title: High level expression of a novel family 3 neutral β-xylosidase from Humicola insolens Y1 with high tolerance to D-xylose.
abstract: A novel β-xylosidase gene of glycosyl hydrolase (GH) family 3, xyl3A, was identified from the thermophilic fungus Humicola insolens Y1, which is an innocuous and non-toxic fungus that produces a wide variety of GHs. The cDNA of xyl3A, 2334 bp in length, encodes a 777-residue polypeptide containing a putative signal peptide of 19 residues. The gene fragment without the signal peptide-coding sequence was cloned and overexpressed in Pichia pastoris GS115 at a high level of 100 mg/L in 1-L Erlenmeyer flasks without fermentation optimization. Recombinant Xyl3A showed both β-xylosidase and α-arabinfuranosidase activities, but had no hydrolysis capacity towards polysaccharides. It was optimally active at pH 6.0 and 60°C with a specific activity of 11.6 U/mg. It exhibited good stability over pH 4.0-9.0 (incubated at 37°C for 1 h) and at temperatures of 60°C and below, retaining over 80% maximum activity. The enzyme had stronger tolerance to xylose than most fungal GH3 β-xylosidases with a high Ki value of 29 mM, which makes Xyl3A more efficient to produce xylose in fermentation process. Sequential combination of Xyl3A following endoxylanase Xyn11A of the same microbial source showed significant synergistic effects on the degradation of various xylans and deconstructed xylo-oligosaccharides to xylose with high efficiency. Moreover, using pNPX as both the donor and acceptor, Xyl3A exhibited a transxylosylation activity to synthesize pNPX2. All these favorable properties suggest that Xyl3A has good potential applications in the bioconversion of hemicelluloses to biofuels.
authors: College of Animal Science, Zhejiang University, Hangzhou 310058, P. R. China; Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P. R. China.
authors: Wei
authors: W
authors: Xia
authors: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P. R. China.
authors: Pengjun
authors: P
authors: Shi
authors: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P. R. China.
authors: Xinxin
authors: X
authors: Xu
authors: College of Animal Science, Zhejiang University, Hangzhou 310058, P. R. China.
authors: Lichun
authors: L
authors: Qian
authors: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P. R. China.
authors: Ying
authors: Y
authors: Cui
authors: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P. R. China.
authors: Mengjuan
authors: M
authors: Xia
authors: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P. R. China.
authors: Bin
authors: B
authors: Yao
mesh_terms: D000595:Amino Acid Sequence; D001203:Ascomycota / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D015966:Gene Expression Regulation, Fungal; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010843:Pichia / Q000378:metabolism; D055550:Protein Stability; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature; D014994:Xylose / Q000378:metabolism*; D014995:Xylosidases / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D014994:Xylose; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase