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

本科学研究B的目的是通过酶法合成各种具有生物活性的糖缀合物，提高内糖苷酶的转糖基化活性，并对酶反应进行分析，建立制备方法。以下是本研究的结果。重组酶的大规模制备：利用大肠杆菌线圈克隆重组毛霉-β- n -乙酰氨基葡萄糖苷酶（Endo- m）基因，在低温无iptg诱导下进行生长，成功大规模生产重组毛霉-β- n -乙酰氨基葡萄糖苷酶（Endo- m）。获得具有较高转糖基化活性的突变酶：我们通过对ento - m .3的推定催化区残基进行定点诱变，获得了具有较高转糖基化活性的Y217F突变酶。获得具有糖合酶样活性的突变酶：我们通过定点诱变获得具有糖合酶样活性的N175A突变体。该突变体没有水解和转糖基化活性。然而，当将恶唑啉形式的糖链作为糖链供体加入到反应混合物中进行转糖基化时，酶表达了转糖基化活性，并且转糖基化产物的增加没有被水解。利用该突变酶，我们可以高产地合成具有抗hiv活性的生物活性糖肽。利用双歧杆菌重组内do-α- n -乙酰半乳糖胺酶，将粘蛋白型糖链（半乳糖β 1,3n -乙酰半乳糖胺）添加到人粘蛋白部分肽Muc1a的丝氨酸或苏氨酸残基上，除游离丝氨酸和苏氨酸外，还成功将粘蛋白型糖链（半乳糖β 1,3n -乙酰半乳糖胺）添加到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.