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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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Chen, Shuo, Huang, Ziliang, Wu, Jingjun, Chen, Yin, Ye, Fengchun, Zhang, Chong, Yatsunami, Rie, Nakamura, Satoshi and Xing, Xin-Hui (2012) 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, Volume 97 (Issue 7). pp. 2907-2916. doi:10.1007/s00253-012-4145-6 ISSN 0175-7598.
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Official URL: http://dx.doi.org/10.1007/s00253-012-4145-6
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.
Item Type: | Journal Article | ||||
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Divisions: | Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- ) | ||||
Journal or Publication Title: | Applied Microbiology and Biotechnology | ||||
Publisher: | Springer | ||||
ISSN: | 0175-7598 | ||||
Official Date: | April 2012 | ||||
Dates: |
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Volume: | Volume 97 | ||||
Number: | Issue 7 | ||||
Page Range: | pp. 2907-2916 | ||||
DOI: | 10.1007/s00253-012-4145-6 | ||||
Status: | Peer Reviewed | ||||
Publication Status: | Published | ||||
Access rights to Published version: | Restricted or Subscription Access |
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