title: Influence of a family 29 carbohydrate binding module on the activity of galactose oxidase from Fusarium graminearum.
abstract: BACKGROUND
  Galactose oxidase (GaO) selectively oxidizes the primary hydroxyl of galactose to a carbonyl, facilitating targeted chemical derivatization of galactose-containing polysaccharides, leading to renewable polymers with tailored physical and chemical properties. Here we investigate the impact of a family 29 glucomannan binding module on the activity and binding of GaO towards various polysaccharides. Specifically, CBM29-1-2 from Piromyces equi was separately linked to the N- and C-termini of GaO.
  
  
  RESULTS
  Both GaO-CBM29 and CBM29-GaO were successfully expressed in Pichia pastoris, and demonstrated enhanced binding to galactomannan, galactoglucomannan and galactoxyloglucan. The position of the CBM29 fusion affected the enzyme function. Particularly, C-terminal fusion led to greatest increases in galactomannan binding and catalytic efficiency, where relative to wild-type GaO, kcat/Km values increased by 7.5 and 19.8 times on guar galactomannan and locust bean galactomannan, respectively. The fusion of CBM29 also induced oligomerization of GaO-CBM29.
  
  
  CONCLUSIONS
  Similar to impacts of cellulose-binding modules associated with cellulolytic enzymes, increased substrate binding impeded the action of GaO fusions on more concentrated preparations of galactomannan, galactoglucomannan and galactoxyloglucan; this was especially true for GaO-CBM29. Given the N-terminal positioning of the native galactose-binding CBM32 in GaO, the varying impacts of N-terminal versus C-terminal fusion of CBM29-1-2 may reflect competing action of neighboring CBMs.
  
  
  CONCLUSIONS
  This study thoroughly examines and discusses the effects of CBM fusion to non-lignocellulytic enzymes on soluble polysaccharides. Herein kinetics of GaO on galactose containing polysaccharides is presented for the first time.
authors.affiliation: Department of Biotechnology and Chemical Technology, Aalto University, 00076 Aalto, Finland.
authors.forename: Filip
authors.initials: F
authors.lastname: Mollerup
authors.affiliation: Department of Food and Environmental Sciences, University of Helsinki, P.O. Box 27, Helsinki 00014, Finland.
authors.forename: Kirsti
authors.initials: K
authors.lastname: Parikka
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, Ontario, M5S 3E5, Canada.
authors.forename: Thu V
authors.initials: TV
authors.lastname: Vuong
authors.affiliation: Department of Food and Environmental Sciences, University of Helsinki, P.O. Box 27, Helsinki 00014, Finland.
authors.forename: Maija
authors.initials: M
authors.lastname: Tenkanen
authors.affiliation: Department of Biotechnology and Chemical Technology, Aalto University, 00076 Aalto, Finland; Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, Ontario, M5S 3E5, Canada. Electronic address: emma.master@utoronto.ca.
authors.forename: Emma
authors.initials: E
authors.lastname: Master
mesh_terms: D000595:Amino Acid Sequence; D004795:Enzyme Stability; D005670:Fusarium / Q000201:enzymology*; D005690:Galactose / Q000737:chemistry / Q000031:analogs & derivatives; D005692:Galactose Oxidase / Q000737:chemistry / Q000378:metabolism*; D008351:Mannans / Q000737:chemistry*; D008969:Molecular Sequence Data
chemical_list: D008351:Mannans; C443804:galactoglucomannan; C012990:galactomannan; C022901:(1-6)-alpha-glucomannan; D005692:Galactose Oxidase; D005690:Galactose
keywords: Carbohydrate binding module; Fusion proteins; Galactoglucomannan; Galactomannan; Galactose oxidase; Galactoxyloglucan