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

doi:10.7668/hbnxb.20193140

摘要/Abstract

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Ca²⁺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。

关键词: 苎麻, 果胶裂解酶, 生物脱胶, 原核表达, 生物信息学

Abstract:

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-handed β-helix structure,belonging to Pec lyase C family.The Ca²⁺ 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.

Key words: Ramie, Pectate lyase, Biodegumming, Prokaryotic expression, Bioinformatics

徐欢, 冯湘沅, 郑科, 杨琦, 段盛文, 成莉凤. 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[J]. 华北农学报, 2023, 38(1): 196-201. doi: 10.7668/hbnxb.20193140.

XU Huan, FENG Xiangyuan, ZHENG Ke, YANG Qi, DUAN Shengwen, CHENG Lifeng. Expression and Sequence Analysis of a Novel Pectate Lyase Gene in Escherichia coli[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(1): 196-201. doi: 10.7668/hbnxb.20193140.

http://www.hbnxb.net/CN/Y2023/V38/I1/196

图/表 8

图1 pET28a-pel419质粒构建

Fig.1 Plasmid construction of pET28a-pel419

图2 pel419基因电泳检验 A.PCR扩增:M.DNA Marker Ⅲ;1.扩增的pel419基因。B.阳性克隆鉴定:M.DNA Marker Ⅲ,1—3.3个阳性克隆的PCR产物。

Fig.2 pel419 electrophoresis test A.PCR amplification identification: M.DNA Marker Ⅲ;1.Amplified pel419 gene.B.Positive clone identification: M.DNA Marker Ⅲ,1—3.PCR products of three positive clones.

图3 Pel419表达效果分析 A.SDS-PAGE:M.Protein Marker;1.阴性对照;2—6.工程菌诱导样品。B.Western Blot:M.Protein Marker;1.工程菌诱导样品。

Fig.3 Expression identification of Pel419 A.SDS-PAGE: M.Protein Marker;1.Negative control;2—6.Induced samples.B.Western Blot:M.Protein Marker;1.Induced samples.

表1 果胶裂解酶活力测定

Tab.1 Determination of pectate lyase activity

菌株 Strains	果胶裂解酶活力/ (U/mL) Pectatelyase activity
pET28a/BL21	0
pEASY-Blunt E1/BL21	0
pET28a-pel419/BL21	434.4
pEASY-Blunt E1-pel419/BL21	160.9

图4 Pel419最适钙离子浓度

Fig.4 Optimal concentration of Ca²⁺ of Pel419

图5 PEL氨基酸序列进化树

Fig.5 Evolutionary tree of amino acid sequence of PEL

图6 Pel419二级结构预测

Fig.6 Secondary structure prediction of Pel419

图7 Pel419三维结构 A.Pel419三维结构,紫色条表示的4个CYS形成2个二硫键;B.橙色球体为Ca²⁺,绿色条表示的ASP151、ASP153和ASP192为Ca²⁺结合位点;C.Ca²⁺结合位点位于蛋白沟表面,分别以橙色、红色和蓝色表示。

Fig.7 Three-dimensional structure of Pel419 A.Pel419 three-dimensional structure,four C represented by purple bars form two disulfide bonds;B.The orange sphere is Ca²⁺,and ASP151,ASP153 and ASP192 represented by the green bar are Ca²⁺ binding sites;C.Ca²⁺ binding sites are located on the surface of the protein groove and are shown in orange,red and blue,respectively.

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