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活性肽的影响。我们采用化学酶法合成了糖基化的交配因子。用固相法合成了N-乙酰氨基葡萄糖α-交配因子。然后，利用Mucor hiemalis endo-β-N-aoetylglucosaminidase的转糖基化活性，我们合成了具有谷氨酰胺连接的唾液酸复合型寡糖的糖基化α-交配因子。用分泌型蛋白酶缺陷的S.啤酒。结果表明，糖基化α-交配因子的生物活性低于天然α-交配因子。然而，糖基化的α-a交配因子比天然的α-交配因子具有更高的抗蛋白水解能力.发现N-乙酰氨基葡萄糖α-交配因子的生物活性高于α-乙酰氨基葡萄糖 ...更多信息 内切-α-N-乙酰半乳糖胺酶（endo-α-GaINAc-ase）催化多种动物源粘蛋白和粘蛋白型糖蛋白中β 1，3-N-乙酰半乳糖胺（Galβ1, 3GalNAc）与丝氨酸或苏氨酸残基之间的O-糖苷α-键的水解。我们在各种双歧杆菌的细胞提取物中发现了这种酶的活性。为了制备人工粘蛋白复合物，我们试图克隆编码这种酶的基因。在长双歧杆菌（Bifidobacterium longum）NCC 2705基因组数据库的基础上，通过对各种来源的内切-α-GalNAc-酶基因数据库的检索，尝试获得B中的内切-α-GalNAc-酶基因。longum JCM1217.我们发现，一个开放的阅读火焰似乎是内切α-GalNAc-酶基因。实际上，通过聚合酶链反应扩增了推定的基因，克隆并测序。在大肠杆菌中表达的重组酶经镍柱纯化后具有酶活性。该酶具有转糖基化活性，利用其转糖基化活性，我们制备了包括Galβ1 - 3GalNAc在内的一些新化合物。

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