Centro de Ciências Exatas e Tecnológicas, Universidade Estadual do Oeste do Paraná, Rua Universitária, 2069, Cascavel, Paraná, 85814-110, Brazil.
Appl Biochem Biotechnol. 2012 Dec;168(8):2218-29. doi: 10.1007/s12010-012-9931-1. Epub 2012 Oct 11.
In the present work, the gene xynB2, encoding a β-xylosidase II of the Glycoside Hydrolase 39 (GH39) family, of Caulobacter crescentus was cloned and successfully overexpressed in Escherichia coli DH10B. The recombinant protein (CcXynB2) was purified using nickel-Sepharose affinity chromatography, with a recovery yield of 75.5 %. CcXynB2 appeared as a single band of 60 kDa on a sodium dodecyl sulfate polyacrylamide gel and was recognized by a specific polyclonal antiserum. The predicted CcXynB2 protein showed a high homology with GH39 β-xylosidases of the genus Xanthomonas. CcXynB2 exhibited an optimal activity at 55 °C and a pH of 6. CcXynB2 displayed stability at pH values of 4.5-7.5 for 24 h and thermotolerance up to 50 °C. The K (M) and V (Max) values were 9.3 ± 0.45 mM and 402 ± 19 μmol min(-1) for ρ-nitrophenyl-β-D-xylopyranoside, respectively. The purified recombinant enzyme efficiently produced reducing sugars from birchwood xylan and sugarcane bagasse fibers pre-treated with a purified xylanase. As few bacterial GH39 family β-xylosidases have been characterized, this work provides a good contribution to this group of enzymes.
在本工作中，克隆了新月柄杆菌的基因 xynB2，该基因编码糖苷水解酶 39（GH39）家族的β-木糖苷酶 II。成功在大肠杆菌 DH10B 中过表达了重组蛋白（CcXynB2）。使用镍琼脂糖亲和层析法纯化重组蛋白，回收率为 75.5%。SDS-PAGE 上 CcXynB2 呈现出 60 kDa 的单一带，并被特异性的多克隆抗血清识别。预测的 CcXynB2 蛋白与黄单胞菌属的 GH39 β-木糖苷酶具有高度同源性。CcXynB2 在 55°C 和 pH6 时表现出最佳活性。CcXynB2 在 pH4.5-7.5 下稳定 24 小时，热稳定性高达 50°C。Km 和 Vmax 值分别为 9.3±0.45 mM 和 402±19 μmol min-1，用于 ρ-硝基苯-β-D-木吡喃糖苷。纯化的重组酶可有效地从预处理过的桦木木聚糖和甘蔗渣纤维中产生还原糖。由于很少有细菌 GH39 家族的β-木糖苷酶被表征，因此这项工作为这组酶提供了很好的贡献。
