title: Extracellular overexpression of chitosanase from Bacillus sp. TS in Escherichia coli.
abstract: 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. Mn(2+) was an activator of the recombinant chitosanase, while Co(2+) was an inhibitor. Hg(2+) and Cu(2+) 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.
authors: National Engineering Laboratory for Industrial Enzymes and Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, Tianjin, 300308, China, zhou_zp@tib.cas.cn.
authors: Zhanping
authors: Z
authors: Zhou
authors: Shuangzhi
authors: S
authors: Zhao
authors: Shouquan
authors: S
authors: Wang
authors: Xiaoman
authors: X
authors: Li
authors: Li
authors: L
authors: Su
authors: Yanhe
authors: Y
authors: Ma
authors: Jian
authors: J
authors: Li
authors: Jiangning
authors: J
authors: Song
mesh_terms: D001407:Bacillus / Q000201:enzymology*; D002686:Chitin / Q000378:metabolism; D048271:Chitosan / Q000378:metabolism*; D003001:Cloning, Molecular; D003470:Culture Media; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005285:Fermentation; D015964:Gene Expression Regulation, Bacterial; D006026:Glycoside Hydrolases / Q000096:biosynthesis* / Q000235:genetics; D006868:Hydrolysis; D012988:Soil Microbiology; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D003470:Culture Media; D002686:Chitin; D048271:Chitosan; D006026:Glycoside Hydrolases; C019868:chitosanase