Conjugation of Agrobacterium radiobacter epoxide hydrolase with ficoll: Catalytic, kinetic and thermodynamic analysis
Epoxide hydrolase-mediated biocatalysis has a great prospective in the biosynthesis of optically pure epoxides. The present work targets toward the thermo-stabilization of epoxide hydrolase by covalent conjugation with polysaccharide. An epoxide hydrolase from Agrobacterium radiobacter (ArEH) was modified by covalent coupling to seven oxidized polysaccharides with different chemical structures and characteristics. Among all conjugates, ArEH with ficoll exhibited both the highest specific activity (494 U/mg) and half-life at 60 °C ( t 1/2 = 183 min). The conjugated enzyme also displayed wider optimum pH and temperature ranges, higher catalytic number ( k cat ), and catalytic efficiency ( k cat / K m ) as compared to its native counterpart. The conjugation significantly decreased the enthalpy of activation (Δ H *), free energy of transition state binding (Δ G * E − T ), and free energy of activation (Δ G *) for epichlorohydrin hydrolysis. Moreover, the conjugated ArEH showed enhanced thermal stability as evidenced by its longer half-life ( t 1/2 ), lower thermal deactivation constant ( k d ), and higher D values at 50–70 °C. Furthermore, the enzymatic activity of conjugated ArEH elevated significantly by Ca 2+ and it showed increased tolerance against Co 2+ , Fe 3+ and EDTA inhibitors. Finally, fluorescence and circular dichroism spectroscopic analysis were performed to confirm the noticeable conformational changes in ArEH structure after conjugation with ficoll, which could be responsible for the observed catalytic and stability enhancement.
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