Study On The Extraction Of Papain With Aqueous Two-phase System

As a result of strong protein hydrolysis ability and has wide adaptability at pH and temperature, papain has a wide application in food, medicine, daliy chemical and so on. So an in-depth study on the separation and purification of papain, and extract to get high quality papain has important application value. There were some problems in the traditional extraction method of papain, so it is necessary to find a new method of preparation of high quality, highly active papain. Aqueous two-phase system (ATPS) is a new type of biological chemical separation technology and has great application prospects in recent years. Now, the study of papain aqueous two-phase extraction mainly concentrated in the PEG/(NH4)2SO4and PEG/phosphate aqueous two-phase system, PEG/Na2SO4aqueous two-phase system, metal chelate affinity aqueous two-phase system, ion liquid aqueous two-phase system on extraction of papain has less reports. So the experiment adopted the three kinds of aqueous two-phase systems extraction papain, and comparison results.Firstly, PEG/Na2SO4aqueous two-phase system is used to extract papain. The influence of reagent and pH value on activity of papain were investigated, in the meantime, the influence of PEG with different molecular weights, the mass fraction of Na2SO4and PEG, pH value and enzyme added on distribution behavior in aqueous two-phase system was discussed. The separation condition of papain in aqueous two-phase system was optimizated by response surface method, and amplified in laboratory scale. The results showed that the mass fraction of Na2SO4on partitioning behavior significantly, the influence of the second is the mass fraction of PEG, the influence of pH on partitioning behavior was not significant. Get the best extraction conditions for:16.0%(w/w) PEG6000,18.0%(w/w) Na2SO4, pH7.0, the dosage of papain is1.0mg/g (enzyme system of quality/total quality). In this condition, the partitioning coefficient of papain in the two-phase system up to3.1, papaya protease activity recovery of82.6%can be obtained. Experiments show:the response surface method to optimize the aqueous two-phase system extraction conditions of papain is feasible, can be used for practical prediction.Secondly, the extraction of papain by metal chelating affinity partitioning in aqueous two-phase systems was studied. The effect of different inorganic salt and concentration, PEG molecular weight and concentration, the addition of affinity ligands, pH value, the dosage of papain on the affinity partition was investigated. Furthermore, the extraction effect of aqueous two-phase system with and without affinity ligands was compared, and the response surface method is used to optimize the metal chelating affinity aqueous two-phase extraction conditions of papain. The results showed that the distribution of papain in PEG phase was improved after the affinity ligands join in. Response surface experiment results show that various factors influence on papaya protease extracted order: PEG4000mass fraction, Na2SO4mass fraction, PEG-IDA-Cu(II) content, the dosage of papain, pH. Get the best extraction conditions for:pH6.50, PEG4000mass fraction is17.25%(w/w), PEG-IDA-Cu(Ⅱ) content is0.75%(w/w), Na2SO4mass fraction is18.0%(w/w), the dosage of papain was2.50mg/g. Under this condition enzyme activity recovery rate was93.42%, the purification factor of1.55was received. The result laid the experimental basis for further research and scale production. Results show that metal chelating affinity partitioning in aqueous two-phase systems was feasible for the separation of papain.Thirdly, ion liquid aqueous two-phase system was used to extract papain. Firstly, the influence of pH value, temperature, reagents to the activity of papain have been investigated. Secondly, effects of different Ionic liquid aqueous two-phase systems, alkyl chain length and concentration of the ionic liquid, the dosage of papain, pH value, temperature to the partitioning behavior of papain were discussed concretely, the optimal separation condition of ionic liquids aqueous two phase extraction of papain by response surface method, and the structure of papain were characterized before and after the extraction. Various factors on the influence of the aqueous two-phase extraction papain sequence as follows:the concentration of K2HPO4,[C4mim] Cl concentration, the dosage of papain, pH. The activity recovery of enzyme achieve98.1%and the purification factor achieve1.6under the optimum conditions:0.20g/mL [C4mim]Cl, K2HPO40.30g/mL, pH7.0, the dosage of papain was2.0mg/mL,30℃. The result of UV-vis and FT-IR demonstrated that the structure of papain before and after extraction was nearly no change, means the extraction condition of ionic liquid aqueous two-phase system was mild. The result laid the experimental basis for further research and scale production.