Purification and Some Properties of Thermotoga neapolitana Thermostable Xylanase B Expressed in E. coli Cells

T. V. Velikodvorskaya,^1,2 I. Yu. Volkov,¹ V. T. Vasilevko,¹ V. V. Zverlov,¹ and E. S. Piruzian¹

¹Institute of Molecular Genetics, Russian Academy of Sciences, pl. Kurchatova 46, Moscow, 123182 Russia; fax: (7-095) 196-02-21; E-mail: uimg@img.rc.ac.ru

²To whom correspondence should be addressed.

Submitted July 25, 1996; revision submitted October 1, 1996.

A xylanase was purified from the recombinant strain E. coli TG1 carrying the pTT32 vector with a fragment of the Thermotoga neapolitana chromosomal DNA. The enzyme was purified 419-fold with 36% yield after heating at 70°C and pH 4.5 and subsequent ion-exchange chromatography. By polyacrylamide gel electrophoresis in the presence of SDS, the molecular weight of the apparently homogenous protein is 39 kD. By isoelectric focusing, the protein is of a single form with pI = 5.9. The optimal pH for hydrolysis is 5.5, and the optimal temperature is 90°C. The xylanase is stable to heating at 70°C for 4 h. The enzyme is inactivated by 50% at 80, 90, and 100°C after 227, 162, and 30 min, respectively. Enzyme activity was tested using xylans and glucans as substrates. By thin-layer chromatography of the xylan hydrolysis products, the enzyme was classified as an endoxylanase.

KEY WORDS: xylanase, cloning, substrate specificity, xylooligosaccharides, xylan, Thermotoga neapolitana.

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