Enhancing the Comprehensive Stability of Candida parapsilosis Carbonyl Reductase in Biphasic Systems through Rational Protein Design

The carbonyl reductase from Candida parapsilosis 7330 (CpCR) is a valuable biocatalyst for synthesizing chiral alcohols and pharmaceutical intermediates. However, its industrial application is hindered by poor stability in biphasic systems. To address this limitation, we heterogeneously expressed CpCR in E. coli BL21(DE3) and employed a rational design strategy. Using Discovery Studio software, we performed virtual saturation mutagenesis to predict mutations that would enhance overall protein stability. Four beneficial mutations were selected and combined, yielding the combinatorial mutant G262N/A98N. This variant exhibited significantly improved stability: in a methyl-tert-butyl ether/phosphate buffer (MTBE/PBS) biphasic system at 30°C, its half-life (t₁/₂) was 64 min, which is 2.48-fold longer than that of the wild-type (25.8 min). The G262N/A98N mutant also showed a 2-fold increase in shear stress stability. Furthermore, the single mutant G262N demonstrated enhanced oxidative stability (t₁/₂ = 11.87 h, a 1.4-fold improvement) and superior cofactor-mediated stabilization, achieving a maximum half-life of 22.4 h in the presence of 0.2 mM NADPH. Molecular dynamics simulations revealed that the G262N/A98N mutation resulted in a more compact overall protein spatial structure and the distance between Gly351 and Gly95 in the two α-helix loop regions decreased from the original 11.37 Å to 9.45 Å, an effect potentially attributed to the G262N mutation's proximity to the coenzyme-binding pocket and enhanced the binding force between the coenzyme NADPH and the CpCR enzyme. These findings underscore the critical role of residues A98 and G262 in enhancing the comprehensive stability of CpCR, providing a robust foundation for its industrial application in biphasic biocatalytic systems.