Mutation Breeding Of Agrobacterium Tumefaciens For Succinoglycan Production And Study On Its Structure And Properties

Succinoglycan is an acidic microbial exopolysaccharide produced by Rhizobium,Agrobacterium,Alcaligenes and Pseudomonas.Succinoglycan is composed of octasaccharide repeating units in which galactose and glucose monomers occur in a molar ratio of 1 to 7 with?-1,3,?-1,4 and?-1,6 glycosidic linkages,in addition,succinate,pyruvate and acetate are commonly found as nonsaccharidic substituents.Due to its unique and stable rheological properties,succinoglycan can be used as emulsifier,gelling agent,stabilizer and thickener in cosmetics,daily use,pharmaceutical products,food and petroleum industry.At present,succinoglycan has been commercialized in foreign countries,although some reports have already been published about its fermentation in China,few research has been described for its structural and rheological properties.In this paper,firstly,succinoglycan producing strain of Agrobacterium tumefaciens AT01 was used as starting strain,and two high-yield strains 52N-11 and 15N-108 were screened by NTG mutagenesis.Then the fermentation medium component of 52N-11strain was optimized.Finally,the chemical structure and rheological properties of succinoglycan were studied.The main results are as follows:(1)Mutagenesis and selection of succinoglycan high-producing strainsUsing AT01 as the starting strain,the method of NTG mutagenesis was established,the time of seed culture was 14 h,the OD₆₀₀ of seed solution was diluted to about 0.3,NTG concentration was 0.5 mg/m L.After three rounds of NTG mutagenesis,two high-yield strains with good genetic stability were screened from 1324 strains.The succinoglycan yields of the two strains were 36.6±1.95 g/L and 38.5±0.87 g/L,respectively,which was 163.3%and 176.9%higher than that of the starting strain AT01.16S r DNA comparison confirmed that the high-yield strain 52N-11 was homologous to Agrobacterium tumefaciens C58 strain.After 72 h of shake flask fermentation,the succinoglycan yield of 52N-11 was 35.5±1.36 g/L,while AT01 was 12.8±0.3 g/L.In the 15 L fermenter,AT01 and 52N-11 both reached the highest succinoglycan yield at60 hours,and the yields of 72 h were 23.4 g/L and 31.0 g/L,respectively.Thus 52N-11strain which was selected by NTG mutagenesis exhibited better ability to synthesize succinoglycan than AT01 strain.The resequencing of 52N-11strain was carried out with the whole genome of AT01 strain as the reference genome,261 SNP and 4 Indel sites were collected.The results of KEGG annotation for the mutant genes in the exonic region indicated that most genes involved in carbohydrate metabolism,energy metabolism,signal transduction,nucleotide metabolism,and amino acid metabolism.(2)Optimization of fermentation medium for 52N-11 strainThe fermentation medium of high-yield strain 52N-11 was optimized,and the optimal medium components were obtained by single factor experiment,Plachett-Burman and response surface Box-Behnken Design:sucrose 64.8 g/L,yeast extract5.95 g/L,K₂HPO₄ 0.0975 g/L,Ca CO₃ 5.0 g/L.The fermentation conditions were also optimized:inoculum volumn was 1%and the initial p H was 7.2.The succinoglycan yield of 52N-11 was 40.5 g/L in shake flask and 34.1 g/L in 15 L fermenter after fermentation for 72 h with optimized medium.Compared to the initial medium,the yield increased by 13.1%(35.8 g/L)and 10%(31.0 g/L)in shake flasks and 15 L fermenter,respectively.(3)Analysis of structural characteristics of succinoglycanThe structural properties of the commercial grade succinoglycan(SG),starting strain produced succinoglycan(SG-A),and high-yield strain produced succinoglycan(SG-N)were analyzed.Infrared spectroscopy results showed similar characteristic absorption peaks of the three succinoglycan samples,and was consistent with the infrared spectrum of succinoglycan reported in the literature;the characteristic signals of succinate and pyruvate were detected by ¹H NMR and ¹³C NMR analysis;The molar ratios of glucose to galactose in SG,SG-A and SG-N samples were 6.63,6.65 and 6.94,which was analyzed by PMP pre-column derivatization high performance liquid chromatography.The molar ratio of 7:1 indicated the structural characteristics of octasaccharide repeating unit.These results illustrated that SG-A and SG-N were indeed succinoglycan.The contents of the substituents in the succinoglycan samples were determined by reversed-phase high performance liquid chromatography.The results showed that the succinate contents in SG-A and SG-N were 68.5 mg/g and 71.2 mg/g,respectively,which was significantly higher than 34.8 mg/g of commercial sample SG;in SG-A and SG-N the acetate substituent contents were 0.28 mg/g and 0.34 mg/g,which was significantly lower than 1.50 mg/g of the SG sample.Monosaccharide composition analysis showed that except galactose and glucose,SG-A and SG-N samples contained1.15%and 0.94%mannose,respectively,while SG samples contained mannose,glucosamine,ribose,rhamnose,galacturonic acid and arabinose and xylose,indicating that the SG-N produced by the high-yield strain and the SG-A produced by the starting strain were slightly different from the structure of the commercial sample SG.High performance gel filtration chromatography(HPGFC)showed that SG-A contained two components,and the Mw was 1.55×10⁷ Da and 1.26×10⁶ Da,respectively.SG-N contained only one peak,and the Mw was 1.01×10⁷ Da.SG had a high molecular weight peak and the Mw was 1.55×10⁷ Da.The melting temperature of the three solid succinoglycan samples was determined by DSC,and the melting temperatures for SG,SG-A and SG-N were 124.01?,134.13°C and 117.93°C,respectively.The crystallinities of SG,SG-A and SG-N were 16.44%,21.96%and 16.09%,respectively,and this result was consistent with the melting temperature of each succinoglycan sample.The TGA results indicated that the degradation temperature of the SG sample was about 260°C,while the degradation temperatures of SG-A and SG-N were both270°C.(4)Study on rheological properties of succinoglycan solutionsThe rheological properties for different concentration solutions of the commercial grade succinoglycan(SG),starting strain produced succinoglycan(SG-A),and high-yield strain produced succinoglycan(SG-N)were analyzed,and the three succinoglycan solutions exhibited similar rheological behavior.The results of oscillating sweep showed that succinoglycan solutions have viscoelasticity,and the viscoelasticity increased with the increase of concentration.62°C was the conformational transition temperature of succinoglycan solution,once the temperature exceeded the conformational transition temperature,viscosity,modulus and pseudoplasticity of succinoglycan solutions decreased sharply,and when cooled to room temperature,the modulus could recover even beyond the pre-heating level.Steady-state rheology experiments proved that the succinoglycan solution was pseudoplastic non-Newtonian fluid with high viscosity.In addition,the viscosity and shear thinning phenomenon of the SG-N solutions were not affected by p H value or the type and concentration of inorganic salt solution.