PMID:16095606
| Citation |
Parikh, MR and Matsumura, I (2005) Site-saturation mutagenesis is more efficient than DNA shuffling for the directed evolution of beta-fucosidase from beta-galactosidase. J. Mol. Biol. 352:621-8 |
|---|---|
| Abstract |
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. |
| Links |
PubMed PMC2014722 Online version:10.1016/j.jmb.2005.07.020 |
| Keywords |
Base Sequence; DNA Shuffling; DNA, Bacterial/genetics; Directed Molecular Evolution/methods; Escherichia coli/enzymology; Escherichia coli/genetics; Kinetics; Models, Molecular; Mutagenesis, Site-Directed; Protein Engineering/methods; Recombinant Proteins/chemistry; Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Substrate Specificity; alpha-L-Fucosidase/chemistry; alpha-L-Fucosidase/genetics; alpha-L-Fucosidase/metabolism; beta-Galactosidase/chemistry; beta-Galactosidase/genetics; beta-Galactosidase/metabolism |
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