Site saturation mutagenesis: Methods and applications in protein engineering

Mutagenesis strategies have been applied to fine-tune different enzyme properties including substrate specificity, thermostability, enantioselectivity or simply to increase activity. The mechanistic understanding of how a particular enzyme operates often dictates suitable mutagenesis strategies. Site saturation mutagenesis is used to substitute targeted residues to any other naturally occurring amino acid. Here, we review several aspects of this mutagenesis approach, comparing different molecular techniques to produce libraries of single-residue substitutions. A discussion of experimental design and combinatorial mutagenesis in light of screening capabilities is provided. Several examples of this mutagenesis strategy applied to directed evolution and structure–function studies are also discussed.

► In this review we discuss methodologies and experimental design for site saturation mutagenesis.
► We demonstrate applications of site saturation mutagenesis for directed evolution and structure–function studies.
► Several examples of experiments involving this technique are presented and discussed.