Scientia Agricultura Sinica ›› 2013, Vol. 46 ›› Issue (22): 4774-4783.doi: 10.3864/j.issn.0578-1752.2013.22.015

• ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT • Previous Articles     Next Articles

Peptide Linkers Optimized Recombinant Enzyme Gene of XynB-ManA and Its Co-expression in pK15 Cells

 ZHANG  Xian-Wei, ZHANG  Guan-Guan, WU  Zhen-Fang, MENG  Fan-Ming, LIU  De-Wu, ZHANG  Mao, XU  Wei-Hua, ZHENG  En-Qin, HE  Xiao-Yan, LI  Zhen, LI  Zi-Cong   

  1. College of Animal Science, South China Agricultural University/National Engineering Research Center for Breeding Swine Industry, Guangzhou 510642
  • Received:2013-04-03 Online:2013-11-15 Published:2013-07-12

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Abstract: 【Objective】This study was aimed at constructing a fusion enzyme of xylanase and mannase which functioned well in mammalian expression system for the research of transgenic animals and potential utilization in the industry of feed enzyme.【Method】Eleven chimeric genes of xylanase and mannase differed in linkers were constructed by SOE-PCR, and then subcloned into pcDNA3.1 vector. The cell free culture supernatants were analyzed by the dinitrosalicylic (DNS) acid method after the recombinant plasmids were transiently transformed into PK15 cells .【Result】Compared with the parental enzymes，the fusion enzymes with Linkers a3, pS3, and “self-cleavage”T2A displayed significantly higher catalytic efficiency. Specifically, the XynB-a3-ManA was increased by 54.06% for the xylanase and of 104.40% for the mannase in enzyme activity, and worked well at pH3.0-7.0 and was partially resistant in pH3.0-8.0.【Conclusion】The results demonstrated that the firstly obtained enhanced bifunctional fusion XynB-ManA enzyme which could be expressed and secreted in mammalian expression system by optimizing the peptide Linkers between two parental genes.

Key words: XynB , ManA , Linker , Fusion enzyme , enzyme assay

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