Design of an activity and stability improved carbonyl reductase from Candida parapsilosis

The carbonyl reductase from Candida parapsilosis (CPCR2) is an industrially attractive biocatalyst for producing chiral alcohols from ketones. The homodimeric enzyme has a broad substrate spectrum and an excellent stereoselectivity, but is rapidly inactivated at aqueous–organic interfaces. The latter limits CPCR2's application in biphasic reaction media.Reengineering the protein surface of CPCR2 yielded a variant CPCR2-(A275N, L276Q) with 1.5-fold increased activity, 1.5-fold higher interfacial stability (cyclohexane/buffer system), and increased thermal resistance (ΔT50 = +2.7 °C). Site-directed and site-saturation mutagenesis studies discovered that position 275 mainly influences stability and position 276 governs activity. After single site-saturation of position 275, amino acid exchanges to asparagine and threonine were discovered to be stabilizing. Interestingly, both positions are located at the dimer interface and close to the active site and computational analysis identified an inter-subunit hydrogen bond formation at position 275 to be responsible for stabilization.Finally, the variant CPCR2-(A275S, L276Q) was found by simultaneous site-saturation of positions 275 and 276. CPCR2-(A275S, L276Q) has compared to wtCPCR2 a 1.4-fold increased activity, a 1.5-fold higher interfacial stability, and improved thermal resistance (ΔT50 = +5.2 °C).

► Two mutational hot spots were identified in a carbonyl reductase: position 275 (stability); position 276 (activity).
► The variant A275T establishes an inter-subunit hydrogen bond explaining stabilization.
► Variants at position 276 confer up to twofold increased activity due to interaction with the substrate in the active site of the other monomer.
► Double mutants of 275/276 are even more stable.