Structure-Guided Directed Evolution of Glycosidases: A Case Study in Engineering a Blood Group Antigen-Cleaving Enzyme.

ABSTRACT

Directed evolution is an incredibly powerful strategy for engineering enzyme function. Applying this approach to glycosidases offers enormous potential for the development of highly specialized tools in chemical glycobiology. Performing enzyme directed evolution requires the generation, by random mutagenesis, of mutant libraries from which large numbers of variant enzymes must be screened in high-throughput assays. A structure-guided "semirational" method for library creation allows researchers to target specific amino acid positions for mutagenesis, concentrating mutations where they might be most effective in order to produce mutant libraries of a manageable size, minimizing screening effort while maximizing the chances of finding improved mutants. Well-designed assays, which may use specially prepared substrates, enable efficient screening of these mutant libraries. This chapter will detail general methods in the structure-guided directed evolution of glycosidases, which have previously been employed in engineering a blood group antigen-cleaving enzyme.