title: Combination of site-directed mutagenesis and calcium ion addition for enhanced production of thermostable MBP-fused heparinase I in recombinant Escherichia coli.
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.
authors.affiliation: Department of Chemical Engineering, Tsinghua University, Beijing, 100084, China.
authors.forename: Shuo
authors.initials: S
authors.lastname: Chen
authors.forename: Ziliang
authors.initials: Z
authors.lastname: Huang
authors.forename: Jingjun
authors.initials: J
authors.lastname: Wu
authors.forename: Yin
authors.initials: Y
authors.lastname: Chen
authors.forename: Fengchun
authors.initials: F
authors.lastname: Ye
authors.forename: Chong
authors.initials: C
authors.lastname: Zhang
authors.forename: Rie
authors.initials: R
authors.lastname: Yatsunami
authors.forename: Satoshi
authors.initials: S
authors.lastname: Nakamura
authors.forename: Xin-Hui
authors.initials: XH
authors.lastname: Xing
mesh_terms: D019943:Amino Acid Substitution; D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism; D003067:Coenzymes / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D019763:Heparin Lyase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
chemical_list: D002413:Cations, Divalent; D003067:Coenzymes; D011993:Recombinant Fusion Proteins; D019763:Heparin Lyase; D002118:Calcium