Directed evolution: From a staphylococcal lipase to a phospholipase

The lipase of Staphylococcus aureus (SAL) is able to degrade lipids and p-n itrophenylesters but is not active on phospholipid substrates. Interestingl y, the homologous lipase from Staphylococcus hyicus is highly active on pho spholipids. In order to investigate the molecular basis for this difference in substrate specificity, phospholipase activity was introduced into SAL b y directed evolution strategy. In this approach, sequential rounds or error -prone PCR were performed in combination with a screening of the resulting mutant libraries. The Screening was based on a high-throughput plate assay and a subsequent chromogenic assay in 96-well plate format to accurately de termine the enzymatic activities in cell lysates of a selected number of cl ones. After 4 rounds of error-prone PCR, two products were obtained, displa ying a 7.8- and 9.2-fold increase in absolute phospholipase activity and a 5.9- and 6.9-fold increase in phospholipase/lipase activity ratio. A final round of DNA shuffling with these two products and wildtype (WT)-SAL was pe rformed to combine beneficial mutations and to eliminate neutral or deleter ious mutations. This procedure yielded a best variant containing 6 amino ac id mutations displaying a 11.6-fold increase in absolute phospholipase acti vity and a 11.5-fold increase in phospholipase/lipase ratio as compared to the starting point. The character of the mutations and their possible effec ts on substrate specificity are discussed.