Preparation Of Aptamer-functionalized PH Double Responsive Block Polymer For Separation,purification And Immobilization Of Cytochrome C

Thermo-responsive polymer is a kind of polymer which is precipitated from aqueous solution by the temperature change.Due to the easy realization of temperature change,Thermo-responsive polymers have played an important role in the field of enzyme separation,purification and immobilization,but there are still problems of limited functional monomers and low response efficiency,which lead to low selectivity of separation and purification and low efficiency of immobilized enzymes.This work designed and prepared aptamer functionalization temperature/pH double-responsive block polymers to achieve efficient,selective separation and purification of enzymes and improve the efficiency of immobilized enzyme recycling.On the one hand,the simultaneous separation,purification and immobilization of cytochrome c were realized through the specific recognition and high affinity of aptamer to solve the production cost increase and activity loss caused by step completion of enzyme purification and immobilization at present.On the other hand,through the synergistic effect of temperature and pH dual stimulation response,the immobilized cytochrome c homogeneous catalysis,heterogeneous high efficiency and rapid recovery are realized.The main research contents are as follows:(1)The temperature/pH double-responsive block polymers(PEG-b-PAAm-b-PAAc)was prepared by Reversible Addition-Fragmentation Chain Transfer Polymerization(RAFT)method.And then effects of block ratio and hydrophobic monomer on the temperature and pH response of the polymer were observed to get a temperature/pH double-responsive block polymer PEG₄₅-b-PAAm₁₇₆-b-P(GMA₂₁-co-AAc₁₄₀)with UCST of 26.6?and responsive pH value of 3.0.The micro self-assembly behavior studies of PEG₄₅-b-PAAm₁₇₆-b-P(GMA₂₁-co-AAc₁₄₀)shows that it can form spherical micelles with PAAm₁₇₆-b-P(GMA₂₁-co-AAc₁₄₀)as core and PEG₄₅ as shell,and has good cyclic reversibility.PEG₄₅-b-PAAm₁₇₆-b-PAAc₁₄₀ required 12 h to complete phase separation under single temperature stimulation.However,PEG₄₅-b-PAAm₁₇₆-b-P(GMA₂₁-co-AAc₁₄₀)can precipitate completely under the synergistic effect of temperature and pH double stimulation as long as 2 h.Construction of this polymer provides a method for efficient and rapid separation of UCST polymers.(2)The temperature/pH double responsive block polymer PEG₄₅-b-PAAm₁₇₆-b-P(GMA₂₁-co-AAc₁₄₀)is hydrophilic at temperature above UCST(26.6?),pH above 3.0 and is hydrophobic below UCST temperature below 3.0.Based on the characteristics of high efficiency and rapid precipitation,it is used for covalently immobilized cytochrome c.The Cyt c immobilization rate is as high as 97.1%at the optimal immobilization conditions.The covalent immobilization of the polymer Cyt c improve the temperature and pH stability of the Cyt c.And it also shows the advantages of homogeneous catalysis,heterogeneous high efficiency and rapid recovery at the same time.Upon completion of the reaction,the immobilization Cyt c still maintaining 81%of the initial activity after 5 cycles under the dual stimulation response of temperature and pH.(3)The aptamer functionalized temperature/pH double-responsive block polymer PEG₄₅-b-PAAm₁₇₆-b-P(GMA₂₁-co-AAc₁₄₀)-Apt was constructed for simultaneous separation,purification and immobilization of cytochrome c(Cyt c)in crude extracts of porcine cardiomyocytes.It is determined that the maximum separation efficiency is 90.2%through the optimization of simultaneous separation and purification and immobilization conditions.The structural studies of immobilized Cyt c shows that PEG₄₅-b-PAAm₁₇₆-b-P(GMA₂₁-co-AAc₁₄₀)-Apt can keep the Cyt c conformation unchanged when it is used for simultaneous separation and purification,and make it have substrate affinity and catalytic reaction rate close to free Cyt c.The catalytic kinetics of the immobilized Cyt c and the circular dichroism analysis of the Cyt c showed that PEG₄₅-b-PAAm₁₇₆-b-P(GMA₂₁-co-AAc₁₄₀)-Apt could better maintain the Cyt c conformation when used for simultaneous separation,purification and immobilization.The pH and storage stability of the immobilized Cyt c were improved,and the initial activity was still maintained by 80%after 5 cycles.This aptamer functionalized temperature/pH double-responsive block polymer provides a new way for the simultaneous separation,purification and immobilization of enzymes.