title: Conversion of a cyclodextrin glucanotransferase into an alpha-amylase: assessment of directed evolution strategies.
abstract: Glycoside hydrolase family 13 (GH13) members have evolved to possess various distinct reaction specificities despite the overall structural similarity. In this study we investigated the evolutionary input required to effeciently interchange these specificities and also compared the effectiveness of laboratory evolution techniques applied, i.e., error-prone PCR and saturation mutagenesis. Conversion of our model enzyme, cyclodextrin glucanotransferase (CGTase), into an alpha-amylase like hydrolytic enzyme by saturation mutagenesis close to the catalytic core yielded a triple mutant (A231V/F260W/F184Q) with the highest hydrolytic rate ever recorded for a CGTase, similar to that of a highly active alpha-amylase, while cyclodextrin production was virtually abolished. Screening of a much larger, error-prone PCR generated library yielded far less effective mutants. Our results demonstrate that it requires only three mutations to change CGTase reaction specificity into that of another GH13 enzyme. This suggests that GH13 members may have diversified by introduction of a limited number of mutations to the common ancestor, and that interconversion of reaction specificites may prove easier than previously thought.
authors.affiliation: Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.
authors.forename: Ronan M
authors.initials: RM
authors.lastname: Kelly
authors.forename: Hans
authors.initials: H
authors.lastname: Leemhuis
authors.forename: Lubbert
authors.initials: L
authors.lastname: Dijkhuizen
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002240:Carbohydrate Sequence; D020134:Catalytic Domain / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D019020:Directed Molecular Evolution / Q000379:methods; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*