Lipase-catalyzed synthesis of isoamyl butyrate. A kinetic study

Kinetics of lipase-catalyzed esterification of butyric acid and isoamyl alcohol have been investigated. The reaction rate could be described in terms of the Michaelis-Menten equation with a Ping-Pong Bi-Bi mechanism and competitive inhibition by both the substrates. No evidence of any significant diffusional limitations was detected that could affect the kinetics. The values of the apparent kinetic parameters were computed as: V(max)=11.72 micromol/min/mg; K(M, Acid)=0.00303 M; K(M, Alcohol)=0.00306 M; K(i, Acid)=1.05 M; and K(i, Alcohol)=6.55 M. This study indicates a competitive enzyme inhibition by butyric acid during lipase-catalyzed esterification reaction. Butyric acid, being a short-chain polar acid, concentrates in the microaqueous layer and causes a pH drop in the enzyme microenvironment leading to enzyme inactivation. Butyric acid binds to acyl-enzyme complex unproductively to yield a dead-end intermediate that can no longer give rise to an ester. High concentration of butyric acid gave rise to inactivation of the biocatalyst in addition to dead-end inhibition.