Biochemical analysis and kinetic modeling of the thermal inactivation of MBP‐fused heparinase I: Implications for a comprehensive thermostabilization strategy

Abstract

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.

Structure-based engineering of heparinase I with improved specific activity for degrading heparin

“…Other residues in Ph-HepI, which also interacted with the substrate, shared no obvious counterparts in the template. The presence of a Ca 2+ ion is crucial for heparinase I activity [30, 31]. From molecular docking, a Ca 2+ binding site was also found in the active pocket of Ph-HepI.…”

Foreword to ‘Multiscale structural biology: biophysical principles and mechanisms underlying the action of bio-nanomachines’, a special issue in Honour of Fumio Arisaka’s 70th birthday

“…Within the phage-related area work on bacterial homologs of phage proteins (Uchida et al 2014), mechanistic action of the gp5 trimer in the cell puncturing device (Nishima et al 2011), and determination of the molecular architecture of the T4 phage neck region (Fokine et al 2013) (all reviewed in Arisaka and Kanamaru 2013). Further to this, being a Professor did not slow down Fumio's own experimental output, with his engagement in a range of collaborative projects on topics as diverse as receptor biology (Mio et al 2010), assembly of nanotechnology components (Yokoi et al 2010;Inaba et al 2012;Sanghamitra et al 2014), hemoglobin allostery (Arisaka et al 2011), crop protein analysis (Wadahama et al 2012;Sato et al 2015;Yamniuk et al 2015), autophagy (Araki et al 2013, amyloid analysis (Zhao et al 2016;Hall et al 2016), biopharmaceutical development (Iwura et al 2014a(Iwura et al , 2014b, AUC methodological development (Zhao et al 2015) and thermostable enzymes (Tamakoshi et al 2011;Chen et al 2011;Ozawa et al 2012) among many others (Huq et al 2010;Yamasaki et al 2010;Kanazawa et al 2010;Sawada et al 2011;Akita et al 2012;Nomura et al 2012;Ishii et al 2012;Nozawa et al 2013;Seio et al 2013;Takei et al 2014;Kumazaki et al 2014;Owa et al 2014;Iwura et al 2014aIwura et al ,...…”

Enzyme activity enhancement of chondroitinase ABC I from Proteus vulgaris by site-directed mutagenesis

“…Protein concentration was detected by Bradford Protein Assay Kit (Bio-rad). One international unit was dened as the amount of protein that could form 1 mmol L À1 4,5-unsaturated uronic acid per minute at 37 C. 28…”