Determination of Activation Energy and Ping-Pong Kinetic Model Constants of Enzyme-Catalyzed Self-Epoxidation of Free Fatty Acids using Micro-reactor

Abstract

This work reports bioconversion of unsaturated free fatty acids present in soapstock, inherent waste of edible oil manufacture, to the epoxidized fatty acids using Candida rugose lipase in a micro-reactor. Activation energy of the epoxidation reaction was found to be 32.27 kJ mol⁻¹ while the enzyme deactivation was observed at higher temperature than 35 °C with an activation energy of 91.18 kJ mol⁻¹. The low hydrodynamic retention time in the micro-reactor provides a proper condition to measure of initial rates of the enzymatic reaction. The kinetics study was performed at ten different molar ratios of H₂O₂/C=C, and constants of the Ping-Pong bi bi model were estimated as follows: the maximum specific enzyme activity (V_max) of 1.453 × 10⁵ mM min⁻¹ g_enzyme⁻¹, dissociation constant for enzyme-FFA complex (K_A) of 58.83 mM g_enzyme⁻¹, Michaelis constant for FFAs (K_mA) of 1.382 × 10³ mM g_enzyme⁻¹, Michaelis constant for hydrogen peroxide (K_mB) of 3.279 × 10⁶ mM g_enzyme⁻¹, and the deactivation constant due to hydrogen peroxide (K_IB) of 59.25 mM g_enzyme⁻¹. The cross-linked enzyme aggregate technology provided a simple method to produce the robust biocatalyst for this bioconversion where more than 89% of original activity was maintained after 9 cycles in the micro-reactor.

Graphical Abstract