| Cytochrome P450 BM-3 (CYP102) from Bacillus megaterium catalyzes the subterminal hydroxylation of long-chain fatty acids (C12 to C20) at the positions ω-1, ω-2, ω-3 and the epoxidation of unsaturated fatty acids. Artificial mutants of P450 BM-3 also hydroxylate substrates like short-chain fatty acids (C8 to C10), alkanes, cycloalkanes, heteroarenes and polycyclic aromatic hydrocarbons, which offers potential applications in the pharmaceutical industry, environmental measurement and bioremediation and chemical industry.Firstly, The synthesis of p-nitrophenoxydecanoic acid used in P450 BM-3 assay was studied. 10-bromodecanoic acid was reacted with methanol to get methyl 10-bromodecanoate, which was reacted with sodium p-nitrophenolate to produce ester, then hydrolyzed with immobilized lipase to obtain p-nitrophenoxydecanoic acid. Structure of final product was confirmed by UV-IV spectrum, 1H-NMR, 13C- NMR and application to enzyme assay. p-Nitrophenoxydecanoic acid was successfully synthesized by using this method with an yield rate of 71.6%.Then, the plasmid containing CYP102 was transformed into E.coli. DH5α and the activity of enzyme solution precipitated by (NH4)2SO4 and cell extract was compared. After precipitation, the activity, protein content and specific activity were 2.73, 1.72 and 1.61 folds, and the crude enzyme was prepared as immobilization metarial.The preparation device and method for NaCS-PDMDAAC microcapsule as well as the effects of preparation conditions (molecular weight of PDMDAAC and gelation time) on the diameter, membrane thickness and compression intensity of the microcapsule were studied. The optimal operation conditions for microcapsule preparation were determined at molecular weight of PDMDAAC of 400,000-500,00 and gelation time of 40~60min.The feasibility of using NaCS-PDMDAAC microcapsule for P450 BM-3 immobilization was explored. Results showed that no permeation of bovine albumin bovine (BSA) from solution into the microcapsule was observed, and co-factor NADH and substrate 10-pNCA can diffuse into the microcapsule successfully. Also NaCS-PDMDAAC microcapsule has a good biocompatibility to P450 BM-3.The conditions of preparation for P450 BM-3 immobilization with NaCS-PDMDAAC were investigated. The results of optimal conditions were that pH was8.0, ionic strength was 50mM Tris-HCl buffer liquid, the mount of enzyme was 0.5-1mgPro/(mL NaCS), the molecular weight of PDMDAAC was 400,000-500,000, the concentration of NaCS was 4.0% (w/w) and the gelation time was 40~60min. In this condition, residual protein reached 85-90%, relative activity reached 30-35% and residual activity reached about 30%.The enzyme characteristics of immobilized P450 BM-3 was also investigated. It was found that the optimum temperature for immobilized and free P450 BM-3 were 42℃and 37℃ respectively, and the optimum pH for immobilized and free P450 BM-3 were 7.8 and 8.0 respectively. Furthermore, The immobilized P450 BM-3 was more stable to organic solvent, pH and temperature than free enzyme. Investigations on long-term storage stability revealed advantages of the immolibized P450 BM-3. At 4℃, the immobilized enzyme has a half-life of 60~100days, but the free enzyme only 5~7days.
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