[reaction: see text] The epoxide hydrolase (EH) from Aspergillus niger, which shows a selectivity factor of only E = 4.6 in the hydrolytic kinetic resolution of glycidyl phenyl ether, has been subjected to directed evolution for the purpose of enhancing enantioselectivity. After only one round of error-prone polymerase chain reaction (epPCR), enantioselectivity was more than doubled (E = 10.8). The improved mutant enzyme contains three amino acid exchanges, two of which are spatially far from the catalytically active center.

Directed evolution has emerged as a key technology to generate enzymes with new or improved properties that are of major importance to the biotechnology industry. A directed evolution approach starts with the identification of a target enzyme to be optimized and the cloning of the corresponding gene. An efficient expression system is needed before the target gene is subjected to random mutagenesis and/or in vitro recombination, thereby creating molecular diversity. Subsequently, improved enzyme variants are identified, preferably after being secreted into culture medium, by screening or selection for the desired property. The genes encoding the improved enzymes are then used to parent the next round of directed evolution. Enantioselectivity is a biocatalyst property of major biotechnological importance that is, however, difficult to deal with. We discuss recent examples of creating enantioselective biocatalysts by directed evolution.

A new and practical method for gene recombination with formation of libraries of mutant genes is presented. The method is based on the assembly of appropriately prepared oligonucleotides whose design is guided by sequence information. High recombination frequency with formation of full-length products is achieved by controlled overlapping of the designed oligomers. This process (ADO) minimizes self-hybridization of parental genes, which constitutes a significant advantage over conventional family shuffling as used in the directed evolution of functional enzymes. ADO was applied to the recombination of two lipase family genes from Bacillus subtilis (LipA and LipB). In a library of 3000 lipase variants created by this method, several were found that display increased enantioselectivity in a model reaction involving the hydrolysis of a meso-diacetate.