Enzyme Engineering by Directed Evolution

This chapter treats exclusively directed evolution of proteins, the primary focus being on recent methodology developments. During the past 25 years, numerous gene mutagenesis methods have been developed. It is difficult even for experts to make the optimal choice, because comparative studies focusing on relative efficiency are rare. A few select studies in directed evolution are reviewed in the chapter, because they allow conclusions regarding the relative merits of different mutagenesis methods and strategies. In a landmark study, single-gene DNA shuffling was applied to turn Escherichia coli β-galactosidase (BGAL) into a β-fucosidase, thereby changing the substrate scope. The enzyme hydrolyzes β-galactosyl linkages such as the β (1,4)-linkage in lactose. In principle, epPCR and DNA shuffling are independent of structural data, whereas saturation mutagenesis generally needs such knowledge to make a decision regarding the randomization sites. Alternatively, saturation mutagenesis can be applied systematically at every single amino acid position, as was reported in the thermostabilization of a xylanase. Subsequent to the first directed evolution study regarding the enantioselectivity of enzymes, which involved four cycles of epPCR in the hydrolytic kinetic resolution of a chiral ester catalyzed by a lipase, numerous academic and industrial studies have appeared that contribute to the generalization of this new approach to asymmetric catalysis. Subsequent investigations addressed the reasons for the apparent efficacy of iterative combinatorial active-site saturation testing as a form of iterative saturation mutagenesis (ISM).