鸟肠球菌(Enterococcus avium)中α-L-鼠李糖苷酶基因的克隆表达及酶学性质

Cloning, expression and enzymology properties of α-L-rhamnosidase gene from Enterococcus avium

摘要:

[背景] 前期工作中筛选出一株产α-L-鼠李糖苷酶的细菌，经分子生物学方法鉴定为鸟肠球菌（Enterococcus avium）。α-L-鼠李糖苷酶能够从天然类黄酮化合物中特异性切割末端鼠李糖，在食品生产、医药加工和化工等方面具有极大的开发前景和应用价值。[目的] 克隆、表达鸟肠球菌中α-L-鼠李糖苷酶基因，进一步对重组蛋白的酶学性质进行研究。[方法] 以鸟肠球菌（Enterococcus avium） strain 352基因组中推定的α-L-鼠李糖苷酶基因序列为基础，设计特异性引物扩增其编码区序列。以pET-28a（+）为载体构建重组表达质粒，将重组蛋白在Escherichia coli BL21（DE3）感受态细胞中进行诱导表达。使用镍亲和层析纯化重组蛋白，以pNPR为底物测定重组蛋白的酶学性质。[结果] 重组蛋白EaRha1分子量大小约为130 kDa。以pNPR为底物，EaRha1最适pH是7.0，最适温度为50 ℃，在pH 5.0-8.0稳定性较好，在40 ℃以下能保持较高酶活。金属离子对EaRha1有不同程度的促进或抑制作用。甲醇对EaRha1有抑制作用，并且抑制作用随着甲醇浓度的增大而增强。酶动力学常数K_m和V_max分别为0.35 mmol/L和4.2 μmol/（mg·min） （R²=0.999）。EaRha1能催化水解新橙皮苷、柚皮苷和芦丁。[结论] 通过对重组蛋白EaRha1酶学性质的研究，确定了该蛋白对黄酮类化合物的水解特性，为黄酮类化合物的生物转化奠定了理论基础。

Abstract:

[Background] A bacterium strain producing α-L-rhamnosidase was screened and identified as Enterococcus avium by molecular biological methods in the preliminary work. α-L-rhamnosidase can specifically cut terminal rhamnose from natural flavonoid compounds, which has great development prospect and application value in food production, pharmaceutical processing and chemical industry. [Objective] The α-L-rhamnosidase gene from E. avium was cloned and expressed, and the enzymatic properties of the recombinant protein were further studied. [Methods] Based on the putative α-L-rhamnosidase gene sequence in the genome of Enterococcus avium strain 352, specific primers were designed to amplify its coding sequence. Recombinant expression plasmid was constructed using pET-28a(+) as vector and the recombinant protein was expressed in Escherichia coli BL21(DE3) competent cells. The recombinant protein was purified by nickel affinity chromatography, and the enzymatic properties were determined using pNPR as a substrate. [Results] The molecular weight of the fusion protein EaRha1 is about 130 kDa. The optimal pH of EaRha1 is 7.0, the optimal temperature is 50℃, EaRha1 is stable at pH 5.0-8.0 and can maintain higher enzyme activity below 40℃. Metal ions can promote or inhibit EaRha1 in different degrees. Methanol has inhibitory effect on EaRha1, and the inhibitory effect increases with the increase of methanol concentration. The kinetic characteristic constants K_m and V_max of EaRha1 were 0.35 mmol/L and 4.2 μmol/(mg·min) (R²=0.999) respectively. The recombinant EaRha1 could catalyze the hydrolysis of neohesperidin, naringin and rutin. [Conclusion] In this study, the hydrolysis characteristics of the protein to flavonoids was determined by studying the enzymatic properties of recombinant protein EaRha1, which laid a theoretical foundation for the biotransformation of flavonoids.