Research On Aqueous Two-phase Extraction And Purification Of Ginger Protease

The research was conducted with ginger in Shandong Agricultural University during the year of 2009 to 2011. Ex traction technology of ginger protease by Aqueous Two-phase System was studied. And the effects of ex traction conditions on partitioning of ginger protease were analyzed. The isoelectric point of ginger protease and a three-step process of Aqueous Two-phase ex traction and purification were obtained to use of the technology. The main results are as fo11ows:1. Establishment of single salt Aqueous two-phase ex)traction systemThe optical single salt Aqueous Two-phase ex traction factors are: the optimum PEG molecular weight was 4000, PEG concentration was 30%,sort of salts was (NH₄)₂SO₄, (NH₄)₂SO₄ concentration was 10%, pH of system was 8.0, operation temperature was room temperature, concentration of electrolyte(NaCl) was 2% and the sample adding to the system was 3 mL. Under the above ex traction conditions, the GP activity recovery and purification factor of top phase in aqueous two-phase system reached max imums at 87.24% and 1.85.2. Establishment of buffer salt Aqueous two-phase ex traction systemThe optical buffer salt Aqueous Two-phase ex traction factors are: the optimum PEG molecular weight was 4000, PEG concentration was 20%, pH of buffer salt was 6.5 and buffer salt concentration was 10%. Under the above ex)traction conditions, the GP activity recovery and purification factor of top phase in aqueous two-phase systems reached max)imums at 94.99% and 1.86.3. Linear equations of phases equilibrium relations of PEG/ (NH₄)₂SO₄ system and PEG/ (NaH₂PO₄-Na₂HPO₄) systemThe linear equations of phases equilibrium relations of different molecular weights of PEG/ (NH₄)₂SO₄ are y=613.33e^-0.2139x ;y=260.66e^-0.2036x ;y=178.46e^-0.1969x ;y=79.91e-0.174x) ; y=124.52e^-0.2492x ; y=282.31e^-0.4302x . The linear equations of phases equilibrium relations of different molecular weights of PEG/ (NaH₂PO₄-Na₂HPO₄) are y=327.41e^-0.2706x; y=170.9e^-0.247x;y=105.9e^-0.2508x;y=91.385e^-0.3029x ;y=106.04e^-0.4173x; y=73.272e^-0.3836x.4. Determination of isoelectric point of GP by cross partitioningResult indicates that the isoelectric points of GP are 4.1 and 6.4 which prove that GP have two components and they are acid proteases.5. Establishment of three-step process of Aqueous Two-phase extraction and purification.Three-step process of Aqueous Two-phase extraction and purification is established and the salt concentration is 20%, quantity added to the system is 10 mL on the second step and the salt concentration is 12%, quantity added to the system is 15 mL on the third step.6. Purification effect test of these two Aqueous Two-phase SystemsThere are two separately protein peaks using Sephadex G-50 molecular sieve chromatography to separate GP crude enzyme extract and there is only one separately protein peak to separate Aqueous Two-phase extracted enzyme extract. However, the protease activity loss was little compared to the GP crude enzyme extract.