Synthesis Of Mimic Chymotrypsin Enzyme And It's Immobilizition Research

The artificial enzyme was defined as a supramolecular compounds, which shows the better catalyse ability and environment stability than bio-enzyme. The magnetic nanoparticles(MNPs) were a new functional complex material, they were always prepared by magnetic Fe3O4 or y-Fe2O3 as core, coated or embedded with polymer matrix or surfactant. In this research we synthesized the mimic enzyme of the chymotrypsin contributed to use theβ-cyclodextrin as the matrix which was coated the iminazole on its C2 position. The mimic enzyme of the chymotrypsin immobilized on the surface of the magnetic nanoparticles was linked covalently with the hydroxy of the other positions of the cyclodextrin and the carboxyl of the the carboxyl functional magnetic nanopariticles and oleic acid coated nanoparticles which have synthesized before in our group. At last, the product was the complex of the artificial enzyme/magnetic nanoparticles which had a good dispersibility in the water and could be reused in mangnetic field. There were 5 parts in this article:1. The opposite-methyl-phenyl sulfon on the C2 position and C6 position of cyclodextrin had been synthesized, the compounds could be simulated the process of the catalyze as the chymotrypsin after coated the imidazole, the catalytic activity of the mimic enzyme substrated reaction on C6 was kcat:3.01×10-3s-. Meanwhile, the mimic enzyme substrated reaction on C6 was kcat:2.7×10-4s-. The mimic enzyme which was displaced with the imidazole at C2 was been selected as magnetic immobiliztion.2. The optimal conditions for the immobilization of carboxylated magnetic nanoparticles were 1mL,5mg/mL the carboxylated magnetic nanoparticles and the enzyme 0.1g when immobilizing 36h in 25℃,pH=7.0. The optimal conditions for the immobilization of oleic acid functional magnetic nanoparticles were 1mL,10mg/mL the carboxylated magnetic nanoparticles and the enzyme 0.15g when immobilizing 36h in 40℃,pH=7.5.3. Take the preparation and characterization of the carboxylgroup functional magnet nanoparticles immobilized mimic enzyme and oleic acid functional magnet nanoparticles immobilized mimic enzyme. TEM image showed that the carboxylgroup functional magnet nanoparticles immobilized mimic enzyme was homogeneous distribution, the average diameter was lOnm, occurred inconspicuous aggregation. Meanwhile, the average diameter of the oleic acid functional magnet nanoparticles immobilized mimic enzyme was 25nm, and its dispersancy effectiveness was better than carboxylgroup functional magnet nanoparticles immobilized mimic enzyme which was no aggregation and obvious shell/core stucture. The FT-IR proved that the mimic enzyme had been immobilized on carboxylgroup functional magnet nanoparticles and oleic acid functional magnet nanoparticles successfully.4. The optimal catalyze reaction condition for mimic enzyme magnet nanoparticles immobilized mimic enzyme was reaction temperature 25℃, pH 7.0. The surplus enzyme activity of immobilized enzyme was 43% of the incipient enzyme activity after resused for 10 times which was bellowed the free mimic enzyme's. The optimal catalyze reaction condition for oleic acid functional magnet nanoparticles immobilized mimic enzyme was reaction temperature 40℃, pH 7.5. The surplus enzyme activity of immobilized enzyme was 82% of the incipient enzyme activity after resused for 10 times which was bellowed the free mimic enzyme's. After analyzing of the enzyme activity, the result showed that the activity of the oleic acid functional magnet nanoparticles immobilized mimic enzyme which was kcat:2.60×10-3s- was larger than the activity of the mimic enzyme magnet nanoparticles immobilized mimic enzyme which was kcat:1.98×10-3s-; meanwhile, the oleic acid functional magnet nanoparticles immobilized mimic enzyme was better than the mimic enzyme magnet nanoparticles immobilized mimic enzyme in the dispersion stability and the pH endurance, however, both of them have lower enzyme activity than the free enzyme which was kcat:3.01×10-3s-. Compared with the two methods of immobilization, the immobilization of magnet nanoparticles has simple operation and short period, however, the immobilization of oleic acid functional magnet nanoparticles has difficult operation and long period.The mimic chymotrypsin enzyme has favourable magnetic response。...