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.

Comparative characterization of xylanases XylA and XylE from Penicillium canescens fungi

“…1) according to which the molecular weights of XylA and XylE were about 30 and 40 kDa. These molecular weights are in good agreement with the published data [9,11]. Both enzymes were identified η cor η extract η buffer -( ) / η buffer C × ( ) , = by MALDI mass spectrometric peptide mapping.…”

“…In general, the specific activities of XylA and XylE against glucuronoxylan and arabinoxylan and such important characteristics as the pH and temperature optima of activity were in satisfactory agreement with the corre sponding parameters described previously for these xylanases in the literature [9,11]. However, the values of the Michaelis constant of XylA and XylE for both types of xylans were somewhat higher, which may be caused by the differences in the structures of the xylans used in the different studies.…”

“…The xylanases XylA and XylE of glycoside hydrolase family 10 were previously isolated from Penicillium canescens fungi; the properties of these enzymes were partially studied and the respective genes were sequenced [8][9][10][11]. However, some parameters impor tant for the practical use of these enzymes, e.g., their resistance to the inhibitor proteins from cereals, have never been studied.…”

Insight into the functional roles of Glu175 in the hyperthermostable xylanase XYL10C-ΔN through structural analysis and site-saturation mutagenesis

“…The crystal structure of XYL10C-ΔN was solved at a high resolution of 1.6 Å by molecular replacement using the structure of XylE (PDB code: 4F8X) from Penicillium scopiformis [ 31 ] as a search model. The structure determination procedures are listed in Additional file 1 : Table S1.…”

“…In the present study, after removal of this N-terminal sequence, the variant XYL10C-ΔN retained its hyperthermophilic characteristic but showed a 1.8- and 2.7-fold improvement in catalytic efficiency and specific activity, respectively. XYL10C-ΔN shares high-sequence identity with the Talaromyces leycettanus xylanase Tl Xyn10A (54%) [ 40 ] and Penicillium canescens xylanase XylE (53%) [ 31 ], but has a higher specific activity (8700 vs. 2240 and 50 U/mg) and catalytic efficiency (8800 vs. 1626 and 92 mL/s/mg). Moreover, XYL10C-ΔN exhibits similar thermostability to Tl Xyn10A (no enzyme activity loss after 1 h-incubation at 80 °C), which is better than XylE (completely inactive after 30 min incubation at 70 °C).…”