Production of Novel Bioactive Compounds Using Microbial Endoglycosidases and Its Enzymatic Analyses

本科研B的目的是用酶法合成提高内切糖苷酶转糖基化活性的各种生物活性糖偶联物，并对酶反应进行分析，以确定制备方法。以下是本研究的成果。结论：1.重组酶的大量制备：利用大肠杆菌克隆β-M基因，在不经IPTG诱导的条件下低温生长，成功地大量生产了重组冬毛霉Endo-M。获得具有较高转糖基化活性的突变酶：我们通过对Endo-M.3可能催化区域的残基进行定点突变，获得了具有较高转糖基化活性的Y217F突变酶。获得具有糖合成酶样活性的突变体酶：我们通过定点突变获得了具有糖合成酶样活性的突变体N175A。该突变体不显示任何水解性和转糖基化活性。然而，当糖链的恶唑啉形式作为糖链的供体加入到反应混合物中时，该酶表现出转糖基化活性，并且转糖基化产物的增加不会发生水解。利用该突变酶可以高产率地合成具有抗HIV活性的生物活性糖肽。利用来自双歧杆菌的重组内切α-N-乙酰半乳糖苷酶在多肽的丝氨酸或苏氨酸残基上添加粘蛋白糖链：除了游离丝氨酸和苏氨酸外，我们还成功地将粘蛋白糖链(半乳糖β1，3N-乙酰半乳糖胺)添加到部分人粘蛋白的多肽Muc1a的丝氨酸或苏氨酸残基上。

The purposes of this Scientific Research B is to enzymatically synthesize various bioactive glycoconjugates compounds rising transglycosylation activity of endoglycosidases and also to analyze the enzyme reaction in order to establish the preparation method. The followings are the results of this research.1. Large preparation of the recombinant enzyme : We succeeded in the large production of recombinant Endo-β-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) by cloning the gene of Endo-M using Escherichia coil and its growing at low temperature without induction by IPTG.2. Obtaining the mutant enzyme having higher transglycosylation activity : We obtained Y217F mutant enzyme having higher transglycosylation activity by site-directed mutagenesis of the residues in the putative catalytic region of Endo-M.3. Obtaining mutant enzyme having glycosynthase-like activity : We obtained N175A mutant having glycosynthase-like activity by site-directed mutagenesis. This mutant shows no activities of hydrolysis and transglycosylation. However, the enzyme expressed the transglycosylation activity when the oxazoline form of sugar chain was added to the reaction mixture as donor of sugar chain in transglycosylation, and the increase of transglycosylation product continued without its hydrolysis. Using this mutant enzyme, we could synthesize the bioactive glycopeptide having anti-HIV activity with high yield.4. Addition of mucin type sugar chain to serine or threonine residue of the peptide using the recombinant endo-α-N-acetylgalactosaminidase from Bifidobacterium: We succeeded in the addition of mucin type sugar chain (galactosylβ1,3N-acetylgalactosamine) to serine or threonine residue of Muc1a which is the peptide of a part of human mucin, in addition to free serine and threonine.