Study On Enzymatic Properties Of Poultry Sialic Acid Aldolase And Its Application In The Synthesis Of KDN

Sialic acid is an important component of glycoproteins and glycolipids in many organisms.It is very important for the regulation of related protein and lipid functions.Therefore,synthetic sialic acid used in food processing has become a hot research topic in recent years.There are many ways to synthesize sialic acid.Among them,natural extraction and chemical synthesis usually has a low yield.The conditions are more demanding,the toxic substances are easily produced.In contrast,enzymatic synthesis can easily and efficiently obtain sialic acid that meets the production requirements.Sialyl aldolase is a very key enzyme in the synthesis pathway,but it is usually more expensive.In addition,poultry products have a high content of sialic acid,which is one of the most abundant natural sources,and the sialic acid cycle of poultry has an important relationship with the prevalence of viruses.Therefore,it is of great significance to explore the new type of poultry-derived sialic acid aldolase and apply it to the synthesis of sialic acids.This research successfully excavated three poultry sources encoding sialic acid aldolase gene sequences from Anas platyrhynchos（Mallard duck）,Columba livia（Rock pigeon）,Gallus（chicken）.After gene synthesis,it was successfully induced and expressed by E.coli expressing cells BL21（DE3）.The enzyme activity was tested and the active site was studied,we explored the enzymatic properties of the three sialic acid aldolase and catalyze the sialic acid analogues.This study explored the sialic acid aldolase from poultry for the first time,and obtained the sialic acid aldolase that is easier to express,higher purity and stronger activity and used it to synthesize KDN.1.Activity test of NPL and research on the active site of NPLThe results of SDS-PAGE electrophoresis showed that all three sialic acid aldolases can be successfully induced and expressed by E.coli expressing cells BL21（DE3）and purified by affinity chromatography with Ni²⁺-NTA.The color reaction after TLC was used to detect the enzyme activity,it was concluded that all three enzymes can catalyze the formation of corresponding monosaccharides in the decomposition reaction,and according to the color of the band on TLC.It can be concluded that the activity of Ap NPL is the strongest among three enzymes;however,the lower concentration of the substrate in the synthesis direction is not prone to the corresponding enzymatic reaction.Subsequently,in order to further explore the active sites of the three enzymes,compared with Ec NPL derived from E.coli,we made the mutation from lysine at position 172 into alanine successfully.And we increased the substrate concentration in the synthesis reaction,measured in the same way to explore the key active site of NPL.From the results of SDS-PAGE,it can be concluded that the three mutant enzymes can still be successfully induced and expressed by E.coli expressing cells BL21（DE3）and can be purified by affinity chromatography;it can be seen from the results:The mutant aldolase has a certain activity in the decomposition reaction,but in the synthesis reaction,the mutant enzyme has no activity.This further verifies the key active site of three enzymes is lysine at position172,which can significantly affect its catalytic activity.2.Research on enzymatic properties and its application in the Synthesis of KDNIn order to have a better understanding of most basic biochemical properties of three enzymes,used Neu5Ac as the substrate to carry out the enzymatic reaction,explore the basic enzymatic characteristics of the most suitable reaction temperature,p H and metal ions of the three enzymes.After the reaction,the conclusion can be drawn according to the microplate reader:The optimal reaction temperature condition of three enzymes is 42°C;the optimal reaction p H of Ap NPL and Cl NPL is 6,and the optimal reaction p H of Gg NPL is 7.5;the activities of the three enzymes are not dependent on metal ions,but magnesium ions can promote enzyme activity to the greatest extent.Compared with other metal ions,ferric ions and manganese ions will inhibit the activities of Cl NPL and Gg NPL.As an unusual sialic acid,KDN can be used as a carcinoete antigen and neuraminidase inhibitor for disease prevention and treatment,and the three sialic acid aldolase explored in this experiment can synthesize KDN.Therefore,by increasing the substrate concentration,used Ap NPL to synthesize KDN,and the products were detected by HPLC and MALDI-TOF-MS.Finally,by calculation,the yield of KDN synthesized from mannose was 42.4%;the yield of 5’EPI-KDN synthesized from galactose was 12.6%.