Site-saturation mutagenesis is more efficient than DNA shuffling for the directed evolution of β-fucosidase from β-galactosidase

Protein engineers use a variety of mutagenic strategies to adapt enzymes to novel substrates. Directed evolution techniques (random mutagenesis and high-throughput screening) offer a systematic approach to the management of protein complexity. This sub-discipline was galvanized by the invention of DNA shuffling, a procedure that randomly recombines point mutations in vitro. In one influential study, Escherichia coli beta-galactosidase (BGAL) variants with enhanced beta-fucosidase activity (tenfold increase in k(cat)/K-M in reactions with the novel para-nitrophenyl-beta-(D)-fucopyranoside substrate; 39- fold decrease in reactivity with the native para-nitrophenyl-beta-(D)-galactopyranoside substrate) were evolved in seven rounds of DNA shuffling and screening. Here, we show that a single round of site-saturation mutagenesis and screening enabled the identification of beta-fucosidases that are significantly more active (180-fold increase in k(cat)/ K-M in reactions with the novel substrate) and specific (700,000-fold inversion of specificity) than the best variants in the previous study. Site saturation mutagenesis thus proved faster, less resource-intensive and more effective than DNA shuffling for this particular evolutionary pathway. (c) 2005 Elsevier Ltd. All rights reserved.