Sao Carlos Institute of Physics, University of Sao Paulo, São Carlos, São Paulo, Brazil.
School of Biological Sciences, University of Canterbury, New Zealand.
N Biotechnol. 2019 Nov 25;53:57-64. doi: 10.1016/j.nbt.2019.07.002. Epub 2019 Jul 9.
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 °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 β-d-xylosidase. Using 4-nitrophenyl β-d-xylopyranoside (pNPX) as a substrate, the K, Vmax, k and k/K kinetic parameters were determined as 0.25 ± 0.03 mM, 889.47 ± 28.54 U/mg, 39.20 s and 156.8 mM s, 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 β-d-xylosidase.
从需氧嗜热菌 Thermogemmatispora sp. T81（A4R35_07040 基因座）的基因组中鉴定到糖苷水解酶家族 5 (GH5)亚家族 22 基因，命名为 T81Xyl5_22A。该基因在大肠杆菌中被克隆并异源表达，并对其进行了生化特性分析。重组酶在 pH5.0 和 65°C 时具有最佳的催化活性，对山毛榉木聚糖和黑麦阿拉伯木聚糖均具有活性。它水解山毛榉木聚糖只产生木糖分子，表明它是 GH5 家族的β-d-木糖苷酶。使用 4-硝基苯基β-d-木吡喃糖苷（pNPX）作为底物，确定了 K、Vmax、k 和 k/K 动力学参数分别为 0.25±0.03mM、889.47±28.54U/mg、39.20s 和 156.8mM•s。小角 X 射线散射（SAXS）数据能够重建酶的低分辨率分子包络，并表明它在溶液中形成二聚体。据我们所知，这是首次描述耐热细菌 GH5 家族的β-d-木糖苷酶。
