Purification and some properties of Thermotoga neapolitana thermostable xylanase B expressed in E. coli cells.
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.
Velikodvorskaya
T V
TV
Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, Russia.
Volkov IYu
Vasilevko
V T
VT
Zverlov
V V
VV
Piruzian
E S
ES
D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D043364:Endo-1,4-beta Xylanases; D004926:Escherichia coli / Q000235:genetics; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology*; D007525:Isoelectric Focusing; D007700:Kinetics; D013379:Substrate Specificity; D014995:Xylosidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D014995:Xylosidases; D043364:Endo-1,4-beta Xylanases