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Extracellular Overexpression of Chitosanase from Bacillus sp. TS in Escherichia coli

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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. 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.

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Acknowledgments

This study was supported, in part, by grants from the Hundred Talents Program of the Chinese Academy of Sciences (CAS), the National Natural Science Foundation of China (11250110508, 31350110507), the Knowledge Innovation Program of CAS (KSCX2-EW-G-8), and the Tianjin Municipal Science & Technology Commission (10ZCKFSY05600). JL is an Australian National Health and Medical Research Council (NHMRC) Senior Research Fellow. JS is an NHMRC Peter Doherty Fellow and recipient of the Hundred Talents Program of CAS. The authors also would like to thank the Technology Support Center of Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, for providing the instrument for MALDI-TOF and mass spectrometry (MS) analysis.

Competing Interests

The authors declare financial competing interests. The content of this manuscript was partially patented by Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences (Application Number 201410230852.X).

Authors’ Contributions

ZZ carried out the cloning, the purification of chitosanase, the heterologous expression, and the biochemical characterization of protein as well as drafted the manuscript. SZ and SW contributed to the experiments of the hydrolysis of chitosan by chitosanase. LS, JL, YM, and JS revised the manuscript. JS supervised the study. All authors have read and approved the final manuscript.

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Authors and Affiliations

  1. 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

    Zhanping Zhou, Xiaoman Li, Yanhe Ma & Jiangning Song

  2. Shandong Food & Fermentation Industry Research & Design Institute, Jinan, 250013, China

    Shuangzhi Zhao, Shouquan Wang & Li Su

  3. Shandong Provincial Key Laboratory of Food and Fermentation Engineering, Jinan, 250013, China

    Shuangzhi Zhao, Shouquan Wang & Li Su

  4. Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, 3052, Australia

    Jian Li

  5. Monash Bioinformatics Platform and Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, 3800, Australia

    Jiangning Song

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  1. Zhanping Zhou
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  2. Shuangzhi Zhao
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  5. Li Su
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Correspondence to Jiangning Song.

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Zhou, Z., Zhao, S., Wang, S. et al. Extracellular Overexpression of Chitosanase from Bacillus sp. TS in Escherichia coli . Appl Biochem Biotechnol 175, 3271–3286 (2015). https://doi.org/10.1007/s12010-015-1494-5

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  • DOI: https://doi.org/10.1007/s12010-015-1494-5

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