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

Applied Microbiology and Biotechnology
Shuo ChenXin-Hui Xing

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 thermostabil...Continue Reading

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Citations

Nov 20, 2013·Applied Microbiology and Biotechnology·Haiquan YangJian Chen
Aug 30, 2016·Journal of Biotechnology·Zhenya ChenQipeng Yuan
Jan 5, 2020·Preparative Biochemistry & Biotechnology·Cai-Yun LiuYe-Wang Zhang
Sep 23, 2018·Applied Microbiology and Biotechnology·Yi-Xin HuoZhenya Chen

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