Study on Some Properties of Calcium-dependent α-Amylase from Bacillus subtilis through Submerged Fermentation of Wheat Bran

M. Irfan, M. Nadeem, Q. Syed, H. Abdullah Shakir and J. Iqbal Qazi

Abstract
This study reveals the production of α-amylase through Bacillus subtilis by submerged fermentation of wheat bran as a substrate. Various conditions were optimized and maximum production was observed at initial medium pH of 7.0, incubation temperature of 35 °C with agitation speed of 140 rpm for 48 h of fermentation period. Peptone, ammonium sulphate and soluble starch favored enzyme production as nitrogen and carbon source, respectively. The crude α-amylase was purified 2.03-fold by column (sephadex G-100) chromatography having specific activity of 480.0 U mg–1. The purified enzyme had molecular mass of 54.18 kDa as determined by 12 % SDS-PAGE. The optimum pH and temperature of the enzyme was 8.5 and 60 °C, respectively. Ca2+ played an important role in the stability of the enzyme retaining relative activity of 87 % and 67 % at pH 9.5 and 80 °C, revealing the Ca2+ dependency respectively. The enzyme was highly stable towards surfactants and oxidizing agents. Kinetic study indicated that the enzyme exhibit Km and Vmax value of 4.4 mg mL–1 and 714.2 U mL–1 using soluble starch as a substrate respectively.

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Keywords
α-amylase, purification, characterization, Bacillus sp.