Epoxide hydrolase activity in the aqueous extracts of vegetable flours and application to the stereoselective hydrolysis of limonene oxide

The aqueous buffered suspensions of some vegetable flours were tested in the reaction with 1,2-epoxide deriving from (4R)- or (4S)-limonene as well as with the standard substrate methyl oleate epoxide and epoxide hydrolase activity was mainly detected in the flours from soybean, mung bean and red mung bean. A simple protocol was applied for obtaining a raw freeze-dried enzymatic preparation from flours, which maintained most of the activity of the fresh suspension and offered advantages in easy handling, reproducibility and stability over the time. Preferential hydrolysis of one of the diastereoisomers present in the starting (4R)- or (4S)-limonene oxide (cis/trans mixture) was observed and the reaction converged toward the formation of a predominant diol, resulting from the attack on the most (C1)- or less (C2)-substituted carbon of the oxirane ring depending on the cis/trans stereochemistry of the substrate. Biocatalyzed hydrolysis of (S)-limonene oxide proceeded at higher reaction rate compared to the (R)-isomer and with superior stereoconvergence toward a single diol product. The kinetic separation of the slow-reacting diastereoisomers of (R)- and (S)-limonene oxides was optimized by using the enzymatic preparations from red mung bean and soybean, respectively, and trans-(4R)-limonene oxide and cis-(4S)-limonene oxide were obtained in good yield and stereochemical purity.