Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University, 114 Sukhumvit 23, Bangkok 10110, Thailand.
Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan.
Enzyme Microb Technol. 2018 May;112:22-28. doi: 10.1016/j.enzmictec.2018.01.009. Epub 2018 Jan 31.
We previously described thermotolerant Streptomyces sp. SWU10, which produced four endo-xylanases and one xylosidase able to digest xylan backbones. To achieve arabinoxylan degradation, the swu62A gene was cloned and overexpressed in Escherichia coli, and the recombinant enzyme, termed SWUAbf62A, was characterized. The 438 amino acids of SWUAbf62A revealed Glyco_hydro_62 and closely related with putative α-l-arabinofuranosidases belonging to glycoside hydrolase family 62. SWUAbf62A was purified in two steps, Ni-affinity and size-exclusion column chromatographies, and its molecular mass without signal peptide was determined to be 49 kDa. SWUAbf62A showed optimum activity at pH 5.0 and 50 °C, and more than 70% of its initial enzymatic activity remained after incubation at pH 4.1-10.5, while SWUAbf62A lost all activity after 1 h at 60 °C. SWUAbf62A activity was stimulated by Ba, Ca, and Mn and decreased by Ag, Cu, Fe, and EDTA. SWUAbf62A had no activity towards p-nitrophenyl-α-l-arabinofuranoside or p-nitrophenyl-β-d-xylopyranoside synthetic substrates. On the other hand, SWUAbf62A had the highest activity against wheat arabinoxylan, with a specific activity of 1.29 U/mg, and was also active against sugar beet arabinan, with a specific activity of 0.14 U/mg; these results indicated that SWUAbf62A is an arabinoxylan arabinofuranohydrolase. Using H-NMR analysis, SWUAbf62A was found to release l-arabinofuranoses singly linked to O-3 of wheat arabinoxylan. In addition, SWUAbf62A acted synergistically with endo-xylanase (XynSW3) and α-l-arabinofuranosidase, which releases arabinose linked to O-3 of double-substituted xylose residues on arabinoxylan, to digest the wheat arabinoxylan. SWUAbf62A is an important debranching enzyme for hydrolysis of hemicelluloses to monosaccharides and can be applied in various industrial biotechnologies.
我们之前曾描述过一种耐热链霉菌 SWU10，它能产生四种内切木聚糖酶和一种木糖苷酶，能够消化木聚糖骨架。为了实现阿拉伯木聚糖的降解，我们克隆了 swu62A 基因并在大肠杆菌中过表达，得到了一种重组酶，称为 SWUAbf62A，并对其进行了表征。SWUAbf62A 的 438 个氨基酸揭示了 Glyco_hydro_62，与属于糖苷水解酶家族 62 的假定 α-l-阿拉伯呋喃糖苷酶密切相关。SWUAbf62A 通过 Ni-亲和和分子筛层析两步进行纯化，其不带有信号肽的分子质量被确定为 49 kDa。SWUAbf62A 在 pH 5.0 和 50°C 时表现出最佳活性，在 pH 4.1-10.5 孵育后仍保留超过初始酶活的 70%，而在 60°C 孵育 1 小时后则完全丧失活性。SWUAbf62A 的活性被 Ba、Ca 和 Mn 刺激，而被 Ag、Cu、Fe 和 EDTA 抑制。SWUAbf62A 对 p-硝基苯基-α-l-阿拉伯呋喃糖苷或 p-硝基苯基-β-d-吡喃木糖苷合成底物没有活性。另一方面，SWUAbf62A 对小麦阿拉伯木聚糖的活性最高，比活为 1.29 U/mg，对糖甜菜阿拉伯聚糖也有活性，比活为 0.14 U/mg；这些结果表明 SWUAbf62A 是一种阿拉伯木聚糖阿拉伯呋喃水解酶。通过 H-NMR 分析发现，SWUAbf62A 能够单独释放与小麦阿拉伯木聚糖 O-3 相连的 l-阿拉伯呋喃糖。此外，SWUAbf62A 与内切木聚糖酶（XynSW3）和 α-l-阿拉伯呋喃糖苷酶协同作用，后者释放与阿拉伯木聚糖双取代木糖残基 O-3 相连的阿拉伯糖，从而消化小麦阿拉伯木聚糖。SWUAbf62A 是一种重要的支链酶，可用于水解半纤维素生成单糖，可应用于各种工业生物技术。
