APPLICATION OF FSQ SPECTROPHOTOMETRIC MULTICOMPONENT ANALYSIS TO BIOCONVERSION MONITORING

Spectrophotometric multicomponent analysis using the novel FSQ algorit hm (Full Spectrum Quantitation) allows the rapid and accurate quantita tion 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 t oluene to toluene-cis-glycol (TCG) by Pseudomonas putida (UV4) served as a model system for this study. The reaction is representative of th e increasingly important group of bioconversions of a toxic, poorly wa ter-soluble substrate. Previous work has shown that it is crucial to b e able to control the toluene concentration to avoid irreversible dama ge to the biocatalyst. After establishing a suitable analytical wavele ngth range (215-340 nm) and determining the linear range for absorbanc e, FSQ calibration was carried out with standard mixtures of the compo unds. Three different systems were tested: toluene/TCG as a two-compon ent system and toluene/TCG/bovine serum albumin and toluene/TCG/cell l ysate as three-component systems. The latter accounted for UV-absorbin g compounds released into the bioconversion medium due to any lysis of the biocatalyst. It was found that accurate quantitation of toluene a nd TCG could be achieved, even in the presence of contaminating protei n or cell lysate. When monitoring bioconversions, TCG could be accurat ely determined up to 18 g/L. The operability range for toluene quantit ation was very narrow due to the low levels of toluene in the reactor and requirements for sample dilution (at high TCG concentrations). Add itionally 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 t ool 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.