Purification And Identification Of Pseudomonas QL212and Curdlan Application

Curdlan is a straight-chain homotype β-1,3-glucan developed by the fermentation ofmicrobes, which has distinctive physics and chemistry characterization and immune irritancyactivity. In food, medicine, agriculture, chemical industry, construction and other fields, itgives a wide prospect in development and application. Curdlan’s polymerization degree isabout450, and the molecular weight is bigger. These features restrict the practical applicationof Curdlan in many respects.The length of Curdlan molecular chain and degree ofpolymerization is of great significance for its application in medicine. Curdlan with largermolecular weight does not dissolve in water or the aqueous solution viscosity is higher, thatinhibit its biological activity. Curdlan with low molecular weight, which is modified, hassatisfactory anti-tumor, anti-HIV and immune activity. Î'-1,3-glucan generally appears in thecell wall or cytomembrane of fungi, bacteria, yeast and algae and higher plants. So far, onlythe agrobacterium and alcaligenes are reported that they can produce the straight-chainhomotype β-1,3-glucan. However, different sources of β-1,3-glucan have different physicalproperties. Take facts as the basis, a strain pseudomonas that can produce Curdlan wasselected by our group. What’s more, we found that the produced Curdlan has goodwater-solubility and a good prospect in application.In this thesis, a strain chashi3-1with high yield of exopolysaccharide was screened fromthe soil as a starting strain. Through microscopy and analysis on16S rDNA sequence, weidentified of the strain chashi3-1. We also explored the extraction, separation and purificationtechnology of Curdlan after fermentation. After that, infrared spectrum and multi-angle laserlight scattering technology was used to analys Curdlan’s molecular structure and molecularweight after the separation and purification. Then, further experiments single factorexperiment and response surface method was applied to optimize the fermentation mediumand culture conditions. The main content is:First,18strains with higher yield of suger were screened from soil. Then, throughmicroscopy and solid medium containing the aniline blue staining, we obtained the strainssecreting Curdlan–chashi3-1. After that, we have accomplished following work: we amplify the16S rDNA sequence with PCR, connect it with T carrier into E. coli DH5α, use blue-whiteselection and eventually obtain the strain which is successfully transfected. Then, we useprove that the strain chashi3-1belong to pseudomonas sp.by comparing with the knownsequence of the NCBI database.Curdlan produced by pseudomonas QL212cultured in the fermentation medium. Thenwe obtained Curdlan crude products by organic solvent grading precipitation, and removedimpurities and other little molecular carbohydrates by dialysis method. After furtherpurification by ion exchange chromatography and molecular sieve, we obtained high qualifiedand pure Curdlan. Pure Curdlan-a kind of white powder-can dissolve in water. Throughmulti-angle laser light scattering analysis the molecular weight of Curdlan was identified as618KDa. Infrared spectrum analysis showed the features of the β-bond: the absorption peak atthe wavelength of890cm-1and no specific absorption peak at840cm-1. So alpha-bond doesnot exist.By the single factor experiment analysis, sucrose and yeast powder was identified as theoptimum carbon source and nitrogen source for the fermentation of pseudomonas QL212. Wealso analyzed the influences of Curdlan fermentation, and then selected three main impactfactors for BBD experiment design. Through optimization, we obtained the optimalconditions of Curdlan fermentation: sucrose is30.11g/L, yeast powder5.94g/L, calciumcarbonate3g/L, inoculation quantity10%, inoculum age12h, fluid volume and shakingspeed75mL and180rpm respectively, initial pH value8.03, fermentation period96h,fermentation temperature30-32℃. Under these conditions, the strains can reach their highestCurdlan productivity.Microbial flocculant has some specific features such as non-toxic, no harmless, nosecondary pollution, which is more promising than ordinary flocculants. This article alsoexplored the flocculating effect of Curdlan, and optimized the factors affecting theflocculation. We used Curdlan for the flocculation of dyeing and finishing effluents, YellowRiver water and juice, obtained better results with flocculating rate of97.67%,93.87%,94.17%and93.98%respectively.