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

Abstract

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, NaNO 3; 10.0, K 2HPO 4; 5.0, MgSO 4.7H 2O; 0.3, CaCl 2.2H 2O; 0.2, NaCl; 5.0 and Na 2CO 3; 10.0 at pH 10.0. The cultures were incubated at 55 °C 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 NaNO 3 (12780) and KNO 3 were found best nitrogen sources. The optimum temperature and pH for protease activity was 70 °C and 11.0, respectively. The addition of 10 mM Ca 2+ enhanced the optimum temperature 80 °C and retained 78% activity even after 1 h heat treatment at 80 °C. Proteolytic activity was completely inhibited by 1 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+, Mg 2+, Cu 2+ and Co 2+ and activity also inhibited in the presence of 10 mM metal ions, such as Fe 3+, Hg 2+ and Zn 2+. The enzyme was stable in the presence of 5% H 2O 2.