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

Cell Surface Display and Enzymatic Property of Codon-optimized α-L-rhamnosidase Expressed in Saccharomyces Cerevisiae

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。

Mulberry is rich in flavonoid glycosides of which bioavailability can be effectively heightened through glycosidase-catalyzed bioconversion,being of great significance in exploring the medicinal and food values of mulberry resources. Although natural glycosidase has high specificity,its expression in heterologous organisms is low. Therefore,in order to meet the practical purpose of efficient catalysis,the present study modified codon preference and adaptation of α-L-rhamnosidase gene（rha） gene from Aspergillus niger,and displayed product of the codon-optimized gene corha on cell surface of Saccharomyces cerevisiae. Compared to the recombinant S. cerevisiae containing the original rha gene,the recombinant S. cerevisiae containing corha gene increased by 2. 9 times in expression of coRHA protein,and the enzymatic activity of coRHA was increased by 2. 7 times after 60 h of culture. The optimal p H and temperature of coRHA were 5. 0 and45 ℃ when 4-nitrophenyl-α-L-rhamnopyranoside（ p NPR）was used as its substrate. A yield of 79. 81% ± 3. 1% was achieved in hydrolyzing rutin to synthesize isoquercitrin by coRHA under p H 5. 0 and 60 ℃. Therefore,codon optimization strategy provides a feasible method for high expresssion of coRHA by using engineering bacteria,which was used in hydrolysis of mulberry flavonoid glycosides.