Purification, biochemical characterization, and structure of recombinant endo-1,4-β-xylanase XylE
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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 wordsPenicillium canescens xylanase homologous expression XylE 3D structure biochemical and physicochemical properties substrate specificity
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- 10.Serebryany, V. A., Vavilova, E. A., and Chulkin, A. M. (2002) Appl. Biochim. Microbiol., 38, 495–501.Google Scholar
- 11.Maisuradze, I. G., Chulkin, A. M., Vavilova, E. A., and Benevolensky, S. V. (2011) Genetika, 47, 1–9.Google Scholar
- 12.Benevolensky, S. V., Vavilova, E. A., Chulkin, A. M., Abyanova, A. R., Maisuradze, I. G., Zatsepin, S. S., Novozhilov, E. V., and Benevolensky, M. S. (2011) RF Patent No. 2412246.Google Scholar
- 13.Nikolaev, I. V., Bekker, O. B., Serebryany, V. A., Chulkin, A. M., and Vinetsky, Yu. P. (1999) Biotekhnologiya, 3, 3–13.Google Scholar
- 14.Maniatis, T., Fritsch, E., and Sambrook, J. (1984) Molecular Cloning: A Laboratory Manual [Russian translation], Mir, Moscow.Google Scholar
- 15.Nikolaev, I. V., and Vinetsky, Yu. P. (1998) Biochemistry (Moscow), 63, 1294–1298.Google Scholar
- 16.Nikolaev, I. V., Vinetsky, Yu. P., Bekker, O. B., and Serebryany, V. A. (1999) RF Patent No. 2126049.Google Scholar
- 18.Long, F., Vagin, A., Young, P., and Murshudov, G. N. (2008) Acta Cryst., D64, 125–132.Google Scholar
- 19.Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Acta Cryst., D53, 240–255.Google Scholar
- 20.Emsley, P., and Cowtan, K. (2004) Acta Cryst., D60, 2126–2132.Google Scholar
- 21.Winn, M. D., Ballard, C. C., Cowtan, K. D., Dodson, E. J., Emsley, P., Evans, P. R., Keegan, R. M., Krissinel, E. B., Leslie, A. G. W., McCoy, A., McNicholas, S. J., Murshudov, G. N., Pannu, N. S., Potterton, E. A., Powell, H. R., Read, R. J., Vagin, A., and Wilson, K. S. (2011) Acta Cryst., D53, 235–242.Google Scholar