title: Directed evolution of new glycosynthases from Agrobacterium beta-glucosidase: a general screen to detect enzymes for oligosaccharide synthesis.
abstract: BACKGROUND
  Oligosaccharide synthesis is becoming increasingly important to industry as diverse therapeutic roles for these molecules are discovered. The chemical synthesis of oligosaccharides on an industrial scale is often prohibitively complex and costly. An alternative, that of enzymatic synthesis, is limited by the difficulty of obtaining an appropriate enzyme. A general screen for enzymes that catalyze the synthesis of the glycosidic bond would enable the identification and engineering of new or improved enzymes.
  
  
  RESULTS
  Glycosynthases are nucleophile mutants of retaining glycosidases that efficiently catalyze the synthesis of the glycosidic linkage by condensing an activated glycosyl fluoride donor with a suitable acceptor sugar. A novel agar plate-based coupled-enzyme screen was developed (using a two-plasmid system) and used to select an improved glycosynthase from a library of mutants.
  
  
  CONCLUSIONS
  Plate-based coupled-enzyme screens of this type are extremely valuable for identification of functional synthetic enzymes and can be applied to the evolution of a range of glycosyl transferases.
authors.affiliation: Protein Engineering Nework of Centres of Excellence of Canada, Department of Chemistry, University of British Columbia, Vancouver.
authors.forename: C
authors.initials: C
authors.lastname: Mayer
authors.forename: D L
authors.initials: DL
authors.lastname: Jakeman
authors.forename: M
authors.initials: M
authors.lastname: Mah
authors.forename: G
authors.initials: G
authors.lastname: Karjala
authors.forename: L
authors.initials: L
authors.lastname: Gal
authors.forename: R A
authors.initials: RA
authors.lastname: Warren
authors.forename: S G
authors.initials: SG
authors.lastname: Withers
mesh_terms: D019020:Directed Molecular Evolution; D008403:Mass Screening / Q000379:methods; D009154:Mutation / Q000235:genetics* / Q000502:physiology; D009844:Oligosaccharides / Q000096:biosynthesis*; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering / Q000639:trends; D012231:Rhizobium / Q000201:enzymology* / Q000235:genetics; D001617:beta-Glucosidase / Q000235:genetics* / Q000378:metabolism*
chemical_list: D009844:Oligosaccharides; D001617:beta-Glucosidase