Study On Paenibacillus Sp.FP01 Producing Levan And Its Levansucrase

Fructans,a class of fructose-based water-soluble polysaccharides produced by plants and microorganisms,possess various biological activities,such as regulating blood sugar and lipids,anti-tumor,immunomodulatory and as prebiotics to regulate the intestinal environment,thus have potential applications in the food,feed and pharmaceutical industries.Based on the linkage type of fructofuranosyl residues,fructans are divided into two main types,levan and inulin.Compared with inulin which usually extracted from plants,levan is mainly derived from microorganisms with high production costs and purification costs,which hinder the research and application of levan.In this thesis,we isolated a new fructan-producing strain from industrial wastewater,characterized the structural,rheological and prebiotic properties of the fructan.Furthermore,we also cloned the levansucrase gene from FP01 strain and determined the enzymatic characteristics of the recombinant levansucrase.One fructan-producing strain FP01 were isolated and identified as Paenibacillus sp.by using phylogenetic analysis based on 16S r DNA gene sequences.The exopolysaccharide(EPS)was identified as fructan by TLC,named Plev.The fermentation medium for producing Plev was optimized as(g/L):sucrose 180,KNO₃3,Na H₂PO₄ 1,Ca Cl₂ 0.07,Mg Cl₂ 0.2,Fe SO₄·7H₂O 0.0125,Mn SO₄ 0.003,Zn Cl₂0.0075,H₂O 1 L,p H 7.0.Under the optimal culture temperature of 30°C for 60 h,the maximum EPS yield was 89.5 g/L.GPC,IR,GC,NMR and methylation analysis indicated that the EPS from FP01 was levan-type fructan with a molecular weight of 3-4×10~6 Da.The rheological properties of Plev were investigated under different concentrations,p H levels,temperatures and inorganic salts.Aqueous solutions of Plev exhibited a non-Newtonian shear-thinning behavior at the concentration of 3-5%(w/v)and a gradual decline in the apparent viscosity with increasing temperature.No obvious change in the viscosity was observed over a wide range of p H 3-11,indicating good acid-and alkali-stable properties of Plev.Heating and freezing pretreatment also had no obvious effects on the apparent viscosity,indicating good thermostability and freeze-thaw stability of Plev.Besides,addition of common salts(Na⁺,K⁺,Ca²⁺,Mg²⁺)at 0.2M could slightly increase the viscosity of Plev solutions.The prebiotic activities of Plev were evaluated based on intestinal microbiome and metabolism analysis of the fecal samples from mice fed with 10%levan diet for 30 days.Compared with the control group,Plev-fed mice exhibited higher food intake and resulted in slightly higher body weight gain.The total cecum weight,cecum wall weight and cecum content weight also increased by 42.42%,47.83%and 20.00%,respectively.Histopathological observation showed no abnormalities in the intestinal tissue of mice in both groups.16S r DNA high-throughput sequencing coupled with quantitative RT-PCR revealed that intake of Plev could improve the relative levels of beneficial bacteria.~1H-NMR based metabolomics coupled with ion chromatography analysis showed that intervention of Plev elevated the levels of short-chain fatty acids and lactate in feces of mice.To investigate the enzymatic synthesis of Plev,a new gene encoding levansucrase from Paenibacillus sp.FP01 was cloned and expressed in E.coli.The purified recombinant levansucrase was obtained by IPTG induction,ultrasonication and Ni column purification.With sucrose as the substrate,the optimal temperature and p H of the recombinant levansucrase were 30°C and 4.0,respectively.The K_m and V_m values were 0.151 mol·L^-1and 1.126?mol·min^-1·m L^-1,respectively.The recombinant levansucrase had good stability at p H 3-8.Non-ionic surfactants(Span 80,Triton X-100)could significantly improve the enzymatic activities.The levansucrase could catalyze the synthesis of levan with sucrose or raffinose as substrates,and could catalyze the synthesis of new fructose-based oligosaccharides with sucrose as the fructosyl donor and other disaccharides(lactose,maltose or trehalose)as acceptor.In conclusion,Paenibacillus sp.FP01 is a high-efficient levan-producing strain.The levan Plev produced by FP01 exhibited excellent rheological properties and prebiotic activities,could be considered as a potential thickener and prebiotic in the food industry.The recombinant levansucrase derived from FP01 strain can be used to synthesize levan and new fructose-based oligosaccharides,which provides research basis for the subsequent studies of levansucrase and fructose-based oligosaccharides.