密码子优化α-L-鼠李糖苷酶基因在酿酒酵母细胞的表面展示表达及产物的酶学性质

发布时间：2018-03-17 03:30

  本文选题：α-L-鼠李糖苷酶　切入点：基因密码子　出处：《蚕业科学》2017年04期 　论文类型：期刊论文

【摘要】：桑树富含黄酮苷类化合物,利用糖苷酶生物转化可有效提高其生物利用度,对挖掘桑树资源的药食用价值具有重要意义。天然来源的糖苷酶催化选择性强,但异源表达量低。以源自黑曲霉(Aspergillus niger)的α-L-鼠李糖苷酶基因rha为目的基因,改善该基因的密码子偏爱性和适应性,将密码子优化后的corha基因展示表达到酿酒酵母细胞表面,以达到高效催化的实用目的。与转入原始rha基因的重组酿酒酵母菌株相比,转入密码子优化corha基因的重组酿酒酵母菌株表面展示coRHA蛋白的表达量提高了2.9倍,培养60 h后coRHA的酶活力提高了2.7倍。以对硝基苯-α-L-鼠李糖苷(p NPR)为底物检测含corha基因重组菌株生产coRHA的酶活力,其最适p H及温度分别为5.0和45℃;以芦丁为底物,在p H 5.0、60℃条件下,coRHA水解芦丁合成异槲皮苷的得率为79.81%±3.1%。采用系统密码子优化策略,为利用工程菌工业化生产coRHA,用于催化桑树黄酮苷类化合物定向水解提供了可行的方法。
[Abstract]:Mulberry is rich in flavonoid glycosides. The bioconversion of glycosidase can effectively improve the bioavailability of mulberry, which is of great significance for exploiting the edible value of mulberry resources. The 伪 -L- rhamnosidase gene (rha) from Aspergillus Niger was used as the target gene to improve the codon preference and adaptability of the gene. The optimized corha gene was expressed on the surface of Saccharomyces cerevisiae cells. Compared with the recombinant Saccharomyces cerevisiae strain with the original rha gene, the expression of coRHA protein on the surface of the recombinant Saccharomyces cerevisiae strain with codon optimized corha gene was increased 2.9 times. The enzyme activity of coRHA was increased by 2.7 times after 60 h culture. The enzyme activity of the recombinant strain containing corha gene was detected by using p-nitrobenzen- 伪 -rhamnoside as substrate. The optimum pH and temperature were 5.0 and 45 鈩，

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