title: Activity of three β-1,4-galactanases on small chromogenic substrates.
abstract: β-1,4-Galactanases belong to glycoside hydrolase family GH 53 and degrade galactan and arabinogalactan side chains of the complex pectin network in plant cell walls. Two fungal β-1,4-galactanases from Aspergillus aculeatus, Meripileus giganteus and one bacterial enzyme from Bacillus licheniformis have been kinetically characterized using the chromogenic substrate analog 4-nitrophenyl β-1,4-d-thiogalactobioside synthesized by the thioglycoligase approach. Values of k(cat)/K(m) for this substrate with A. aculeatus β-1,4-galactanase at pH 4.4 and for M. giganteus β-1,4-galactanase at pH 5.5 are 333M(-1)s(-1) and 62M(-1)s(-1), respectively. By contrast the B. licheniformis β-1,4-galactanase did not hydrolyze 4-nitrophenyl β-1,4-d-thiogalactobioside. The different kinetic behavior observed between the two fungal and the bacterial β-1,4-galactanases can be ascribed to an especially long loop 8 observed only in the structure of B. licheniformis β-1,4-galactanase. This loop contains substrate binding subsites -3 and -4, which presumably cause B. licheniformis β-1,4-galactanase to bind 4-nitrophenyl -1,4-β-d-thiogalactobioside non-productively. In addition to their cleavage of 4-nitrophenyl -1,4-β-d-thiogalactobioside, the two fungal enzymes also cleaved the commercially available 2-nitrophenyl-1,4-β-d-galactopyranoside, but kinetic parameters could not be determined because of transglycosylation at substrate concentrations above 4mM.
authors.affiliation: Department of Chemistry, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Søs
authors.initials: S
authors.lastname: Torpenholt
authors.forename: Jérôme
authors.initials: J
authors.lastname: Le Nours
authors.forename: Ulla
authors.initials: U
authors.lastname: Christensen
authors.forename: Michael
authors.initials: M
authors.lastname: Jahn
authors.forename: Stephen
authors.initials: S
authors.lastname: Withers
authors.forename: Peter Rahbek
authors.initials: PR
authors.lastname: Ostergaard
authors.forename: Torben V
authors.initials: TV
authors.lastname: Borchert
authors.forename: Jens-Christian
authors.initials: JC
authors.lastname: Poulsen
authors.forename: Leila
authors.initials: L
authors.lastname: Lo Leggio
mesh_terms: D002240:Carbohydrate Sequence; D002855:Chromatography, Thin Layer; D002863:Chromogenic Compounds / Q000737:chemistry* / Q000378:metabolism*; D005690:Galactose / Q000737:chemistry / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism*; D008969:Molecular Sequence Data; D013379:Substrate Specificity
chemical_list: D002863:Chromogenic Compounds; D006026:Glycoside Hydrolases; C094192:arabinogalactan endo-1,4-beta-galactosidase; D005690:Galactose