Preparation Of Lubcan And Research On Genes Related To Its Biosynthesis

Microbial extracellular polysaccharides(EPS)have complex chemical structures with excellent biological functions,such as antitumor,antioxidant,moisturizing,immunomodulatory,hypoglycemic,and anticoagulant activity.In this paper,we mainly studied the preparation technology of exopolysaccharide from strain ZX1905,analyzed the chemical structure,physicochemical properties and related genes of the polysaccharide,and explored the biological activity and potential application prospects of the polysaccharide.The main results were as follows:1.Preparation and structure identification of EPS from strain ZX1905.To determine the phylogenetic affiliation of the strain ZX1905,BLAST analysis comparing the 16S r RNA sequence of ZX1905 with other bacteria in Gen Bank,and a phylogenetic tree was constructed.The strain ZX1905 clustered with the genus of Paenibacillus and the novel strain named Paenibacillus sp.ZX1905.ZX1905 was gram-positive and could grow at 25-37?,p H 6.0-9.0.ZX1905 was able to utilize glucose,maltose,sucrose,soluble starch,and corn starch as the carbon source,and KNO₃,Na NO₃,peptone,and yeast extract as the nitrogen source to produce the EPS.The highest EPS production of 15 g/L was achieved in a medium containing soluble starch,peptone,and inorganic salts after 28-32 h fermentation at 28?and 230 rpm.Native EPS was obtained by ethanol precipitation,and the chemical compositions of the native EPS were polysaccharide 71.21%,protein 13.16%,ash 8.40%,and moisture 6.10%.The purified polysaccharide exhibited excellent skin lubricating properties and was named lubcan.The average molecular weight of lubcan was about3.27×10⁶ Da by gel permeation chromatography.Based on the monosaccharide compositions,FT-IR,methylation,and NMR analysis,the predicted structure of lubcan was[4)-?-D-Manp-(1?4)-?-D-Glcp-(1?3)-?-D-Galp-(1?3)-?-L-Rhap-(1?3)-?-D-Galp-(1?]n,and the branch?-D-Glcp(4,6-pyr)(1?4)-?-D-Glcp A(1?4)-?-D-Glcp A(1?3)-?-L-Rhap-(1?3)-?-D-Glcp-(1?was linked with the C6 of?4)?-D-Glcp(1?residue.Lubcan was an acidic polysaccharide with a novel structure.2.Identification of genes related to lubcan biosynthesis.The genome of ZX1905was analyzed and the lubcan biosynthesis gene cluster was predicted.The genome of ZX1905 consisted of 8,392,216 base pairs with an average G+C content of 56.35%.The predicted lubcan synthesis gene cluster,named as eps gene cluster,was composed of 17 ORFs co-located in a 16,322 bp region.The eps DEFGM were putative glycosyltransferases(GTs).The eps A and eps B were likely to regulate and determinate the length of polysaccharide chains.The eps N was probably polysaccharide pyruvyl transferase.The esp O was predicted to encode flippase.The eps CHIJKLPQ were probably involved in the synthesis of nucleoside sugar precursors.The knockout vector p JQKAN::eps N was inserted into the eps N gene by homologous recombination and the knockout strain?eps N obtained did not produce polysaccharide anymore.The insertion of the plasmid in the eps gene cluster affected its expression,resulting in the inability to synthesize lubcan.The result indicated that the predicted eps gene cluster involved in lubcan synthesis.3.Research on the rheological properties of lubcan solutions.The rheological properties of lubcan over the concentrations,temperature,p H,and salts were investigated.Lubcan solutions(0.5%,1.0%,and 1.5%,w/w)had typical shear-thinning pattern which fitted the Cross model well(R²>0.99).With increasing frequency,the storage and loss moduli G?and G?of 1.0%and 1.5%lubcan solution increased continuously,and there was always G?>G?which showed an elastic behavior.While a crossover of G?and G?was observed at the high frequency of 0.5%lubcan solution,which indicated that elasticity of 0.5%lubcan was dominant at a lower frequency,and viscosity was dominant at a higher frequency.The lubcan solution exhibited stability at p H ranging from 5.0 to 7.0 and temperature between 5and 50?.Both monovalent(Na⁺and K⁺)and divalent(Ca²⁺and Mg²⁺)ions increased the apparent viscosity,thermo-stability,and viscoelasticity of lubcan solutions.4.Bioactivity analysis of lubcan.The moisture absorption rates of lubcan were16.98%and 40.41%under the conditions of 43%and 81%relative humidity,which were close to that of HA(17.28%and 41.20%,respectively).The moisture holding rates of lubcan and HA were 29.34%and 25.16%,respectively.These results indicated that the moisture absorption and holding capacities of lubcan were almost the same as that of HA.Lubcan had an obvious effect on reducing the friction coefficient on the skin in a dose-dependent manner.Lubcan could reduce friction and improve lubrication performance.MTT assay showed that there was no cytotoxicity of lubcan(50-500?g/m L)on a variety of cells(such as Haca T,HFF-1,NIH3T3,and RAW264.7).Conclusion:it was found that lubcan produced by Paenibacillus sp.ZX1905 was an acidic polysaccharide with a new structure consisted of glucuronic acid,glucose,mannose,galactose,and rhamnose.The biosynthesis gene cluster of lubcan was predicted and verified.Lubcan had excellent rheological properties,moisture holding and absorption characteristics,lubrication characteristics,and had no cytotoxicity to skin and immune cells.Lubcan may become a good alternative to more expensive hyaluronic acid in cosmetics,and has the potential to be applied in biomedical materials as a novel structure polysaccharide.