黑曲霉α-L-鼠李糖苷酶的同源表达与特性分析及其在黄酮类化合物转化中的应用。
Homologous Expression and Characterization of α-L-rhamnosidase from Aspergillus niger for the Transformation of Flavonoids.
机构信息
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China.
Department of Fundamental Medicine, Xinyu University, Xinyu, 338004, People's Republic of China.
出版信息
Appl Biochem Biotechnol. 2022 Aug;194(8):3453-3467. doi: 10.1007/s12010-022-03894-9. Epub 2022 Apr 2.
Aspergillus niger has been used for homologous and heterologous expressions of many protein products. In this study, the α-L-rhamnosidase from A. niger (Rha-N1, GenBank XP_001389086.1) was homologously expressed in A. niger 3.350 by Agrobacterium tumefaciens-mediated transformation. The enzyme activity of Rha-N1 was 0.658 U/mL, which was obtained by cultivation of engineered A. niger in a 5-L bioreactor. Rha-N1 was purified by affinity chromatography and characterized. The optimum temperature and optimum pH for Rha-N1 were 60 °C and 4.5, respectively. Enzyme activity was promoted by Al, Li, Mg, and Ba and was inhibited by Mn, Fe, Ca, Cu, and organic solvents. The result indicated that rutin was the most suitable substrate for Rha-N1 by comparison with the other two flavonoid substrates hesperidin and naringin. The transformed products of isoquercitrin, hesperetin-7-O-glucoside, and prunin were identified by LC-MS and H-NMR.
黑曲霉已被用于许多蛋白质产物的同源和异源表达。在这项研究中，通过根癌农杆菌介导的转化，在黑曲霉 3.350 中同源表达了黑曲霉α-L-鼠李糖苷酶（Rha-N1，GenBank XP_001389086.1）。通过在 5-L 生物反应器中培养工程化的黑曲霉，获得了 0.658 U/mL 的酶活。Rha-N1 通过亲和层析进行纯化和表征。Rha-N1 的最适温度和最适 pH 分别为 60°C 和 4.5。酶活受 Al、Li、Mg 和 Ba 的促进，受 Mn、Fe、Ca、Cu 和有机溶剂的抑制。通过与另外两种类黄酮底物橙皮苷和柚皮苷比较，结果表明芦丁是 Rha-N1 最适宜的底物。通过 LC-MS 和 H-NMR 鉴定了异槲皮苷、橙皮素-7-O-葡萄糖苷和樱黄素的转化产物。
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