Enzymatic Synthesis Of Homogeneous Chondroitin Oligosaccharides

Sugar(carbohydrate)is one of the vital biological macromolecules in life.It is not only an indispensable component of organisms,but also carries many key biological functions.In vivo,Sugar exists in many forms,and the glycoconjugates combined with sugar and non-sugar substances(such as peptides,proteins and lipids etc.)play an important role in the occurrence and development of many physiological and pathological processes such as tumors and inflammatory reactions.The occurrence of the disease is accompanied by changes in the glycan profiles,making it a marker for the diagnosis of many diseases.Although many functional sugar has been developed into health products and pharmaceuticals,the mystery of sugar has not yet been clearly clarified.One of the reasons is that the structural complexity of the sugar chains leads to the obstruction of their structure-activity relationship studies.Because of the complexity of the sugar chain,there is no systematically mature pathway to obtain it.In vitro,enzymatic synthesis using glucotransferases or glucosidases is a major method of sugar chain preparation.The Leloir-type glycosyltransferase is a powerful tool for synthesizing sugar chains,which efficiently synthesizes target sugar chains using the activated form of monosaccharide-sugar nucleotides as a donor substrate.The sugar nucleotide is a derivative formed by linking the hemiacetal hydroxyl group at the reducing end of the monosaccharide to the terminal phosphate group of nucleoside diphosphate or nucleoside monophosphate.Due to the cumbersome protection and deprotection steps,the chemical synthesis of sugar nucleotides is extremely challenging.As the recovery and expression of the sugar nucleotide synthetic related enzymes in the salvage pathway in vivo,enzymatic synthesis of sugar nucleotides in vitro has been widely utilized.In the second chapter of the thesis,the enzymes involved in sugar nucleotide synthesis were fermented in fermentor,and the expressions of enzymes BlNahK,AGX1,AtGlcAK and AtUSP required for the sugar nucleotide synthesis in salvage pathway were completed.The expression level and enzyme activity were also determined.Subsequently,two kinds of sugar nucleotides UDP-GalNAc,UDP-GlcA required for chondroitin production were synthesized in large amounts,which laid the foundation for the study of the preparation of homogeneous chondroitin.Chondroitin sulfate(CS)is a type of glycosaminoglycan with sulfation modification,which is composed of disaccharide repeating units of[glucuronic acid-β1,3-N-acetylgalactosamine-β 1,4-].Owing to its unique physicochemical properties,commercial CS has been widely used in the fields of food and medicine.Recently,In recent years,studies have found that CS not only exists in the extracellular matrix,plays an important role as a signaling molecule that affects lung cancer cell metastasis,osteoblast differentiation,and neuronal regeneration,but also is a receptor molecule on the cell surface that regulates physiological processes by interacting with various key molecules such as growth factors,cytokines,chemokines,adhesion molecules,and lipoproteins.CS is closely related to major diseases,and CS participates in the development of tumors by directly regulating cell function or indirectly regulating the effector molecules such as growth factors or cytokines.CS has anti-inflammatory,anti-oxidant,anti-tumor,anti-infection and other effects can be developed as a drug with more functions.More importantly,CS is a receptor molecule of pathogenic microorganisms in cell surface and plays an important role in bacterial adhesion and infection.However,the microbial surface capsule components of certain pathogenic microorganisms contain chondroitin polysaccharides,which is a molecular camouflage to escape the immune response of the host cells,such as the Eschierichia coli K4 strain and the Pasteurella multocida type F strain.At present,the production technology of commercial CS is mainly the animal tissue extraction method.The CS structure produced by this method has a big difference.The raw material growth cycle is long,and there is a risk of animal virus infection.The production of chondroitin by the microbial fermentation method has a low yield,and the product is difficult to purify and has endotoxin,accounting,and protein contamination.The CS structure is complex and special,making chemical synthesis of homogeneous CS extremely difficult.These positive explorations cannot satisfy the precision preparation of homogeneous CS,and limit the research on structure-activity relationship of CS.The thesis focused on the preparation of homogenous chondroitin.Firstly,we expressed and purified the related enzymes involved in sugar nucleotide synthesis,We studied the enzymatic synthesis of UDP-GlcA and UDP-GalNAc,and established the material basis for subsequent studies.Afterwards,We studied the mechanism of the catalytic reaction of chondroitin synthase(PmCS)derived from Pasteurellamultocida type F strains.A two-step coupled enzymatic synthesis strategy for homogeneous chondroitin was developed by synthesizing chondroitin oligosaccharides fragments one by one and synchronizing the synthesis of homogenous chondroitin in one-pot method.In the second chapter of the dissertation,we initially used fermentation tank to explore the fermentation of enzymes involved in sugar nucleotide synthesis.Based on the experience with shaking flask to express proteins,the growth patterns of the bacteria and the expression conditions of the proteins were explored.We expressed four enzymes:AtGlcAK,AtUSP,BlNahK,and AGX1,which can be purified to obtain AtGlcAK 51 mg/L,AtUSP 26.88 mg/L,BlNahK 24 mg/L,and AGX1 29 mg/L.Subsequently,we used a two-step coupled-enzymatic synthesis of sugar nucleotides to obtain UDP-GlcA 89 mg in a yield of 38.36%and UDP-GalNAc 210 mg in a yield of 43.2%.Providing a material basis for the enzymatic synthesis of chondroitin.In the third chapter of the dissertation,We firstly selected GlcA derivatives modified witih C1 position(GlcA-pNP and GlcA-N3)as the starting substrate for the synthesis of chondroitin oligosaccharides.When the synthesis of chondroitin tetrasaccharide was initiated with GlcA-pNP,a series of chondroitin polysaccharides were prepared by changing the stoichiometric ratio of the receptor/donor respectively using GlcA-pNP,CH2-pNP,CH3-pNP and CH4-pNP as receptor substrates,using UDP-GalNAc and UDP-GlcA as donor substrates.However,CH2-N3 was not obtained when chondroitin oligosaccharides were initially synthesized with GlcA-N3.Subsequently,the effect of the starting monosaccharide receptor on the catalytic synthesis of chondroitin disaccharide by PmCS was investigated.It was found that the reaction rate and conversion ratio of PmCS with GalNAc as the acceptor substrate and UDP-GlcA as the donor substrate for the synthesis of chondroitin disaccharide were both greater than the reaction rate and conversion ratio of chondroitin disaccharide synthesized using GlcA as the receptor substrate and UDP-GalNAc as the donor substrate.With the synthesis,isolation,purification and NMR analysis of GlcAβ1-3GalNAcα(β)-N3,it was proved that the influence of the α linker or the β linker on the glycosyltransferase-PmCS is negligible because the broad substrate specificity of the enzyme.In the study of the speed of PmCS synthesis of chondroitin glycans,it was found that PmCS has an initial rate-limiting step in the synthesis of new chondroitin glycans with different lengths of oligosaccharide receptors as the starting substrate.Synthesis of chondroitin glycans with trisaccharide as a receptor is shorter than synthesis of chondroitin glycans with monosaccharide as a receptor.In the homogeneity study of PmCS synthesis chondroitin,it was found that chondroitin trisaccharide is the minimum receptor substrate that PmCS catalyzes the synthesis of homogeneous chondroitin polysaccharides;Further study found that the proportion of receptor/donor molarity in the reaction system is a key factor affecting the length of the chondroitin sugar chain in the case of sufficient enzyme and sufficient reaction time.Through the stoichiometric control of the reaction system,homogeneous chondroitin sugar chain length controlled enzymatic synthesis was realized.