Study On The Enzymatic Synthesis Of Glucuronic Oligosaccharides And N-Acetyl Hexosamine Oligosaccharides

Oligosaccharides with different types and origins which widely exist in nature have different biological activities. Glycosaminoglycans (GAGs) which are mainly made up of repeating disaccharides carrying alternating hexuronic acids and N-acetyl hexosamine are thought to play major roles in diverse biological events such as cell recognition, antiblood coagulation, cell growth, anti-inflammatory and antitumor. Therefore, research of the GAGs has become interesting issue in the area of glycobiology. To obtain a clearer insight into biological activity and structure of the GAGs, using β-D-glucuronidase and β-D-galactosidase to synthesis GAG fragments such as glucuronyl oligosaccharides and N-acetyl amino oligosaccharides is essential. The following three aspects have been systematically studied:the enzymatic synthesis of oligosaccharides with bovine β-glucuronidase B. circulansβ-D-galactosidases; isolation, purification and structure characterization of pure components oligosaccharides; optimization of synthetic conditions of oligosaccharides.Firstly, β-glucuronidase from bovine liver was used to catalyst with pNP-P-D-GlcA as donor and pNP-β-D-GlcA,pNP-α-D-Glc and pNP-a-D-Gal as acceptors, respectively. The reaction products could be detected in the mixture by HPLC and isolated using AKTA purifier combined with Zorbax SB-C18semi-preparative column. Then four kinds of oligosaccharides were obtained and were identified by ESI-TOF-MS,1H-NMR and13C-NMR. When we used pNP-β-D-GlcA and pNP-a-D-Glc as acceptors respectively, the glucuronidase gave β(1→3)-linked disaccharides. The products’structure were p-NP β-D-GlcA-(1→3)-β-D-GlcA and p-NP a-D-GlcA-(1→3)-p-D-Glc respectively. A different regioselectivity was observed using pNP-α-D-Gal as an acceptor leading to the synthesis of p-NP α-D-GlcA-(1→2)-β-D-Gal and of the regioisomerp-NP α-D-GlcA-(1→4)-P-D-Gal.Secondly, the effects of different factors on the formation of glucuronyl oligosaccharides were investigated, such as temperatue, pH, substrate concentration, the molar ratio of the donor/acceptor and enzyme concentration. We found that the major reaction conditions were temperature, pH, substrate concentration and molar ratio of donor/acceptor, whereas enzyme concentration was secondary condition. The optimum conditions for all kinds of products were listed as follows:(1) p-NP α-D-GlcA-(1→3)-β-D-Glc:35℃, pH=6.0,13U/μL enzyme concentration,13%substrate concentration (pNP-β-D-GlcA:pNP-β-D-Glc=3:1), the yield of product was about28.8%.(2)p-NP a-D-GlcA-(1→2)-β-D-Gal and p-NP a-D-GlcA-(1→4)-β-D-Gal:35℃, pH=6.0,12U/μL enzyme concentration,15%substrate concentration (pNP-β-D-GlcA: pNP-β-D-Gal=2:1), the yield of products were about35.4%and31.7%, respectively.Thirdly, β-D-galactosidase from B. circulans was used to catalyst with lactose as donor and pNP-β-D-GlcNAc and pNP-β-D-GalNAc as acceptors, respectively. The reaction products could be detected in the mixture by HPLC and isolated using medium pressure liquid chromatography combined with Toyopearl HW-40S column. Then seven kinds of oligosaccharides were obtained and were identified by by ESI-TOF-MS,1H-NMR and13C-NMR. It proved that disaccharides with β-(1→4) linkage were obtained as the major products together with β-(1→6) isomers. Degree of polymerization (DP) of the transfer products was2to4. When we used pNP-β-D-GlcNAc as an acceptor, we got four kinds of products. The structures were listed as follows: p-NP P-D-Gal-(1→4)-β-D-GlcNAc,p-NP β-D-Gal-(1→6)-β-D-GlcNAc,p-NP β-D-Gal-(1→4)-β-D-Gal-(1→4)-β-D-GlcNAc and p-NP β-D-Gal-(1→4)-β-D-Gal-(1→4)-β-D-Gal-(1→4)-β-D-GlcNAc. When we used pNP-β-D-GalNAc as an acceptor, we got three kinds of products. The structures were listed as follows:p-NP β-D-Gal-(1→4)-β-D-GalNAc, p-NP β-D-Gal-(1→6)-β-D-GalNAc and p-NP β-D-Gal-(1→4)-p-D-Gal-(1→4)-β-D-GalNAc.