein hydrolysis Test (i) Clear zone around the growth due to production of protease enzyme (ii) Media Used: Skim Milk Agar Media Batch fermentation was carried out with E. coli in eight different media (M1 to M8) separately containing 100ml medium into 250ml conical flask for 72 hours at 37 o C (Figure 2). Figure 2 Eight different fermentation media (100ml each) (i) Sterilized at 15psi, 121 o C for 15 minutes (ii) Inoculated with E. coli for production of alkaline protease. It is observed from Graph 1 that medium M8 was found best among these media for alkaline protease production while minimum production was observed in M3 fermentation media. Graph 1 Estimation of alkaline protease produced from E. coli (a) Maximum yield was observed in M8 fermentation media (b) Minimum yield in M3 fermentation media B

4 The isolated enzyme was partially purified by gel filtration chromatography viz. sephadex G- 75 different fraction were collected which was store at 4 o C for further use and the molecular weight of isolated protein from E. coli was found to be 42kd through SDS-PAGE.Further the purified enzyme was optimized at different parameters to study the maximum yield. All the eight different fermentation media inoculated with E. coli were optimized at different ph range using Sodium carbonate and sodium bicarbonate buffer solution (adjusted at different ph range) to study the maximum activity of alkaline protease. The maximum activity was observed in M7 (3.25±0.25U/ml), M8 (6.11±0.46U/ml), M7 (6.32±0.6U/ml), M7 (6.38±0.56U/ml) and M2 (5.32±0.46U/ml) at ph3, ph5, ph7, ph9 and ph11 respectively. Among these highest activity was at ph9 in M7 (6.38±0.46U/ml) media as shown in Graph 2. Graph 2 Optimization of alkaline protease production from E. coli at different ph (a) Maximum yield was observed in M7 at ph 9 (b) Minimum yield in M6 at ph11. All the eight different fermentation media inoculated with E. coli were optimized at different temperature range to study the maximum activity of alkaline protease. The maximum activity was observed in M7 (10.89±0.66U/ml), M2 (11.58±0.60U/ml), M2 (15.73±0.66U/ml), M4 (15.47±0.76U/ml) and M2 (13.72±0.66U/ml) at 40 o C, 50 o C, 60 o C, 70 o C and 80 o C respectively. Among these highest activity was at 60 o C in M2 (15.73±0.66U/ml) media as shown in Graph3. B

5 Graph 3 Optimization of alkaline protease production from E. coli at different temperature (a) Maximum yield was observed in M2 at 60oC (b) Minimum yield in M7 at 40 o C All the eight different fermentation media inoculated with E. coli were optimized by different carbon source to study the maximum activity of alkaline protease. The maximum activity was observed in M1 (4.40±0.66U/ml), M1 (4.40±0.66U/ml), M4 (5.56±0.69U/ml), M3 (3.96±0.46U/ml) and M3 (3.93±0.46U/ml) in lactose, sucrose, fructose, maltose and dextrose as carbon source respectively. Among these highest activity was in fructose in M4 (5.56±0.69U/ml) media as shown in Graph 4. Graph 4 Optimization of alkaline protease production from E. coli in different carbon source (lactose, sucrose, fructose, maltose & dextrose) (a) Maximum yield was observed in M4 viz. fructose (b) Minimum yield in M3 viz. dextrose All the eight different fermentation media inoculated with E. coli were optimized by different nitrogen source to study the maximum activity of alkaline protease. The maximum B

6 activity was observed in M2 (4.41±0.66U/ml), M1 (4.66±0.66U/ml), M1 (4.56±0.66U/ml), M4 (4.01±0.66U/ml) and M4 (4.37±0.66U/ml) in gelatin, ammonium chloride, urea, ammonium nitrate and sodium nitrate as a nitrogen source respectively. Among these highest activity was in ammonium chloride in M1 (4.56±0.66U/ml) media as shown in Graph 5. Graph 5 Optimization of alkaline protease production from E. coli in different nitrogen source (a) Maximum yield was observed in M1 viz. ammonium chloride (b) Minimum yield in M4 viz. ammonium nitrate. All the eight different fermentation media inoculated with E. coli were optimized by different metal ions to study the maximum activity of alkaline protease. The maximum activity was observed in M7 (1.28±0.22U/ml), M8 (0.98±0.22U/ml), M8 (0.83±0.22U/ml), M8 (0.65±0.22U/ml) and M8 (0.43±0.22U/ml) in sodium chloride, potassium chloride, calcium chloride, magnesium chloride, and zinc chloride as metal ion source respectively.among these highest activity was in sodium chloride in M7 (1.28±0.22U/ml) media as shown in Graph 6. Graph 6 Optimization of alkaline protease production from E. coli in different nitrogen source (a) Maximum yield was observed in M7 viz. sodium chloride (b) Minimum yield in M8 viz. zinc chloride. B

7 The effect of inhibitors was studied on all the eight different fermentation media inoculated with E. coli to study the maximum inhibitory effect on alkaline protease production. The maximum inhibition was observed in M7 (0.33U/ml), M1 (0.18U/ml), M7 (0.19U/ml), M5&M8 (0.24U/ml) and M1&4 (0.16U/ml) in copper sulphate, EDTA, SDS, ferric nitrate and β-mercapto-ethanol as protease inhibitors respectively. Among these highest inhibition was in copper sulphate in M7 (0.33U/ml) media as shown in Graph 7. Graph 7 Effect of inhibitors on alkaline protease production from E. coli (copper sulphate, EDTA, SDS, ferric nitrate and β-mercaptoethanol) (a) Maximum inhibition was observed in M7 viz. copper sulphate (b) Minimum inhibition in M5 viz. β-mercaptoethanol. CONCLUSION In the present study E. coli was used for the production of alkaline protease in eight different fermentation media. This bacterium was isolated from soil sample and screened for protease production. The enzyme production from E. coli was estimated using casein as substrate and its molecular weight was determined by SDS-PAGE which was found to be 42kd. The enzyme activity was also studied at different parameters like ph (maximum activity at ph9 in M7 i.e., 6.38±0.46u/ml), temperature (maximum activity at 60oC in M2 i.e., 15.73±0.66u/ml), carbon source (maximum activity in fructose in M4 i.e., 5.56±0.69u/ml), nitrogen source (maximum activity in ammonium chloride in M1 i.e., 4.56±0.66u/ml), metal ions concentration (maximum activity in sodium chloride in M7 i.e., 1.28±0.22u/ml) and effect of inhibitors (maximum inhibition in copper sulphate in M7 i.e., 0.33u/ml). ACKNOWLEDGEMENT I am highly obliged and thankful to my guide Dr. O. P. Verma under whose constant sharp and creative guidance, I have completed my thesis. I really admire him for his gravity and maturity and in giving out his hoard of cognizance.i wish to express my deep sense of gratitude and sincere thanks to Er. Sujeet Kumar Singh, Director (Admin) of CytoGene Research & Development for providing me with all necessary requirements during my thesis and for his kind support. B

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