Kinetics of acyl transfer reactions in organic media catalysed by Candida antarctica lipase B

Abstract

The acyl transfer reactions catalysed by Candida antarctica lipase B in organic media followed a bi-bi ping-pong mechanism, with competitive substrate inhibition by the alcohols used as acyl acceptors. The effect of organic solvents on V m and K m was investigated. The V m values in acetonitrile was 40–50% of those in heptane. High K m values in acetonitrile compared to those in heptane could partly be explained by an increased solvation of the substrates in acetonitrile. Substrate solvation caused a 10-fold change in substrate specificity, defined as ( V m/ K m)ethyloctanoate/( V m/ K m)octanoicacid, going from heptane to acetonitrile. Deacylation was the rate determining step for the acyl transfer in heptane with vinyl- and ethyl octanoate as acyl donors and ( R)-2-octanol as acyl acceptor. With 1-octanol, a rate determining deacylation step in heptane was indicated using the same acyl donors. Using 1-octanol as acceptor in heptane, S-ethyl thiooctanoate had a 25- to 30-fold lower V m/ K m value and vinyl octanoate a 4-fold higher V m/ K m value than that for ethyl octanoate. The difference showed to be a Km effect for vinyl octanoate and mainly a K m effect for S-ethyl thiooctanoate. The V m values of the esterification of octanoic acid with different alcohols was 10–30-times lower than those for the corresponding transesterification of ethyl octanoate. The low activity could be explained by a low pH around the enzyme caused by the acid or a withdrawing of active enzyme by nonproductive binding by the acid.