?-Fructosyltransferase Gene Of Aspergillus Oryzae Bioinformatics Analysis And Its Gene Cloning And Expression In Pichia Pastoris

Fruto-oligosaccharide?FOS?has good food processing properties,thus,can promote the proliferation of intestinal bacteria,regulate the immune system and improve the immunity of the body,promote mineral absorption and other unique physiological functions.Commonly,FOS was produced used the sucrose as raw material and catalysised by?-fructosyltransferase?FTase,2.4.1.9 FTase,EC?in a variety of microorganisms or plants.Aspergillus oryzae has a high ability to secrete enzymes with high stability,and is one of the international certification of GRAS?Recognized as Safe Generally?strains.Pichia pastoris is an excellent eukaryotic expression system,and was used to express exogenous protein in industrial applications.Pichia pastoris,as a single cell type of methanol nutrition,can control and regulate the expression of protein secretion with organellae,thus,it can modify the translation-protein,such as the folding of the polypeptide,methylation,acetylation,glycosylation,etc.In this paper,6?-fructosyltransferase-producing Aspergillus oryzae strains were activated and cultured at shake flask fermentation.In this study,high efficiency liquid chromatography technology was applied to gauging the enzyme activity of?-fructosyltransferase,and the enzyme activity of?-fructosyltransferase produced by 6 different Aspergillus oryzae strains were contrasted,in order to screening the efficient?-fructosyltransferase-producing Aspergillus oryzae strains.Based on the efficient expressing Aspergillus oryzae strain A-F04,the DNA and RNA of strain A-F04 were extracted,PCR and RT-PCR strategies were used to obtain?-FTase sequences,thus,?-FTase gene bank was constructed.Evolutionary tree was built,and physicochemical property including formula weight,isoelectric point and amino acid composition were predicted,the possible glycosylation sites were predicted.SWISS MODEL tool was used to predict?-FTase tertiary structure.The FTase gene was cloning and recombined with Pichia pastoris eukaryotic expression system,explore the Pichia pastoris recombinant expression.The results of the Aspergillus oryzae FTase gene study were summarized as follows.1.Screening and identification of efficient?-fructosyltransferase-producing Aspergillus oryzae and gene cloning,identificationIn this study,Aspergillus oryzae A-F04 strain has the highest enzyme activity after cuLtured for 96 h.?-FTase gene was successfully identified in strain A-F04.Strain A-F04's coding region was 1630 bp,including an intron,between173 bp and224bp;CDS were 1578 bp.2.Construction of pPIC9K-FTase vector and bioinformatics analysis of?-fructosyltransferase from Aspergillus oryzae A-F04In this study,a pair of primers was designed,and pPIC9K-FTase vector was constructed successful.Bioinformatics analysis program analysis show that?-FTase was hydrophilic stable protein,its formula weight was 57.4653 kDa,isoelectric point was 4.83,composed by 525 amino acids,within 6 possible glycosylation sites.Amino acid residues with positive charge?Asp+Glu?was 54,and the negative?Arg+Lys?was 31.The molecular formula was C₂₅₉₇H₃₈₃₄N₆₇₀O₈₀₂S₆,it was stable protein with instability index 34.37.FTase has no transmembrane region.Thus,FTase was hydrophilic protein with fat soluble index 66.65 and total average hydrophilicity-0.292.Signal P program show that the amino acids at 1 to 17 sites were signal peptide;The tertiary structure model constructed by SWISS-MODEL contributes to the?-FTase's structure and function exploration.3.Construction and expression of the Pichia pastoris GS115 recombinantIn this study,pPIC9K-FTase-His6 vector was constructed by the method of seamless and homologous cloning.The FTase gene was shifted to Pichia pastoris GS115 by electroporation after digested with Sal?,screened the high copy positive Pichia pastoris recombinant strain,then induced by 0.25%,0.5%,0.75%and 1%methanol to express the?-FTase,gauged the enzyme activity with high efficiency liquid chromatography technology from different fermentation time?24 h,48 h,72 h,96 h and 120 h?,and identified the?-FTase produced by recombinant successful,the enzyme activity of the fermentation broth of 72 h reached 18.84 U/mL induced by0.5%methanol.