Studies on production of thermostable alkaline protease from thermophilic and alkaliphilic Bacillus sp JB-99 in a chemically defined medium

The thermophilic and alkaliphilic Bacillus sp. JB-99 was isolated from sugarcane molasses and was Cultured in 250 ml Erlenmeyer flasks containing 50 ml of synthetic medium consisting of (g/l): citric acid; 10.0, NaNO3; 10.0, K2HPO4,; 5.0 MgSO4.7H(2)O; 0.3, CaCl2.2H(2)O: 0.2, NaCl. 5.0 and Na2CO3; 10.0 at pH 10.0. The cultures were incubated at 55 C-circle with agitation (180 rpm) for 24 h. To study the effect of different carbon and nitrogen sources on enzyme yield (U/ml): citric acid (12780), soluble starch (12480), fructose (11760) and raffinose (11650) were found best carbon sources, while NaNO3 (12780) and KNO3 were found best nitrogen sources. The optimum temperature and pH for protease activity was 70 C-circle and 11.0, respectively. The addition of 10 mM Ca2+ enhanced the optimum temperature 80 degreesC and retained 78% activity even after I h heat treatment at 80 degreesC. Proteolytic activity was completely inhibited by I mM PMSF and TPCK showed that it seems to be trypsin like serine alkaline protease. The enzyme activity was enhanced in the presence of 10 mM metal ions namely Mn-2, Mg2+, Cu2- and Co2+ and activity also inhibited in the presence of 10 mM metal ions. such as Fe3+, Hg2+ and Zn2+. The enzyme was stable in the presence of 5% H2O2. (C) 2001 Elsevier Science Ltd. All rights reserved.