| Aqueous two-phase extraction technology (ATPS) was a bioseparation technology that is pollution-free and facilitate. It could make the target product enrich to single phase, meanwhile, it also could make the target product achieve the purpose of purification, and didn’t destroy the biological activity of the target product. Glutamate decarboxylase(GAD) is a kind of endoenzyme of Escherichia coli AS1.505(E.coli). In order to get GAD from E.coli, E.coli cell disruption by Ultrasonic was necessarily. But it was difficult to get high purity of GAD because of some culture and cell debris as same as impurities in broken fluid of E.coli cells. In order to conquer this problem, a novel separation technology——aqueous two-phase technology was used to purify and separate GAD which was treated by Ultrasonic cell disruption in the study. However, because the target product was extracted to a single phase, it produced a difficult problem that target product couldn’t be separated from phase material. This problem limited the development of ATPS. In order to solve the bottleneck problem, in this paper, GAD was extracted from broken fluid of E.coli cells into PEG phase by ATPS. Then the method of centrifugation ultrafiltration was used to separate GAD from PEG and recover them respectively. The contents of this research are as follows:(1) To set up the aqueous two-phase system. Phase diagrams of different molecular weight PEG and sodium citrate were researched. To choose suitable concentration of PEG and sodium citrate, the phase diagrams of PEG-sodium citrate was researched, and then the ATPS of PEG-sodium citrate was established.(2) Using PEG-sodium citrate aqueous two-phase system to purify and separate GAD. Extracting GAD from Ultrasonic wall-breaking liquid of E.coli GAD by ATPS of PEG-sodium citrate. In this part, the effects of the molecular weight of PEG, the mass fraction of PEG, the mass fraction of sodium citrate, pH and the adding amount of NaCl in ATPS of PEG-sodium citrate on partition coefficient of GAD, partition coefficient of protein, the yield of GAD and purification fold in the extraction process of E.coli GAD were researched. Orthogonal design was used to optimize extraction technological parameter.(3) Separating GAD from PEG and recovering them respectively by the ultrafiltration centrifugal. Separation GAD from PEG and recovery of GAD and PEG respectively by the ultrafiltration centrifugal were preliminary studied. The effects of centrifugation ultrafiltration was discussed. Response surface methodology with three-factor and three-level design was applied to design experiments, Box-Behnken design was used with response value of GAD’s recovery ratio to optimize process parameters of centrifugal ultrafiltration.The results showed that PEG and sodium citrate could build phase diagram of ATPS, PEG-sodium citrate aqueous two-phase system could extract E.coli GAD from PEG effectively. The optimal levels of phase material were PEG1000(29%, w/w)-sodium citrate(14%, w/w) aqueous two-phase system and2%(w/w) NaCl addition got a purification fold of4.52. According to the results of the response surface analysis, with a centrifugal ultrafiltration rotational speed10000r/min, centrifugal ultrafiltration time20min and4.5dilution multiple for process parameters of centrifugal ultrafiltration, the actual average recovery of GAD was90.87%and the recovery of PEG was91.49%.In this study the method of centrifugal ultrafiltration was used to separate and recover of GAD and PEG. It was advantageous to get high purity of GAD, to reduce reagent consumption, the cost and environmental pollution. ATPS combined with the method of centrifugal ultrafiltration to solve the problem that GAD was extracted by ATPS easily but was separated from phase material difficultly. It facilitated to extract GAD in industry, and offered practical application feasibility for extracting enzyme in industry... |
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