Study On Separation And Purification Of γ-Polyglutamic Acid From Fermentation Broth And Its Preliminary Identification

As a kind of macromolecular polymer, y-polyglutamic (y-PGA) is biocompatibility, biodegradability, non-toxicity and non-pollution. Compared with chemical synthesis, enzymatic conversion, extaction, microbial fermentation, a simple process and high yield method, has become the mian pathway for producing y-PGA. However, y-PGA broth composition is very complex and the vicoity is overhigh. So seeking method for separation and purification of y-PGA from broth has become hot spot and difficulty.Traditional methods for separation and purification of y-PGA include precipition method of organic solvents, chemical precipition and membrane separation. The first two methods have disadvantages of low extraction efficiency and low purity. The membrane was easy to plug and expensive. To improve these shortages, the paper intended to extract effectively y-PGA from broth by choosing suitable aqueous two-phase system (ATPS). Extracting y-PGA from broth with PEG-potassium phosphate ATPS, propyl alcohol-Na2CO3 ATPS and [C4mim]Cl-K2HPO4 ATPS, the influence of process and environmental conditions on partition coefficient K and extraction efficiency Y were studied. And y-PGA extracted from fermentation broth was characterized preliminarily. The main content and results are as follows:The phase separation and extraction ability of different ATPSs of PEG-salt were determined to extract y-PGA, the result showed extraction efficiency of PEG2000-potassium phosphate ATPS was highest. By single-factor experiments and orthogonal experiment, the process and environmental conditions of extracting γ-PGA were optimized. The optimum conditions were as follows: 22%(w/w) PEG2000,14%(w/w) potassium phosphate, and 1.2%(w/w) NaC 1, put less than 5 minutes at pH 7.5 and room temperature, the two phases changed into clear. The partition coefficient and extraction efficiency were 0.10 and 96.02%.The phase separation and extraction ability of different ATPSs of organic solvent-salt were determined to extract γ-PGA, the result showed extraction efficiency of propyl alcohol-Na2CO3 ATPS was highest. By single-factor experiments and orthogonal experiment, the process and environmental conditions of extracting γ-PGA were optimized. The optimum conditions were as follows:18%(w/w) propyl alcohol,14%(w/w) Na2CO3, put 2.5 hours at pH 10 and 40 ℃, the two phases changed into clear. The partition coefficient and extraction efficiency were 0.23 and 98.07%.The phase separation and extraction ability of different ATPSs of ionic liquids-salt were determined to extract γ-PGA, the result showed extraction efficiency of [C4mim]Cl-K2HPO4ATPS was highest. The paper investigated influence of [C4mim]Cl concentration, K2HPO4 amount, pH and temperature on γ-PGA extraction efficiency. The response surface experimental design method of three factors and three levels were used to optimize the impact factors of extracting γ-PGA, and picked up the best conditions:[C4mim]Cl concentration was 404.58mg/mL, K2HPO4 amount was 1.51g, pH was 8.96. Under the best conditions, the extraction efficiency of γ-PGA was 97.45%. Based on actual production, extraction conditions were adjuted:[C4mim]Cl concentration was 404mg/mL, K2HPO4 amount was 1.5g, pH was 9.0. To verify the predicition results, we found that the extraction efficiency was 96.63% which closed to the theoretical prediction. That demonstrated the credibility of the model is high. According to the three-dimensional response surface chart, we concluded that the order of influencing factors on the extraction efficiency was pH> K2HPO4 amount> [C4mim]Cl concentration.Considering extraction efficiency, cost and industrial application feasibility, propyl alcohol-Na2CO3 ATPS was chosen to extract γ-PGA from fermentation broth. After bottom phase solution dialyzing and freezing drying, ivory powder was obtained. Using ninhydrin colorimetry, we found the purity of γ-PGA was 56.85%. With paper chromatography to assay the composition, we found that γ-PGA was made of pure glutamic acid, not containing free amino acid. And SDS-PAGE electrophoresis was used to measure molecular weight of γ-PGA, the molecular weight ranged from 669kDa-700kDa, thus, γ-PGA had polymer molecular weight.From the study on water-retaining property and hygroscopicity of y-PGA, we found that y-PGA had the water-retaining property truly, and the water-retaining property increased with the concentration increasing. Hygroscopicity would enhance if it underwent the ultraviolet irradiating, and the irradiation time was longer, the hygroscopicity was stronger.