Reetz_2006_Angew.Chem.Int.Ed.Engl_45_1236

Recently, we introduced a practical method in directed evolution called a combinatorial active site saturation test (CAST). CAST was originally developed with the purpose of expanding the range of substrate acceptance of an enzyme. Based on the 3D structure of the enzyme, two or three amino acids, whose side chains reside next to the binding pocket, are identified and the respective positions are then randomized simultaneously with the creation of relatively small libraries of mutants. Through the use of this method, it was possible to obtain mutant lipases that catalyze the hydrolysis of esters derived from sterically demanding acids that are normally not at all accepted as substrates. CASTing thus means the systematic design and screening of focused libraries around the complete binding pocket. Owing to the fact that two or three amino acids (rather than just one) are simultaneously randomized at each site, high catalyst diversity is possible, therefore theoretically allowing the possibility of cooperative effects. The method is a useful alternative to epPCR as the starting point of directed evolution studies. However, it is not, in itself, an evolutionary process because only the initial libraries are considered. In our original paper, we suggested that, for future work, Darwinistic character, which is central to directed evolution, can be introduced through the performance of further rounds of mutagenesis/screening. This can be performed by using any one of the conventional methods, such as epPCR or DNA shuffling after CASTing, which may lead to remote effects. As an alternative, we herein propose iterative cycles of CASTing.