Construction Of Two-copy Engineered Yeast Of Feruloyl Esterase A And Immunogenicity Of Its Yeast Expression Product In Mice

Feruloyl esterase plays an important role in hydrolysis of plant cell walls and releases ferulic acid.Furthermore,feruloyl esterase had been secreted and purified from various microorganisms.Feruloyl esterase was limited by their low activities and expression level in industrial applications.In this study,we constructed two-copy engineered strains of feruloyl esterase A for high expression of feruloyl esterase A.In addition,we also investigated the immunogenicity of feruloyl esterase A in mice.The main research contents and the results were listed as following:1.Constructed of two-copy engineered yeast of feruloyl esterase A and its enzymatic propertiesThe full-length cDNA sequence of feruloyl esterase A(AnFaeA)gene was successfully amplified from A.niger GIM3.452 by PCR techniques.The full-length cDNA of AnFaeA gene was 846 bp which encoded 282 amino acids.NCBI Blast results showed that the gene sequence shared up to 90%similarity to AnFaeA gene which was reported in GenBank.The sequence reported in this study has been deposited in the GenBank database under accession NO.KP126605.The cDNA encoding the mature AnFaeA without signal peptide was ligated into Pichia pastoris expression vectors pPIC9K and pPICZaA,repectively.The resulting recombinant expression plasmids,named PIC9K-AnFaeA and pPICZaA-AnFaeA,repectively.The recombinant expression plasmid PIC9K-AnFaeA was linearized by Pme I and transformed into Pichia pastoris GS115 competent cells by electroporation.After a screen in shaker flask,a strain having the highest feruloyl esterase activity of 2.4 U/mL was obtained,and named GSKFA3.Then,the recombinant plasmid pPICZ?A-AnFaeA was linearized with Sac I and transformed into GSKFA3 competent cells,and the transformants were grown on YPDS plates with antibiotic Zeocin.After cultivation,a highly expressed two-copy engineered strain named GSKZaFA20 was got(its enzyme activity was 10.6 U/mL).PCR analysis showed that the recombinant transformants were stably integrated into the Pichia pastoris genome and the recombinant strains which containing AnFaeA gene had good genetic stability.SDS-PAGE and Western-blot analysis showed that the expressed product was AnFaeA glycosylated protein with an apparent molecular weight of about 40 kDa.By optimizing the culture conditions of the recombinant protein,the result showed that AnFaeA activity reached 15.49 U/mL when it induced by 1%methanol for 7 d.The reAnFaeA showed optimum activity at 50 ? and pH 6.0,and was stable at temperature below 45? and at the pH range of 4.0?6.0.2.Study on immunogenicity of feruloyl esterase A in miceA total of 63 male Kunming mice at 3 weeks of age which were similar weight and health,were randomly assigned to 3 groups,and each group included 21 mice.In the experiment group,the mice were subcutaneously injected with 0.1 mg feruloyl esterase A protein at the 5th,15th and 25th day,and in the empty vector group,the mice were injected with the same dose of the expression supernatant of pPICZaA trans X-33 yeast.The feed intake and body weight of mice were recorded.The contents of IFN-?,IL-4 and IgG in serum samples were detected by ELISA technology.The results showed that in vivo injection of feruloyl esterase A had no significant effect on the growth of mice(P>0.05).But feruloyl esterase A could significantly increase the contents of IFN-?,IL-4 and IgG in the serum(P<0.05),and there was no significant difference between the control group and empty vector group(P>0.05).Western-blot analysis showed that feruloyl esterase A had good reactogenicity.The above results indicated that the feruloyl esterase A had good immunogenicity and reactogenicity,could stimulate cellular and humoral immunities,and affected the immune level of the mice body.In conclusion,we obtained the highest feruloyl esterase A level by construction of two-copy engineered yeast and by optimization of the culture conditions.The feruloyl esterase A from A.niger GIM3.452 had good immunogenicity and reactogenicity,could stimulate cellular and humoral immunities and affected theimmune level in the mice.This study will provide valuable reference material for the development of high-level expression of feruloyl esterase A in the future.At the same time,it will provide a theoretical basis for the further study on the function of feruloyl esterase A.