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Combination of site-directed mutagenesis and calcium ion addition for enhanced production of thermostable MBP-fused heparinase I in recombinant Escherichia coli

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Abstract

Heparinase I (HepI), which specifically cleaves heparin and heparan sulfate, is one of the most extensively studied glycosaminoglycan lyases. Low productivity of HepI has largely hindered its industrial and pharmaceutical applications. Loss of bacterial HepI enzyme activity through poor thermostability during its expression and purification process in production can be an important issue. In this study, using a thermostabilization strategy combining site-directed mutagenesis and calcium ion addition during its production markedly improved the yield of maltose-binding protein-fused HepI (MBP–HepI) from recombinant Escherichia coli. Substitution of Cys297 to serine in MBP–HepI offered a 30.6 % increase in the recovered total enzyme activity due to a mutation-induced thermostabilizing effect. Furthermore, upon addition of Ca2+ as a stabilizer at optimized concentrations throughout its expression, extraction, and purification process, purified mutant MBP–HepI showed a specific activity of 56.3 IU/mg, 206 % higher than that of the wild type obtained without Ca2+ addition, along with a 177 % increase in the recovered total enzyme activity. The enzyme obtained through this novel approach also exhibited significantly enhanced thermostability, as indicated by both experimental data and the kinetic modeling. High-yield production of thermostable MBP–HepI using the present system will facilitate its applications in laboratory-scale heparin analysis as well as industrial-scale production of low molecular weight heparin as an improved anticoagulant substitute.

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (Nos. 20836004 and 20676071). Shuo Chen was supported by the Tokyo Tech–Tsinghua University Joint Graduate Program.

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Author notes

  1. Shuo Chen

    Present address: Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan

  2. Yin Chen

    Present address: School of Life Sciences, University of Warwick, Coventry, CV4 7AL, United Kingdom

Authors and Affiliations

  1. Department of Chemical Engineering, Tsinghua University, Beijing, 100084, China

    Shuo Chen, Ziliang Huang, Jingjun Wu, Yin Chen, Fengchun Ye, Chong Zhang & Xin-Hui Xing

  2. Department of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, 226-8501, Japan

    Shuo Chen, Rie Yatsunami & Satoshi Nakamura

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  1. Shuo Chen
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Correspondence to Xin-Hui Xing.

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Standard curve of OD600 vs. CFU/ml of E. coli TB1 [pMHS] strain cultivated in the M9-based medium (PDF 25.5 kb)

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Chen, S., Huang, Z., Wu, J. et al. Combination of site-directed mutagenesis and calcium ion addition for enhanced production of thermostable MBP-fused heparinase I in recombinant Escherichia coli . Appl Microbiol Biotechnol 97, 2907–2916 (2013). https://doi.org/10.1007/s00253-012-4145-6

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  • DOI: https://doi.org/10.1007/s00253-012-4145-6

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