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.