Study On Screening Hyaluronic Acid-degrading Bacteria And Characterizing The Hyaluronate Lyase

Hyaluronic acid is a macromolecular glycosaminoglycan that exists in living organisms.Hyaluronic acid oligosaccharides have certain biological immune activity,good antioxidant properties,and promote tissue blood flow,angiogenesis and wound healing.Hyaluronidases are a class of glycosidases that can degrade hyaluronic acid into small molecules of hyaluronic acid oligosaccharides.It is an effective method for preparing hyaluronic acid oligosaccharides.It develops new types of hyaluronidases with high activity and excellent properties,obtaining bioactive hyaluronic acid oligosaccharide has important research significance.In this study,we screened hyaluronic acid-degrading active strains from the soil to obtain a hyaluronate lyase,and studied the heterologous expression,purification,functions,and enzymatic properties of the enzyme.The main research contents and results are as follows:1.Screening and identification of hyaluronic acid degrading bacteriaBy controlling carbon source enrichment,preliminary screening by turbidity method,and secondary screening by colorimetric method,13 strains of active bacteria that degrade hyaluronic acid were isolated from 80 soil and sludge samples.Further,by producing enzymes under the same fermentation conditions,The enzyme activity of each strain was measured and compared,and the most active strain BN3-1 was selected.The 16S rDNA phylogenetic tree analysis and bacterial physiological and biochemical reaction experiments were performed on BN3-1.The strain was identified as Citrobacter freundii,named YNLX,and stored in the Yunnan University Microbiology Institute Center,deposit number is YMF3.01173.2.Cloning and heterologous expression of hyaluronate lyase from strain YNLXA genome sequencing analysis of strain YNLX revealed a hyaluronate lyase YNLX-hyl.Blast alignment showed that there is no experimental data to reveal the functional properties of its highly homologous sequence.YNLX-hyl is cleaved with hyaluronic acid that has revealed function The highest similarity of the enzymes was only 30.9%.YNLX-hyl was successfully expressed and purified in E.coli,and was also expressed in Pichia pastoris.E.coli expresses YNLX-hyl with a greater yield,with a specific activity of 12816 U/mg.3.Functions and enzymatic properties of recombinant hyaluronate lyase YNLX-hylUltraviolet spectrophotometry was used to test the properties of hyaluronate lyase.The optimum reaction pH of YNLX-hyl is pH 5.5,which has high stability at pH5.0⁷.0.The optimal reaction temperature is 35?,at 0?,10?and 20?,there are still about 10%,40%,and 55%activity.When the temperature is higher than 40?,the enzyme isn't stabale,indicating that the enzyme is a typical low-temperature enzyme.1 mmol/L of AlCl₃?FeCl₃?CTAB?SDS?Tween 80 significantly inhibited the activity of the enzyme,while?-mercaptoethanol significantly promoted the activity of the enzyme;at 5.0²0.0%?w/v?Among Na₂SO₄ and?NH₄?₂SO₄,YNLX-hyl has high activity and certain stability.Enzyme kinetic analysis showed that V_max=13836 U/mg,K_m=1.44 mM,K_cat=9.85 s^-1.YNLX-hyl can degrade hyaluronic acid and chondroitin sulfate,has a weak ability to degrade polygalacturonic acid,and has no degrading activity to other polysaccharides.As detected by thin-layer chromatography,the product of YNLX-hyl complete degradation of hyaluronic acid and chondroitin sulfate is an unsaturated disaccharide.By controlling the reaction conditions of the enzyme,other small molecular polysaccharides can be generated.Enzymatic hydrolysis products of hyaluronic acid and chondroitin sulfate were obtained by ultrafiltration separation with ultrafiltration tube.It was found that the enzymatic hydrolysis products of hyaluronic acid and chondroitin sulfate have scavenging hydroxyl free radicals,nitrogen free radicals,superoxide anion free radicals and anti-ABTS oxidation ability,and this ability is significantly higher than undegraded hyaluronic acid and chondroitin sulfate.Nitrogen radical scavenging rates of Mixed-hyaluronic acid,hyaluronic acid disaccharide,Mixed-chondroitin sulfate,and chondroitin sulfate disaccharide obtained by enzymatic hydrolysis are 68.42%,73.21%,74.06%,77.73%,and hydroxyl radicals,respectively.The scavenging rates are 68.42%,78.74%,42.22%,and 52.55%,the superoxide anion free radical scavenging rates are 62.28%,63.70%,63.75%,and 64.89%,and the anti-ABTS oxidation ability are 62.24%,61.39%,56.41%,57.43%.Small molecule glycosaminooligosaccharides have high antioxidant properties and have good application prospects in cosmetics,health products,medical treatment and other fields.