Purification, biochemical characterization, and structure of recombinant endo-1,4-ß-xylanase XylE.

The gene xylE encoding endo-1,4-ß-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 ß-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-ß-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.

[Multigene families of endo-(1-4)-beta-xylanases of Penicillium canescens].

Four novel genes of the enzymes of the endoxylanase (EC 3.2.1.8) families found in the mycelial fungus Penicillium canescens have been cloned. The xylB, xylC, and xylD genes encode endoxylanases of glycosyl hydrolase family 11; the xylEgene, those of family 10. In the promoter region of the xylB, xylC, and xylD genes, the binding sequences for the protein activator of xylanolytic gene transcription have been found; the promoter region of the xylB gene contains the binding sequences for the catabolite repression protein. Since the TATAA sequence, which is an element of the minimal eukaryotic promoter, has not been found in the promoter region of the xylC gene, in contrast to those of the xylB and xylD genes, it may be assumed that this gene is silent. Comparative phylogenetic analysis has shown that the cloned genes are highly homologous to some endoxylanase genes of mycelial fungi of the genera Penicillium and Aspergillus. However, within the species P. canescens, they exhibit a low homology both within and between families, and they diverge into different branches of the phylogenetic tree, which suggest divergence of the genes of this group at an early stage of evolution.