Expression Of A Hyalronidase And Preparation Oligomers Hyaluronate By Enzymolysis

Hyaluronan(hyaluronic acid,HA)is a high-molecular mass polysaccharide,high and low molecular weight forms of HA exhibit opposite effects on biological activity.Hyaluronidase,is a class of glycosidases that predominately degrade hyaluronic acid(HA)to HA oligosaccharides.The microbial hyaluronidases(EC.4.2.2.1)also called hyaluronate lyases,they degrade HA by a(3-eliminate reaction to yield unsaturated disaccharide.Discovering A hyalurondiase with high activity and solubility is of great importance to industrially produce low degree of polymerization HA by enzymatic reaction.In this paper,Streptococcus zooepidemicus Hyaluronidase HylB was as a study object.The main results were as follows:(1)Expression of the hyaluronate lyase.PCR amplified the the hylb gene fragment from the S.zooepidemicus ATCC39920 genome,which encodes a 83 kDa protein,then constructed the recombinant expression vector pET28a-hylb and expressed it in Escherichia.coli BL21.After optimized the induction condition,we finded that 0.1 mM IPTG and 20? produce a positive effect,the level of the HylB expression is higher than other conditions and the enzyme is a soluble protein.The recombinant protein HylB was purified by Ni-NTA affinity chromatography and eluted by imidazole gradiently,then analysised the enzymatic characters by using HA as substrate.The results showed that the optimum temperature and pH were 37?and 6.5,metal ions such as Ca2+ and Mg2+ could increase the enzyme activity in varying degrees,the purified enzyme activity was 4655 U/mg at the optimum condition,and the half-life of the enzyme at 37 ? was 2.4 h.(2)To prepare the low molecular weight HA.We successful prepared the low molecular weight HA from 1 ×106Da to 6×103 Da by using this enzyme to catalystic and digest the high molecular weight HA,even lower molecular weight or HA oligosaccharides if we extended the reaction time or increased the amount of the enzyme.Determination of the effects of the enzymatic digested by agarose gel electrophoresis,the molecular weight of HA changes constantly and the distribution range got much narrower.After that,the chemical structure of the products were confirmed by ultraviolet spectrum(UV)and infrared spectrum(FTIR),the results showed that the catalytic products come appear a specific absorption at 232 nm,which accord to the(3-eliminate reaction produce a double C=C unsaturated bond and the chemical structure remain intactly.(3)To prepare and purifie the final product unsaturated disaccharide.The enzymatic reaction was ensured completed by increasing the amount of the enzyme,filter the product using polysulfone membrane whose interception was 600 Da,then gathered the filtered solution and purified it.Finally,we prepared the disaccharide.Then compared several gels for purifying other molecular weight oligosaccharides.