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

doi:10.7668/hbnxb.20193140

Abstract

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

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.

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Figures/Tables 8

Fig.1 Plasmid construction of pET28a-pel419

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.

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.

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

Fig.4 Optimal concentration of Ca²⁺ of Pel419

Fig.5 Evolutionary tree of amino acid sequence of PEL

Fig.6 Secondary structure prediction of Pel419

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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