title: Cloning of a xylanase gene from the ruminal fungus Neocallimastix patriciarum 27 and its expression in Escherichia coli.
abstract: An endo-beta-1,4-xylanase gene was cloned from Neocallimastix patriciarum 27 in the bacteriophage vector lambda gtWES lambda B and was subcloned into the plasmid vectors pUC18 and pUC19 in which xylanase activity was expressed in both orientations. The xylanase was located in the periplasmic space of the host, Escherichia coli HB101. The pH and temperature optima for periplasmic xylanase activity were 6.2 and 40 degrees C, respectively, and the Km for oat spelt xylan hydrolysis was 0.89 mg.mL-1. It also exhibited hydrolytic activity on carboxymethyl cellulose that was equivalent to 28% of the activity exhibited by the enzyme on xylan. It bound to crystalline cellulose, but lacked hydrolytic activity on amorphous cellulose. SDS-PAGE followed by zymogram analysis showed active bands of 68, 58, and 51 kDa. Isoelectric focusing in gels combined with zymogram analysis showed one band of xylanase activity with a pI of 3.6.
authors.affiliation: Department of Microbiology, University of Guelph, Ont., Canada.
authors.forename: J M
authors.initials: JM
authors.lastname: Lee
authors.forename: Y
authors.initials: Y
authors.lastname: Hu
authors.forename: H
authors.initials: H
authors.lastname: Zhu
authors.forename: K J
authors.initials: KJ
authors.lastname: Cheng
authors.forename: P J
authors.initials: PJ
authors.lastname: Krell
authors.forename: C W
authors.initials: CW
authors.lastname: Forsberg
mesh_terms: D000818:Animals; D002417:Cattle; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005658:Fungi / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012417:Rumen / Q000382:microbiology*; D043325:Xylan Endo-1,3-beta-Xylosidase
chemical_list: D006026:Glycoside Hydrolases; D043325:Xylan Endo-1,3-beta-Xylosidase