一种新型果胶裂解酶基因在大肠杆菌中的表达及序列分析  

Expression and Sequence Analysis of a Novel Pectate Lyase Gene in Escherichia coli

作　　者：徐欢 冯湘沅[1] 郑科[1] 杨琦[1] 段盛文[1] 成莉凤[1] XU Huan;FENG Xiangyuan;ZHENG Ke;YANG Qi;DUAN Shengwen;CHENG Lifeng(Institute of Bast Fiber Crops,Chinese Academy of Agricultural Sciences,Changsha 410205,China)

机构地区：[1]中国农业科学院麻类研究所,湖南长沙410205

出　　处：《华北农学报》2023年第1期196-201,共6页Acta Agriculturae Boreali-Sinica

基　　金：麻类所岳麓青年人才培养计划项目(IBFC-YLQN-202102);中央级公益性科研院所基本科研业务费专项(1610242021002);国家自然科学基金(31700438,31871675);国家创新工程(ASTIP-IBFC);国家现代农业产业技术体系建设专项(CARS-16-E22)。

摘　　要：为了获得高酶活力的苎麻脱胶果胶裂解酶,为后续分子改造和新型苎麻生物脱胶酶制剂复配提供理论依据。将果胶裂解酶基因pel419从重组质粒pEASY-Blunt E1-pel419更换至pET28a载体中,转化大肠杆菌BL21(DE3)诱导表达;用SDS-PAGE和Western Blot检验pel419基因的表达效果;用生物信息学软件分析Pel419理化表征和系统发育,预测其二级结构、高级结构及关键氨基酸位点。结果表明,工程菌pET28a-pel419/BL21的果胶裂解酶活力为434.4 U/mL,较pEASY-Blunt E1-pel419/BL21提高了1.7倍;Pel419含375个氨基酸,N末端前22个氨基酸为信号肽,理论pI值为6.59,有4个半胱氨酸,不稳定系数为18.20;其蛋白结构域含有典型的右手β-螺旋结构,属于Pec lyase C家族;Pel419的Ca^(2+)结合位点为ASP151、ASP153和ASP192,定位发现3个保守位点均暴露在三维空间的表面,并处于底物结合空间口袋。大幅度提高了Pel419表达量,并获得了Pel419关键结构信息。In order to obtain ramie degumming pectate lyase with high enzymatic activity,and provide a theoretical basis for the subsequent molecular transformation and the compounding of new ramie biodegumming enzyme preparations.The pectate lyase gene pel419 was replaced from the recombinant plasmid pEASY-Blunt E1-pel419 into pET28a vector and transformed into Escherichia coli BL21(DE3)to induce expression.The expression effect of pel419 gene was tested by SDS-PAGE and Western Blot.The physicochemical characterization and phylogeny of Pel419 were analyzed by bioinformatics software,and its secondary structure,higher order structure and key amino acid positions were predicted.The results showed that the pectate lyase activity of the engineered strain pET28a-pel419/BL21 was 434.4 U/mL,which was 1.7 times higher than that of pEASY-Blunt E1-pel419/BL21.Pel419 contained 375 amino acids,the first 22 amino acids of the N-terminal were signal peptides,the theoretical pI value was 6.59,there were 4 cysteines,and the instability coefficient was 18.20.The protein domain contained a typical right-handedβ-helix structure,belonging to Pec lyase C family.The Ca^(2+)binding sites of Pel419 were predicted to be ASP151,ASP153 and ASP192,and the three conserved sites were found to be exposed to the surface of three-dimensional space and in the pocket of substrate binding space.This study greatly increased the expression of Pel419,and obtained the amount of structural information of Pel419.

关 键 词：苎麻 果胶裂解酶 生物脱胶 原核表达 生物信息学 

分 类 号：Q78[生物学—分子生物学]