【摘要】 克隆表达乳酸片球菌AS1.2696菌株中的α-L-鼠李糖苷酶基因,研究重组酶的酶学性质。分析乳酸片球菌AS1.2696菌株的全基因组序列,聚合酶链式反应扩增目的基因,以p SE380为表达载体构建重组质粒p SE-prha2和p SE-prha3,在大肠杆菌Escherichia coli XL-blue进行表达,使用镍亲和层析纯化重组蛋白,研究重组酶PRHA2、PRHA3的酶学性质。结果表明,重组酶PRHA2的最适pH值和温度分别为5.0和60℃,Km值为(3.039±0.581)mmol/L,Vm a x值为(2.032±0.186)μmol/(min·mg);PRHA3的最适p H值和温度分别为5.5和45℃,Km值为(2.797±0.132)mmol/L,Vmax值为(113.35±1.485)μmol/(min·mg)。PRHA2和PRHA3能够水解人工底物对硝基苯基-α-L-吡喃鼠李糖苷(p-nitrophenyl-α-L-rhamnopyranoside,p NPR)以及α-1,6键的橙皮苷、芦丁;PRHA2对4-硝基苯基-β-D-阿拉伯糖苷(4-nitrophenyl-β-L-arabinoside,p NPA)有一定的水解活性;PRHA3对淫羊藿苷、淫羊藿次苷I、朝藿定A、朝藿定B、朝藿定C这几种物质C-7位的葡萄糖糖苷键有较弱的水解作用。此外,PRHA3能够水解朝藿定C的C-3位以α-Rha(2→1)α-Rha连接的外侧的鼠李糖苷键,在食品及医疗方面具有一定的应用价值。更多还原

【Abstract】 The aim of this study was to evaluate the enzymatic properties of recombinant α-L-rhamnosidases PRHA2 and PRHA3 from Pediococcus acidilactici AS1.2696. The whole genome sequence of P. acidilactici AS1.2696 was analyzed and the two genes encoding α-L-rhamnosidase were amplified by PCR and ligated into expression vector pSE,yielding recombinant plasmids pSE-prha2 and pSE-prha3. The recombinant plasmids were separately transformed and expressed in E.coli XL-blue. The recombinant proteins PRHA2 and PRHA3 were purified by Ni-NTA affinity column chromatography and their enzymatic characteristics were investigated in detail. The results showed the optimal pH and temperature of PRHA2 were 5.0 and 60 ℃, respectively. Its Km and Vmax values were(3.039 ± 0.581) mmol/L and(2.032 ±0.186) μmol/(min·mg), respectively. The optimal pH and temperature of PRHA3 were 5.5 and 45 ℃, and its Km and Vmax values were(2.797 ± 0.132) mmol/L and(113.35 ± 1.485) μmol/(min·mg), respectively. PRHA2 and PRHA3 could not only hydrolyze the artificial substrate p-nitrophenyl-α-L-rhamnopyranoside(p NPR) but could also hydrolyze the α-1,6 glucosidic linkages of the natural substrates hesperidin and rutin. PRHA2 had hydrolytic activity on 4-nitrophenyl-β-L-arabinoside(p NPA). PRHA3 weakly hydrolyzed the glucosidic linkages at the C-7 position of icariin, icariside I, epimedin A, epimedin B and epimedin C. In addition, PRHA3 could hydrolyze the outer rhamnosidic linkage to the C-3 position of epimedin C through α-Rha(2→1)α-Rha, leading to potential applications in the food and medicinal fields.更多还原