Studies On The Expression Of Alpha 1-2 Fucosyltransferase From Treponema Primitia And For The Synthesis Of Fucosylated Oligosaccharide LNFPⅠ In Human Milk

The human milk oligosaccharides(HMOs)have the functions of promoting the intestinal probiotic microbiota,resisting the invasion of pathogenic bacteria and viruses,and regulating immunity in newborn infants.The content of HMOs in human milk can be up to 22-24 g/L.Neutral human milk oligosaccharides were mainly fucosylated and account for about 90%,of which alpha 1,2-fucosylated oligosaccharides account for 73%.Its synthesis is realized by fucosyltransferase catalysis.The ways of enzymatic synthesis of fucosylated oligosaccharides possess various characteristics with high efficiency and specificity.Fucosyltransferases derived from bacteria are often more prone to expression with high efficiency.It has great advantages in the bio-enzymatic synthesis of human milk fucosylated oligosaccharides.In this paper,alpha 1-2 fucosyltransferases genes derived from Syntrophus aciditrophicus,Bacteroides salanitronis,Helicobacter cinaedi,Vibrio parahaemolyticus,Treponema primitia,Thermosynechococcus sp.were cloned and imported into prokaryotic expression vector pET15b,and then construct Escherichia coli BL21(DE3)recombinant expression strain.The expression of alpha 1-2 fucosyltransferase and its synthesis of fucosylated oligosaccharide LNFPI in human milk were also studied.The results showed that α1-2 fucosyltransferases derived from T.primitia and Thermosynechococcus could be expressed.The assay of activity screening with 10 kinds of substrates had been found that α1-2 fucosyltransferases from T.primitia(Tpα1,2FT)and Thermosynechococcus sp.(Tsα1,2FT)have obvious catalytic activities on Lacto-N-tetraose,Galβ1,3 GalNAcaProN3,Galβ1,3GalNAcβProN3,Galβ1,3GlcNAcaProN3,and Galβ1,3GlcNAcβProN3.The homology of amino acid sequence between Tpα1,2FT and Teα1,2FT,which has been reported with highest catalytic activity,were 19.6%,and homology with other reported enzymes is also low.The result showed that Tpα1,2FT had good novelty.The expression and purification of recombinant protein Tpα1,2FT were optimized.The yield of Tpα1,2FT was 6.2 mg/L when the condition of induction temperature at 16℃,IPTG concentration with 0.05 mmol/L.The optimum temperature and pH of Tpα1,2FT were 40℃and 8.0,respectively.Mg2+Can improve its catalytic efficiency.Lacto-N-fucopentaose Ⅰ(LNFPI)synthesized by Lacto-N-tetraose as receptor could observably promote the growth of intestinal probiotic Bifidobacterium longum subsp.infantis ATCC15697.At present,only seven prokaryotic α1-2 fucosyltransferases have been reported.The results have expanded the library of α1-2 fucosyltransferases and provided a strong support for the synthesis and biological research of human milk fucosylated oligosaccharides.