Time-course kinetic model for the enzyme-limited hydrolysis of methyl esters elucidates the reaction mechanism and inhibition challenges for the production of fatty acids

Fatty acid methyl ester (FAME) and fatty acid (FA) are important oleochemicals. In our previous work, we have shown that FAME can be hydrolyzed to FA using free Candida antarctica lipase B (CALB) in a simple one-pot batch process. However, under enzyme-limited conditions, low conversions inadequate for industrial applications were obtained. Moreover, the reaction mechanism still eludes complete understanding to facilitate further optimization efforts. For the first time, we propose a time-course kinetic model for the enzyme-limited hydrolysis of FAME catalyzed by free CALB to shed light on the reaction mechanism and its associated inhibition challenges. The model was developed based on the ping-pong bi-bi mechanism integrated with several inhibitory mechanisms involving the reaction substrates and by-product. Noteworthily, the model demonstrates a satisfactory fit to the experimental data at water-to-FAME molar ratios between 1:1 and 120:1, as characterized by the high R² and low root-mean-square deviation values averaging at 0.97 and 0.025, respectively. Additionally, the results suggest that the model output conforms with the reaction's stoichiometry and does not show any dependence on the carbon chain length of FAME. Beyond the goodness-of-fit, the model reveals that the enzyme experiences insignificant levels of inhibition by the FAME substrate. Furthermore, the model affirms the enzyme's vulnerability to inhibition induced by the methanol by-product, which reduces with increasing water-to-FAME molar ratio. The model also unravels that a heavy excess of water could inhibit the enzyme. Ultimately, this work reveals that the reaction can be performed under enzyme-limited conditions, and the insights acquired from this model could potentially inspire forthcoming endeavors to improve the process.