Enzymatic esterification of eugenol and benzoic acid by a novel chitosan-chitin nanowhiskers supported Rhizomucor miehei lipase: Process optimization and kinetic assessments

A biotechnological route via enzymatic esterification was proposed as an alternative way to synthesize the problematic anti-oxidant eugeriy benzoate. The new method overcomes the well-known drawbacks of the chemical route in favor of a more sustainable reaction process. The present work reports a Box-Behnken design (BBD) optimization process to synthesize eugenyl benzoate by esterification of eugenol and benzoic acid catalyzed by the chitosan-chitin nanowhiskers supported Rhizomucor miehei lipase (RML-CS/CNWs). Effects of four reaction parameters: reaction time, temperature, substrate molar ratio of eugenol: benzoic acid and enzyme loading were assessed. Under optimum conditions, a maximum conversion yield as high as 66% at 50 degrees C in 5 h using 3 mg/mL of RML-CS/CNWs, and a substrate molar ratio (eugenol: benzoic acid) of 3:1. Kinetic assessments revealed the RML-CS/CNWs catalyzed the reaction via a ping-pong bi-bi mechanism with eugenol inhibition, characterized by a V-max of 3.83 mM min(-1). The Michaelis-Menten constants for benzoic acid (K-m,K-A) and eugenol (K-m,K-B) were 34.04 and 138.28 mM, respectively. The inhibition constant for eugenol (K-i,K-B) was 438.6 mM while the turnover number (k(cat)) for the RML-CS/CNWs-catalyzed esterification reaction was 40.39 min(-1). RML-CS/CNWs were reusable up to 8 esterification cycles and showed higher thermal stability than free RML.