Can directed evolution give a staphylococcal lipase phospholipase activity?

Details

Staphylococcus aureus lipase (SAL) degrades lipids and p-nitrophenylesters but is not active on phospholipid substrates. The homologous lipase from Staphylococcus hyicus, by contrast, is highly active on phospholipids. To investigate the molecular basis of this difference in substrate specificity, researchers used directed evolution to introduce phospholipase activity into SAL.

They performed sequential rounds of error-prone PCR and screened the resulting mutant libraries. Screening used a high-throughput plate assay followed by a chromogenic assay in a 96-well plate format to accurately determine enzyme activities in cell lysates from a selected number of clones.

After four rounds of error-prone PCR, two products showed 7.8- and 9.2-fold increases in absolute phospholipase activity and 5.9- and 6.9-fold increases in the phospholipase-to-lipase activity ratio, respectively. A final round of DNA shuffling used these two products and wild-type (WT) SAL to combine beneficial mutations and eliminate neutral or deleterious mutations. The best resulting variant contained six amino acid mutations and showed an 11.6-fold increase in absolute phospholipase activity and an 11.5-fold increase in the phospholipase-to-lipase ratio compared with the starting point. The researchers discuss the character of the mutations and their possible effects on substrate specificity.