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 eugenyl 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 °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 Vmax of 3.83 mM min-1. The Michaelis–Menten constants for benzoic acid (Km,A) and eugenol (Km,B) were 34.04 and 138.28 mM, respectively. The inhibition constant for eugenol (Ki,B) was 438.6 mM while the turnover number (kcat) 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