KINETICS OF ACYL TRANSFER-REACTIONS IN ORGANIC MEDIA CATALYZED BY CANDIDA-ANTARCTICA LIPASE-B

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 accepters. 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 going from heptane to acetonitrile. Deacylation was the rate determining specificity, defined as (V-m/K-m)(ethyl) (octanoate)/(V-m/K-m)(octanoic) (acid), step for the acyl transfer in heptane with vinyl- and ethyl octanoate as acyl donors and (R)-2-octanol as acyl acceptor. With I-octanol, a rate determining deacylation step in heptane was indicated using the same acyl donors. Using I-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 K-m 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.