Natural Diversity to Guide Focused Directed Evolution

Simultaneous multiple site-sturation mutagenesis was performed at four activ-site positions of an esterase form pseudomonas flouresecens to imporve its ability to convert 3-phenlbutric acid ester (3-PBA) in an enantioselective manner. Based on an appropriate codon choice derived form a structural alignment of 1751 sequences alpha/beta-hydrolse fold enzymes, only those amino acids were considered for library creation that appeared frequently in structurally equivlaent position. Thus, the nimber of mutants to be screened could be sub-stantilly reduced while the number of functionally inatvariants was incresed. Whereas the wild-type esterase showed only marginal activity and poor enantioselctivity (E-true-3.2) towards 3-PBA-ethyl ester, a significant number of hits with imporved rate (up to 240-fold and entioselectivities (up to E-ture = 80) were identified in these 'smart' libraries.