A total of 28,000 bacterial isolates were encountered during 2008-2011 from north eastern region of India, which is falling under Indo-Burma biodiversity hot-spots and were preserved in glycerol stock solution at -80°C in deep freezer. 19,834 isolates were biochemically characterized and screened for protease activity in culture conditions further 1,579 isolates were found to be good amount of protease producing bacteria where, the zone of inhibition was observed in the range between 20-45 mm on skim milk agar medium at different pH range i.e. 4.0, 7.0 and 10.0. Out of 1,579 protease producing bacteria, 425 isolates produced protease activity at acidic pH-4.0, 547 bacteria at alkaline pH-10.0 and 607 bacteria at neutral pH-7.0. Compared to acidic and neutral pH, the highest proteolytic activity was exhibited by the isolates which were grown in alkaline medium. In this study, 5 isolates forming larger zone as a result of casein hydrolysis were further studied for quantitative production of extracellular alkaline protease activity. Isolate no. B-2 was observed as a highest potential protease producer bacterium. Upon 16S rRNA analysis, it displayed maximum similarity with Bacillus sp. and the sequences were deposited in GenBank database. Different cultural parameters like effect of pH, temperature, time and inoculum sizes were optimized for maximal enzyme production. Maximum yield of enzyme was obtained at a pH of 10.0 with 1 ml of inoculum in the medium after 48 hours of incubation and maintained at a temperature of 37ºC. The present investigation indicates the potential use of these microorganisms as biotechnological tools for various industrial activities.
Key words: Alkaline proteases, Bacillus sp., 16S rRNA gene sequence, proteolytic activity, optimization
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