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

针对唾液酸糖的酶促合成，对微生物神经氨酸酶的性质进行了研究。使用小单孢菌、梭状芽孢杆菌和链球菌的神经氨酸酶(分别命名为M、C和S)。考察了底物和添加剂对酶活力和反应平衡的影响。M对可可酸的亲和力最高，是C的200倍。乙醇(5-10%)对酶有抑制作用，而聚乙二醇200-600对酶有120-200%的激活作用。氯化钠降低了活性，表明静电相互作用对反应是重要的，但不是关键的。当以神经节苷脂为底物时，当乙醇浓度为2-5%时，C活性下降到10%，而在20-40%乙醇浓度下，C活性恢复到130%。另一方面，在2-20%的聚乙二醇组分时，酶活为200%。聚乙二醇和乙醇的作用差异可能是由于底物胶束的状态不同所致。酶的活性受底物和添加剂的复杂调控。在唾液糖的合成中，加入2-20%的聚乙二醇酯可以有效地提高反应速度和产物的产率。唾液酰乳糖的合成收率仅为2%，但合成成本太低，难以实现工业化生产。然而，在这项研究中，获得了酶法合成唾液酸糖的基本信息。这里使用的酶是不稳定的，它们的稳定性是未来需要解决的问题。

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.