Prepare Of PH-sensitive Polymer And Its Application In Purification Of Papain

Papain is widely used in food and feed fields. However, domestic papain with high purity is largely dependent on the import, so the use of papain in biological pharmacy and research fields are limited due to its high cost. Therefore, the processing of high purity papain with low costs is hot. Affinity precipitation is a new technology in affinity purity. Generally, affinity precipitation is a method that uses polymers with reversible precipitation characteristics as the carrier. A pH sensitive polymer with high recovery rate was prepared and connected to the affinity'ligand of Reactive Red120, Cu2+, and Fe3^to purify papain. In the meantime, the FTIR, pI, recovery rate, ligand density, absorption and desorption conditions of all of them were determined and optimized. The results are shown below:1. Prepare of pH sensitive polymer. MAA. MMA. DMAEMA and N-MAM was polymerized through free radical polymerization to a pH sensitive polymer. FTIR was used to verify its structure and its pI and recovery rate were5.15and98%, respectively.2. Connection of affinity ligand. The affinity ligands(Reactive Red120, Cu2+, Fe3+)was connect to this polymer and the ligand density was investigated. The ligand density of polymer with Reactive Red120achieved43.00?mol/g under initial ligand amount45.00mg and its pI and recovery rate were5.12and96%, respectively. The ligand density of polymer with Cu2+and polymer with Fe3+were3.30mmol/g and2.92mmol/g under initial ligand amount20mmol and0.4ml ECH. The pI and recovery rate of polymer with metal ion were5.08and95, respectively.3. Purification of papain. The polymer with affinity ligand was used in the purification of papain and the optimized adsorption condition wan investigated. About10.84mg papain was absorbed on polymer with Reactive Red120under the conditions: contact time150min, pH8.5. At the same time,5.08mg papain was absorbed on polymer with Cu2+under the conditions:contact time120min, pH7.0and NaCl1.00mol/L. And polymer with Fe3+had absorbed4.59mg papain under conditions:contact time210min, pH7.0and NaCl1.OOmol/L. For the best elution result,85.87%papain was desorbed from polymer with Reactive Red120and its activity recovery rate was76.18%under0.1moL/L pH7.0Tris-HCl+1.5mol/L NaCl. For polymer with Cu2+,91.71%papain and88.69%activity recovery rate were determined under conditions:0.1mol/L pH7.0Tris-HCl+lmol/L NaSCN. With the same conditions,74.46%papain and72.87%activity recovery rate were determined for polymer with Fe3+.The SDS-PAGE showed that the papain purified by polymer with Cu2+had achieved electrophoretic pure.The purification results lay a foundation to the development of the separation and purification of high purity papain using affinity precipitation.