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Biochemistry (Moscow)

, Volume 77, Issue 10, pp 1190–1198 | Cite as

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

  • T. V. Fedorova
  • A. M. Chulkin
  • E. A. Vavilova
  • I. G. Maisuradze
  • A. A. Trofimov
  • I. N. Zorov
  • V. P. Khotchenkov
  • K. M. Polyakov
  • S. V. Benevolensky
  • O. V. KorolevaEmail author
  • V. S. Lamzin
Article

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 τ1/2 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.

Key words

Penicillium canescens xylanase homologous expression XylE 3D structure biochemical and physicochemical properties substrate specificity 

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Copyright information

© Pleiades Publishing, Ltd. 2012

Authors and Affiliations

  • T. V. Fedorova
    • 1
  • A. M. Chulkin
    • 1
  • E. A. Vavilova
    • 1
  • I. G. Maisuradze
    • 1
  • A. A. Trofimov
    • 1
    • 2
  • I. N. Zorov
    • 1
  • V. P. Khotchenkov
    • 1
  • K. M. Polyakov
    • 1
    • 2
  • S. V. Benevolensky
    • 1
  • O. V. Koroleva
    • 1
    Email author
  • V. S. Lamzin
    • 3
  1. 1.Bach Institute of BiochemistryRussian Academy of SciencesMoscowRussia
  2. 2.Engelhardt Institute of Molecular BiologyRussian Academy of SciencesMoscowRussia
  3. 3.European Molecular Biology Laboratory (EMBL)HamburgGermany

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