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

利用由13个氨基酸组成的酿酒酵母α交配因子研究了糖基化对生物活性多肽的影响。采用化学-酶法合成了糖基化交配因子。采用固相法合成了N-乙酰氨基葡萄糖α交配因子。然后，利用毛霉内切β-N-乙酰氨基葡萄糖苷酶的糖基化活性，合成了谷氨酰胺连接的唾液酸络合型寡糖糖基化α交配因子。用分泌型蛋白酶缺陷的酿酒酵母a细胞的生长停滞实验检测了α交配因子衍生物的生物活性。结果表明，糖基化α交配因子的生物活性低于天然交配因子。然而，糖基化的α-a交配因子表现出比天然α-交配因子更高的抗蛋白水解性。结果表明，N-乙酰氨基葡萄糖α交配因子的生物活性高于…内切-α-N-乙酰半乳糖胺酶(Endo-α-GainAc-α)催化半乳糖基β1，3-乙酰半乳糖胺(Galβ1 3GalNAc)与各种动物来源的粘蛋白和粘蛋白类型糖蛋白中的丝氨酸或苏氨酸残基之间的O-糖苷半乳糖键的水解。我们在各种双歧杆菌的细胞提取液中发现了这种酶的活性。为了制备人工粘蛋白化合物，我们试图克隆该酶的编码基因。本研究以长双歧杆菌基因组数据库NCC2705为基础，通过检索各种含有endo-α-GalNAc-ase的微生物来源数据库，获得了该菌株的endo-α-GalNAc-ase基因。我们发现，一个开放的阅读火焰似乎是endo-a GalNAc-ase基因。实际上，这个可能的基因是通过聚合酶链式反应扩增、克隆和测序的。在大肠杆菌中表达的重组酶具有较强的酶活性，并用镍柱进行纯化。该酶具有较强的糖基化活性，并利用其较低的糖基化活性合成了半乳糖β1-3GalNAc等新化合物

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