title: Heterologous expression and kinetic characterisation of Neurospora crassa β-xylosidase in Pichia pastoris.
abstract: To degrade plant hemicelluloses fungi employ β-xylosidases to hydrolyse xylooligosaccharides, released by endo-xylanases, into xylose. We have expressed the β-xylosidase from Neurospora crassa in Pichia pastoris under the control of alcohol oxidase 1 (AOX1) promoter. The recombinant enzyme is optimally active at 50 °C and pH 5.0 with Km and Vmax values of 8.9 mM and 1052 μmol min⁻¹ mg⁻¹ respectively against 4-nitrophenyl β-xylopyranoside. Xylose is a non-competitive inhibitor with a K(i) of 1.72 mM. The enzyme is characterised to be an exo-cutting enzyme releasing xylose from the non-reducing ends of β-1,4 linked xylooligosaccharides (X₂, X₃ and X₄) but also capable of transxylosilation. Catalytic conversion of X₂, X₃ and X4 decreases (V(max) and k(cat)) with increasing chain length.
authors.affiliation: Division of Food Sciences, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK.
authors.forename: N
authors.initials: N
authors.lastname: Kirikyali
authors.affiliation: Division of Food Sciences, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK. Electronic address: ian.connerton@nottingham.ac.uk.
authors.forename: I F
authors.initials: IF
authors.lastname: Connerton
mesh_terms: D000595:Amino Acid Sequence; D001673:Biodegradation, Environmental; D018533:Biomass; D002473:Cell Wall / Q000378:metabolism; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D007218:Industrial Microbiology; D007700:Kinetics; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D010944:Plants / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase
keywords: Enzyme kinetics; Neurospora crassa; Protein expression; Xylose; β-Xylosidase