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

In this work, benzoylformate decarboxylase (BFD, EC 4.1.1.7) from Pseudomonas putida has been applied for the asymmetric synthesis of 2-hydroxy ketones which are important building blocks for pharmaceutical products. Thereby, this present study mainly focussed on the establishment of a suitable online monitoring technique to enable direct process analysis in order to provide reduced time delay for sample preparation and as well as a huge concentration data basis for subsequent evaluation such as kinetic modelling. In view of this, the synthesis of 2-hydroxy ketones catalysed by wtBFD as well as mutants thereof (BFD A460I, BFD F464I and BFD A460I–F464I) was followed by infrared- and 2D-fluorescence spectroscopy. For both methods chemometrical analysis was applied to handle the huge multivariate data. The FT–MIR measurements gave reproducible results with low RMSEP values (2-HPP 1.5 mM (2.9%)). 2D-Fluorescence spectroscopy was applied for monitoring carboligations yielding 2-hydroxy ketones for the first time and it was shown that the synthesis of 2-HPP (main product) and benzoin (by-product) as well as the conversion of benzaldehyde (substrate) could successfully be monitored. Reaction components were predicted with low RMSEP values (e. g. 2-HPP 0.57 mM (1.52%), BZ 0.01 mM (2.11%), BA 0.49 mM (1.31%)). The obtained online data were further used for estimation of kinetic parameters of the proposed kinetic model including two KM values for benzaldehyde as a donor and as an acceptor aldehyde. Thus, in total five kinetic parameters were successfully determined with low standard deviations for wtBFD (e. g. Vmax,2-HPP = 42.7 ± 2.6 U2-HPP/mg), BFD A460I–F464I (e. g. Vmax,2-HPP = 28.7 ± 1.0 U2-HPP/mg) and BFD F464I (e. g. Vmax,2-HPP = 14.1 ± 2.7 U2-HPP/mg) catalysis. Furthermore, fluorescence spectroscopy was evaluated as technique for online reaction analysis under high pressure conditions, which can give new insights into the characteristics of enzymes and the postulated mechanism of biotransformations. Here, FT–MIR spectroscopy was not applicable since the stability of the ATR-probe was limited at a pressure of 30 MPa. Therefore, in the last part of this study, the 2D-fluorescence spectroscopy was applied for the monitoring of BFD-catalysed carboligations at 100 MPa. Although fluorescence is very sensitive and therefore resulted in high noises in the spectra; the general trend in the reaction progress could be monitored.