TY - JOUR
T1 - Cellulose-Inducible Xylanase Xyl10A from Acremonium cellulolyticus: Purification, Cloning and Homologous Expression
AU - Kishishita, Seiichiro
AU - Yoshimi, Miho
AU - Fujii, Tatsuya
AU - Taylor, Larry E.
AU - Decker, Stephen R.
AU - Ishikawa, Kazuhiko
AU - Inoue, Hiroyuki
PY - 2014
Y1 - 2014
N2 - 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, 53 kDa, and rXyl10A-II, 51 kDa) 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 232-251 U/mg for birchwood xylan. The molecular weights of N-deglycosylated rXyl10As were consistent with that of wild-type Xyl10A (wtXyl10A, 51 kDa).
AB - 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, 53 kDa, and rXyl10A-II, 51 kDa) 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 232-251 U/mg for birchwood xylan. The molecular weights of N-deglycosylated rXyl10As were consistent with that of wild-type Xyl10A (wtXyl10A, 51 kDa).
KW - Glycoside hydrolase family 10
KW - Glycosylation
KW - Hemicellulose
KW - Homologous expression
KW - Thermostable xylanase
UR - http://www.scopus.com/inward/record.url?scp=84888594034&partnerID=8YFLogxK
U2 - 10.1016/j.pep.2013.10.020
DO - 10.1016/j.pep.2013.10.020
M3 - Article
C2 - 24211645
AN - SCOPUS:84888594034
SN - 1046-5928
VL - 94
SP - 40
EP - 45
JO - Protein Expression and Purification
JF - Protein Expression and Purification
ER -