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

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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.