title: Cellulose-inducible xylanase Xyl10A from Acremonium cellulolyticus: Purification, cloning and homologous expression.
abstract: Cellulose-inducible endo-β-1,4-xylanase (Xyl10A) from the mesophilic fungus Acremonium cellulolyticus was purified, characterized, and expressed by a homologous expression system. A. cellulolyticus CF-2612 produces a high level of xylanase upon induction by Solka-Floc cellulose. To identify this xylanase, the major fraction showing xylanase activity was purified from the CF-2612 culture supernatant, and its gene was identified from the genome sequence. Amino acid sequence homology of Xyl10A revealed that the purified xylanase, designated Xyl10A, exhibited significant homology to family 10 of the glycoside hydrolases (GH10), possessing a cellulose-binding module 1 in the C-terminal region. The xyl10A gene was cloned and expressed in A. cellulolyticus under the control of a glucoamylase promoter. Two recombinant Xyl10As (rXyl10A-I, 53kDa, and rXyl10A-II, 51kDa) were purified that have slightly different molecular weights based on SDS-PAGE. The rXyl10As had the same physicochemical and enzymatic properties as wtXyl10A: high thermostability (Tm 80.5°C), optimum pH 5.0 and specific activity 232-251U/mg for birchwood xylan. The molecular weights of N-deglycosylated rXyl10As were consistent with that of wild-type Xyl10A (wtXyl10A, 51kDa).
authors.affiliation: Biomass Refinery Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 3-11-32 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046, Japan.
authors.forename: Seiichiro
authors.initials: S
authors.lastname: Kishishita
authors.affiliation: Biomass Refinery Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 3-11-32 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046, Japan.
authors.forename: Miho
authors.initials: M
authors.lastname: Yoshimi
authors.affiliation: Biomass Refinery Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 3-11-32 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Fujii
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, CO 80401, USA.
authors.forename: Larry E
authors.initials: LE
authors.lastname: Taylor
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, CO 80401, USA.
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Decker
authors.affiliation: Biomass Refinery Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 3-11-32 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046, Japan.
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Ishikawa
authors.affiliation: Biomass Refinery Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 3-11-32 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046, Japan. Electronic address: inoue-h@aist.go.jp.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Inoue
mesh_terms: D000164:Acremonium / Q000201:enzymology*; D002482:Cellulose / Q000737:chemistry; D003001:Cloning, Molecular; D043364:Endo-1,4-beta Xylanases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D015966:Gene Expression Regulation, Fungal; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics; D011401:Promoter Regions, Genetic; D017386:Sequence Homology, Amino Acid; D014990:Xylans / Q000737:chemistry / Q000378:metabolism*
chemical_list: D014990:Xylans; D002482:Cellulose; D005087:Glucan 1,4-alpha-Glucosidase; D043364:Endo-1,4-beta Xylanases
keywords: AA; CMC; Glycoside hydrolase family 10; Glycosylation; Hemicellulose; Homologous expression; SF; Solka-Floc cellulose; TSA; Thermostable xylanase; amino acids; carboxymethylcellulose; thermal shift assay