Aqueous Two-phase Extraction Of Uricase And The Glycerol Phosphate Oxidase

Aqueous two-phase extraction is a purification technique basing on the matter'sdistribution, the principle is similar to aqueous-organic two-phase extraction.Aqueous two-phase system is compeosed of high polymers or high polymers andsalts with proper concentration. Proteins won't denaturalize in the systerm becauseof moisture; quality transfers easily in the systerm because surface tension is low;the systerm is prone to form two phases and extend sequentially, environment isbenign. In view of the merits, aqueous two-phase system was used of extractinguricase and L-α-Glycerophosphate oxidase, proper system had been founded, thefactors affecting extraction had been researched(such as component proportion; PH;salts; enzyme concentration). Besides uricase and GPO lind been purified ulteriorlyand identified. The results were as follows:1.PEG and (NH₄)₂SO₄ had been chosed as the compositions of Aqueoustwo-phase sysytem. PEG of different mocular weight and (NH₄)₂SO₄ composedthree Aqueous two-phase systems, and the rang of concentration made certain.2.Uricase had been extracted by three kinds of Aqueous two-phase systems, thebest mocular weight of PEG which extracted enzyme was 2000. Many kinds offactors affecting purification had been discussed, and the prime conditions ofextraction that PEG2000 25%, (NH₄)₂SO₄ 9%, PH=7.5, NaCl 2% crude enzyme 5%had been confirmed, it is proved that the purification multiple was 10 and thereclaim ration of uricase was 90%. Comparing the proteins of before and aferextraction, it is found that the proteins reduced evidencely. The enzyme was purifiedby DEAE-sepharose.FF continuously, and detected by SDS-PAGE.3. L-α-Glycerophosphate oxidase was extracted by the three kinds of Aqueoustwo-phase sysytem, it was proved that the best mocular weight of PEG was 2000.Factors affeting extraction had been researched, the greatest conditions were asfollows, PEG2000 16.5%, (NH₄)₂SO₄ 13.2%, PH=7.5, crude enzyme 30%. Then thepurification multiple was 7.0 and the reclaim ration of L-α-Glycerophosphateoxidase was 90%. Comparing the proteins of before and afer extraction,it was foundthat purity had improved significantly. DEAE-sepharose.FF was used for GPO purification, SDS-PAGE for identification, the result was single belt.