Purification and characterization of a thermostable beta-xylosidase from Thermoanaerobacter ethanolicus.
A highly thermostable beta-xylosidase, exhibiting similarly high activities for arylxylose and arylarabinose, was purified (72-fold) to gel electrophoretic homogeneity from the ethanologenic thermophilic anaerobe Thermoanaerobacter ethanolicus. The isoelectric point is pH 4.6; the apparent molecular weight is around 165,000 for the native enzyme (gel filtration and gradient polyacrylamide gel electrophoresis) and 85,000 for the two subunits (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The enzyme exhibited the highest affinity towards p-NO2-phenyl xyloside (pNPX) (substrate concentration for half-maximal activity = 0.018 mM at 82 degrees C and pH 5.0) but the highest specific activity with p-NO2-phenylarabinofuranoside. T(opt), 5 min, the temperature for the maximum initial activity in a 5-min assay of the purified enzyme, was observed around pH 5.9 and 93 degrees C; however at 65 and 82 degrees C, the pH optimum was 5.0 to 5.2, and at this pH the maximal initial activity was observed at 82 degrees C (pH 5.0 to 5.5). The pH curves and temperature curves for arylxylosides as substrates differed significantly from those for arylarabinosides as substrates. An incubation for 3 h at 82 degrees C in the absence of substrate reduced the activity to around 75%. At 86 degrees C the half-life was around 15 min. With pNPX as the substrate, an Arrhenius energy of 69 kJ/mol was determined. The N-terminal sequence did not reveal a high similarity to those from other published enzyme sequences.
Shao
W
W
Department of Microbiology, University of Georgia, Athens 30602.
Wiegel
J
J
D000595:Amino Acid Sequence; D001421:Bacteria, Anaerobic / Q000201:enzymology*; D001426:Bacterial Proteins; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000096:biosynthesis / Q000302:isolation & purification*; D006027:Glycosides / Q000378:metabolism; D016988:Gram-Positive Asporogenous Rods, Irregular / Q000201:enzymology* / Q000254:growth & development; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011489:Protein Denaturation; D013379:Substrate Specificity; D014995:Xylosidases / Q000096:biosynthesis / Q000302:isolation & purification*
D001426:Bacterial Proteins; D006027:Glycosides; C010807:xylosides; D006026:Glycoside Hydrolases; C410514:XARB protein, Thermoanaerobacter ethanolicus; D014995:Xylosidases