Synthesis of eugenyl benzoate catalyzed by rhizomucor miehei lipase immobilized on reinforced chitosan-chitin nanowhiskers support

Abstract

The chemical route to produce esters has several drawbacks associated with the utilization of homogeneous acid catalysts that require high reaction temperatures, hence is energy intensive, not reusable, creates dissolved solids and involves laborious separation processes. Hence, a biotechnological route via enzymatic esterification has been proposed as an alternative way to synthesize the problematic anti-oxidant, eugenyl benzoate. This is because an enzyme-catalyzed synthesis offers favorable advantages such as the use of a more sustainable reaction process with high yields and purity, as well as the biocatalyst reusability. An ecofriendly support was prepared from chitosan-chitin nanowhiskers (CS-CNWs) for covalent immobilization of Rhizomucor miehei lipase (RML) to be used as the biocatalysts. Analyses on the RML-CS/CNWs using Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis-differential thermogravimetry (TGA-DTG), field emission scanning electron microscopy (FESEM), X-ray diffraction (XRD) and fluorescence microscopy affirmed the successful covalent immobilization of RML onto the surface of CS-CNWs. The resultant RML-CS/CNWs biocatalysts were studied for catalyzing synthesis of eugenyl benzoate for various reaction parameters. One-Variable-at-A-Time (OVAT) study revealed that under optimal experimental conditions of 50 °C at 250 rpm with catalyst loading of 3 mg/mL and 3:1 molar ratio of eugenol to benzoic acid, the maximum yield that reached 62.1% was attained after 5 h as compared to free RML (50.7%). The RML-CS-CNWs also demonstrated good operational stability, whereby the biocatalysts retaining 50% of its initial activity for up to eight successive esterification cycles. The present work also reports a response surface methodology (RSM) with Box-Behnken design (BBD) optimization process to synthesize eugenyl benzoate. The effects of four reaction parameters: reaction time, temperature, the substrate molar ratio of eugenol to benzoic acid and enzyme loading were assessed based on OVAT findings. Under optimum conditions, a maximum conversion yield of 66.8% was attained at 50 °C in 5 h using 3.75 mg/mL of the RML-CS-CNWs, and substrate molar ratio (eugenol:benzoic acid) of 3:1. Meanwhile, kinetic assessments revealed the RMLCS/ CNWs catalyzed the reaction via a Ping-pong Bi Bi mechanism with eugenol inhibition, characterized by a Vmax of 3.83 mM min-1 and turnover number (kcat) of 40.39 min-1 under an optimized experimental condition. Based on the findings, it can be concluded that the use of the RML-CS-CNWs biocatalysts was promising in affording relatively satisfactory yield of eugenyl benzoate within a reasonably short time. Aside from improving enzymatic operational activity and stability, the immobilization strategy can facilitate rapid and easy removal of the RML-CS-CNWs from the reaction mixture when completed.