Action mechanism of microbial neuraminidases and their application to the enzymatic synthesis of sialo-saccharides

微生物神经氨酸酶的作用机制及其在唾液酸酶法合成中的应用

基本信息

批准号：
05660091
负责人：
INOUYE Kuniyo
金额：
$ 1.02万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05660091/
关键词：
neuraminidase sialidase sialo-saccharide sialic acid ganglioside enzymatic synthesis ノイラミニダーゼ

项目摘要

Properties of microbial neuraminidases have been studied aiming at enzymatic synthesis of sialo-saccharides. Neuraminidases of Micromonospora, Clostridium, and Streptococcus (named as M,C,and S,respectively) were used. Effects of substrates and additives on the enzyme activity and reaction equilibrium were examined. M shows the highest affinity to colominic acid, and it is 200 times as high as that of C.Ethanol (5-10%) inhibits the enzymes, while polyethylene glycol (PEG) #200-600 activates them to 120-200%. NaCl decreases the activity, suggesting that electrostatic interaction is significant but not crucial for the reaction. When gangliosides are substrates, the C activity decreases to 10% at 2-5% ethanol, but recovers to 130% at 20-40% ethanol. On the other hand, at 2-20% PEG,the activity is 200%. Difference in the effect of PEG and ethanol may be due to the state of substrate micelles. The activity of the enzymes was shown to be regulated complicatedly by substrates and additives. In the sialo-saccharide synthesis, addition of 2-20% PEG may be effective to enhance the reaction rate and yield of the products. Sialyl-lactose was synthesized with a yield of 2%, being too low to be practical. However, basic information for the enzymatic synthesis of sialo-saccharides has been obtained in this study. The enzymes used here are unstable, and their stabilization is the problem to be solved in future.

已经研究了微生物神经氨酸酶的特性，目的是针对唾液酸糖的酶促合成。使用了微孔孢子，梭状芽胞杆菌和链球菌（分别为M，C和S）的神经氨酸酶。检查了底物和添加剂对酶活性和反应平衡的影响。 M显示了对属属酸的最高亲和力，并且它高的200倍（5-10％）抑制酶，而聚乙烯乙二醇（PEG）＃200-600将它们激活至120-200％。 NaCl降低了活性，表明静电相互作用是显着的，但对于反应而言并不重要。当神经节剂是底物时，C活性在2-5％乙醇时降至10％，但在20-40％的乙醇中恢复到130％。另一方面，在2-20％的PEG下，活动为200％。 PEG和乙醇的作用差异可能是由于底物胶束的状态。该酶的活性被证明是由底物和添加剂调节的。在唾液 - 糖的合成中，添加2-20％PEG可能有效提高产物的反应速率和产量。 siAllyl-乳糖的产量为2％，太低而无法实用。但是，在本研究中已经获得了唾液 - 糖酶合成的基本信息。这里使用的酶是不稳定的，它们的稳定是将来要解决的问题。