Kinetics of acyl transfer reactions in organic media catalysed by Candida antarctica lipase B.
The acyl transfer reactions catalysed by Candida antartica 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 Vm and Km was investigated. The Vm values in acetonitrile was 40-50% of those in heptane. High Km 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 (Vm/Km)ethyl octanoate/(Vm/Km)octanoic acid, 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 Vm/Km value and vinyl octanoate a 4-fold higher Vm/Km value than that for ethyl octanoate. The difference showed to be a Km effect for vinyl octanoate and mainly a Km effect for S-ethyl thiooctanoate. The Vm 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.
Martinelle
M
M
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
Hult
K
K
D002175:Candida / Q000201:enzymology*; D002210:Caprylates; D003470:Culture Media; D007700:Kinetics; D008049:Lipase / Q000378:metabolism*; D000442:Octanols; D012997:Solvents
D002210:Caprylates; D003470:Culture Media; D000442:Octanols; D012997:Solvents; C067943:2-octanol; D008049:Lipase; C031492:octanoic acid