Aqueous Two Phase Extraction Coupled With Boric Acid Affinity Adsorption In The Separation And Purification Of Horseradish Peroxidase

Horseradish peroxidase?HRP?is a glycoprotein composed of colorless protein and brown iron porphyrin.At present,HRP is widely used in sewage treatment,food industry and catalytic reactions.However,HRP come from the plant and it is easy to inactivate in the purification process,which makes its production cost is generally high.These have seriously hindered the application of proteins in industrial production.Therefore,improving the efficiency of proteins separation and purification is a hot topic in the field of bioengineering.In this paper,the aqueous two-phase extraction coupled with boric acid magnetic graphene affinity adsorption in the separation and purification of horseradish peroxidase.The specific work contents are as follows:?1?The 17R4/L35-salt aqueous two-phase system was applied to the separation and purification horseradish peroxidase.The concentration of 17R4/L35,salt mass fraction,electrolyte and pH on protein distribution efficiency were investigated.Under the optimal conditions?17R4,?NH₄?₂SO₄,T=20?.And adding electrolyte:C₆H₅Na₃O₇,pH=7.0,T=30??,the recovery rate of HRP reached 70-80%.To simulate the unknown substances and environment in horseradish,a model experiment was established and the distribution of 9 substances?saccharides,pigments,proteins?were studied.The best extraction conditions were obtained by adjusting pH value,changing temperature and adding electrolytes.The feasibility of the model was verified by the separation and purification of HRP in horseradish crude enzyme solution using 17R4-?NH₄?₂SO₄ aqueous two-phase system.The maximum extraction recovery of HRP crude enzyme solution reached 69.47%,and saccharides,pigments,hybrid protein and solid impurities were removal,the preliminary separation and purification of HRP were achieved.?2?PEI and 4-formylphenylboronic acid were used as ligands to synthesize dendrimeric phenylboronic acid-affinitive magnetic graphene oxide nanoparticles?d-PBA-GO@Fe₃O₄@PEI?for further selective enrichment of HRP.The properties of enriched HRP such as optimal temperature,pH,adsorption ratio,time,reusability and stability of structure were studied.Under the optimal conditions?the optimal adsorption ratio of d-PBA-GO@Fe₃O₄@PEI to horseradish peroxidase was 2:3,pH=8.0,T=35?,60 min?,and the optimal adsorption amount reached 546.39 mg/g.The HRP adsorption rate reached 88%.At the same time,the selectivity experiment showed that strong selectivity of d-PBA-GO@Fe₃O₄@PEI to HRP.The separation and enrichment of HRP in horseradish was studied.The purification factor was 1.92,the specific enzyme activity was 28.93 U·mg^-1,and the extraction recovery was65.20%.The adsorption performance of d-PBA-GO@Fe₃O₄@PEI on horseradish peroxidase was evaluated by equilibrium adsorption experiment.The affinity adsorbent d-PBA-GO@Fe₃O₄@PEI was recovered and reused,which effectively reduced the cost and material consumption of the system.Finally,FT-IR and circular dichroism?CD?spectra were determined to investigate the structure of enzyme before and after purification process.The results showed that the aqueous two-phase preliminary extraction coupled with magnetic material further adsorption could be successfully applied in the separation and purification of horseradish peroxidase,displaying good prospect for application.