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 ofα-L-rhamnosidase gene from Enterococcus avium

作　　者：郑鼎玉 陈婕 郑紫云 卢丹丹 杨官娥[1] ZHENG Dingyu;CHEN Jie;ZHENG Ziyun;LU Dandan;YANG Guan’e(School of Pharmacy,Shanxi Medical University,Taiyuan 030001,Shanxi,China)

机构地区：[1]山西医科大学药学院,山西太原030001

出　　处：《微生物学通报》2022年第1期49-60,共12页Microbiology China

基　　金：山西省国际科技合作项目(201803D421065);中央引导地方科技发展基金(YDZX20201400001443);国家自然科学基金(30672621,81173473);太原市科学技术发展计划(120247-08)。

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p ET-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p NPR为底物测定重组蛋白的酶学性质。【结果】重组蛋白Ea Rha1分子量大小约为130 k Da。以p NPR为底物,Ea Rha1最适p H是7.0,最适温度为50℃,在p H 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Ea Rha1有不同程度的促进或抑制作用。甲醇对Ea Rha1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_(max)和V_(max)分别为0.35 mmol/L和4.2μmol/(mg·min)(R^(2)=0.999)。Ea Rha1能催化水解新橙皮苷、柚皮苷和芦丁。【结论】通过对重组蛋白Ea Rha1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。[Background]A bacterium strain producingα-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]Theα-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 putativeα-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 p ET-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 p NPR as a substrate.[Results]The molecular weight of the fusion protein Ea Rha1 is about 130 k Da.The optimal p H of Ea Rha1 is 7.0,the optimal temperature is50℃,Ea Rha1 is stable at p H 5.0-8.0 and can maintain higher enzyme activity below 40℃.Metal ions can promote or inhibit Ea Rha1 in different degrees.Methanol has inhibitory effect on Ea Rha1,and the inhibitory effect increases with the increase of methanol concentration.The kinetic characteristic constants K_(max)and V_(max)of Ea Rha1 were 0.35 mmol/L and 4.2μmol/(mg·min)(R^(2)=0.999)respectively.The recombinant Ea Rha1 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 Ea Rha1,which laid a theoretical foundation for the biotransformation of flavonoids.

关 键 词：α-L-鼠李糖苷酶 基因克隆 蛋白表达 酶学性质 

分 类 号：Q936[生物学—微生物学]