title: Purification, biochemical characterization, and structure of recombinant endo-1,4-β-xylanase XylE.
abstract: The gene xylE encoding endo-1,4-β-xylanase from the 10th family of glycosyl hydrolases produced by the mycelial fungus Penicillium canescens has been expressed under the control of the strong promoter of the bgaS gene encoding β-galactosidase from P. canescens. As a result, a strain-producer of endoxylanase XylE was developed. The recombinant enzyme was isolated and purified to homogeneity with specific activity of 50 U/mg. The physicochemical and biochemical properties of the endoxylanase were studied. The maximal enzymatic activity was observed at pH 6.0 and 70°C. Endoxylanase XylE was shown to be a highly thermostable enzyme with half-inactivation period τ(1/2) of 7 h at 60°C. The kinetic parameters were 0.52 mg/ml (K(m)) and 75 µmol/min per mg (V(max)) using birch xylan as the substrate. Crystals of endoxylonase XylE were obtained, and the 3D structure was solved at 1.47 Å resolution. The 3D structure of an endo-1,4-β-xylanase from the 10th family containing carbohydrate and unique cyclic structure located at the C-terminus of the polypeptide chain was obtained for the first time.
authors.affiliation: Bach Institute of Biochemistry, Russian Academy of Sciences, 119071 Moscow, Russia.
authors.forename: T V
authors.initials: TV
authors.lastname: Fedorova
authors.forename: A M
authors.initials: AM
authors.lastname: Chulkin
authors.forename: E A
authors.initials: EA
authors.lastname: Vavilova
authors.forename: I G
authors.initials: IG
authors.lastname: Maisuradze
authors.forename: A A
authors.initials: AA
authors.lastname: Trofimov
authors.forename: I N
authors.initials: IN
authors.lastname: Zorov
authors.forename: V P
authors.initials: VP
authors.lastname: Khotchenkov
authors.forename: K M
authors.initials: KM
authors.lastname: Polyakov
authors.forename: S V
authors.initials: SV
authors.lastname: Benevolensky
authors.forename: O V
authors.initials: OV
authors.lastname: Koroleva
authors.forename: V S
authors.initials: VS
authors.lastname: Lamzin
mesh_terms: D018360:Crystallography, X-Ray; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010407:Penicillium / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D027981:Symporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D027981:Symporters; C033903:xylE protein, E coli; D043364:Endo-1,4-beta Xylanases