Online monitoring of biocatalytic 2-hydroxy ketone synthesis under ambient and high pressure

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This study focuses on the application of benzoylformate decarboxylase (BFD) from Pseudomonas putida for the asymmetric synthesis of 2-hydroxy ketones, key building blocks in pharmaceuticals. The research emphasizes the establishment of an online monitoring technique for direct process analysis, reducing sample preparation time and providing extensive concentration data for kinetic modeling. The synthesis of 2-hydroxy ketones, catalyzed by wild-type BFD and its mutants, was monitored using infrared and 2D-fluorescence spectroscopy, with chemometric analysis applied to manage the multivariate data. FT–MIR measurements yielded reproducible results with low RMSEP values, while 2D-fluorescence spectroscopy successfully tracked the synthesis of 2-hydroxy ketones and by-products for the first time. Reaction components were predicted with low RMSEP values, and the online data facilitated the estimation of kinetic parameters, including two KM values for benzaldehyde. Five kinetic parameters were determined with low standard deviations across different BFD variants. Additionally, fluorescence spectroscopy was evaluated for online reaction analysis under high pressure, providing insights into enzyme characteristics and biotransformation mechanisms, despite FT–MIR being unsuitable at pressures above 30 MPa. The study concluded with 2D-fluorescence monitoring of BFD-catalyzed reactions at 100 MPa, demonstrating the technique's sensitivity