title: Cloning, expression and characterization of β-xylosidase from Aspergillus niger ASKU28.
abstract: β-Xylosidases catalyze the breakdown of β-1,4-xylooligosaccharides, which are produced from degradation of xylan by xylanases, to fermentable xylose. Due to their important role in xylan degradation, there is an interest in using these enzymes in biofuel production from lignocellulosic biomass. In this study, the coding sequence of a glycoside hydrolase family 3 β-xylosidase from Aspergillus niger ASKU28 (AnBX) was cloned and expressed in Pichia pastoris as an N-terminal fusion protein with the α-mating factor signal sequence (α-MF) and a poly-histidine tag. The expression level was increased to 5.7 g/l in a fermenter system as a result of optimization of only five codons near the 5' end of the α-MF sequence. The recombinant AnBX was purified to homogeneity through a single-step Phenyl Sepharose chromatography. The enzyme exhibited an optimal activity at 70°C and at pH 4.0-4.5, and a very high kinetic efficiency toward a xyloside substrate. AnBX demonstrated an exo-type activity with retention of the β-configuration, and a synergistic action with xylanase in hydrolysis of beechwood xylan. This study provides comprehensive data on characterization of a glycoside hydrolase family 3 β-xylosidase that have not been determined in any prior investigations. Our results suggested that AnBX may be useful for degradation of lignocellulosic biomass in bioethanol production, pulp bleaching process and beverage industry.
authors.affiliation: Interdisciplinary Graduate Program in Genetic Engineering, Faculty of Graduate School, Kasetsart University, Bangkok 10900, Thailand.
authors.forename: Khuanjarat
authors.initials: K
authors.lastname: Choengpanya
authors.affiliation: Department of Biochemistry and Special Research Unit for Protein Engineering and Protein Bioinformatics, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.
authors.forename: Siriphan
authors.initials: S
authors.lastname: Arthornthurasuk
authors.affiliation: Department of Chemistry, Center of Excellence for Innovation in Chemistry and Special Research Unit for Advanced Magnetic Resonance, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.
authors.forename: Pakorn
authors.initials: P
authors.lastname: Wattana-amorn
authors.affiliation: Department of Applied Chemistry, College of Science, National Chiao Tung University, Hsinchu 300, Taiwan.
authors.forename: Wan-Ting
authors.initials: WT
authors.lastname: Huang
authors.affiliation: Department of Biochemistry and Special Research Unit for Protein Engineering and Protein Bioinformatics, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.
authors.forename: Wandee
authors.initials: W
authors.lastname: Plengmuankhae
authors.affiliation: Department of Applied Chemistry, College of Science, National Chiao Tung University, Hsinchu 300, Taiwan.
authors.forename: Yaw-Kuen
authors.initials: YK
authors.lastname: Li
authors.affiliation: Interdisciplinary Graduate Program in Genetic Engineering, Faculty of Graduate School, Kasetsart University, Bangkok 10900, Thailand; Department of Biochemistry and Special Research Unit for Protein Engineering and Protein Bioinformatics, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand; Center for Advanced Studies in Tropical Natural Resources, NRU-KU, Kasetsart University, Bangkok 10900, Thailand. Electronic address: fscippt@ku.ac.th.
authors.forename: Prachumporn T
authors.initials: PT
authors.lastname: Kongsaeree
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology*; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014990:Xylans; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D003062:Codon; D005656:Fungal Proteins; D011994:Recombinant Proteins; D014990:Xylans; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase
keywords: Aspergillus niger; Codon optimization; Glycoside hydrolase family 3; Pichia pastoris; Xylan hydrolysis; β-Xylosidase