Carbonyl reductase of Candida parapsilosis – Stability analysis and stabilization strategy

Abstract

The homodimeric Candida parapsilosis carbonyl reductase 2 (CPCR2) is an industrially attractive biocatalyst due to its broad substrate range and high stereoselectivity. In addition, CPCR2 is reasonably stable in monophasic organic solvents (e.g. alcohols) but apparently instable in biphasic organic systems. Hence, we conducted first a thorough quantitative inactivation study of CPCR2, using both wild-type and stability improved variants, in an attempt to identify critical factors influencing the enzyme stability. Possible inactivation phenomena including oxidation, shear forces, dissociation and adsorption at interfaces were assessed on a microliter scale using quantitative kinetic assays. Our results demonstrate that interface interactions and dimer dissociation are the main reasons for inactivation of CPCR2. Shear forces seems to enhance these inactivation processes whereas oxidation plays no role in CPCR2 inactivation. Secondly, an attempt was made to find suitable stabilization strategies to utilize CPCR2 in various reaction systems. To minimize the inactivation, bovine serum albumin was used as traditional blocking and crowding agent. The residual activity of the wild-type was successfully increased up to 2.5-fold by addition of 1μgmL−1 bovine serum albumin. To avoid dimer dissociation the cofactor concentration was successively increased. The residual activity was successfully enhanced up to 5-fold, 3-fold and 1.5-fold for the wild-type, single and double mutant, respectively. Further, recently gained data from the enzyme crystal structure were used to interpret the effects of stabilization. We propose conformational change of a flexible region in CPCR2 upon binding of the cofactor leading to internal stabilization of the enzyme. In conclusion, we propose the addition of bovine serum albumin and the cofactor NADH as a suitable stabilization strategy to utilize CPCR2 in various reaction systems.