Esters are important solvents in multiple industries including adhesives, food, and pharmaceuticals. Although esters are biodegradable solvents, the conventional process of producing them is not eco-friendly because they are largely derived from petrochemicals. This has led scientists to consider implementing biological routes in their production process by incorporating heterologous or improving inherent esterification pathways. However, due to inequality in the biosynthesis of esters and their precursors (organic acid and alcohol), a significant amount of precursors are left unconverted, thereby lowering overall esterification efficiency. Therefore, the primary goal of the current research is to improve the ester titers by incorporating one more step of in vitro esterification with the culture broth, thereby esterifying the unconverted precursors using high efficiency commercial enzymes in the presence of compatible organic solvent. In principle, the medium containing the precursors will be treated with the enzyme in presence of organic solvent, where the precursors will be distributed in both the phases, aqueous and organic, based on their polarity, and the enzymatic esterification will happen at the interface. Hence, as a first step, efforts were made to optimize the reaction conditions, beginning with choosing the most efficient organic solvent and corresponding enzyme candidate. Our results showed that, for production of ethyl acetate through this reactive extraction approach, Novozyme435 exhibited significant esterification with chloroform, with almost 85% conversion efficiency. Further optimizations with phase ratios, pH and incubation time showed that the pH 6.0 (3.1 g/L) was the most optimum where ethyl acetate titer was found to improve 10 times than that at pH 7.0 (0.164 g/L) with the phase ratio of 1:1. The kinetic studies further added that the incubation at 37oC gives the maximum ethyl acetate production within 8h. After initial optimization studies, cell broth from E. coli cells transformed to overproduce an esterase was also tested with the reactive extraction method. It was found that there was a ~7.5X decrease in ethyl acetate production in the cell media versus synthetic samples with the same concentration of reactants. Such a large decrease indicates that enzymatic promiscuity or inhibition currently prevent the cell samples from reaching the same conversion as synthetic studies. To characterize the maximum reaction rate (Vmax) and affinity constants of the substrates to Novozym 435, further kinetic studies were performed with one minute of reaction. The mathematical model employed assumes that enzyme kinetics rather than diffusion was the rate limiting step, that the concentrations of reactants at the interface are equivalent to the initial concentration of reactants, and that neither substrate is an inhibitor. Vmax was found to be 18.5 Mmol min-1g-1 (of catalyst used), and the affinity constants were 0.957 M and 0.00557 M for acetic acid and ethanol respectively. Vmax was similar to literature values with Novozym 435, and the affinity constants indicate a much higher binding efficiency of ethanol in comparison to acetic acid, indicating that a cocktail of esters are likely produced from Novozym 435 in cell broth. Overall, moving away from fossil-fuel dependence is necessary to promote sustainable industry standards, and microbial cell factories combined with reactive extraction, if optimized for industrial applications, can replace harmful environmental procedures. By optimizing the reactive extraction process for ester production, biorefineries could become more competitive and economically feasible for numerous applications.
