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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.