Andre Jakoblinnert

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 CPCR2s 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).

Who's Who? Allocation of Carbonyl Reductase Isoenzymes from Candida parapsilosis by Combining Bio- and Computational Chemistry

and high stereoselectivity, it is characterized by high stability in the presence of low-molecular-weight alcohols, such as isopropanol, and a preference for NADH as cofactor rather than the more expensive and less stable NADPH associated with many other alcohol dehydrogenases. As a result, regeneration of the essential cofactor can be achieved either by providing isopropanol as co-substrate b, 3] or by using established co-catalysts such as formate dehydrogenase (FDH; 1.2.1.2) from Candida boidinii. All these features imply a special utility of CPCR for organic synthesis. Nevertheless, even today, the application of CPCR in research or industrial production is limited. This can probably be explained by the uncertainties regarding the biocatalyst’s identity and thus its precise and reproducible synthetic use: Two preparations are currently commercially available, they are isolated either from the native production strain C. parapsilosis (Codexis, USA), or from Escherichia coli as recombinant host organism (X-zyme, Germany). However, the preparations reveal clear differences in their biochemical features and do not exactly coincide with the data defining the original target enzyme. 5] This study describes the elucidation of the molecular identity of CPCR isoenzymes by the interdisciplinary approach of combining classical biochemical methods and methods from computational chemistry. It involves a stepwise identification of two possible enzyme candidates with their amino acid sequences, the establishment of active-site molecular models with subsequent application to the prediction of substrate binding patterns and a final discrimination of enzymes by experimental investigation of their activity towards selected indicator substrates.

Reengineered carbonyl reductase for reducing methyl-substituted cyclohexanones

The carbonyl reductase from Candida parapsilosis (CPCR2) is a versatile biocatalyst for the production of optically pure alcohols from ketones. Prochiral ketones like 2-methyl cyclohexanone are, however, only poorly accepted, despite CPCR2s large substrate spectrum. The substrate spectrum of CPCR2 was investigated by selecting five amino positions (55, 92, 118, 119 and 262) and exploring them by single site-saturation mutagenesis. Screening of CPCR2 libraries with poor (14 compounds) and well-accepted (2 compounds) substrates showed that only position 55 and position 119 showed an influence on activity. Saturation of positions 92, 118 and 262 yielded only wild-type sequences for the two well-accepted substrates and no variant converted one of the 14 other compounds. Only the variant (L119M) showed a significantly improved activity (7-fold on 2-methyl cyclohexanone; vmax = 33.6 U/mg, Km = 9.7 mmol/l). The L119M substitution exhibited also significantly increased activity toward reduction of 3-methyl (>2-fold), 4-methyl (>5-fold) and non-substituted cyclohexanone (>4-fold). After docking 2-methyl cyclohexanone into the substrate-binding pocket of a CPCR2 homology model, we hypothesized that the flexible side chain of M119 provides more space for 2-methyl cyclohexanone than branched L119. This report represents the first study on CPCR2 engineering and provides first insights how to redesign CPCR2 toward a broadened substrate spectrum.