Carbohydrate Enzyme Biotechnology Laboratory, Department of Biosciences and Bioengineering, Indian Institute of Technology, Guwahati, Guwahati, Assam 781039, India.
NZYTech - Genes & Enzymes, Estrada do Paço do Lumiar, Campus do Lumiar, Edifício E - R/C, 1649-038 Lisbon, Portugal; CIISA - Faculdade de Medicina Veterinária, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisbon, Portugal.
Carbohydr Polym. 2024 Sep 15;340:122295. doi: 10.1016/j.carbpol.2024.122295. Epub 2024 May 23.
GH30 xylobiohydrolases, an expanding enzyme category, need deeper insights for optimal use. The primary aim of this study was to characterize a new xylobiohydrolase, AcGH30A of GH30 family from Acetivibrio clariflavus. The gene encoding AcGH30A was cloned using pET28a(+) vector and expressed in E. coli BL21(DE3) cells. AcGH30A was purified by immobilized metal-ion affinity chromatography. SDS-PAGE analysis of AcGH30A showed molecular mass of ~58 kDa. AcGH30A showed optimum temperature 80 °C and optimum pH 7.0. AcGH30A was stable (maintaining >80 % of control activity) in pH range, 4-7 and temperature range, 30 °C -70 °C when incubated for 90 min. AcGH30A displayed melting temperature, 72 °C and half-life, 21 days at 4 °C. The enzyme activity of AcGH30A was enhanced by 10 mM Ca and Mg ions by 25 % and 21 %, respectively, whereas 10 mM Co, Zn, Fe, and Cu ions significantly reduced it. AcGH30A showed activity against various xylan polysaccharides displaying highest V, 139 U.mg and K, 0.71 mg.ml against 4-O-methyl glucuronoxylan under optimum conditions. TLC, HPLC and LC-MS analyses of AcGH30A hydrolyzed products from xylan substrates revealed the release of sole product, xylobiose, confirming it as an obligate xylobiohydrolase. AcGH30A being a highly thermostable enzyme can be potentially utlilized in various biotechnological applications.
GH30 木糖苷水解酶是一个不断扩展的酶类家族，需要更深入的了解才能实现最佳应用。本研究的主要目的是对来自阿克氏醋酸杆菌的 GH30 家族新木糖苷水解酶 AcGH30A 进行表征。使用 pET28a(+)载体克隆编码 AcGH30A 的基因，并在大肠杆菌 BL21(DE3)细胞中表达。通过固定化金属离子亲和层析对 AcGH30A 进行纯化。SDS-PAGE 分析显示 AcGH30A 的分子量约为 58 kDa。AcGH30A 的最适温度为 80°C，最适 pH 为 7.0。当孵育 90 分钟时，AcGH30A 在 pH 4-7 和温度 30°C-70°C 范围内保持稳定（保持 >80%的对照活性）。AcGH30A 的熔点为 72°C，半衰期为 21 天，在 4°C 下。AcGH30A 的酶活性分别被 10 mM 的 Ca 和 Mg 离子增强 25%和 21%，而 10 mM 的 Co、Zn、Fe 和 Cu 离子则显著降低了它的活性。AcGH30A 对各种木聚糖多糖表现出活性，在最佳条件下对 4-O-甲基葡萄糖醛酸木聚糖的 V 值最高为 139 U.mg 和 K 值最高为 0.71 mg.ml。TLC、HPLC 和 LC-MS 分析 AcGH30A 水解木聚糖底物的产物表明，仅释放出唯一产物木二糖，证实其为一种必需的木糖苷水解酶。AcGH30A 是一种高度热稳定的酶，可潜在地应用于各种生物技术应用。
