ABSTRACT
Aims: The research was done to study the conditions enhancing catalytic activities of alkaline proteases from Vibro sp., Lactobacillus brevis, Zymomonas sp., Athrobacter sp., Corynebacterium sp. and Bacillus subtilis. Methodology and Results: The proteolytic enzymes were purified in 2-step procedures involving ammonium sulphate precipitation and sephadex G-150 gel permeation chromatography. The upper and lower limits for the specific activities of proteases from the selected microorganisms were estimated at 20.63 and 47.51 units/mg protein with Zymomonas protease having the highest specific activity towards casein as its substrate and purification fold of 3.46, while that of Lactobacillus brevis protease was 8.06. The native molecular weights of these active proteins ranged from 30.4 to 45.7 kDa with Athrobacter sp. protease having the highest weight for its subunits. The proteolytic enzymes had optimum pH range of 8 to 10 and temperature range of 50 to 62 ºC accounting for the percentage relative activity range of 75 to 94% and 71 to 84 % respectively. The activities of Lactobacillus brevis and Bacillus subtilis proteases were maximum at pH 9 and 10 respectively. Lactobacillus brevis protease activity was maximum at temperature of 62 ºC, while beyond this value, a general thermal instability of these active proteins was observed. At above 70 ºC, the catalytic activities of Corynebacterium sp., Vibrio sp., Zymomonas sp. and Arthrobacter sp. proteases were progressively reduced over a period of 120 min of incubation, while Bacillus subtlis and Lactobacillus brevis proteases were relatively stable. Effect of metal ions was investigated on the catalytic activity of protease from the microorganisms. Lactobacillus brevis, Zymomonas sp., Arthrobacter sp., Corynebacterium sp. and Bacillus subtilis protease activities were strongly activated by metal ions such as Ca+2 and Mg+2. Enzyme activities were inhibited strongly by Cu2+ and Hg2+ but were not inhibited by ethylene diamine tetra acetic acid (EDTA), while a slight inhibition was observed with K+, Na+ and Fe2+. Conclusion, significance and impact of study: The outcome of this present study indicated useful physico-chemical properties of proteolytic enzymes that could be of biotechnological use in enhancing enzyme catalytic efficiency.
ABSTRACT
Aims: The research was done to study the partial purification and characterization of thermostable alkaline protease from Lactobacillus brevis. Methodology and Results: The enzyme was purified in a two-step procedure involving ammonium sulphate precipitation and Sephadex G-150 gel permeation chromatography. The protease was purified 8.04 fold with a yield of approximately 30% after purification with Sephadex G-150 column. It has a relative molecular weight of 33.2 kDa and optimally active at a temperature of 60 oC and pH 9.0. The maximum velocity Vmax and Michaelis constant Km of the protease produced during the hydrolysis of casein were 66.66 U/mg protein and 3.33 mg/ml. It was strongly activated by Ca2+ and ethylene diamine tetra acetic acid (EDTA), mildly inhibited by Na+, K+, Mg2+ and Fe2+ and strongly inhibited by Cu2+ and Hg2+. The ability of the enzyme to improve the cleansing power of various detergents was also studied. Conclusion, significance and impact of study: The findings in this study suggest that the protease is a suitable candidate for detergent formulation and biotechnological applications.