微胶囊固定化细胞色素P450 BM-3的研究
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摘要
细胞色素P450BM-3来源于Bacillus megaterium,能催化长链脂肪酸(C_(12)-C_(20))的次末端(ω-1,ω-2,ω-3)羟化及不饱和脂肪酸的环氧化,其突变体催化的底物拓展到中链的脂肪酸(C_8-C_(10))、烷烃、环烷烃、杂环烃、多环芳烃等,因此在医药工业、环境检测和修复及化学工业具有广阔的前景。
论文首先合成P450 BM-3的酶活检测试剂10-对硝基苯氧基癸酸(10-pNCA),其合成是以10-溴代癸酸为原料,与甲醇反应生成10-溴代癸酸甲酯,接着对硝基酚钠反应生成10-对硝基苯氧基癸酸甲酯,最后用固定化脂肪酶水解得到10-pNCA。经紫外光谱、~1H-NMR、~(13)C-NMR和酶分析应用表明成功合成出10-pNCA,总收率达到71.6%。
其次将已构建的质粒转入大肠杆菌DH5α中发酵培养,考察了硫酸铵沉淀前后P450 BM-3酶活的变化,结果表明,经硫酸铵沉淀后,粗酶的酶活、蛋白含量和比活分别是破胞上清液的2.73、1.72和1.61倍,硫酸铵沉淀起了初步分离和浓缩的作用,所制粗酶作为后续固定化的原料。
研究了NaCS-PDMDAAC微胶囊的制备装置和制备方法,考察了不同PDMDAAC分子量、成胶囊时间对形成的微胶囊膜的强度、膜厚、粒径等物理性质的影响,并确定了微胶囊的制备条件:PDMDAAC的分子量为400,000～500,000,成胶囊时间为40～60min。
对NaCS-PDMDAAC生物微胶囊作为P450 BM-3固定化材料的一些必需性质进行研究,结果表明,BSA不能从溶液中扩散进入空白的微胶囊,P450 BM-3催化所需的辅酶NADH、底物10-pNCA均能顺利地扩散至微胶囊内部,NaCS-PDMDAAC生物微胶囊与P450 BM-3酶具有良好的生物相容性。这些表明该胶囊体系比较适合P450 BM-3的固定化。
NaCS-PDMDAAC微胶囊固定化P450 BM-3的最佳条件:pH为8.0、50mM的Tris-HCl缓冲液、0.5～1mgPro/(mL NaCS)的加酶量、分子量400,000～500,000的PDMDAAC、4.0%的NaCS溶液、胶囊固化40～60min。在此条件下,蛋白包埋率可达85～90%,相对酶活达30～35%,酶活回收率达30%左右。
对固定化P450 BM-3酶学性质的研究表明,固定化酶的最适温度为42℃,比游离酶37℃有所提高,固定化酶的最适pH为7.8,比游离酶pH8.0略有降低,还发现固定化酶对有机溶剂、pH及热稳定性均比游离酶大大提,而贮存稳定性也有了明显的提高,游离酶在4℃下保存酶活下降50%的时间为5～7天,而用NaCS-PDMDAAC微胶囊固定化的酶则长达60～100天。这表明NaCS-PDMDAAC
微胶囊固定化P450 BM一3具有良好的发展前景。
关键词:细胞色素P450 BM一3;固定化;微胶囊;NaCS一PDMDAAC;10一对硝基
苯氧基癸酸
夕
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 was
8.0, 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.
论文首先合成P450 BM-3的酶活检测试剂10-对硝基苯氧基癸酸(10-pNCA),其合成是以10-溴代癸酸为原料,与甲醇反应生成10-溴代癸酸甲酯,接着对硝基酚钠反应生成10-对硝基苯氧基癸酸甲酯,最后用固定化脂肪酶水解得到10-pNCA。经紫外光谱、~1H-NMR、~(13)C-NMR和酶分析应用表明成功合成出10-pNCA,总收率达到71.6%。
其次将已构建的质粒转入大肠杆菌DH5α中发酵培养,考察了硫酸铵沉淀前后P450 BM-3酶活的变化,结果表明,经硫酸铵沉淀后,粗酶的酶活、蛋白含量和比活分别是破胞上清液的2.73、1.72和1.61倍,硫酸铵沉淀起了初步分离和浓缩的作用,所制粗酶作为后续固定化的原料。
研究了NaCS-PDMDAAC微胶囊的制备装置和制备方法,考察了不同PDMDAAC分子量、成胶囊时间对形成的微胶囊膜的强度、膜厚、粒径等物理性质的影响,并确定了微胶囊的制备条件:PDMDAAC的分子量为400,000～500,000,成胶囊时间为40～60min。
对NaCS-PDMDAAC生物微胶囊作为P450 BM-3固定化材料的一些必需性质进行研究,结果表明,BSA不能从溶液中扩散进入空白的微胶囊,P450 BM-3催化所需的辅酶NADH、底物10-pNCA均能顺利地扩散至微胶囊内部,NaCS-PDMDAAC生物微胶囊与P450 BM-3酶具有良好的生物相容性。这些表明该胶囊体系比较适合P450 BM-3的固定化。
NaCS-PDMDAAC微胶囊固定化P450 BM-3的最佳条件:pH为8.0、50mM的Tris-HCl缓冲液、0.5～1mgPro/(mL NaCS)的加酶量、分子量400,000～500,000的PDMDAAC、4.0%的NaCS溶液、胶囊固化40～60min。在此条件下,蛋白包埋率可达85～90%,相对酶活达30～35%,酶活回收率达30%左右。
对固定化P450 BM-3酶学性质的研究表明,固定化酶的最适温度为42℃,比游离酶37℃有所提高,固定化酶的最适pH为7.8,比游离酶pH8.0略有降低,还发现固定化酶对有机溶剂、pH及热稳定性均比游离酶大大提,而贮存稳定性也有了明显的提高,游离酶在4℃下保存酶活下降50%的时间为5～7天,而用NaCS-PDMDAAC微胶囊固定化的酶则长达60～100天。这表明NaCS-PDMDAAC
微胶囊固定化P450 BM一3具有良好的发展前景。
关键词:细胞色素P450 BM一3;固定化;微胶囊;NaCS一PDMDAAC;10一对硝基
苯氧基癸酸
夕
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 was
8.0, 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.
引文
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