title: Molecular cloning and characterization of the novel acidic xylanase XYLD from Bispora sp. MEY-1 that is homologous to family 30 glycosyl hydrolases.
abstract: We cloned and sequenced a xylanase gene named xylD from the acidophilic fungus Bispora sp. MEY-1 and expressed the gene in Pichia pastoris. The 1,422-bp full-length complementary DNA fragment encoded a 457-amino acid xylanase with a calculated molecular mass of 49.8 kDa. The mature protein of XYLD showed high sequence similarity to both glycosyl hydrolase (GH) families 5 and 30 but was more homologous to members of GH 30 based on phylogenetic analysis. XYLD shared the highest identity (49.9%) with a putative endo-1,6-beta-D-glucanase from Talaromyces stipitatus and exhibited 21.1% identity and 34.3% similarity to the well-characterized GH family 5 xylanase from Erwinia chrysanthemi. Purified recombinant XYLD showed maximal activity at pH 3.0 and 60 degrees C, maintained more than 60% of maximal activity when assayed at pH 1.5-4.0, and had good thermal stability at 60 degrees C and remained stable at pH 1.0-6.0. The enzyme activity was enhanced in the presence of Ni(2+) and beta-mercaptoethanol and inhibited by some metal irons (Hg(2+), Cu(2+), Pb(2+), Mn(2+), Li(+), and Fe(3+)) and sodium dodecyl sulfate. The specific activity of XYLD for beechwood xylan, birchwood xylan, 4-O-methyl-D-glucuronoxylan, and oat spelt xylan was 2,463, 2,144, 2,020, and 1,429 U mg(-1), respectively. The apparent K (m) and V (max) values for beechwood xylan were 5.6 mg ml(-1) and 3,622 micromol min(-1) mg(-1), respectively. The hydrolysis products of different xylans were mainly xylose and xylobiose.
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing, 100081, People's Republic of China.
authors.forename: Huiying
authors.initials: H
authors.lastname: Luo
authors.forename: Jun
authors.initials: J
authors.lastname: Yang
authors.forename: Jiang
authors.initials: J
authors.lastname: Li
authors.forename: Pengjun
authors.initials: P
authors.lastname: Shi
authors.forename: Huoqing
authors.initials: H
authors.lastname: Huang
authors.forename: Yingguo
authors.initials: Y
authors.lastname: Bai
authors.forename: Yunliu
authors.initials: Y
authors.lastname: Fan
authors.forename: Bin
authors.initials: B
authors.lastname: Yao
mesh_terms: D000595:Amino Acid Sequence; D001203:Ascomycota / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005800:Genes, Fungal; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008670:Metals / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature; D014990:Xylans / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D008670:Metals; D011994:Recombinant Proteins; D014990:Xylans; D006867:Hydrolases; D043364:Endo-1,4-beta Xylanases