Studies On Substrate Specificities Of Pasteurella Multocida Type A Hyaluronans Ynthase And Synthesis Of HA Oligosaccharides And TFA Modified Derivatives By One-pot Multienzyme System

Glycosaminoglycans (GAGs) consist of repeating disaccharide units composed of an N-acetylated or N-sulfated hexosamine and either an uronic acid (glucuronic acid or iduronic acid) or galactose, including chondroitin sulfate (CS), heparan sulfate (HS), hyaluronic acid (HA) and etc. HA, a linear polysaccharide consisting of alternative β1,4-linked glucuronic acid (GlcA) and β1,3-linked N-acetylglucosamine (GlcNAc), is an essential polysaccharide in vertebrates and a putative virulence factor in certain microbes. HA widely exists in extracellular matrix in the form of proteoglycans and plays important roles in cellular development, repair and replacement. The chains are vital constituents for the development of many systems in the body, including corneas, cartilageand tendons, skin and connective tissue. HA is applied to joins, ophthalmology and some other aspects in medicine as well as cosmetics and health production because of its good hydrophilic and viscoelastic properties.Class Ⅱ HA synthase, pmHAS, a 972-amino acidresidues membrane-associate HA synthase from Gram-negative Type A Pasteurella multocida, polymerizes the HA chain by the addition of sugar units to the nonreducing terminus. pmHAS1-703 is a soluble, acive HA synthase catalyzing the transfer of both GlcNAc and GlcUA to form the HA polymer.To save cost and simplify the process of reaction as well as purification steps significantly, relative inexpensive substrates can be used to synthesize HA oligosaccharides and its TFA modified HA derivatives by one-pot multienzyme system with pmHAS and several sugar nucleotide synthases. And TFA modified HA derivatives can be used to synthesize sulfated HA which will be studied to verify the inhibition of hyaluronidase to prolong the time of HA injected in vivo. The contents and results of the research as follows:(1) The recombinant expression system of HA synthase from Pasteurella multocida Type A (pmHAS), pET28a-PmHAS1"703, was constructed and transformed into BL21 strain of Escherichia coli. The expression conditions and the purification procedure of the pmHAS were defined.(2)The substrate speciability of pmHAS was preliminaryly analyzed. It was found that, in addition to HA, the CS oligosaccharides were also the receptors of pmHAS because of the same glycosidic linkage as HA; while the HS chains could not serve as a substrate because of the quite different glycosidic linkage with HA.(3) Recombinant pET28a-At/GlcAK was constructed and transformed into BL21 strain of Escherichia coli. The purified recombinant protein At/GlcAK was used to enzymatic synthesis of UDP-GlcUA coupling with UDP-sugar pyrophosphorylase (USP), which could solve the problem of expensive sugar nucleotides.(4) HA oligosaccharides and its TFA modified HA derivatives were synthesized by one-pot multienzyme system with pmHAS and several sugar nucleotide synthases, which helped save cost and simplify the process of reaction as well as purification steps significantly.