ACTA AGRICULTURAE BOREALI-SINICA ›› 2021, Vol. 36 ›› Issue (5): 18-23. doi: 10.7668/hbnxb.20192340

Special Issue: Biotechnology

• Crop Genetics & Breeding·Germplasm Resources·Biotechnology • Previous Articles     Next Articles

High Level Expression of Pectate Lyase Gene from Microbiology for Ramie Degumming and Its Bioinformatics Analysis

XU Huan1, DUAN Shengwen1, FENG Xiangyuan1, ZHENG Ke1, YANG Qi1, WANG Qiming2, CHENG Lifeng1, PENG Yuande1   

  1. 1. Institute of Bast Fiber Crops, Chinese Academy of Agriculture Sciences, Changsha 410205, China;
    2. College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China
  • Received:2021-05-08 Published:2021-10-28

Abstract: In order to efficiently express the pectate lyase PelG403 from ramie biodegumming. This study replaced pel G403 from the recombinant plasmid pEASY-Blunt E1-G403 to the pET28a vector with a higher plasmid copy number, and then introduced Escherichia coli BL21(DE3) to induce expression. The enzyme activity was determined by DNS method, and the expression effect of PelG403 was tested by SDS-PAGE and Western Blot analysis, and the sequence was analyzed and identified. The results showed that the pectate lyase activity of pET28a-G403/BL21 was 335.8 U/mL, which was 3.05 times higher than that of pEASY-Blunt E1-G403/BL21;the molecular weight of the expressed product of pET28a-G403/BL21 was slightly smaller than that of the predicted fusion protein with His tag(43.6 ku);PelG403 contained 387 amino acids, and the first 35 amino acids at the N-terminal were signal peptides;the theoretical pI value was 7.64, 4 cysteines, and the instability coefficient was 27.42;its protein domain contained a typical right-handed β-helix structure and belonged to the Pec lyase C family. Therefore, replacing pelG403 from the recombinant plasmid pEASY-Blunt E1-G403 into the pET28a vector and introducing E.coli BL21(DE3) for inducible expression was a method and approach for high-efficiency expression of the ramie degummed pectate lyase gene pelG403, and its sequence was analyzed and identified. It laid a foundation for the purification of pectate lyase, key site analysis, molecular modification and the study of its degumming functional characteristics.

Key words: Ramie, Biological degumming, Pectate lyase, Prokaryotic expression, Bioinformatics

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Cite this article

XU Huan, DUAN Shengwen, FENG Xiangyuan, ZHENG Ke, YANG Qi, WANG Qiming, CHENG Lifeng, PENG Yuande. High Level Expression of Pectate Lyase Gene from Microbiology for Ramie Degumming and Its Bioinformatics Analysis[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2021, 36(5): 18-23. doi: 10.7668/hbnxb.20192340.

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