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 °C, respectively, and the enzyme was stable below 40 °C. The K m, V max, and specific activity of the enzyme were 1.19 mg mL–1, 674.71 μmol min–1 at 50 °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.