Studies On The Synthesis Of Oligosaccharides By Glycoside Phosphorylases Coupled With Co-factor Regeneration And Their Immobilization

Carbohydrates play structural or functional roles in various biological processes and are the most abundant biopolymers in nature.Due to the special properties of monosaccharide residues and linkages,sugar chains are more diverse,complex and often branched than nucleic acids and proteins.The production of structurally-defined oligosaccharides,polysaccharides,and their conjugates by chemical synthesis,enzymatic synthesis and the combination of both can effectively facilitate the study of the structure and function of glycans,and also serve as a basis for their application in medicine and other fields.In recent years,enzymatic and chemical enzymatic synthesis have flourished and continue to make mutational progress.Leloir glycosyltransferases are the most commonly used tool enzymes for enzymatic sugar synthesis,but the need for expensive sugar nucleotides as donor substrates limits the application of glycosyltransferases in large-scale sugar synthesis.In addition,although the enzymatic sugar synthesis is a sustainable,environmentally friendly and efficient process,there are certain limitations in this method,such as the addition of expensive cofactor ATP for ATP-dependent kinases and the need to purify large amounts of enzymes,which is high cost,time-consuming and labor-intensive,and non-reusable,these limitations make enzymatic glycan synthesis difficult to achieve large-scale application.Therefore,there is an urgent need to develop efficient and cost-effective methods for large-scale production of functional oligosaccharides.Our research group has established the oligosaccharide synthesis system catalyzed by glycoside phosphorylase,using sugar-1-phosphates synthesized by monosaccharide kinase as the donor substrates,which can efficiently synthesize simple oligosaccharides without the need for sugar nucleotides.On this basis,taking the synthesis of lacto-N-biose(LNB),a component of human milk oligosaccharide,as an example,on the basis of the synthetic modules catalyzed by monosaccharide kinase and glycoside phosphorylase above,the ATP regeneration module catalyzed by polyphosphate kinase(PPK)was introduced,and the synthesis conditions of LNB were optimized.Eventually,without the addition of exogenous ATP and sugar nucleotides,PPK 12 from Erysipelotrichaceae bacterium(EbPPK),D-galactosyl-β1,3-N-acetyl-D-hexosamine phosphorylase from Bifidobacterium infantis(BiGHNP)and galactokinase(GalK)derived from Bifidobacterium longum were used to successfully prepare gram-scale LNB with a yield of 92%using mainly simple monosaccharides and polyphosphate as substrates.The successful preparation of LNB verified the feasibility of glycoside phosphorylase combined with an ATP regeneration reaction to prepare oligosaccharides,Then we extended this synthesis strategy to the preparation of other functional oligosaccharides.By coupling seven glycoside phosphorylases with an ATP regeneration reaction,galacto-N-biose,N,N’-diacetylchitobiose and its derivatives,N-glycan core trisaccharide derivatives and a panel of naturally occurring β1,2-,β1,3-,and β1,4-mannosides containing six common β-mannosyl linkages were readily prepared without the addition of exogenous ATP.The yields of products are between 40%and 91%,and the yields of most products are above 50%,some can reach more than 80%.The established three-enzymes system by this work avoids the need for expensive sugar nucleotide donors and cofactor ATP during the process of enzymatic glycan synthesis,thus is practical with simple steps,cost-effectiveness,and suitable for large-scale preparation of some important oligosaccharides or polysaccharides.Subsequently,in order to further reduce the cost of oligosaccharide synthesis and solve the problem of enzyme reusability,the immobilisation study was carried out using the enzymes or recombinant cells involved in LNB synthesis(EbPPK,BiGHNP and GalK)as an example.First,the polyacrylamide embedding method was used to compare the activities of individually-immobilized enzyme vs co-immobilized enzyme and the activities of co-immobilized enzyme vs co-immobilized cells.The results showed that the co-immobilized enzyme was superior to the individually-immobilized enzymes and the co-immobilized cells had comparable activity to the co-immobilized enzymes.Furthermore,the properties of three immobilization methods,namely polyacrylamide embedding method,calcium ion-sodium alginate embedding method and CLCAs(cross-linked cell aggregates)immobilization method were compared.Escherichia coli cells harboring LNB synthesis-related enzymes were co-immobilized,and the optimal co-immobilization conditions of the three methods were investigated.Finally,recyclable co-immobilized cells were successfully prepared under the optimal co-immobilization conditions.The results showed co-immobilized cells prepared by polyacrylamide embedding method and CLCAs could be recycled for more than 6 batches,in contrast to those prepared by calcium ion-sodium alginate embedding method with poor mechanical strength and recycling for 3 batches.Comparing the three methods from the aspects of shape,strength,and manufacturing process,it is found that the CLCAs immobilization method is more advantageous with high protein immobilization rate and simple operation.This study has laid a foundation for further developing the glycoside phosphate-catalyzed synthesis system combined with cofactor regeneration into a greener and more economical method for oligosaccharide synthesis.In summary,the thesis contains two aspects of work centring the enzymatic synthesis of glycan.Firstly,the enzymatic oligosaccharide synthesis method catalyzed by glycoside phosphorylase combining with an ATP regeneration reaction has been established.A variety of functional oligosaccharides were synthesized using monosaccharides and polyphosphates as simple substrates without the addition of expensive donor substrates and exogenous ATP,such as components of human milk oligosaccharide,mucin-type O-glycan core structures,Candida antigenic oligosaccharide and N-glycan core trisaccharide.Secondly,taking LNB synthesis-related enzymes as an example,the immobilization of biocatalysts was investigated.The characteristics of three commonly-used immobilization techniques were compared and the co-immobilized cells,a reusable solid catalyst without purification process,were successfully prepared,which laid the groundwork for future research.