New Methods For Protein Purification And Immobilization By Magnetic Nanoparticles

Biocatalysis is one of the most important techniques in modern industry, and the study of enzymes as catalysts has not stopped ever. In recent years, using the magnetic carriers for enzyme purification and immobilization becomes more and more popular, since the effect of separation is very ideal through a magnetic field. Therefore, in this study, we studied enzyme purification, immobilization and co-immobilization by the combined use of magnetic Fe3O4 nanoparticles and catechol. Detailed work is described as follows:(1) Separation and purification of glutathione S-transferase (GST)-tagged proteinsWe synthesized Fe304@polydopamine (GSH-PD-IONPs) to separate the glutathione S-transferase (GST)-tagged protein. GSH-PD-IONPs showed excellent affinity and selectivity to GST-GFP and proved that GSH-PD-IONPs can be used for separation and purification of GST tagged proteins.(2) Purification and immobilization of His-tag proteinsWe synthesized Ni2+-functionalized Fe3O4@polydopamine magnetic nanoparticles (Ni2+-PD-MNPs) for purification and selectively immobilization of His-tagged protein. The results showed that the Ni2+-PD-MNPs had extraordinary selectivity for His-RFP purification. In addition, a His-tagged transaminase (ω-transaminase BJ110) was selectively immobilized onto the Ni2+-PD-MNPs without purification, and the immobilized enzyme showed improved specific activity, as well as enhanced stability and reusability.(3) Co-immobilization of glucose oxidase and catalaseWe synthesized catechol-chitosan-iron oxide nanoparticles (CCS-IONPs) by coating of IONPs with CCS to co-immobilization of GOD and CAT. Under the optimal conditions, the loading capacity of GOD on CCS-IONPs was about 402.82 mg/g, and its activity recovery was as high as 85.34%. When co-immobilized of GOD and CAT, the loading capacity come to 566.89 mg/g, and the activity recovery of GOD was up to 137.83%, slightly higher than the activity recovery (128.01%) of combined use by respectively immobilized of GOD and CAT. In addition, the co-immobilized enzyme retained more than 40% of its initial activity after 10 repeated reaction cycles using magnetic separation, and also improved the temperature stability and pH stability compared with free enzymes.