title: Expanding the substrate scope of chitooligosaccharide oxidase from Fusarium graminearum by structure-inspired mutagenesis.
abstract: Chitooligosaccharide oxidase from Fusarium graminearum (ChitO) oxidizes N-acetyl-D-glucosamine (GlcNAc) and its oligomers with high efficiency at the C1-hydroxyl moiety while it shows poor or no activity with other carbohydrates. By sequence and structural comparison with other known carbohydrate oxidases (glucooligosaccharide oxidase from Acremonium strictum and lactose oxidase from Microdochium nivale) eleven mutants were designed to redirect the catalytic scope of ChitO for improved oxidation of lactose, cellobiose and maltose. The catalytic properties of the most interesting mutants were further improved by combining single mutations. This has resulted in the creation of a set of ChitO variants that display totally different substrate tolerances. One ChitO variant shows a dramatic improvement in catalytic efficiency towards oxidation of glucose, cellobiose, lactose, and maltose. We also describe a ChitO variant with the highest catalytic efficiency in GlcNAc oxidation so far reported in the literature.
authors.affiliation: Molecular Enzymology Group, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands.
authors.forename: Alessandro R
authors.initials: AR
authors.lastname: Ferrari
authors.forename: Misun
authors.initials: M
authors.lastname: Lee
authors.forename: Marco W
authors.initials: MW
authors.lastname: Fraaije
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism*; D019943:Amino Acid Substitution; D002475:Cellobiose / Q000378:metabolism; D002686:Chitin / Q000031:analogs & derivatives* / Q000378:metabolism; D048271:Chitosan; D005670:Fusarium / Q000201:enzymology*; D005947:Glucose / Q000378:metabolism; D007785:Lactose / Q000378:metabolism; D008320:Maltose / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D009844:Oligosaccharides; C493484:oligochitosan; D002686:Chitin; D002475:Cellobiose; D008320:Maltose; D048271:Chitosan; D010088:Oxidoreductases; D005947:Glucose; D007785:Lactose; D000117:Acetylglucosamine
keywords: carbohydrates; chitooligosaccharides; enzyme engineering; glucooligosaccharides; lactose; oxidase