Effective Production of (S)-α-Hydroxy ketones: An Reaction Engineering Approach

Abstract

The use of enzyme engineering for optimisation of an enzymatic reaction can significantly improve lifetime and performance of the biocatalyst. Reaction optimisation is a second powerful tool to enhance yield and enantiomeric excess (ee) of a desired product. In combination, an effective toolbox for biocatalytic process development is given. Previous rational enzyme engineering of thiamine diphosphate-dependent enzymes provided access to unavailable (S)-hydroxy ketones like phenylpropionylcarbinol (PPC) by carboligation of inexpensive aldehydes, but ee was only moderate (89 %) and yields low. In this work, the strong impact of reaction parameters of both, the performance as well as the stereoselectivity of the asymmetric synthesis of PPC catalysed by an (S)-selective Acetobacter pasteurianus pyruvate decarboxylase variant has been assessed. By combining appropriate substrate choice, reaction-, and solvent engineering the ee of the product could be improved from 89 % up to 98 % (S)-PPC. Simultaneously, specific space–time-yield could be increased up to 61-fold, proving the immense impact of suitable reaction condition on both, catalysts productivity and selectivity. Especially avoidance of organic co-solvents, used as substrate solubility enhancers, as well as the exchange of the donor aldehyde by its corresponding α-keto acid, which is decarboxylated to the aldehyde prior to carboligation by the same enzyme, were essential for the enhancement. On the basis of this successful optimisation it could be further shown, that iterative multi-parametric reaction optimisation and especially the application of Pareto charts for visualisation are valuable tools for effective process optimisation.