A secreted Staphylococcus aureus lipase engineered for enhanced alcohol affinity for fatty acid esterification

Presently the production and use of biodiesel is not cost-effective in comparison to traditional fossil fuels. Naturally occurring enzymes, such as lipases and esterases, can potentially be engineered to lower the cost of certain steps in the biodiesel synthesis process that would otherwise be more costly. However, these enzymes have evolved to perform biologically relevant functions, and not necessarily to manufacture biodiesel under commercially viable conditions. To this end, we have identified, cloned, expressed, purified, and characterized two proteins from the staphylococcal lipase family that are capable of catalyzing the formation of fatty acid alkyl esters. In an effort to explore strategies for improving these fatty acid modifying enzymes (FAMEs), we have engineered a chimeric fusion protein that significantly increases the esterification of free fatty acid with ethanol. The fusion protein, which consists of a staphylococcal FAME fused to a Drosophila ethanol binding protein, demonstrably improves the rate of catalysis by providing an additional substrate binding site and concomitant increase in the local concentration of substrate. This results in greater overall substrate (ethanol) residence in proximity to the catalytic domain, and a faster rate of catalysis, without the necessity of altering the amino acid sequence of the FAME protein.