Expression and characterization of a thermostable beta-xylosidase from the hyperthermophile, Thermotoga maritima.
A thermostable beta-xylosidase from a hyperthermophilic bacterium, Thermotoga maritima, was over-expressed in Escherichia coli using the T7 polymerase expression system. The expressed beta-xylosidase was purified in two steps, heat treatment and immobilized metal affinity chromatography, and gave a single band on SDS-PAGE. The maximum activity on p-nitrophenyl beta-D-xylopyranoside was at 90 degrees C and pH 6.1. The purified enzyme had a half-life of over 22-min at 95 degrees C, and retained over 57% of its activity after holding a pH ranging from 5.4 to 8.5 for 1 h at 80 degrees C. Among all tested substrates, the purified enzyme had specific activities of 275, 50 and 29 U mg(-1) on pNPX, pNPAF, and pNPG, respectively. The apparent Michaelis constant of the beta-xylosidase was 0.13 mM for p NPX with a V (max) of 280 U mg(-1). When the purified beta-xylosidase was added to xylanase, corncob xylan was hydrolized completely to xylose.
Xue
Yemin
Y
College of Life Science, Nanjing Normal University, P.R. China.
Shao
Weilan
W
D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D013696:Temperature; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics*; D014995:Xylosidases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics
D011994:Recombinant Proteins; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase