Extracellular Overexpression of Chitosanase from Bacillus sp TS in Escherichia coli

The chitosanase gene from a Bacillus sp. strain isolated from soil in East China was cloned and expressed in Escherichia coli. The gene had 1224 nucleotides and encoded a mature protein of 407 amino acid residues. The optimum pH and temperature of the purified recombinant chitosanase were 5.0 and 60 A degrees C, respectively, and the enzyme was stable below 40 A degrees C. The K (m), V (max), and specific activity of the enzyme were 1.19 mg mL(-1), 674.71 mu mol min(-1) at 50 A degrees C, and 555.3 U mg(-1), respectively. Mn2+ was an activator of the recombinant chitosanase, while Co2+ was an inhibitor. Hg2+ and Cu2+ inhibited the enzyme at 1 mM. The highest level of enzyme activity (186 U mL(-1)) was achieved in culture medium using high cell-density cultivation in a 7-L fermenter. The main products of chitosan hydrolyzed by recombinant chitosanase were (GlcN)(3-6). The chitosanases was successfully secreted to the culture media through the widely used SecB-dependent type II pathway in E. coli. The high yield of the extracellular overexpression, relevant thermostability, and effective hydrolysis of commercial grade chitosan showed that this recombinant enzyme had a great potential for industrial applications.