Fluorescence spectroscopy as a novel method for on-line analysis of biocatalytic C–C bond formations

On-line analysis of bioprocesses is of increasing interest avoiding the time delay for off-line sample preparation and the following analyses via chromatographic methods. Moreover, continuous monitoring of the reaction components during chemo- or biocatalytic transformations provides a direct control of the process. Since productivity of the processes can be controlled simultaneously, on-line monitoring of the processes is attractive for industrial applications. The reliable in situ monitoring of biocatalyzed reactions has been a challenge where reactions run in aqueous solutions. Limited work has been published on the use of spectroscopic methods for on-line analysis of biocatalytic reactions up to now. However, in this communication two dimensional (2D)-fluorescence spectroscopy has been proved to be an effective tool for on-line monitoring of the carboligation reactions catalyzed by wild type benzoylformate decarboxylase (BFD) from Pseudomonas putida. BFD is a thiamine diphosphate (ThDP)-dependent enzyme that catalyzes the asymmetric C–C bond formation to (S)-2-hydroxypropiophenone ((S)-2-HPP) starting from benzaldehyde and acetaldehyde. The analysis of the fluorescence spectra was achieved by chemometric modeling performing principle component analysis (PCA) and partial least square (PLS) regression. The derived chemometric models were used for the validation of concentrations of yielded 2-HPP and the substrate benzaldehyde with low root mean square error of calibration (RMSEC).