Enzymes-Assisted Generation of Thiols from Thioacetates

Thiols are important constituents of the flavour of many foods and beverages. They were produced efficiently by enzymatic hydrolysis of thioacetates, which are commercially available or easily accessible, by reaction of thioacetic acid with alkenes. Enzymatic reactions were performed in water or phosphate buffer resulting in good overall yields of the target thiols. Introduction Natural flavours are defined as biologically derived aroma chemicals generated by microbial fermentation and/or by the action of endogenous or technical enzymes (Bakker et al., 1994). Lipases and esterases are the main biocatalysts used in the production of flavouring compounds, particularly esters that are widely present in fmit flavours. Among these esters, thioacetates have received little attention and very few studies deal with the generation and enzymatic hydrolysis of thioacetates to produce thiols (Sproull et al., 1997). These thiols play, as potent odorants, a key role in many food flavours. They occur in low concentrations and contribute significantly to characteristic aroma notes due to their low odour thresholds (Blank, 2002). However, thiols are very unstable and easily oxidise and polymerise upon storage, even at low temperature (Hofmann et al., 1996). To overcome this drawback, we investigated the feasibility of enzymatic hydrolysis of thioacetates, which are much more stable, into the corresponding thiols. This could be an interesting approach if the reaction rate and yield are satisfactory. The thiols could be stored in their stable thioacetate before release by enzymatic hydrolysis. In this study, we report on the generation of 2-methyl-3 -furanthiol and 2-furfurylthiol by enzymatic hydrolysis of their thioacetates. In addition, we describe a chemoenzymatic approach to produce 4-mercapto4-methyl-2-pentanone and 3-mercaptohexanal. Experimental Chemicals and enzymes The chemicals were of analytical grade. S-2-Furfuryl thioacetate, trans-2-hexenal and 4-methyl-3-penten-2-one were purchased from Aldrich. Thioacetic acid was from Fluka and S-3-(2-methylfuryl) thioacetate from Oxford chemicals. Lipase from Candida rugosa and esterase from porcine liver were purchased from Sigma. Synthesis of thioacetates 10 mmol of trans-2-hexenal or 4-methy-3-penten-2-one were added to a solution of thioacetic acid in n-hexane (11 mmol, 30 mL). Reactions were performed at room temperature under stirring. Samples were withdrawn at various time intervals and analysed by gas chromatography (FID, FPD, MS). After 48 h reaction time, the solvent was evaporated and samples were used for enzymatic hydrolysis. Enzymatic hydrolysis of thioacetates in water mmol) in distilled water or phosphate buffer (0.2 M). Enzymatic reactions were performed 2 2 O. ,O 5