title: Biochemical characterization of the novel endo-β-mannanase AtMan5-2 from Arabidopsis thaliana.
abstract: Plant mannanases are enzymes that carry out fundamentally important functions in cell wall metabolism during plant growth and development by digesting manno-polysaccharides. In this work, the Arabidopsis mannanase 5-2 (AtMan5-2) from a previously uncharacterized subclade of glycoside hydrolase family 5 subfamily 7 (GH5_7) has been heterologously produced in Pichia pastoris. Purified recombinant AtMan5-2 is a glycosylated protein with an apparent molecular mass of 50kDa, a pH optimum of 5.5-6.0 and a temperature optimum of 25°C. The enzyme exhibits high substrate affinity and catalytic efficiency on mannan substrates with main chains containing both glucose and mannose units such as konjac glucomannan and spruce galactoglucomannan. Product analysis of manno-oligosaccharide hydrolysis shows that AtMan5-2 requires at least six substrate-binding subsites. No transglycosylation activity for the recombinant enzyme was detected in the present study. Our results demonstrate diversification of catalytic function among members in the Arabidopsis GH5_7 subfamily.
authors.affiliation: From KTH Royal Institute of Technology, School of Biotechnology, Division of Industrial Biotechnology, AlbaNova University Center, 106 91 Stockholm, Sweden.
authors.forename: Yang
authors.initials: Y
authors.lastname: Wang
authors.affiliation: From Wallenberg Wood Science Center, Department of Fiber and Polymer Technology, KTH Royal Institute of Technology, 100 44 Stockholm, Sweden.
authors.forename: Shoaib
authors.initials: S
authors.lastname: Azhar
authors.affiliation: From KTH Royal Institute of Technology, School of Biotechnology, Division of Industrial Biotechnology, AlbaNova University Center, 106 91 Stockholm, Sweden; From Karolinska Institute, Department of Medical Biochemistry and Biophysics, Scheelelaboratoriet, Scheeles väg 2, 17177 Stockholm, Sweden.
authors.forename: Rosaria
authors.initials: R
authors.lastname: Gandini
authors.affiliation: From KTH Royal Institute of Technology, School of Biotechnology, Division of Industrial Biotechnology, AlbaNova University Center, 106 91 Stockholm, Sweden; From Karolinska Institute, Department of Medical Biochemistry and Biophysics, Scheelelaboratoriet, Scheeles väg 2, 17177 Stockholm, Sweden.
authors.forename: Christina
authors.initials: C
authors.lastname: Divne
authors.affiliation: From KTH Royal Institute of Technology, School of Biotechnology, Division of Industrial Biotechnology, AlbaNova University Center, 106 91 Stockholm, Sweden.
authors.forename: Ines
authors.initials: I
authors.lastname: Ezcurra
authors.affiliation: From KTH Royal Institute of Technology, School of Biotechnology, Division of Industrial Biotechnology, AlbaNova University Center, 106 91 Stockholm, Sweden. Electronic address: henrik.aspeborg@biotech.kth.se.
authors.forename: Henrik
authors.initials: H
authors.lastname: Aspeborg
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics*; D029681:Arabidopsis Proteins; D006868:Hydrolysis; D009844:Oligosaccharides / Q000378:metabolism*; D030781:Organisms, Genetically Modified; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D044902:beta-Mannosidase
chemical_list: D029681:Arabidopsis Proteins; D009844:Oligosaccharides; D044902:beta-Mannosidase; C000611294:mannanase 2, Arabidopsis
keywords: Cell wall; GH5; Glycoside hydrolase; Mannan polysaccharides/oligosaccharides; endo-β-1,4-Mannan hydrolase