Characterization Of The Chemical Structure And Anti-inflammatory Activity Of The Exopolysaccharide From Paenibacillus Edaphicus NU-2

Polysaccharide polymers with physiological activity play an important role in immunomodulation,antitumor,anticoagulation,antioxidation and other aspects.Polysaccharides can be modified by group modification to further change the physiological activity of natural polysaccharides,which provides feasibility for the production of new functional additives or therapeutic drugs.Many Paenibacillus strains have the ability to synthesize exopolysaccharides,and the structures and properties of exopolysaccharide products are diverse.This makes it possible to obtain new structural polysaccharides from Paenibacillus.In this study,a Paenibacillus edaphicus strain NUST16 was used as the starting strain,and a mutant with high yield of exopolysaccharides was obtained by UV mutagenesis,and the rheological properties of the exopolysaccharides were analyzed,and then the chemical structure of the purified polysaccharide was analyzed.Then the sulfated polysaccharides were obtained by group modification,and their physiological activities were determined.Finally,the structure-activity relationship between the chemical structure of polysaccharides and their physiological activities was preliminarily analyzed.The main conclusions are as follows:（1）A mutant strain NU-2 with high exopolysaccharide yield was screened by treating Paenibacillus sp.NUST16 with ultraviolet mutagenesis using nucleoside diphosphate kinase inhibitor c AMP as a screening factor.The exopolysaccharide yield of PNU2 reached 11.9 g/L after 72 h fermentation,which was 27.51%higher than that of the starting strain.The rheological analysis of PNU2 showed that the flow of PNU2 solution followed the Carreau flow model,and the zero shear viscosity increased with the increase of concentration,showing a typical shear dilution behavior at high shear rates.PNU2 has a certain temperature tolerance（Tm≈50°C）,but the temperature tolerance of the solution decreases slightly with the addition of 1%potassium chloride.The viscoelastic characteristics of PNU2 solution vary with the oscillation frequency and concentration.When the oscillation frequency was less than 2.5 rad/s,the 0.5%PNU2 solution was a sol-type solution,and when the oscillation frequency or the polysaccharide concentration was increased,the PNU2 solution was rapidly transformed into a gel-type solution.（2）Analysis of monosaccharide composition showed that the purified PNU2 was composed of D-mannose,D-glucose,D-glucuronic acid,D-galactose and L-fucose in a molar ratio of 3:3:1:1:1.PNU2 has a weight-average molecular weight（M_w）of 2.45×10⁷ Da,a number-average molecular weight（M_n）of 3.16×10⁶ Da,and a polydispersity index（PDI）of7.74.The chemical structure of PNU2 was obtained by FT-IR,methylation-GC/MS and 1D-2D-NMR.A main chain structure in a monosaccharide repeat unit is as follows:→3)-β-D-Man-（1→3）-β-D-Glc-（1→3）-β-D-Man-（1→3）-β-D-Glc-（1→4）-β-D-Glc A-（1→3）-β-D-Man-（1→.The C4 position of the mannose on the far left is linked with Pyruvyl→4,6）-β-D-Glc-(1→,and the C4 position of the mannose in the middle is linked withβ-L-Fuc-（1→3）-β-D-Gal-.（3）Two sulfated polysaccharides with the degree of substitution（DS）of 1.64（S-PNU2₁）and 0.87（S-PNU2₂）were prepared by total substitution and protected-partial substitution,respectively.The weight-average molecular weights of the two sulfated polysaccharides were3.18×10⁵ Da and 1.79×10⁶ Da,respectively.The results of in vitro cell experiment showed that low concentration of polysaccharide could promote cell proliferation,while high concentration of sulfated polysaccharide could weaken the effect of promoting cell proliferation,and the effect of partially substituted S-PNU2₂ was better than that of fully substituted S-PNU2₁.Both S-PNU2₁ and S-PNU2₂ could significantly reduce LPS-induced NO production in macrophages,and the inhibitory effect was positively correlated with polysaccharides concentration.It can inhibit the inflammatory reaction of macrophages induced by LPS,and its effect is better than that of dexamethasone.The results of molecular docking showed that although the two structures（A and B）intercepted different segments of the sugar chain,the strongest binding ability was with the LPS receptors,indicating that the sulfated modified polysaccharides probably produced anti-inflammatory effect by competing with LPS binding sites.The polysaccharide of A conformation could inhibit the inflammatory response by competitive binding with LPS receptor 3VQ2,and the binding sites were speculated to be LYS360,ASN359,HIS429,ASN456,SER480,ASN479 and GLN505.The polysaccharide inhibitory effect of the B conformation may be achieved by competitive binding to the LPS receptor 2Z64,and the binding sites were speculated to be ARG256,HIS96,LYS153,THR231,ARG106,ASP208,HIS178,SER206 and HIS228.Although the predicted binding sites are different,different sugar units in the sugar chain are involved in the binding with the receptor,which proves that the activity of sulfated modified polysaccharides is closely related to their structure.