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Purification, Biochemical Characterization, and Structure of Recombinant Endo-1,4-β-xylanase XylE

T. V. Fedorova1, A. M. Chulkin1, E. A. Vavilova1, I. G. Maisuradze1, A. A. Trofimov1,2, I. N. Zorov1, V. P. Khotchenkov1, K. M. Polyakov1,2, S. V. Benevolensky1, O. V. Koroleva1*, and V. S. Lamzin3

1Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky pr. 33, 119071 Moscow, Russia; fax: (495) 954-2732; E-mail: koroleva@inbi.ras.ru

2Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, 119991 Moscow, Russia; fax: (499) 135-1405; E-mail: isinfo@eimb.ru

3European Molecular Biology Laboratory (EMBL), c/o DESY, Build. 25A, Notkestrasse 85, 22603 Hamburg, Germany; fax: +49(0)4089-902149; E-mail: info@embl-hamburg.de

* To whom correspondence should be addressed.

Received June 20, 2012; Revision received July 7, 2012
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 τ1/2 of 7 h at 60ºC. The kinetic parameters were 0.52 mg/ml (Km) and 75 µmol/min per mg (Vmax) 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.
KEY WORDS: Penicillium canescens, xylanase, homologous expression, XylE, 3D structure, biochemical and physicochemical properties, substrate specificity

DOI: 10.1134/S0006297912100112