초록
<P><B>Abstract</B></P> <P>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 <I>Drosophila</I> 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.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Staphylococcus aureus lipases were isolated and shown to esterify fatty acids. </LI> <LI> The enzymes responsible are a pair of highly similar α/β hydrolases. </LI> <LI> The active sites of these lipases bind small alcohols, such as ethanol, weakly. </LI> <LI> Fusion with a LUSH alcohol-binding domain caused increased rebinding of ethanol. </LI> <LI> Increased rebinding leads to an increased rate of ethanol-based esterification. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>