Key mutation sites for improvement of the enantioselectivity of lipases through protein engineering

Abstract

Lipases can resolve many chiral compounds of commercial interest. However, for some substrates, it has been difficult to find lipases with the necessary chemo-, regio- and enantioselectivities. Searching in nature for new lipases with the desired selectivities is a difficult task and is not always successful. An alternative strategy is to engineer existing lipases. However, until now, there has been no systematic evaluation to identify which positions in the protein molecule should be engineered to obtain variants with the desired selectivities. Such an evaluation is possible because, even though the catalytic clefts of lipases have differing shapes, there is sufficient structural similarity for one to identify the key regions involved. In this review, we analyze representative studies that have engineered bacterial and fungal lipases for increased enantioselectivity for application in racemic resolutions. Based on this analysis, we identify key sites of the catalytic cleft at which mutations should be targeted in attempts to improve the enantioselectivity of lipases. The locations of these key sites depend on whether the chiral substrate is a carboxylic acid or an alcohol.