Synthesis of artificial mucin, which is a barrier for living body, using microbial endo-type glycosidase and its application

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The effect of glycosylation on a bioactive peptide was studied using yeast Saccharomyues cerevisiae α-mating factor, which is composed of 13 amino acids. We prepared glycosylated a mating factor by chemo-enzymatic synthesis. N-Acetylglucosaminyl α-mating factor was chemically synthesized by the solid-phase method. Then, using the transglyoosylation activity of Mucor hiemalis endo-β-N-aoetylglucosaminidase, we synthesized glycosylated α-mating factor with a glutamine-linked sialo complex type oligosaccharide. The biological activity of α-mating factor derivatives was examined by means of a growth arrest assay using secreted-protease-defective a cells of S. cerevisiae. The results showed that the bioactivity of glycosylated α-mating factor was lower than that of native a mating a factor. However, glycosylated α-a mating factor exhibited higher resistance against proteolysis than native α-mating factor. It was found that the bioactivity of N-acetylglucosaminyl α-mating factor was higher t … More han that of native a mating factor.Endo-α-N-acetylgalactosaminidase (endo-α-GaINAc-ase) catalyzes the hydrolysis of O-glyoosidic α-linkage between galactosyl β1,3 N-acetylgalactosamine (Galβ1 3GalNAc) and serine or threonine residue in mucins and mucin-type glycoproteins of various animal sources. We found this enzyme activity in cell extracts of various Bifidobacteria. To prepare the artificial mucin compound, we tried to clone the gene encoding this enzyme. Based on the information of genome database of Bifidobacterium longum NCC2705 and a search of database of various microbial sources having endo-α-GalNAc-ase, we tried to obtain the endo-α-GalNAc-ase gene in B. longum JCM1217. We found that one open reading flame seemed to be the endo-a GalNAc-ase gene. Actually, the putative gene was amplified by polymerase chain reaction, cloned and sequenced. The recombinant enzyme expressed in Escherichia coli was found to, have the enzyme activity, and purified by Nickel column. The enzyme exhibited transglycosylation activity and then we prepared some novel compounds including Galβ1 -3GalNAc using its transglycosy lation activity Less