Purification and some properties of Thermotoga neapolitana thermostable xylanase B expressed in E. coli cells

• Published in: Biochemistry (Moscow) Vol. 62; no. 1; pp. 66 - 70
• Main Authors: Velikodvorskaya, T V, Volkov IYu, Vasilevko, V T, Zverlov, V V, Piruzian, E S
• Format: Journal Article
• Language: English
• Published: United States 01.01.1997
• Subjects: Chromatography, Ion Exchange; Cloning, Molecular; Electrophoresis, Polyacrylamide Gel; Endo-1,4-beta Xylanases; Escherichia coli - genetics; Gram-Negative Anaerobic Bacteria - enzymology; Isoelectric Focusing; Kinetics; Substrate Specificity; Xylosidases - genetics; Xylosidases - isolation & purification; Xylosidases - metabolism
• ISSN: 0006-2979; 1608-3040

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 degrees 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 homogeneous 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 degrees C. The xylanase is stable to heating at 70 degrees C for 4 h. The enzyme is inactivated by 50% at 80, 90, and 100 degrees 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.