Study On The Separation Of Bromelain Using Aqueous Two-Phase Extraction

Aqueous two-phase extraction is a new bioseparation technique developed in recent years. The bioseparation technique has many advantages such as higher biocompatibility, less separation procedures, milder operating condition, easier engineering amplification, less residue of organic solvents in the product and so on. It has been widely used to purify different enzymes, nucleic acids, growth hormone and bioactive component in plants.In this experiment, the effects of several factors on bromelain activity were investigated. These factors included the components of aqueous two-phase system and different operating conditions. The experiment results show that the different molecular masses of polyethylene glycol (PEG) and the condition of PEG1000 in the experiment range have little effect to bromelain activity. In the selected concentration range, (NH4)2SO4, anionic surfactant sodium dodecyl sulfonate (SDS) and cationic surfactant cetyl trimethyl amine bromide(CTAB) all have little effect to bromelain activity. The experiment results also show that the suitable temperature range of bromelain activity is from 20C to 50C and the suitable pH value range of reaction is from 4.0 to 10.0. In the range of experiment, NaCl and NaBr have no obviously effect on enzyme activity, but MgCl2 and CaCl2 all inhibited the bromelain activity.It was investigated that the distribution behaviour and influence factors on distribution of bromelain in the PEG/(NH4)2SO4, SDS/CTAB, PEG/ hydroxypropyl starch aqueous two-phase systems.In the PEG/(NH4)2SO4 aqueous two-phase system, the effects of the PEGwith different molecular masses, concentrations of PEG, concentrations of (NH4)2SO4i and NaCl on the distribution factor of bromelain in two phase, the volume ratio of the top phase to the bottom phase and the recovery of bromelain were investigated. Under the selected separation conditions, a high recovery of bromelain was obtained from the mixed fluid. The experiment results show that the suitable extraction conditions are 17% PEG1000, 14% (NH4)2SO4, pH 7.0, and the distribution coefficient of K =3.9 and the yield of bromelain can reach 91% at room temperature.Through the experiment, the phase diagrams of SDS and CTAB aqueous mixtures were discussed. The influence of temperature and added salt sodium bromide(NaBr) on the aqueous two-phase regions have been observed. Besides, bromelain was extracted under the condition of different temperatures and pH values and added salt. The experiment results show that the yield of bromelain can reach 80% in the system of SDS/CTAB/H2O.Finally, the synthetis method of hydroxypropyl starch using maniocca as raw material and the effect of technological conditions on the substitution were investigated. Using the hydroxypropyl starch and PEG construct aqueous two-phase system, the phase diagrams and distribution behaviour of bromelain in this aqueous two-phase system was determined. The experiment results show that the suitable extraction conditions were 11% PEG1000, 10.5% hydroxypropyl starch, pH 7.0, 0.15 mol/L NaCl, and the distribution coefficient of K =3.23 and the yield of bromelain can reach 71% at room temperature.In this paper, the advantages and the disadvantages of using three aqueous two-phase systems to extract bromelain were analyzed and some suggests to existing questions were put in forward. The project is subsidized by the Young People Fund of Guangxi Science and Technology Department (No 0135029) and the Scientific Research Fund of Guangxi Education Department.