Purification and characterization of a thermostable beta-xylosidase from Thermoanaerobacter ethanolicus

Abstract

A highly thermostable 13-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 towardsp-NO2-phenyl xyloside (pNPX) (substrate concentration for half-maximal activity = 0.018 mM at 82°C and pH 5.0) but the highest specific activity with p-NO2-phenylarabinofuranoside. T0pt, s mInt the temperature for the maximum initial activity in a 5-min assay of the purified enzyme, was observed around pH 5.9 and 93°C; however at 65 and 82°C, the pH optimum was 5.0 to 5.2, and at this pH the maximal initial activity was observed at 82°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°C in the absence of substrate reduced the activity to around 75%. At 86°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.

Genetic organization, sequence and biochemical characterization of recombinant -xylosidase from Thermoanaerobacterium saccharolyticum strain B6A-RI

“…This is consistent with the observation that most of the P-xylosidase activity from T . sacchurolyticum strain BGA-RI was intracellular, as has been found for the closely related thermophilic anaerobes Thermoatzaerobacter strain B6A (Hespell, 1992) (now Thermoanaerobacterium saccharolyticum strain B6A) and Thermoanaerobacter ethanolicus JW200 (Shao & Wiegel, 1992). The deduced amino acid sequence of thermostable T. saccharolyticum strain B6A-RI P-xylosidase exhibited a higher degree of homology to thermostable P-xylosidase from C. saccharolyticum (Luthi et al, 1990) than to those of less thermostable P-xylosidases from Bacillus (Xu et al, 1991) and Butyriuibrio (Sewell et al, 1989) species.…”

“…Relatively little work has been done on P-xylosidases from thermophilic bacteria (Bachmann & McCarthy, 1989;Hudson et al, 1991 ;Nanmori et al, 1990;Shao & Wiegel, 1992). To date, only P-xylosidase genes from Bacillus pumilus (Panbangred et al, 1984;Xu et al, 199 l), Bacillus polymixa (Sandhu & Kennedy, 1984), Bacillus subtilis (Bernier et al, 1983), Butyrivibrio Jibrisolvens (Sewell et al, 1989), and Caldocellum saccharolyticum (Luthi et al, 1990) have been cloned into Escherichia coli.…”

Characterization of a Novel β-Xylosidase, XylC, from Thermoanaerobacterium saccharolyticum JW/SL-YS485

“…If the affinity was determined, most xylosidases, especially those from bacteria, have K i values for xylose of between 2 and 10 mM. Additionally, many xylosidases exhibit high activity against pNP-xyloside as well as some activity against p-nitrophenyl-␣-L-arabinofuranoside (pNPAF) (26). However, XylC from T. saccharolyticum JW/SL-YS485 did not have any arabinosidase activity.…”

“…The ␤-xylosidases from the anaerobic thermophilic Bacteria include those from Thermotoga maritima (35), Thermotoga sp. FjSS3-B.1 and Thermotoga neapolitana (31), and Thermoanaerobacterium brockii and Thermoanaerobacter ethanolicus JW200 (15,26) (all GH 3); Clostridium stercorarium (GH 3 and 39) (22); Thermoanaerobacterium saccharolyticum (GH 39) (11,13); Caldicellulosiruptor saccharolyticus (GH 43) (14); and Thermoanaerobacter italicus and Thermoanaerobacter mathrani (GH 52) (cited at InterPro protein matches [http://www.ebi.ac.uk /interpol/]). The thermostable enzymes are of interest due to their structural features which confer thermostability as well as for potential advantages in industrial processes.…”

Isolation, analysis, and expression of two genes from Thermoanaerobacterium sp. strain JW/SL YS485: a beta-xylosidase and a novel acetyl xylan esterase with cephalosporin C deacetylase activity

“…Recombinant xylosidase activity was measured by using crude E. coli lysates which had been clarified by centrifugation. Xylosidase activity was determined in a 4-min assay performed at 70°C, pH 6.0, with a p-nitrophenyl-␤-D-xylopyranoside (pNPX) substrate as described elsewhere (31). Other p-nitrophenyl (pNP) conjugates, including pNP-␤-D-arabinoside, pNP-␤-D-arabinofuranoside, pNP-␤-D-glucoside, and pNP-␤-D-mannoside, were tested under the same conditions.…”