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 CPCR2's 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 spectru

Recent advances in whole cell biocatalysis techniques bridging from investigative to industrial scale

Recent advances in biocatalysis have strongly boosted its recognition as a valuable addition to traditional chemical synthesis routes. As for any catalytic process, catalyst's costs and stabilities are of highest relevance for the economic application in chemical manufacturing. Employing biocatalysts as whole cells circumvents the need of cell lysis and enzyme purification and hence strongly cuts on cost. At the same time, residual cell wall components can shield the entrapped enzyme from potentially harmful surroundings and aid to enable applications far from natural enzymatic environments. Further advantages are the close proximity of reactants and catalysts as well as the inherent presence of expensive cofactors. Here, we review and comment on benefits and recent advances in whole cell biocatalysis

An Enzymatic 2‐Step Cofactor and Co‐Product Recycling Cascade towards a Chiral 1,2‐Diol. Part II: Catalytically Active Inclusion Bodies

Optimal performance of multi‐step enzymatic one‐pot cascades requires a facile balance between enzymatic activity and stability of multiple enzymes under the employed reaction conditions. We here describe the optimization of an exemplary two‐step one‐pot recycling cascade utilizing the thiamine diphosphate (ThDP)‐dependent benzaldehyde lyase from Pseudomonas fluorescens (PfBAL) and the alcohol dehydrogenase from Ralstonia sp. (RADH) for the production of the vicinal 1,2‐diol (1R,2R)‐1‐phenylpropane‐1,2‐diol (PPD) using both enzymes as catalytically active inclusion bodies (CatIBs). PfBAL is hereby used to convert benzaldehyde and acetalydehyde to (R)‐2‐hydroxy‐1‐phenylpropanone (HPP), which is subsequently converted to PPD. For recycling of the nicotinamide cofactor of the RADH, benzyl alcohol is employed as co‐substrate, which is oxidized by RADH to benzaldehyde, establishing a recycling cascade. In particular the application of the RADH, required for both the reduction of HPP and the oxidation of benzyl alcohol in the recycling cascade is challenging, since the enzyme shows deviating pH optima for reduction (pH 6–10) and oxidation (pH 10.5), while both enzymes show only low stability at pH>8. This inherent stability problem hampers the application of soluble enzymes and was here successfully addressed by employing CatIBs of PfBAL and RADH, either as single, independently mixed CatIBs, or as co‐immobilizates (Co‐CatIBs). Single CatIBs, as well as the Co‐CatIBs showed improved stability compared to the soluble, purified enzymes. After optimization of the reaction pH, the RADH/PfBAL ratio and the co‐solvent content, we could demonstrate that almost full conversion (>90%) was possible with CatIBs, while under the same conditions the soluble enzymes yielded at most >50% conversion. Our study thus provides convincing evidence that (Co‐)CatIB‐immobilizates can be used efficiently for the realization of cascade reactions, i. e. under conditions where enzyme stability is a limiting issue