Enzymatic Synthesis Of Hyaluronan And Its Application

Carbohydrates and glycoconjugates are prevalent in all living organisms ranging from bacteria to plants and animals.They are not only essential structural components,but also exhibit irreplaceable roles as informative molecules in a variety of vital biological processes.Hyaluronan is a linear polysaccharide composed of a repeating disaccharide unit.It has multiple functions in the human body being involved in creating flexible and protective layers in tissues and inmany signalling pathways during embryonic development,wound healing,inflammation,and cancer.Industrial manufacturing of hyaluronan is based on two main processes,the extraction from animal tissues and microbial fermentation using bacterial strains.As in any process for the production of therapeutic compounds from animal sources,there is a potential risk of contamination with proteins and viruses.Bacterial fermentation produces hyaluronan with high molecular weight and purity,but risk of contamination with bacterial endotoxins,proteins,nucleic acids.In the past decade a new technology emerged using isolated HA synthase to catalyze the polymerization of the UDP-sugar monomers.This novel enzymatic technology for hyaluronan synthesis is very versatile allowing producing both high molecular weight hyaluronan and hyaluronan oligosaccharides with defined chain length and low polydispersity.Since sugar nucleotides are expensive substrates their regeneration is a crucial step for the development of an economic process for the production of hyaluronan.In chapter 2,we investigated the roles of several highly conservedr amino acid residues of tGlmU involved in substrate binding and recognition.In order to probe the roles of these highly conserved residues,structure-and sequence-guided mutagenesis studies were carried out to broad substrate specificity and to improve catalytic efficiency for the formation of sugar nucleotides.Out of total 16 mutants designed,tGlmU Q76E mutant which had a novel catalytic activity to convert CTP and GlcNAc-1-P into unnatural sugar nucleotide CDP-GlcNAc was identified.Furthermore,tGlmU Y103F and N169R mutants were also investigated to have enhanced uridyltransferase activities compared with wide-type tGlmU.In chapter 3,we described the cloning and biochemical characterization of A.thaliana GlcAK.GlcAK is a novel plant sugar kinase able to phosphorylate GlcA to GlcA-I-phosphate in the presence of ATP.GlcAK was over-expressed in E.coli BL21(DE3).To demonstrate the application of GlcAK for the synthesis of UDP-GlcA,we performed a large-scale synthesis of GlcA-1-P with yield exceed 95%.In chapter 4,we demonstrated an economic enzymatic approach in which cheaper substrates,nucleotide triphosphates(ATP,UTP)and corresponding monosaccharides(GlcNAc,GlcA),were used as the starting material for hyaluronan production by utilizing sugar nucleotides de novo generation route.We further optimized the amounts of each enzymes involved in the reaction.Finally,large-scale synthesis of HA was performed and 151 mg HA polymers was obtained with 73%yield.In chapter 5,polysaccharides nanoparticle was prepared by using hyaluronan,chitosan and alginate as material and the characteristics of nanoparticle,particle size distribution,were well measured.With the nanoparticles that we obtained,we explored its application in environmental pollution improvement,sterilization,provide a new way to solve the air pollution.