The Enzymatic Synthesis,Purification And Structure Identification Of Sialosides

As an important biological information transmission molecule,sialic acid has very important biological functions on the body.Synthesizing sialosides can be used to study their competition for binding sites of sialic acids.The sialosides also provide an important theoretical basis for further exploration of the mechanism of sialic acid metabolism in living organisms.The current synthesizing methods of sialosides include chemical synthesis,chemical-enzymatic synthesis and extraction from natural materials.But the product yield of chemical synthesis and extraction from the natural materials is really low,the reaction conditions are also quietly harsh,etc.Besides,they will cause considerable pollution to the environment,thus simple natural extraction and chemical synthesis can not meet the production requirements of sialosides.In order to replace the chemical synthesis process in the chemical-enzymatic synthesis pathway,this study explored the role of CmCBDA deacetylase in catalyzing the N-acetylation reaction at 37? without protecting groups,and improve the enzymatic total synthesis pathways of various sialosides.Sialic acid aldolase is a key enzyme during the synthesis pathway of sialic acids and its analogs.At present,the sialic acid aldolase has been found to be mostly inducible,and it is required to add N-acetylneuraminic acid as an inducer in the culture medium.So we cloned genes of sialic acid aldolase from Pedobacter heparinus and constructed its engineering bacteria constructed heterologous expression,then with the function of four enzymes excavated in this laboratory to explore the "one-pot five enzyme" method for synthesis of a variety of sialosides?At present,there are still no relevant studies on the structural identification of sialosides synthesized by various methods.In this paper,the synthesized products will be further purified and analyzed by NMR to fill in the existing gaps and further analyze the whole process.The specific research content and results are as follows:1.The cloning,recombinant expression,purification,enzyme activity detection of Sialic acid Aldolase and acylation of Deacetylase.Using Pedobacter heparinus as a research strain,the gene coding for sialic acid aldolase was found by homology comparison of biological information and named PhNeuLy3300.Constructing the engineering bacteria of PhNeuLy3300 by genetic engineering method,expressed and purified,then did the SDS-PAGE.The result of SDS-PAGE showed that the protein band was single,and the size was basically the same as the theoretical molecular weight,so the pure sialic acid aldolase could be obtained after expression and purification.The result of enzyme activity detection by microplate reader showed that the PhNeuLy3300 has a high activity,so it can use as a vital tool to explore the pathway for the enzymatic synthesis of sialosides.Using the CmCBDA catalyze the N-acetylation of glucosamine and 4 different organic acids(acetic or propionic acid or glycolic acid or thioglycolic acid).The 1H-NMR analysis showed that we synthesed N-acetyl glucosamine,N-propionyl glucosamine,N-glycosyl glucosamine and N-decyl acetyl glucosamine successfully,and the product yield was 69%,68%,77%and 77%,respectively.This demonstrated the CmCBDA can catalyze the N-acetylation reaction of glucosamine and various organic acids successfully,so it also can use as a tool to explore the pathway for the enzymatic synthesis of sialosides.2.The enzymatic total synthesis of 4 sialosides.Using D-glucosamine and 4 different organic acids(acetic acid or propionic acid or glycolic acid or thioglycolic acid)as substrates,the quantitative PhNeuLy3300 enzyme?CmCBDA enzyme(deacetylase)?PhGn2E3295 enzyme(N-acetylglucosamine-2-isomerase)?NmCTT enzyme(CMP-sialic acid synthase)and PdST6 enzyme(sialic acid glycosyltransferase)which were deposited in the laboratory were used for—"one pot five enzymes" synthesis of various sialosides.Detecting the products by HPLC,the results showed that X-Gal-N-acetylneuraminic acid,X-Gal-N-propionylneuraminic acid,X-Gal-N-glycolneuraminic acid and X-Gal-N-thioglycolylneuraminic acid were synthesized successfully by the enzymatic total synthesis of sialosides,their yields were 98%,94%,29%and 92%,respectively.The results indicated that the enzymatic total synthesis of sialosides is feasible and has high product conversion rates.In addition,it can be conducted on the mild reaction conditions and does not require special chemical reaction equipment and toxic reagents,so it will not cause environmental pollution and other problems.3.The Purification and NMR analysis of product.For a further analysis about the method of the enzymetic total synthesis,we still use"one pot five enzymes" to get a large number of X-Gal-N-acetylneuraminic acid,X-Gal-N-propionylneuraminic acid,X-Gal-N-glycolylneuraminic acid and X-Gal-N-thioglycolylneuraminic acid.Then we used C18 solid-phase extraction column and the silica gel column to de-enzyme,salt,and X groups.The results of HPLC showed no aberrant peaks,demonstrated that the products were of high purity and no by-products were generated.After that,analyzed the samples of X-Gal-N-acetylneuraminic acid,X-Gal-N-propionylneuraminic acid and X-Gal-N-glycolylneuraminic acid by 1H-NMR and 13C-NMR.The results showed that the structures of products are consistent with the theoretical target products',which means that this route has the advantages of simple extraction and high product conversion rates.