A novel thermostable GH5 β-xylosidase from Thermogemmatispora sp. T81

A glycoside hydrolase family 5 (GH5) subfamily 22 gene, designated T81Xyl5_22A, was identified in the genome of the aerobic thermophilic bacterium, Thermogemmatispora sp. T81 (locus A4R35_07040). The gene was cloned and heterologously expressed in Escherichia coli and the gene product characterized biochemically. The recombinant enzyme had an optimal catalytic activity at pH5.0 and 65 degrees C, and was active against beechwood xylan and rye arabinoxylan. It yielded only xylose molecules as products of beechwood xylan hydrolysis, indicating that it is a GH5 family beta-D-xylosidase. Using 4-nitrophenyl beta-D-xylopyranoside (pNPX) as a substrate, the K-M, Vmax, k(cat) and k(cat)/K-M kinetic parameters were determined as 0.25 +/- 0.03 mM, 889.47 +/- 28.54 U/mg, 39.20 s(-1) and 156.8 mM(-1) s(-1), respectively. Small-angle X-ray scattering (SAXS) data enabled reconstruction of the enzyme's low-resolution molecular envelope and revealed that it formed dimers in solution. As far as we are aware, this is the first description of a thermostable bacterial GH5 family beta-D-xylosidase.