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Biochemical analysis and kinetic modeling of the thermal inactivation of MBP‐fused heparinase I: Implications for a comprehensive thermostabilization strategy
Author(s) -
Chen Shuo,
Ye Fengchun,
Chen Yang,
Chen Yu,
Zhao Hongxin,
Yatsunami Rie,
Nakamura Satoshi,
Arisaka Fumio,
Xing XinHui
Publication year - 2011
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.23144
Subject(s) - thermostability , chemistry , maltose binding protein , heparin , enzyme , biophysics , biochemistry , kinetics , recombinant dna , combinatorial chemistry , chromatography , fusion protein , biology , gene , physics , quantum mechanics
Enzymatic degradation of heparin by heparin lyases has not only largely facilitated heparin structural analysis and contamination detection, but also showed great potential to be a green and cost‐effective way to produce low molecular weight heparin (LMWH). However, the commercial use of heparinase I (HepI), one of the most studied heparin lyases, has been largely hampered by its low productivity and extremely poor thermostability. Here we report the thermal inactivation mechanism and strategic thermal stabilization of maltose‐binding protein (MBP)‐HepI, a fusion HepI produced in E. coli with high yield, solubility and activity. Biochemical studies demonstrated that the thermal inactivation of MBP‐HepI involves an unfolding step that is temperature‐dependently reversible, followed by an irreversible dimerization step induced by intermolecular disulfide bonds. A good consistency between the kinetic modeling and experimental data of the inactivation was obtained within a wide range of temperature and enzyme concentration, confirming the adequacy of the proposed inactivation model. Based on the inactivation mechanism, a comprehensive strategy was proposed for the thermal stabilization of MBP‐HepI, in which Ca 2+ and Tween 80 were used to inhibit unfolding while site mutation at Cys297 and DTT were employed to suppress dimerization. The engineered enzyme exhibits remarkably improved storage and operational thermostability, for example, 16‐fold increase in half‐life at its optimum temperature of 30°C and 8‐fold increase in remaining activity of 95% after 1‐week storage at 4°C, and therefore shows great potential as a commercial biocatalyst for heparin degradation in the pharmaceutical industry. Biotechnol. Bioeng. 2011; 108:1841–1851. © 2011 Wiley Periodicals, Inc.