OPTIMIZATION, PRODUCTION, PARTIAL PURIFICATION AND CHARACTERIZATION OF NEUTRAL AND ALKALINE PROTEASES PRODUCED BY BACILLUS SUBTILIS

Abstract

Different strains of Bacillus subtilis (Ehrenberg) Cohn, BIOTECH 1056, 1333, 1573 and 1679, UPCC 1295 and USTCMS 1011 from various culture collections in the country were characterized morphologically, biochemically, and assayed for proteolytic activity. Fermentation parameters like pH, temperature, and incubation time were optimized for the production of proteases that were used as gauges to select the best bacterial isolates for possible commercial application. Among the six isolates studied, USTCMS 1011 gave the highest neutral protease activity of 0.647 U/mg protein at pH 7 and at 37oC for72 hours. The same strain yielded the highest alkaline protease activity of 0.495 U/mg protein at pH 9 and at 30oC for 72 hours. Various beans and fruit extracts or honey were tested as possible protein or carbohydrate sources in the medium. Dextrin produced the highest protease activity with 0.647U/mg protein for neutral protease and 0.495 U/mg protein for alkaline protease as carbohydrate source. Garbanzos (chickpea) as protein source, supplemented with yeast extract, gave the highest protease activity at 0.548 U/mg protein for neutral protease and 0.475 U/mg protein for alkaline protease. Both crude proteases were purified by ammonium sulfate precipitation followed by desalting and gel filtration chromatography. Optimal activity of the neutral protease was found at pH 8 and 30oC with an incubation time of 90min while optimal activity of the alkaline protease was at pH 11 and 40oC with only 30min incubation period. Neutral protease showed a single band with molecular weight of 65.4kDa in SDS-PAGE while alkaline protease showed single band with molecular weight of 8kDa.