Purification and properties of an aryl beta-xylosidase from a cellulolytic extreme thermophile expressed in Escherichia coli.
An aryl beta-xylosidase was purified to homogeneity from an Escherichia coli strain containing a recombinant plasmid carrying a beta-xylosidase (EC 3.2.1.37) gene from the extremely thermophilic anaerobic bacterium isolate Tp8T6.3.3.1 ('Caldocellum saccharolyticum'). It has a pI of 4.3 and shows optimal activity at pH 5.7. The enzyme is highly specific, acting on o- and p-nitrophenyl beta-D-xylopyranosides and minimally on p-nitrophenyl alpha-L-arabinopyranoside. It does not act on xylobiose. The Km for p-nitrophenyl beta-D-xylopyranoside at the optimum pH for activity is 10 mM, and at pH 7.0 is 6.7 mM. Xylose is a competitive inhibitor with Ki 40 mM. Thermal inactivation follows first-order kinetics at 65 and 70 degrees C with t1/2 values of 4.85 h and 40 min respectively. The t1/2 at 70 degrees C is increased 3-fold and 4-fold by the addition of 0.5 mg of BSA/ml and 2 mM-dithiothreitol respectively.
Hudson
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Microbial Biochemistry and Biotechnology Research Unit, University of Waikato, Hamilton, New Zealand.
Schofield
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Coolbear
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Daniel
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Morgan
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D001421:Bacteria, Anaerobic / Q000201:enzymology* / Q000235:genetics; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006358:Hot Temperature; D006882:Hydroxyapatites; D007700:Kinetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D006882:Hydroxyapatites; D011994:Recombinant Proteins; D017886:Durapatite; D006026:Glycoside Hydrolases; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase; C026579:exo-1,4-beta-D-xylosidase