Application of FSQ spectrophotometric multicomponent analysis to bioconversion monitoring

Spectrophotometric multicomponent analysis using the novel FSQ algorithm (Full Spectrum Quantitation) allows the rapid and accurate quantitation of complex mixtures of organic compounds. This work investigated the applicability of the FSQ method to the monitoring of substrate and product concentrations during a bioconversion. The hydroxylation of toluene to toluene-cis-glycol (TCG) by Pseudomonas putida (UV4) served as a model system for this study. The reaction is representative of the increasingly important group of bioconversions of a toxic, poorly water-soluble substrate. Previous work has shown that it is crucial to be able to control the toluene concentration to avoid irreversible damage to the biocatalyst. After establishing a suitable analytical wavelength range (215-340 nm) and determining the linear range for absorbance, FSQ calibration was carried out with standard mixtures of the compounds. Three different systems were tested: toluene/TCG as a two-component system and toluene/TCG/bovine serum albumin and toluene/TCG/cell lysate as three-component systems. The latter accounted for UV-absorbing compounds released into the bioconversion medium due to any lysis of the biocatalyst. It was found that accurate quantitation of toluene and TCG could be achieved, even in the presence of contaminating protein or cell lysate. When monitoring bioconversions, TCG could be accurately determined up to 18 g/L. The operability range for toluene quantitation was very narrow due to the low levels of toluene in the reactor and requirements for sample dilution (at high TCG concentrations). Additionally FSQ measurements were able to provide important information about the state of lysis of the biocatalyst. In conclusion, it should be underlined that the FSQ method provides a valuable new analytical tool and its applicability for the case studied is only limited by the nature of the bioconversion itself. Finally its applicability to other types of bioconversion is discussed.