细胞色素P450 BM-3体外定向进化及突变酶性能的研究
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摘要
来自巨大芽孢杆菌(BacillusMegaterium)的细胞色素P450 BM-3是由细胞色素单加氧酶和依赖NADPH的FMN/FAD还原酶组合而成的单链融合蛋白。它的天然底物是长链饱和或不饱和脂肪酸。通过理性设计得到的P450 BM-3(A74G/F87V/L188Q)突变酶能够催化吲哚生成靛蓝和靛玉红。为了进一步提高P450 BM-3催化吲哚生成靛蓝的活性，同时提高其对吲哚的区域选择性减少副产物的生成，本论文采用体外随机诱变的易错PCR和饱和突变相结合的定向进化策略，以P450 BM-3(A74G/F87V/L188Q)作为亲本酶，进行定向进化，通过筛选获得了三个高酶活突变酶，同时对突变酶性质、突变酶活力改变的分子机制等方面进行了较为系统的研究。
首先，建立了E．coli DH5(a)收集突变文库和E．coli BL21表达P450 BM-3的底物活性平板初筛系统，同时在96微孔板高通量筛选方案中提出采用产物靛蓝分析和辅酶NADPH分析相结合的筛选策略。这种底物活性平板和96微孔板双重筛选方法的建立，在降低劳动强度的同时也提高了筛选效率。
其次，采用易错PCR随机突变对编码细胞色素P450 BM-3(F87V/A74G/L188Q)的单加氧酶基因片段进行定向进化操作。通过优化PCR体系中的基因体外突变因子Mn~(2+)浓度，确定0.05mmol/L的Mn~(2+)浓度是进行体外诱变的最适浓度，同时发现Mn~(2+)的浓度不仅影响TagDNA聚合酶的活性，而且直接影响到活性克隆的获得。对此条件下建立的突变文库进行筛选，仅通过一轮进化就得到了四个酶活提高的突变酶(I39V，D168N/A225V/K440N，K434R，E435D)。
第三，在取得的四个突变酶的基础上，根据易错PCR定向进化获得的突变位点信息，进一步采用饱和突变理性定向进化技术对以上六个氨基酸位点进行饱和性分析，采用同样的筛选方案获得了三个既高于亲本酶也高于易错PCR技术得到的突变酶活力的新突变酶(D168H，D168L，E435T)。
第四，对三个高活力突变酶D168H、D168L和E435T的动力学参数以及基本酶学性质进行了分析。实验结果发现，与亲本酶相比，突变酶具有更高的底物亲和力，K_m值分别是：亲本酶为2.2mM，突变酶D168H为1.2mM，突变酶D168L为0.92mM，突变酶E435T为0.78mM，从而使其催化效率(k_(cat)/K_m)与亲本酶相比都提高了5倍以上，说明突变的确影响了酶的催化反应特征，同时酶活性的提高也源于酶对底物亲和力的增强。突变酶的最适作用pH和热稳定性基本酶学性质几乎未发生改变，突变酶D168H、D168L和E435T均在pH8.2附近表现出最大羟基化吲哚生成靛蓝的能力；突变酶D168H，D168L与亲本酶相比，热稳定性略有降低，但突变酶E435T与亲本酶热稳定性几乎相同。
第五．对突变酶催化吲哚的电子耦合率以及区域选择性进行了分析。实验结果发现，突变酶D168H电子耦合率在所有突变酶包括亲本酶中都是最低的，仅为8％，与亲本酶相比降低了2倍之多，而突变酶D168L和E435T的电子耦合率却有一定的提高，说明在168位点当用组氨酸替代天冬氨酸时，将极大地影响电子从FMN氧化还原酶区域到亚铁血红素的传递。突变酶D168H、E435T对吲哚的区域选择性得到了提高，主产物靛蓝分别从亲本酶的72％提高到了93％和85％，这种区域选择性的改变进一步显示出定向进化在改造酶分子功能方面所具有的巨大魅力。
第六，运用计算机三维结构模拟，对具有代表性的突变酶氨基酸置换所导致的酶结构变化和可能的活力提高机制进行了初步分析、探讨，其结果进一步强调了体外定向进化的重要性。
第七，此外，还研究了P450 BM-3催化与吲哚同属芳香烃化合物苯乙烯的反应特性。实验结果表明：P450 BM-3突变酶在水相体系中可以催化苯乙烯合成环氧苯乙烷。当发生E435D突变时，P450 BM-3(E435D)催化苯乙烯的能力与其它P450 BM-3突变酶以及亲本酶相比有一定的提高，进一步强调了435氨基酸残基位点在P450 BM-3结构和功能关系中的重要性。选用P450 BM-3(E435D)作为催化用酶，发现该酶催化苯乙烯反应在20分钟内快速达到平衡；当底物浓度高于8.7mmol/L时，将对酶活性产生很大的影响，产物得率显著下降；P450 BM-3的最适反应温度和pH分别为37℃、pH8.2；在底物浓度为4.4mmol//L，助溶剂DMSO浓度为1.5％时，有助于催化反应的顺利进行。
总之，本研究将体外分子定向进化技术用于改造细胞色素P450 BM-3催化吲哚生成靛蓝功能，获得了若干功能改进的突变酶。具有高活力、高区域选择性的
突变酶的获得为进一步定向进化P450 BM-3的催化性能提供了更好的进化模板。所有的实验结果不仅有助于我们理解突变酶活力提高的分子机制提供了一些线索，并有助于我们进一步了解细胞色素P450 BM-3结构与功能之间的关系。
P450 BM-3 from Bacillus megaterium is a self-sufficient natural fusion protein consisting of a P450 heme monooxygenase and a NADPH-dependent diflavin reductase. The natural products of long-chain saturated and unsaturated fatty acids are the substrates for wild type P450 BM-3. A mutant of cytochrome P450 BM-3(F87V/A74G/L188Q) engineered by rational design can hydroxylate indole into indigo and indirubin. In this paper, in order to further improve its capability in the hydroxylation of indole into indigo as well as in terms of higher regioselectivity to less indirubin production, the triple mutant P450BM-3 (A74G/F87V/L188Q) was subjected to further evolution by error-prone PCR and saturation mutagenesis, three P450 BM-3 variants with higher activity had been found. A systematic research of characteristics of mutant enzymes and the molecular mechanism of the activity change of P450 BM-3 mutants was made.
First, a combination method of collecting the mutant libraries by E.coli DH5(a) and expressing P450 BM-3 by E. coli BL21 was made, which made the pre-selection system based on color formation agar plate feasible. A spectroscopic assay based on absorbance of indigo assay and NADPH assay in 96-well plate reader was proposed. By using the double screening procedure consisting of a pre-selection based on color formation agar plate and a quantitative comparison of catalytic activity on a 96-well plate reader, the screening efficiency was improved and the intensity of labor was also lessen.
Second, the mutagenesis of the monooxygenase domain of the P450 BM-3 (F87V/A74G/L188Q) mutant was performed by error-prone PCR. Mn~(2+) concentration as a gene mutagenesis in vitro was optimized and 0.05 mmol/L Mn~(2+) was found to be optimal in suitable for acquirement of mutant library with appropriate mutation frequency. On this condition, the mutant libraries were made and iteratively screened, four mutants (I39V, K434R, E435D, and D168N/A225V/K440N) based on the triple mutant of P450 BM-3 with a slightly higher hydroxylation activity toward indole than the parental enzyme were obtained only through one round of error-prone PCR
random mutagenesis.
Third, for the identification of target position with a critical effect on P450 BM-3 activity toward indole, the libraries were constructed by saturation mutagenesis based on potential hot spots identified by error-prone PCR. Using this approach, three P450 BM-3 variants (D168H, D168L, E435T) containing A74G, F87V and L188Q substitutions were found to hydroxylate indole into indigo more efficiently than other mutants.
Fourth, comparison was made between three mutants D168H, D168L, E435T and the parental enzyme with respect to primary enzymatic properties and kinetic values. The kinetics analysis indicated that the evolved enzymes exhibited higher affinity for substrate indole than the parental enzyme. Their K_m values were 2.2 mM for the parental enzyme, 1.2 mM for D168H mutant, 0.92 mM for D168L mutant and 0.78 mM for E435T mutant respectively. Correspondingly, the k_(cat)/K_m of all mutants showed up to 5-fold enhancement over that of the parental enzyme, respectively. This enhancement in catalytic eficiency was due to an increase in k_(cat) and a decrease in K_m, which illustrates that the mutations did affect the catalytic features of enzyme. Three mutants also exhibited higher hydroxylation activity at pH of 8.2. The thermostability of the mutants D168H, D168L decreased a little compared with the parental enzyme, but the thermostability of the mutant E435T did not change.
Fifth, the coupling efficiency and regioselectivity of three mutants was investigated. The coupling efficiency of the mutant D168H decreased almost 2 folds compared to the parental enzyme while the coupling efficiency of the mutant D168L, E435T slightly increased. Therefore, exchange at position 168 aspartic acid substituted by hisitidine could influence the interaction between the monooxygenase domain and an FMN-binding reductase domain. The mutants D168H and E435T exhibited a higher regioselectivity forming indigo compared to the parental enzyme P450 BM-3 (A74G/F87V/L188Q), which further emphasized the importance of amino acid 168 and amino acid 435 in the relationship between structure and function of P450 BM-3.
Sixth, based on the 3-D structure modeling of mutant enzymes, the changes in molecular structure of evolved enzymes were probed, and the possible explanations
for the improvement of activity were preliminarily analyzed.
Finally, the epoxidation of styrene with similar structure to indole by P450 BM-3 mutants engineered by error-prone PCR directed evolution were investigated. Experiment indicated that P450 BM-3 mutants can epoxidate styrene into styrene oxidate. The mutant E435D had a higher activity than other mutants and parent enzyme, which also emphasized the importance of amino acid 435 in the relationship between structure and function of P450 BM-3. The mutant E435D was choosed as the model enzyme and the effects of reaction conditions such as substrate concentration, reaction pH, temperature and co-solvent were investigated. The experiments showed that the optimum temperature and pH of this enzyme were about 37°C and 8.2. The yield of styrene oxidate decreased faster when styrene concentration came to 8.7mmol/L. And 1.5% co-solvent DMSO could make for favorably process of catalytic reaction in this reaction based on that substrate concentration 4.4 mM.
In conclusion, we used directed evolution to improve the catalytic activity of P450 BM-3 toward indole. The evolved enzyme with higher activity and higher regioselectivity is a suitable parent for further directed evolution to improve catalytic rates and enhance regioselectivity. Moreover, all of the results obtained here may serve as the basis for further elucidation of the mechanism of substrate activation in this enzyme, which provides some hints for further study of the catalytic mechanism of P450B M-3.
首先，建立了E．coli DH5(a)收集突变文库和E．coli BL21表达P450 BM-3的底物活性平板初筛系统，同时在96微孔板高通量筛选方案中提出采用产物靛蓝分析和辅酶NADPH分析相结合的筛选策略。这种底物活性平板和96微孔板双重筛选方法的建立，在降低劳动强度的同时也提高了筛选效率。
其次，采用易错PCR随机突变对编码细胞色素P450 BM-3(F87V/A74G/L188Q)的单加氧酶基因片段进行定向进化操作。通过优化PCR体系中的基因体外突变因子Mn~(2+)浓度，确定0.05mmol/L的Mn~(2+)浓度是进行体外诱变的最适浓度，同时发现Mn~(2+)的浓度不仅影响TagDNA聚合酶的活性，而且直接影响到活性克隆的获得。对此条件下建立的突变文库进行筛选，仅通过一轮进化就得到了四个酶活提高的突变酶(I39V，D168N/A225V/K440N，K434R，E435D)。
第三，在取得的四个突变酶的基础上，根据易错PCR定向进化获得的突变位点信息，进一步采用饱和突变理性定向进化技术对以上六个氨基酸位点进行饱和性分析，采用同样的筛选方案获得了三个既高于亲本酶也高于易错PCR技术得到的突变酶活力的新突变酶(D168H，D168L，E435T)。
第四，对三个高活力突变酶D168H、D168L和E435T的动力学参数以及基本酶学性质进行了分析。实验结果发现，与亲本酶相比，突变酶具有更高的底物亲和力，K_m值分别是：亲本酶为2.2mM，突变酶D168H为1.2mM，突变酶D168L为0.92mM，突变酶E435T为0.78mM，从而使其催化效率(k_(cat)/K_m)与亲本酶相比都提高了5倍以上，说明突变的确影响了酶的催化反应特征，同时酶活性的提高也源于酶对底物亲和力的增强。突变酶的最适作用pH和热稳定性基本酶学性质几乎未发生改变，突变酶D168H、D168L和E435T均在pH8.2附近表现出最大羟基化吲哚生成靛蓝的能力；突变酶D168H，D168L与亲本酶相比，热稳定性略有降低，但突变酶E435T与亲本酶热稳定性几乎相同。
第五．对突变酶催化吲哚的电子耦合率以及区域选择性进行了分析。实验结果发现，突变酶D168H电子耦合率在所有突变酶包括亲本酶中都是最低的，仅为8％，与亲本酶相比降低了2倍之多，而突变酶D168L和E435T的电子耦合率却有一定的提高，说明在168位点当用组氨酸替代天冬氨酸时，将极大地影响电子从FMN氧化还原酶区域到亚铁血红素的传递。突变酶D168H、E435T对吲哚的区域选择性得到了提高，主产物靛蓝分别从亲本酶的72％提高到了93％和85％，这种区域选择性的改变进一步显示出定向进化在改造酶分子功能方面所具有的巨大魅力。
第六，运用计算机三维结构模拟，对具有代表性的突变酶氨基酸置换所导致的酶结构变化和可能的活力提高机制进行了初步分析、探讨，其结果进一步强调了体外定向进化的重要性。
第七，此外，还研究了P450 BM-3催化与吲哚同属芳香烃化合物苯乙烯的反应特性。实验结果表明：P450 BM-3突变酶在水相体系中可以催化苯乙烯合成环氧苯乙烷。当发生E435D突变时，P450 BM-3(E435D)催化苯乙烯的能力与其它P450 BM-3突变酶以及亲本酶相比有一定的提高，进一步强调了435氨基酸残基位点在P450 BM-3结构和功能关系中的重要性。选用P450 BM-3(E435D)作为催化用酶，发现该酶催化苯乙烯反应在20分钟内快速达到平衡；当底物浓度高于8.7mmol/L时，将对酶活性产生很大的影响，产物得率显著下降；P450 BM-3的最适反应温度和pH分别为37℃、pH8.2；在底物浓度为4.4mmol//L，助溶剂DMSO浓度为1.5％时，有助于催化反应的顺利进行。
总之，本研究将体外分子定向进化技术用于改造细胞色素P450 BM-3催化吲哚生成靛蓝功能，获得了若干功能改进的突变酶。具有高活力、高区域选择性的
突变酶的获得为进一步定向进化P450 BM-3的催化性能提供了更好的进化模板。所有的实验结果不仅有助于我们理解突变酶活力提高的分子机制提供了一些线索，并有助于我们进一步了解细胞色素P450 BM-3结构与功能之间的关系。
P450 BM-3 from Bacillus megaterium is a self-sufficient natural fusion protein consisting of a P450 heme monooxygenase and a NADPH-dependent diflavin reductase. The natural products of long-chain saturated and unsaturated fatty acids are the substrates for wild type P450 BM-3. A mutant of cytochrome P450 BM-3(F87V/A74G/L188Q) engineered by rational design can hydroxylate indole into indigo and indirubin. In this paper, in order to further improve its capability in the hydroxylation of indole into indigo as well as in terms of higher regioselectivity to less indirubin production, the triple mutant P450BM-3 (A74G/F87V/L188Q) was subjected to further evolution by error-prone PCR and saturation mutagenesis, three P450 BM-3 variants with higher activity had been found. A systematic research of characteristics of mutant enzymes and the molecular mechanism of the activity change of P450 BM-3 mutants was made.
First, a combination method of collecting the mutant libraries by E.coli DH5(a) and expressing P450 BM-3 by E. coli BL21 was made, which made the pre-selection system based on color formation agar plate feasible. A spectroscopic assay based on absorbance of indigo assay and NADPH assay in 96-well plate reader was proposed. By using the double screening procedure consisting of a pre-selection based on color formation agar plate and a quantitative comparison of catalytic activity on a 96-well plate reader, the screening efficiency was improved and the intensity of labor was also lessen.
Second, the mutagenesis of the monooxygenase domain of the P450 BM-3 (F87V/A74G/L188Q) mutant was performed by error-prone PCR. Mn~(2+) concentration as a gene mutagenesis in vitro was optimized and 0.05 mmol/L Mn~(2+) was found to be optimal in suitable for acquirement of mutant library with appropriate mutation frequency. On this condition, the mutant libraries were made and iteratively screened, four mutants (I39V, K434R, E435D, and D168N/A225V/K440N) based on the triple mutant of P450 BM-3 with a slightly higher hydroxylation activity toward indole than the parental enzyme were obtained only through one round of error-prone PCR
random mutagenesis.
Third, for the identification of target position with a critical effect on P450 BM-3 activity toward indole, the libraries were constructed by saturation mutagenesis based on potential hot spots identified by error-prone PCR. Using this approach, three P450 BM-3 variants (D168H, D168L, E435T) containing A74G, F87V and L188Q substitutions were found to hydroxylate indole into indigo more efficiently than other mutants.
Fourth, comparison was made between three mutants D168H, D168L, E435T and the parental enzyme with respect to primary enzymatic properties and kinetic values. The kinetics analysis indicated that the evolved enzymes exhibited higher affinity for substrate indole than the parental enzyme. Their K_m values were 2.2 mM for the parental enzyme, 1.2 mM for D168H mutant, 0.92 mM for D168L mutant and 0.78 mM for E435T mutant respectively. Correspondingly, the k_(cat)/K_m of all mutants showed up to 5-fold enhancement over that of the parental enzyme, respectively. This enhancement in catalytic eficiency was due to an increase in k_(cat) and a decrease in K_m, which illustrates that the mutations did affect the catalytic features of enzyme. Three mutants also exhibited higher hydroxylation activity at pH of 8.2. The thermostability of the mutants D168H, D168L decreased a little compared with the parental enzyme, but the thermostability of the mutant E435T did not change.
Fifth, the coupling efficiency and regioselectivity of three mutants was investigated. The coupling efficiency of the mutant D168H decreased almost 2 folds compared to the parental enzyme while the coupling efficiency of the mutant D168L, E435T slightly increased. Therefore, exchange at position 168 aspartic acid substituted by hisitidine could influence the interaction between the monooxygenase domain and an FMN-binding reductase domain. The mutants D168H and E435T exhibited a higher regioselectivity forming indigo compared to the parental enzyme P450 BM-3 (A74G/F87V/L188Q), which further emphasized the importance of amino acid 168 and amino acid 435 in the relationship between structure and function of P450 BM-3.
Sixth, based on the 3-D structure modeling of mutant enzymes, the changes in molecular structure of evolved enzymes were probed, and the possible explanations
for the improvement of activity were preliminarily analyzed.
Finally, the epoxidation of styrene with similar structure to indole by P450 BM-3 mutants engineered by error-prone PCR directed evolution were investigated. Experiment indicated that P450 BM-3 mutants can epoxidate styrene into styrene oxidate. The mutant E435D had a higher activity than other mutants and parent enzyme, which also emphasized the importance of amino acid 435 in the relationship between structure and function of P450 BM-3. The mutant E435D was choosed as the model enzyme and the effects of reaction conditions such as substrate concentration, reaction pH, temperature and co-solvent were investigated. The experiments showed that the optimum temperature and pH of this enzyme were about 37°C and 8.2. The yield of styrene oxidate decreased faster when styrene concentration came to 8.7mmol/L. And 1.5% co-solvent DMSO could make for favorably process of catalytic reaction in this reaction based on that substrate concentration 4.4 mM.
In conclusion, we used directed evolution to improve the catalytic activity of P450 BM-3 toward indole. The evolved enzyme with higher activity and higher regioselectivity is a suitable parent for further directed evolution to improve catalytic rates and enhance regioselectivity. Moreover, all of the results obtained here may serve as the basis for further elucidation of the mechanism of substrate activation in this enzyme, which provides some hints for further study of the catalytic mechanism of P450B M-3.
引文
1. Arnold F H. Directed evolution: creating biocatalysts for the future. Chem. Eng. Sci., 1996, 51: 5091-5102.
2. Kuchner O, Arnold F H. Directed evolution of enzyme catalysts. Trends in Bioteehnology, 1997, 15:523-530.
3. Zhao H, Giver L, Shao Z, Arnold F H. Molecular evolution by staggered extension proeess(STEP) in vitro recombination. Nat. Biotechnol., 1998, 16: 258-261.
4. Shao Z, Zhao H, Arnold F H. Random-priming in vitro recombination: an effective tool for directed evolution. Nucleic. Adds. Res., 1998, 26:681-683
5. Coco WM, Levinson WE, Crist MJ, Hektor HJ, Darzins A, Pienkos PT, Squires CH, Monticello DJ. DNA Shuffling method for generating highly recombined genes and evolved enzymes. Nat. Biotechnol., 2001, 19:354-359
6. Pelletier J N. A RACHITT for our toolbox. Nat. Biotechnol., 2001, 19:314-315
7. Ostermeier M, Nixon A E, Shim J H, Benkovic SJ. Combinatorial protein engineering by incremental truncation. Proc. Nat. Acad. Sci., 1999, 96:3562 -35671
8. Sieber V, Martinez C A, Arnold F H. Libraries of hybrid proteins from distantly related sequence. Nat. Biotechnol., 2001, 19:456-46
9. Ryota F, Motomitsu K, Kiyoshi H S. RAISE: a simple and novel method of generating random insertion and deletion mutations. Nucleic. Acids. Research., 2006, 34:e30
10．徐卉芳，中国科学院博士学位论文：大肠杆菌碱性磷酸酶的体外分子定向进化．中国科学院微生物研究所，北京，2002
11. Leung D W, Chen E, Goeddel D V. A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique., 1989, 1: 11-15
12. Cadwell R C, Joyce G F. Randomization of genes by PCR mutagenesis. PCR. Methods. Appl., 1992, 2:28-33
13. Chen K, Arnold F H. Enzyme engineering for non-aqueous solvent: random mutagenesis to enhance activity ofsublisin E in polar organic media. Biotechnol., 1991, 9:1073-1077
14. Vandeyar MA, Weiner MP, Hutton CJ, Batt CA. A simple and rapid method for the selection of oligodeoxy-nucleotide-directed mutants. Gene, 1988, 65:129-133.
15. Stemmer W P C. Rapid evolution of a protein in vitro by DNA Shuffling. Nature, 1994, 370:389-391
16. Stemmer W P C. DNA Shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution. Proc. Natl. Acad. Sci. USA, 1994, 91:10747-10751
17. Zhao H, Arnold F H. Optimization of DNA shuffling for high fidelity recombination. Nucleic. Acids. Res., 1997, 25:1307-1308
18. Zhao H, Arnold F H. Functional and non-functional mutations distinguished byrandom recombination of homologous genes. Proc. Natl. Acad. Sci. USA, 1997,94:7997-8000
19. Zhao HM, Arnold F H. Combinatorial protein design: strategies for screening protein libraries. Current Opinion in Structural Biology, 1997,7:480 - 485.
20. Cohen N, Abramov S, Dror Y. Freeman A. In vitro enzyme evolution: the screening challenge of isolating the one in a million. Trends in Biotechnology, 2001,19: 507-510
21. Stemmer W P C. DNA Shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution. Proc. Natl. Acad. Sci. USA., 1994,91:10747-10751
22. Meyer A, Schmid A, Held M, Westphal A H, Rothlisberger M, Kohler H P E, van Berkel W J H, Witholt B. Changing the substrate reactivity of 2-hydroxybiphenyl 3-monooxygenase from Pseudomonas azelaica HBP1 by directed evolution. J. Biol. Chem., 2002, 277:5575-5582
23. Glieder A, Farinas E T, Arnold F H. Laboratory evolution of a soluble, self sufficient, highly active alkane hydroxylase. Nat. Biotechnol., 2002,20:1135-1139
24. Lingen B, Grotzinger J, Kolter D, Kula M R, Pohl M. Improving the carboligase activity of benzoylformate decarboxylase from Pseudomonas putida by a combination of directed evolution and site-directed mutagenesis. Protein. Eng., 2002,15:585-593
25. Broo K, Larsson A K, Jemth P, Mannervik B: An ensemble of theta class glutathione transferases with novel catalytic properties generated by stochastic recombination of fragments of two mammalian enzymes. J. Mol. Biol., 2002, 318:59-70
26. Kauffmann I, Schmidt-Dannert C. Conversion of Bacillus thermocatenulatus lipase into an efficient phospholipase with increased activity towards long-chain fatty acyl substrates by directed evolution and rational design. Protein. Eng., 2001,14: 919-928
27. Ni J F, Sasaki Y, Tokuyama S, Sogabe A, Tahara Y. Conversion of a typical catalase from Bacillus sp TE124 to a catalase-peroxidase by directed evolution. J. Biosci. Bioeng., 2002, 93:31-36
28. Samuelson J C, Xu S Y. Directed evolution of restriction endonuclease BstYI to achieve increased substrate specificity. J. Mol. Biol., 2002, 319:673-683
29. Sio C F, Riemens A M, Van der Laan J M, Verhaert R M D, Quax W J. Directed evolution of a glutaryl acylase into an adipyl acylase. Eur. J. Biochem., 2002, 269:4495-4504
30. Flores H, Ellington A D. Increasing the thermal stability of an oligomeric protein, beta-glucuronidase. J. Mol. Biol., 2002,315:325-337
31. Oh K H, Nam S H, Kim H S. Improvement of oxidative and thermostability of N-carbamyl -D-amino acid amidohydrolase by directed evolution. Protein. Eng., 2002, 15: 689-695
32. Song J K, Rhee J S. Enhancement of stability and activity of phospholipase A_1 in organic solvents by directed evolution. Biochim. Biophys. Acta., 2001, 1547: 370-378
33. Morawski B, Quan S, Arnold F H. Functional expression and stabilization of horseradish peroxidase by directed evolution in Saccharomyces cerevisiae. Biotechnol. Bioeng., 2001, 76: 99-107
34. Jaeger K E, Eggert T, Eipper A, Reetz M T. Directed evolution and the creation of enantioselective biocatalysts. Appl. Microbiol. Biotechnol., 2001, 55:519-530
35. May O, Nguyen P T, Arnold F H. Inverting enantioselectivity by directed evolution of hydantoinase for improved production of L-methionine. Nat. Biotechnol., 2000,18:317-320
36. Wong S, Machajewski T D, Mak C C, Wong C. Directed evolution of D-2-keto-3 deoxy-6-phosphogluconate aldolase to new variants for the efficient synthesis of D- and L-sugars. Chem. Biol., 2000, 7:873-883
37. Crameri A, Raillard S A , Bermudez E, Stemmer W PC. DNA Shuffling of a family of genes from diverse species accelerates evolution. Nature, 1998,391:288-291
38. Kikuchi M, Ohnishi K, Harayama S. An efective family shuffling method using single-stranded DNA. Gene, 2000, 243:133-137
39. Abecassis V, Pompon D, Truan G. High efficiency family shuffling based on multi-step PCR and in vivo DNA recombination in yeast. Nucleic Acids Res, 2000, 28: e88
40. Kim G J, Cheon Y H, Kim H S. Directed evolution of a novel N-carbamylase /D-hydantoinase fusion enzyme for functional expression with enhanced stability. Biotechnol. Bioeng., 2000,68:211-217
41. Hertzberg R P, Pope A J. High-throughput screening: new technology for the 21st century. Curr.Opin. Chem. Biol., 2000,4 : 445-451
42. Matsumura I, Ellington A D. In vitro evolution of betaglucuronidase into a beta-galactosidase proceeds through non-specific intermediates. J. Mol. Biol., 2001, 305:331 -339
43. Raillard S, Krebber A, Chen Y, Ness J E, Bermudez E, Trinidad R, Fullem R, Davis C, Welch M, Seffernick J et al. Novel Enzyme activities and functional plasticity revealed by recombining highly homologous enzymes. Chem.Biol., 2001 8:891-899
44. Meyer A, Schmid A, Held M, Westphal AH, Rothlisberger M, Kohler H P E, van Berkel W J H, Witholt B. Changing the substrate reactivity of 2-hydroxybiphenyl 3-monooxygenase from Pseudomonas azelaica HBP1 by directed evolution. J. Biol Chem., 2002, 277:5575-5582
45. Meyer A, Wursten M, Schmid A, Kohler H P E, Witholt B. Hydroxylation of indole by laboratory-evolved 2-hydroxybiphenyl 3-monooxygenase. J. Biol. Chem., 2002, 277:34161 -34167
46. Iffland A, Tafelmeyer P, Saudan C, Johnsson K. Directed molecular evolution of cytochrome c peroxidase. Biochemistry, 2000, 39:10790-10798
47. Iffland A, Gendreizig S, Tafelmeyer P, Johnsson K. Changing the substrate specificity of cytochrome c peroxidase using directed evolution. Biochem. Biophys. Res. Commun., 2001, 286:126-132
48. Sun L, Bulter T, Alcalde M, Petrounia IP, Arnold FH. Modification of galactose oxidase to introduce glucose 6-oxidase activity. Chembiochem., 2002,3: 781-783
49. Raillard S, Krebber A, Chen Y, Ness J E, Bermudez E, Trinidad R, Fullem R, Davis C, Welch M, Seffernick J et al. Novel enzyme activities and functional plasticity revealed by recombining highly homologous enzymes. Chem. Biol., 2001, 8:891-898
50. Altamirano M M, Blackburn J M, Aguayo C,_Fersht AR. et al. Directed evolution of new catalytic activity using α/β barrel scaffold. Nature, 2000,403: 617 -622
51. Wislocki P G, Miwa G T, Lu A Y H. In Enzymatic Bask of Detoxication. Academic. Press., New York, 1980:135-182,
52. Garfinkel D. Studies on pig liver microsomes. I. Enzymic and pigment composition of different microsomal fractions. Arch. Biochem. Biophys., 1958, 77:493-509
53. Klingenberg M. Pigments of rat liver microsomes. Arch. Biochem. Biophys., 1958, 75:376-386
54. Nelson D R, Koymans L, Kamtaki T, et al. P450 superfamily and the individuality of species. Arch. Biochem. Biophys., 1999,369:1-10
55. Gunsalus IC, Lpederson T C, et al. Oxygenase-catalyzed biological hydroxylations. Annul Rev. Biochem., 1975, 44:377-407
56．冷欣夫，邱星辉 细胞色素P450酶系的结构、功能与应用前景，科学出版社，北京，2001
57. Omura T, Ishimura Y, Fujii K Y. Cytochrome P450. Kodansha. VCH, Tokyo, 1993
58. Porter T D, Coon M J. Cytochrome P450: Multiplicity of Isoforms, Substrates, and catalytic and regulatory mechanisms. J. Biol. Chem.,1991,266: 13459-13472
59. Takemori S, Yamazaki T, Tkushiro S. Cytochrome P450-1iked Electron Transport System in Monooxygenase Reaction. In: Cytoehrome P450. 1993:44-46
60. Munro A W, Lindsay J G. Bacterial Cytochromes P450. Mol. Mierobiol., 1996, 20:1115-1125
61. Porter T D, Coon M J. Cytochrome P450: Multiplicity of lsoforms, Substrates, and catalytic and regulatory mechanisms. J. Biol. Chem., 1991,266:13459-13472
62. Miura Y, Fulco A J. (ω-2) Hydroxylation of Fatty Acids by a Soluble System from Bacillus megaterium. J. Biol. Chem., 1974, 249:1880-1888
63. Narih L O, Fulco A J. Characterization of a Catalytically Self-sufficient 119,000-Dalton Cytochrome P-450 Monooxygenase Induced by Barbiturates in Bacillus Megaterium. J. Biol. Chem., 1986, 261:7160-7169
64. Narih L O, Fulco A J. Identification and Characterization of Two Functional Domains in Cytochrome P-450_(BM-3), a Catalytically Self-sufficient Monooxygenase Induced by Barbiturates in Bacillus Megaterium. J. Biol. Chem., 1987, 262:6683-6690
65. Wen L P, Fulco A J. Cloning of the gene encoding a catalytically self-sufficient cytochrome P-450 fatty acid monooxygenase induced by barbiturates in Bacillus megaterium and its functional expression and regulation in heterologous (Escherichia coli) and homologous(Bacillus megateriura) hosts. J. Biol. Chem., 1987, 262:6676-6682
66. Munro A W, Leys D G, McLean K J. Marshall KR, Ost TW, Daff S, Miles CS, Chapman SK, Lysek DA, Moser CC, Page CC, Dutton PL. P450 BM3: the Very Model of a Modern Flavocytochrome. Trends Biochem. Sci., 2002,27: 250-257
67. Fulco A J, Ruettinger R T. Occurrence of a Barbiturate-Inducible Catalytically Self-Sufficient 119,000 Dalton Cytochrome P450 Monooxygenase in Bacillius. Life Sci., 1987, 40: 1769-1775
68. Ruettinger R T, Wen L P, Fulco A J. Coding Nucleotide, 5' Regulatory, and Deduced Amino Acid Sequences of P450 BM-3, a Single Peptide Cytochrome P-450: NADPH-P-450 Reductase from Bacillus megaterium. J. Biol. Chem., 1989, 264: 10987-10995
69. Li H, Poulos T L. Fatty acid metabolism, conformational change, and electron transfer in cytochrome P450 BM-3. Biochim. Biophys. Acta., 1999,1441: 141-149
70. Urlacher V B, Lutz-Wahl S, Schmid R D. Microbial P450 enzymes in biotechnology. Appl Microbiol. Biotechnol., 2004,64:317-325
71. Urlacher V B, Schmid R D. Protein Engineering of the Cytochrome P450 Monooxygenase from Bacillus megaterium. Methods Enzymology: Protein Engineering, Dan E. Robertson, Joseph P. Noel, eds. 2004, 388: 208-224
72. Maurer S C, Schulze H et al. Immobilisation of P450 BM-3 and an NADP+ CofactorRecycling System: Towards a Technical Application of Heme-Containing Monooxygenasesin Fine Chemical Synthesis, Adv.Synth.Catal., 2003,345: 802-810
73. Schwaneberg U, Appel D, Schmitt J, Schmid R D. P450 in biotechnology:zinc driven ω-hydroxylation of π-nitrophenoxydodecanoic acidusing P450 BM-3 F87A as a catalyst. J. Biotechnol.,2000, 84:249-257
74. Kazlauskaite J, Westlake A C G, Wong L-L, Hill H. Direct electrochemistry of of cytochrome P450cam. Chem. Commun., 1996,18:2189-2190
75. Fang X, Halpert R J. Dithionite-supported hydroxylation of palmitic acid by cytochrome P450 BM-3. Drug. Metab. Dispos., 1996,24:1282-1285
76. Cirino P C, Arnold F H. A self-sufficient peroxide-driven hydroxylation biocatalyst. Angew. Chem. Int. Ed. Engl., 2003,42:3299-330
77. Schwaneberg Schmidt-Dannet C, Schmitt J, Schmid R D. A continuous spectrophotometic assay for P450 BM-3, a fatty acid hydroxylating enzyme,and its mutant F87A. Ana. Biochem., 1999, 269:359-366
78. Schwaneberg U, Otey C, Cirino PC, Farinas E, Arnold FH. Cost-effective whole-cell assay for laboratory evolution of hydroxylases in Excheichia coli. J. Biomol. Screen., 2001,6:111-117
79. Carmichael AB, Wong LL. Protein engineering of Bacillus megaerium C YP102,theoxidation of polycyclic aromatic hydrocarbons. Eur. J .Biochem., 2001, 268:3117-3125
80. Li Q-S, Schwaneberg U, Fischer M, Schmitt J, Pleiss J, Lutz-Wahl S, Schmid RD. Rational evolution of a medium chain-specific cytochrome P450 BM-3 variant. Biochim. Biophys. Acta., 2001, 1545:114-121
81. Li Q-S, Schwaneberg U, Fischer M, Schmitt J, Pleiss J, Lutz-Wahl S , Schmid R D. Rational evolution of a medium chain-specific cytochrome P-450 BM-3. Biochimica. Biophysica. Acta., 2001, 1545: 114-121
82. Li Q S, Ogawa J, Schmid R D, Shimizu S. Engineering cytochrome P450 BM-3 for oxidation of polycyclic aromatic hydrocarbons. Appl. Environ. Microbial., 2001, 67:5735-5739
83. Lentz O, Li Q S, Schwaneberg U, Lutz-Wahl S, Fischer P, Schmid R D. Modification of the fatty acid specificity of cytochrome P450 BM-3 from Bacillus megaterium by directed evolution: a validated assay. Journal of Molecular Catalysis, 2001, 15:123-133
84. Appel D, Lutz-Wahl S, Fischer P, Schwaneberg U, Schmid R D. A P450 BM-3 mutant hydroxylates of alkanes, cycloalkanes, arenes and heteroarenes. J. Biotechnology, 2001, 88: 167-171
85. Glieder A, Farinas E T, Arnold F H. Laboratory evolution of a soluble, self-sufficient, highly active alkane hydroxylase. Nat. Biotechnol., 2002,20:1135-1139
86. Peters M W, Meinhold P, Glieder A, Arnold F H. Regio- and enantioselective alkane hydroxylation with engineered cytochromes P450 BM-3. J. Am. Chem. Soc, 2003, 125:13442-13450
87. Cirino P C, Arnold F H . Regioselectivity and activity of cytochrome P450 BM-3 and mutant F87A in reactions driven by hydrogen peroxide. Adv. Synth. Catal., 2002, 344:932-937
88. Tuck S W, Arnold F H, Schwaneberg U. Laboratory Evolution of CytochromeP450 BM-3 Monooxygenase for Organic Cosolvents. Biotechnol. Bioeng.,2004, 85:351-358
89. Otey C R, Bandara G, Lalonde J, Takahashi K, Arnold F H. Preparation of human metabolites of propranolol using laboratory-evolved bacterial cytochromes P450. Bioteehnol. Bioeng., 2006, 93:494-499
90. Kubo T, Peters M W, Peter M, Arnold F H. Enantioselective Epoxidation of Terminal Alkenes to (R)- and (S)-Epoxidesby Engineered Cytochromes P450 BM-3. Chem. Eur. J., 2006, 12: 1216-1220
91．姚昕，秦文，齐春梅等，花生四烯酸的生理活性及其应用，粮油加工与食品机械．2004，5：57-59
92. Capdevila J H, Wei S, Helvig C, Falck J R, Belosludtsev Y, Truan G, Graham-Lorence S E, Peterson J A. The Highly Stereoselective Oxidation of Polyunsaturated Fatty Acids by Cytochrome P450 BM-3. J. Biol. Chem., 1996, 271:22663-22671
93. Falck J R, Reddy Y K, Haines D C, Roman RJ. Practical, Enantiospecific Syntheses of 14, 15-EET and Leukotoxin B (Vernolic Acid). Tetrahedron. Lett., 2001, 25:4131-4133
94. Harford-Cross C F, Carmichael A B, Allan F K, England PA, Rouch DA, Wong LL. Protein Engineering of Cytochrome P450cam (CYP101) for the Oxidation of Polycyclic Aromatic Hydrocarbons. Protein. Eng., 2000, 13:121-128
95. Joo H, Lin Z, Arnold F H. Laboratory Evolution of Peroxide Mediated Cytochrome P450 Hydroxylation. Nature, 1999, 339:670-673
96. Shou M, Gonzalez F J, Gelboin H V. Stereoselective Epoxidation and Hydration at the K-region of Polycyclic Aromatic Hydrocarbons by cDNA-expressed Cytochromes P450 1Al, lA2, and Epoxide Hydrolase. Biochemistry, 1996, 35:15807-15813
97．潘湘波，梁静娟，刘雄民，熊德元，李飘英．15-羟基十五烷酸的脂肪酶催化合成环十五内酯，应用化学，2004，21：850-852
98. Strobel J, Groger D. Biochemie und Physioloie der Panzen. 1989,184:321
99. Koehler C S W. Synthetic dyes and the German chemical industry. Today's Chemist at Work, 1999, 8: 85-91.
100．化学史编写组．化学发展简史．北京：科学出版社，1980：185．
101. Abbott D. The Biographical Dictionary of Scientists-Chemists. Frome &Lond: Batler &Tanner Ltd., 1983:13～14.
102. Murdock D, Ensley B D, Serdar C, and Thalen M. Construction of metabolic operons catalyzing the de novo biosynthesis of indigo in Eschedchia coli. Bio.Teehnology, 1993, 11：381-386
103．吴祺，拜耳与合成靛蓝．化学通报，2001，8：527-528
104．任吉远，王文涛，李红．近期靛蓝生产工艺研究进展．燃料染料工业，1997，4：22-24
105. Hill R R, Illing N, Kirby R, Woods DR. Cloning and expression of Rhodococcus genes encoding pigment production in Escherichia coli. Journal of General Microbiology, 1989, 135:1507-1520
106. Eyal J, Spencer, Michael G. Natural blue pigment. United States Patent, 5077201, 1991
107. Hart T, Kirby P, David R. Structure of a Rhodococcus gene encoding pigment production in Escherichia coli. Journal of General Microbiology, 1990, 136:1357-1363
108. Hart T, Woods D R. Construction of an insertional-inactivation cloning vector for Eschedchia coli using a Rhodococcus gene for indigo production. Journal of General Microbiology, 1992, 138:205-214
109. Drewlo S, Christian O. Bramer, Mohamed Madkour, Frank Mayer, and Alexander Steinbüchel. Cloning and expression of a Ralstonia eutropha HF39 gene mediating indigo formation in Escherichia coli. Applied and Environmental Microbiolog, 2001, 67:1964-1969
110. Elizabeth M. J, Anna M A, Lisa M N, Kim D, Mundkowski R G, Alexander A. Volkov, Arnold F H, Soucek P, DeVoss J J, Peter F. Guengerich. Formation of indigo by recombinant mammalian cytochrome P450. Bioehem. Biophys. Res. Commun., 1999, 265:469-472
111. Gillam EM, Notley LM, Cai H, De Voss JJ, Guengerich FP. Oxidation of indole by cytochrome P450 enzymes. Biochemistry, 2000, 39:13817-13824
112. Katsunori N, Martha V M, Peter G. Random mutagenesis of human cytochrome P450 2A6 and screening with indole oxidation products. Arch. Biochem. Biophys., 2001, 395:25-31
113. Li Q S, Schwaneberg U, Schmid R D, Urlacher V B. Directed evolution of the fatty-acid hydroxylase P450 BM-3 into an indole-hydroxylating catalyst. Chemistry-a European Journal, 2000, 6:1531-1536
114. Miller V P, Fruetel J A, Ortiz de Montellano P R. Cytochrome P450cam-catalyzed oxidation of a hypersensitive radical probe. Arch. Biochem. Biophys., 1992, 298:697-702
115. Martin P. M, Vytautas R, Marcia J h, Vincent L V. Improving the Cytochrome P450 Enzyme System for Electrode-Driven Biocatalysis of Styrene Epoxidation. Biotechnol. Prog., 2000, 16:610-616
116. Nickerson D P, Harford-Cross C F, Fulcher S R, Wong L L.The catalytic activity of cytochrome P450cam towards styrene oxidation is increased by site-specific mutagenesis. FEBS Letters, 1997,405:153-156
117. Farinas E T, Alcalde M, Arnold F H. Alkene epoxidation catalyzed by cytochrome P450 BM-3 139-3. Tetrahedron, 2004,605:25-528
118. Soumillion P, Fastrez J. Novel concepts for selection of catalytic activity. Curr. Opin. Biotech, 2001,12: 387-394
1. Li Q S, Schwaneberg U, Fischer P, Schmid RD. Directed evolution of the fatty-acid hydroxylase P450 BM-3 into an indole-hydroxylating catalyst. Chem., 2000, 6(9):1531-1536
2．J．萨姆布鲁克，E．F．弗里奇，T．曼尼阿蒂斯，Molecular Cloning：A laboratory Manual(分子克隆实验指南)，科学出版社，北京，1999
3. Vandeyar M A, Weiner M P, Hutton C J, Batt C A. A simple andrapid method for the selection of oligodeoxynucleotide-directedmutants. Gene., 1988, 65:129-133
4. Budde M, Maurer S C, Schmid R D, Urlacher V B. Cloning, expression and characterization of CYP102A2, a self-sufficient P450 monooxygenase from Bacillus subtility. Appl. Microbiol. Bioteehnol., 2004, 66(2):180-186
5．汪家政，范明．蛋白质技术手册(第一版)．科学出版社，2001
6. Omura T, Sato R. The carbon monoxide-binding pigment of liver microsomers. J. biol. Che., 1964, 239(7): 2370～2378
7. Li Q-S, Schwaneberg U, Fischer M, Schmitt J, Pleiss J, Lutz-Wahl S, Schmid R D. Rational evolution of a medium chain-specific cytochrome P-450 BM-3. Biochimica Biophysica Acta, 2001, 1545:114-121
8. Maurer S, Schmid R D, Urlacher V B. Immobilization of P450 BM-3 and an NADP+ Cofactor Recycling System: Towards a Technical Appication of Heine-containing Monooxygenases i Fine Chemical Synthesis. Adv. Synth. Coral., 2003, 345:802-810
9．pET系列操作手册(第十版，中译本)，Novagen Inc．
10. Gorsky L D, Koop D R, Coon M J. On the stoichiometry ofthe oxidase and monooxygenase reactions catalyzed by liver microsomal cytochrome P-450. Products of oxygen reduction. J. Biol. Chem., 1984, 259:6812-6817
11. Leung D W, Chen E, Goeddel, DV. A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique, 1989, 1:11-15
12．吴乃虎．基因工程原理：上册，北京：科学出版社，1999
13. Moore J C, Jin H M, Kuchner O, Arnold FH. Strategies for the in vitro evolution of protein function: enzyme evolution by random recombination of improved sequences. J. Mol. Biol., 1997, 272:336--347
14. Arnold F H. When blind is better: protein design by evolution. Nat. Bioteehnol., 1998, 16:617-618
15. Arnold F H. Enzyme engineering reaches the boiling point. Proc. Natl. Aead. Sei. USA., 1998, 95:2035-2036
16. Kocks C, Rajewsky K. Step wise intraclonal maturation of antibody affinity through somatic hypermutation. Proc. Natl. Aead. Sci. USA., 1988,85:8206-8210
17. Hanczyc M M, Dorit R L. Replicability and recurrence in the experimental evolution of a group Ⅰ ribozyme. Mol. Biol. Evol., 2000, 17:1050-1060
18. Miyazaki K, Arnold F H. Exploring non-natural evolutionary pathways by saturation mutagenesis: rapid improvement of protein function. J. Mol. Evol., 1999, 49(6): 716-736
19. Kast P, Hilvert D. 3D structural information as a guide to protein engineering using genetic selection. Curt. Opin. Struet. Biol., 1997, 7:470-479
20. Stemmol-Ler W P C, Minshull J. DNA shuffling of subogenomic sequences of subtilisin. Nat. Struet. Biol., 1999,17:893-896
21．陈曾樊等．生物化学实验，中国科技大学出版社，1994
22. Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Biotechnology, 1992, 24:104-108.
23．沈同，王镜岩．《生物化学》(第三版)．高等教育出版社，北京，2000
24. Gillam EM, Notley LM, Cai H, De Voss JJ, Guengerich FP. Oxidation of indole by cytochrome P450 enzymes. Biochemistry, 2000, 39:13817-13824
1. Vandeyar M A, Weiner M P, Hutton C J, Batt CA. A simple and rapid method for the selection of oligodeoxy-nucleotide-directed mutants. Gene., 1988, 65: 129-133
2. Miyazaki K, Arnold F H. Exploring nonnatural evolutionary pathways by saturation mutagenesis: rapid improvement of protein function. J. Mol. Evol., 1999, 49(6): 716-736
3. Appel D, Lutz-Wahl S, Fischer P, Schwaneberg U, Schmid R D. A P450 BM-3 mutant hydroxylates alkanes, cycloalkanes, arenes and heteroarenes. J. Biotechnol., 2001, 88:167-171
1. Urlacher V B, Schmid R D. Protein Engineering of the Cytochrome P450 Monooxygenase from Bacillus megaterium. Methods Enzymology, 2004, 388: 208-224.
2. Budde M, Maurer S C, Schmid R D, Urlacher V B. Cloning, expressionand characterisation of CYPI02A2, a self-sufficient P450 monooxygenase from Bacillus subtility. Appli. Mierobiol. Bioteehaol., 2004, 66:180-186
3. Narhi I Q, Fuko A J. Characterization of a catalytically self-sufficient 119,000 Dalton cythrome P450 monoxygenase induced by harbiturates in Bacillus megaserium. J. Biol. Chemmol., 1989, 264:10987-10995
4. Li H, Poulos T L. The structure of the cytoehrome P450 BM-3 haem domain complexed with the fatty acid substrate, palmitoleicacid. Nat. Struet. Biol., 1997, 4:140-146
5．冷欣夫，邱星辉．细胞色素P450酶系的结构功能和应用前景．科学出版社，北京，2001
6．沈同，王镜岩．《生物化学》(第三版)．高等教育出版社，北京，2000
7. Appel D, Lutz-Wahl S, Fischer P, Schwaneberg U, Schmid R D. A P450 BM-3 mutant hydroxylates alkanes, cycloalkanes, arenes and heteroarenes. J.Bioteehnol., 2001, 88:167-171
8. Hart T, Woods D R. Construction of an insertional-inactivation cloning vector for Escherichia coli using a Rhodococcus gene for indigo production. J. Gen. Mierobiol., 1992, 138:205-209
9. L Q-S, Ulrich S, Peter F, Fischer P, Schmid RD. Directed evolution of the fatty-acid hydroxylase P450 BM-3 into an indole- hydroxylating catalyst, J. Chem. Eur., 2000, 6: 1531-1536
10．夏伟．细胞色素P450的研究进展．国外医学卫生学分册，2000，27：41-47
11. Gorsky LD, Koop D R, Coon M J. On the stoichiometry of the oxidase and monooxygenase reactions catalyzed by liver microsomal cytochrome P 450 Products of oxygen reduction. J. Biol. Chem., 1984, 259:6812-6817
1. Li Q S, Schwaneberg U, Fischer P, Schmid R D. Directed evolution of the fatty-acid hydroxylase P450 BM-3 into an indole-hydroxylating catalyst. Chem., 2000, 6:1531-1536
2. Li Q S, Ogawa J, Schmid R D, Shimizu S. Engineering Cytochrome P450 BM-3 for Oxidation of Polycyclic Aromatic Hydrocarbons. Appl. Environ. Microbiol., 2001, 67:5735-5739
3. Li Q S, Ogawa J, Schmid R D, Shimizu S. Residue size at position 87 of cytochrome P450 BM-3 determines its stereoselectivity in propylbenzene and 3-chlorostyrene oxidation. FEBS. Lett., 2001, 508: 249-252
4. Farinas E T, Alcalde M, Arnold F H. Alkene epoxidation catalyzed by cytochrome P450 BM-3 139-3. Tetrahedron., 2004, 605:25-528
5. Richard A, Friesner. Computational Methods for Protein Folding: Advances in Chemical Physics. John Wiley & Sons, Inc. ISBNs: 0-471-20955-4 (Hardback); 0-471-22442-1 (Electronic). 2002, 120
6. Thompson JD, Higgins DG, Gibson TJ.CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res., 1994,22:4673-80
7. Peitsch MC. Protein modeling, Bio.Technology., 1995, 13: 658-660
8. Guex N, Peitsch MC SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modeling. Electrophoresis. 1997,18: 2714-2723
9. Schwede T, Kopp J, Guex N. Peitsch MCSWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res., 2003, 31: 3381-3385
10. Van Gunsteren W F, Billeter S R, Eising A A. In Biomolecular simulation: the GROMOS96 manual and user guide. Vdf Hochschulverlag ETHZ. 1996
11. Elizabeth M. J, Anna M A, Lisa M N, Kim D, Mundkowski R G, Alexander A. Volkov, Arnold F H, Soucek P, DeVoss J J, Guengerich P F. Formation of indigo by recombinant mammalian cytochrome P450. Biochem. Biophys. Res. Commun., 1999,265: 469-472
12. Gillam EM, Notley LM, Cai H, De Voss JJ, Guengerich FP. Oxidation of indole by cytochrome P450 enzymes. Biochemistry, 2000,39: 13817-13824
13. Katsunori N, Martha V M, Peter G. Random mutagenesis of human cytochrome P450 2A6 and screening with indole oxidation products. Arch. Bioehem. Biophys., 2001, 395:25-31
14．季海涛，张万年，周有骏．细胞色素P450超家族4种蛋白质晶体结构比较研究．生物化学与生物物理学报，1998，30：420-428
15. Li H, Poulos T L. The structure of the cytochrome P450 BM-3 haem domain complexed with the fatty acid substrate, palmitoleic acid. Nat. Struct. Biol., 1997, 4(2):140-146
16. Ravichandran K G, Sekhar S, Boddupalli S, Hasemann C A, Peterson J A, Deisenhofer J. Crystal structure of hemoprotein domain of P450BM-3, a prototype for microsomal P450's. Science, 1993,261:731-736
17. Modi S, Sutcliffe M J, Primrose WU, L.Y. lian, Roberts G C K, Nat.Struct.Biol., 1996, 3:414-417
18. Wong S, Machajewski T D, Mak C C, Wong C. Directed evolution of D-2-keto-3 deoxy-6-phosphogluconate aldolase to new variants for the efficient synthesis of D- and L-sugars. Chem. Biol., 2000, 7:873-883
19. Carmichael A B, Wong L L. Protein engineering of Bacillus megaterium CYP102. The oxidation of polycyclic aromatic hydrocarbons. Eur. J. Biochem., 2001 268:3117-3125
20. Cowart L A, Falck J R, Capdevila J H. Structural determinants of active site binding affinity and metabolism by cytochrome P450 BM-3. Arch. Biochem. Biophys., 2001, 387:117-124
21. Graham-Lorence S, Truan G, Peterson J A, Falck JR, Wei S, Helvig C, Capdevila JH . An active site substitution, F87V, converts cytochrome P450 BM-3 into a regio- and stereoselective (14S,15R)-arachidonic acid epoxygenase.J. Biol. Chem., 1997,272: 1127-1135
22. Urlacher V B, Makhsumkhanov A, Schmid R D, Biotransformation of ionones by engineered cytochrome P450 BM-3. Appl. Microb.Biotechnol., 2005, 70:53-59
1．邢其毅、徐瑞秋，周政 裴伟伟．基础有机化学上册(第二版)，高等教育出版社，北京，2000
2．徐成华，吕绍洁，邱发礼．苯乙烯环氧化制环氧化苯乙烷的研究．石油与天然气化工，1998，27(2)：72-77
3．沈同，王镜岩．生物化学上册(第二版)，高等教育出版社，北京，1991
4. Schwaneberg U. Schmitt J A, Schmid R D. P450 in biotechnology: zinc driven w-hydroxylation of p-nitrophenoxydodecanoic acid using P450 BM-3 F87A as a catalyst. J. Biotechnol., 2000, 84:249-257
5. Seng Wong T, Arnold FH, Schwaneberg U. Laboratory evolution of cytochrome p450 BM-3 monooxygenase for organic cosolvents. Biotechnol. Bioeng., 2004,85:351-358
6. Schwaneberg U, Schmidt-Dannert C, Schmid R D. A continuous spectrophotometric assay for P450 BM-3, a fatty acid hydroxylating enzyme, and its mutant F87A. Anal. Biochem., 1999, 269:359-366
7．冷欣夫，邱星辉 细胞色素P450酶系的结构、功能与应用前景，科学出版社，北京，2001
2. Kuchner O, Arnold F H. Directed evolution of enzyme catalysts. Trends in Bioteehnology, 1997, 15:523-530.
3. Zhao H, Giver L, Shao Z, Arnold F H. Molecular evolution by staggered extension proeess(STEP) in vitro recombination. Nat. Biotechnol., 1998, 16: 258-261.
4. Shao Z, Zhao H, Arnold F H. Random-priming in vitro recombination: an effective tool for directed evolution. Nucleic. Adds. Res., 1998, 26:681-683
5. Coco WM, Levinson WE, Crist MJ, Hektor HJ, Darzins A, Pienkos PT, Squires CH, Monticello DJ. DNA Shuffling method for generating highly recombined genes and evolved enzymes. Nat. Biotechnol., 2001, 19:354-359
6. Pelletier J N. A RACHITT for our toolbox. Nat. Biotechnol., 2001, 19:314-315
7. Ostermeier M, Nixon A E, Shim J H, Benkovic SJ. Combinatorial protein engineering by incremental truncation. Proc. Nat. Acad. Sci., 1999, 96:3562 -35671
8. Sieber V, Martinez C A, Arnold F H. Libraries of hybrid proteins from distantly related sequence. Nat. Biotechnol., 2001, 19:456-46
9. Ryota F, Motomitsu K, Kiyoshi H S. RAISE: a simple and novel method of generating random insertion and deletion mutations. Nucleic. Acids. Research., 2006, 34:e30
10．徐卉芳，中国科学院博士学位论文：大肠杆菌碱性磷酸酶的体外分子定向进化．中国科学院微生物研究所，北京，2002
11. Leung D W, Chen E, Goeddel D V. A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique., 1989, 1: 11-15
12. Cadwell R C, Joyce G F. Randomization of genes by PCR mutagenesis. PCR. Methods. Appl., 1992, 2:28-33
13. Chen K, Arnold F H. Enzyme engineering for non-aqueous solvent: random mutagenesis to enhance activity ofsublisin E in polar organic media. Biotechnol., 1991, 9:1073-1077
14. Vandeyar MA, Weiner MP, Hutton CJ, Batt CA. A simple and rapid method for the selection of oligodeoxy-nucleotide-directed mutants. Gene, 1988, 65:129-133.
15. Stemmer W P C. Rapid evolution of a protein in vitro by DNA Shuffling. Nature, 1994, 370:389-391
16. Stemmer W P C. DNA Shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution. Proc. Natl. Acad. Sci. USA, 1994, 91:10747-10751
17. Zhao H, Arnold F H. Optimization of DNA shuffling for high fidelity recombination. Nucleic. Acids. Res., 1997, 25:1307-1308
18. Zhao H, Arnold F H. Functional and non-functional mutations distinguished byrandom recombination of homologous genes. Proc. Natl. Acad. Sci. USA, 1997,94:7997-8000
19. Zhao HM, Arnold F H. Combinatorial protein design: strategies for screening protein libraries. Current Opinion in Structural Biology, 1997,7:480 - 485.
20. Cohen N, Abramov S, Dror Y. Freeman A. In vitro enzyme evolution: the screening challenge of isolating the one in a million. Trends in Biotechnology, 2001,19: 507-510
21. Stemmer W P C. DNA Shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution. Proc. Natl. Acad. Sci. USA., 1994,91:10747-10751
22. Meyer A, Schmid A, Held M, Westphal A H, Rothlisberger M, Kohler H P E, van Berkel W J H, Witholt B. Changing the substrate reactivity of 2-hydroxybiphenyl 3-monooxygenase from Pseudomonas azelaica HBP1 by directed evolution. J. Biol. Chem., 2002, 277:5575-5582
23. Glieder A, Farinas E T, Arnold F H. Laboratory evolution of a soluble, self sufficient, highly active alkane hydroxylase. Nat. Biotechnol., 2002,20:1135-1139
24. Lingen B, Grotzinger J, Kolter D, Kula M R, Pohl M. Improving the carboligase activity of benzoylformate decarboxylase from Pseudomonas putida by a combination of directed evolution and site-directed mutagenesis. Protein. Eng., 2002,15:585-593
25. Broo K, Larsson A K, Jemth P, Mannervik B: An ensemble of theta class glutathione transferases with novel catalytic properties generated by stochastic recombination of fragments of two mammalian enzymes. J. Mol. Biol., 2002, 318:59-70
26. Kauffmann I, Schmidt-Dannert C. Conversion of Bacillus thermocatenulatus lipase into an efficient phospholipase with increased activity towards long-chain fatty acyl substrates by directed evolution and rational design. Protein. Eng., 2001,14: 919-928
27. Ni J F, Sasaki Y, Tokuyama S, Sogabe A, Tahara Y. Conversion of a typical catalase from Bacillus sp TE124 to a catalase-peroxidase by directed evolution. J. Biosci. Bioeng., 2002, 93:31-36
28. Samuelson J C, Xu S Y. Directed evolution of restriction endonuclease BstYI to achieve increased substrate specificity. J. Mol. Biol., 2002, 319:673-683
29. Sio C F, Riemens A M, Van der Laan J M, Verhaert R M D, Quax W J. Directed evolution of a glutaryl acylase into an adipyl acylase. Eur. J. Biochem., 2002, 269:4495-4504
30. Flores H, Ellington A D. Increasing the thermal stability of an oligomeric protein, beta-glucuronidase. J. Mol. Biol., 2002,315:325-337
31. Oh K H, Nam S H, Kim H S. Improvement of oxidative and thermostability of N-carbamyl -D-amino acid amidohydrolase by directed evolution. Protein. Eng., 2002, 15: 689-695
32. Song J K, Rhee J S. Enhancement of stability and activity of phospholipase A_1 in organic solvents by directed evolution. Biochim. Biophys. Acta., 2001, 1547: 370-378
33. Morawski B, Quan S, Arnold F H. Functional expression and stabilization of horseradish peroxidase by directed evolution in Saccharomyces cerevisiae. Biotechnol. Bioeng., 2001, 76: 99-107
34. Jaeger K E, Eggert T, Eipper A, Reetz M T. Directed evolution and the creation of enantioselective biocatalysts. Appl. Microbiol. Biotechnol., 2001, 55:519-530
35. May O, Nguyen P T, Arnold F H. Inverting enantioselectivity by directed evolution of hydantoinase for improved production of L-methionine. Nat. Biotechnol., 2000,18:317-320
36. Wong S, Machajewski T D, Mak C C, Wong C. Directed evolution of D-2-keto-3 deoxy-6-phosphogluconate aldolase to new variants for the efficient synthesis of D- and L-sugars. Chem. Biol., 2000, 7:873-883
37. Crameri A, Raillard S A , Bermudez E, Stemmer W PC. DNA Shuffling of a family of genes from diverse species accelerates evolution. Nature, 1998,391:288-291
38. Kikuchi M, Ohnishi K, Harayama S. An efective family shuffling method using single-stranded DNA. Gene, 2000, 243:133-137
39. Abecassis V, Pompon D, Truan G. High efficiency family shuffling based on multi-step PCR and in vivo DNA recombination in yeast. Nucleic Acids Res, 2000, 28: e88
40. Kim G J, Cheon Y H, Kim H S. Directed evolution of a novel N-carbamylase /D-hydantoinase fusion enzyme for functional expression with enhanced stability. Biotechnol. Bioeng., 2000,68:211-217
41. Hertzberg R P, Pope A J. High-throughput screening: new technology for the 21st century. Curr.Opin. Chem. Biol., 2000,4 : 445-451
42. Matsumura I, Ellington A D. In vitro evolution of betaglucuronidase into a beta-galactosidase proceeds through non-specific intermediates. J. Mol. Biol., 2001, 305:331 -339
43. Raillard S, Krebber A, Chen Y, Ness J E, Bermudez E, Trinidad R, Fullem R, Davis C, Welch M, Seffernick J et al. Novel Enzyme activities and functional plasticity revealed by recombining highly homologous enzymes. Chem.Biol., 2001 8:891-899
44. Meyer A, Schmid A, Held M, Westphal AH, Rothlisberger M, Kohler H P E, van Berkel W J H, Witholt B. Changing the substrate reactivity of 2-hydroxybiphenyl 3-monooxygenase from Pseudomonas azelaica HBP1 by directed evolution. J. Biol Chem., 2002, 277:5575-5582
45. Meyer A, Wursten M, Schmid A, Kohler H P E, Witholt B. Hydroxylation of indole by laboratory-evolved 2-hydroxybiphenyl 3-monooxygenase. J. Biol. Chem., 2002, 277:34161 -34167
46. Iffland A, Tafelmeyer P, Saudan C, Johnsson K. Directed molecular evolution of cytochrome c peroxidase. Biochemistry, 2000, 39:10790-10798
47. Iffland A, Gendreizig S, Tafelmeyer P, Johnsson K. Changing the substrate specificity of cytochrome c peroxidase using directed evolution. Biochem. Biophys. Res. Commun., 2001, 286:126-132
48. Sun L, Bulter T, Alcalde M, Petrounia IP, Arnold FH. Modification of galactose oxidase to introduce glucose 6-oxidase activity. Chembiochem., 2002,3: 781-783
49. Raillard S, Krebber A, Chen Y, Ness J E, Bermudez E, Trinidad R, Fullem R, Davis C, Welch M, Seffernick J et al. Novel enzyme activities and functional plasticity revealed by recombining highly homologous enzymes. Chem. Biol., 2001, 8:891-898
50. Altamirano M M, Blackburn J M, Aguayo C,_Fersht AR. et al. Directed evolution of new catalytic activity using α/β barrel scaffold. Nature, 2000,403: 617 -622
51. Wislocki P G, Miwa G T, Lu A Y H. In Enzymatic Bask of Detoxication. Academic. Press., New York, 1980:135-182,
52. Garfinkel D. Studies on pig liver microsomes. I. Enzymic and pigment composition of different microsomal fractions. Arch. Biochem. Biophys., 1958, 77:493-509
53. Klingenberg M. Pigments of rat liver microsomes. Arch. Biochem. Biophys., 1958, 75:376-386
54. Nelson D R, Koymans L, Kamtaki T, et al. P450 superfamily and the individuality of species. Arch. Biochem. Biophys., 1999,369:1-10
55. Gunsalus IC, Lpederson T C, et al. Oxygenase-catalyzed biological hydroxylations. Annul Rev. Biochem., 1975, 44:377-407
56．冷欣夫，邱星辉 细胞色素P450酶系的结构、功能与应用前景，科学出版社，北京，2001
57. Omura T, Ishimura Y, Fujii K Y. Cytochrome P450. Kodansha. VCH, Tokyo, 1993
58. Porter T D, Coon M J. Cytochrome P450: Multiplicity of Isoforms, Substrates, and catalytic and regulatory mechanisms. J. Biol. Chem.,1991,266: 13459-13472
59. Takemori S, Yamazaki T, Tkushiro S. Cytochrome P450-1iked Electron Transport System in Monooxygenase Reaction. In: Cytoehrome P450. 1993:44-46
60. Munro A W, Lindsay J G. Bacterial Cytochromes P450. Mol. Mierobiol., 1996, 20:1115-1125
61. Porter T D, Coon M J. Cytochrome P450: Multiplicity of lsoforms, Substrates, and catalytic and regulatory mechanisms. J. Biol. Chem., 1991,266:13459-13472
62. Miura Y, Fulco A J. (ω-2) Hydroxylation of Fatty Acids by a Soluble System from Bacillus megaterium. J. Biol. Chem., 1974, 249:1880-1888
63. Narih L O, Fulco A J. Characterization of a Catalytically Self-sufficient 119,000-Dalton Cytochrome P-450 Monooxygenase Induced by Barbiturates in Bacillus Megaterium. J. Biol. Chem., 1986, 261:7160-7169
64. Narih L O, Fulco A J. Identification and Characterization of Two Functional Domains in Cytochrome P-450_(BM-3), a Catalytically Self-sufficient Monooxygenase Induced by Barbiturates in Bacillus Megaterium. J. Biol. Chem., 1987, 262:6683-6690
65. Wen L P, Fulco A J. Cloning of the gene encoding a catalytically self-sufficient cytochrome P-450 fatty acid monooxygenase induced by barbiturates in Bacillus megaterium and its functional expression and regulation in heterologous (Escherichia coli) and homologous(Bacillus megateriura) hosts. J. Biol. Chem., 1987, 262:6676-6682
66. Munro A W, Leys D G, McLean K J. Marshall KR, Ost TW, Daff S, Miles CS, Chapman SK, Lysek DA, Moser CC, Page CC, Dutton PL. P450 BM3: the Very Model of a Modern Flavocytochrome. Trends Biochem. Sci., 2002,27: 250-257
67. Fulco A J, Ruettinger R T. Occurrence of a Barbiturate-Inducible Catalytically Self-Sufficient 119,000 Dalton Cytochrome P450 Monooxygenase in Bacillius. Life Sci., 1987, 40: 1769-1775
68. Ruettinger R T, Wen L P, Fulco A J. Coding Nucleotide, 5' Regulatory, and Deduced Amino Acid Sequences of P450 BM-3, a Single Peptide Cytochrome P-450: NADPH-P-450 Reductase from Bacillus megaterium. J. Biol. Chem., 1989, 264: 10987-10995
69. Li H, Poulos T L. Fatty acid metabolism, conformational change, and electron transfer in cytochrome P450 BM-3. Biochim. Biophys. Acta., 1999,1441: 141-149
70. Urlacher V B, Lutz-Wahl S, Schmid R D. Microbial P450 enzymes in biotechnology. Appl Microbiol. Biotechnol., 2004,64:317-325
71. Urlacher V B, Schmid R D. Protein Engineering of the Cytochrome P450 Monooxygenase from Bacillus megaterium. Methods Enzymology: Protein Engineering, Dan E. Robertson, Joseph P. Noel, eds. 2004, 388: 208-224
72. Maurer S C, Schulze H et al. Immobilisation of P450 BM-3 and an NADP+ CofactorRecycling System: Towards a Technical Application of Heme-Containing Monooxygenasesin Fine Chemical Synthesis, Adv.Synth.Catal., 2003,345: 802-810
73. Schwaneberg U, Appel D, Schmitt J, Schmid R D. P450 in biotechnology:zinc driven ω-hydroxylation of π-nitrophenoxydodecanoic acidusing P450 BM-3 F87A as a catalyst. J. Biotechnol.,2000, 84:249-257
74. Kazlauskaite J, Westlake A C G, Wong L-L, Hill H. Direct electrochemistry of of cytochrome P450cam. Chem. Commun., 1996,18:2189-2190
75. Fang X, Halpert R J. Dithionite-supported hydroxylation of palmitic acid by cytochrome P450 BM-3. Drug. Metab. Dispos., 1996,24:1282-1285
76. Cirino P C, Arnold F H. A self-sufficient peroxide-driven hydroxylation biocatalyst. Angew. Chem. Int. Ed. Engl., 2003,42:3299-330
77. Schwaneberg Schmidt-Dannet C, Schmitt J, Schmid R D. A continuous spectrophotometic assay for P450 BM-3, a fatty acid hydroxylating enzyme,and its mutant F87A. Ana. Biochem., 1999, 269:359-366
78. Schwaneberg U, Otey C, Cirino PC, Farinas E, Arnold FH. Cost-effective whole-cell assay for laboratory evolution of hydroxylases in Excheichia coli. J. Biomol. Screen., 2001,6:111-117
79. Carmichael AB, Wong LL. Protein engineering of Bacillus megaerium C YP102,theoxidation of polycyclic aromatic hydrocarbons. Eur. J .Biochem., 2001, 268:3117-3125
80. Li Q-S, Schwaneberg U, Fischer M, Schmitt J, Pleiss J, Lutz-Wahl S, Schmid RD. Rational evolution of a medium chain-specific cytochrome P450 BM-3 variant. Biochim. Biophys. Acta., 2001, 1545:114-121
81. Li Q-S, Schwaneberg U, Fischer M, Schmitt J, Pleiss J, Lutz-Wahl S , Schmid R D. Rational evolution of a medium chain-specific cytochrome P-450 BM-3. Biochimica. Biophysica. Acta., 2001, 1545: 114-121
82. Li Q S, Ogawa J, Schmid R D, Shimizu S. Engineering cytochrome P450 BM-3 for oxidation of polycyclic aromatic hydrocarbons. Appl. Environ. Microbial., 2001, 67:5735-5739
83. Lentz O, Li Q S, Schwaneberg U, Lutz-Wahl S, Fischer P, Schmid R D. Modification of the fatty acid specificity of cytochrome P450 BM-3 from Bacillus megaterium by directed evolution: a validated assay. Journal of Molecular Catalysis, 2001, 15:123-133
84. Appel D, Lutz-Wahl S, Fischer P, Schwaneberg U, Schmid R D. A P450 BM-3 mutant hydroxylates of alkanes, cycloalkanes, arenes and heteroarenes. J. Biotechnology, 2001, 88: 167-171
85. Glieder A, Farinas E T, Arnold F H. Laboratory evolution of a soluble, self-sufficient, highly active alkane hydroxylase. Nat. Biotechnol., 2002,20:1135-1139
86. Peters M W, Meinhold P, Glieder A, Arnold F H. Regio- and enantioselective alkane hydroxylation with engineered cytochromes P450 BM-3. J. Am. Chem. Soc, 2003, 125:13442-13450
87. Cirino P C, Arnold F H . Regioselectivity and activity of cytochrome P450 BM-3 and mutant F87A in reactions driven by hydrogen peroxide. Adv. Synth. Catal., 2002, 344:932-937
88. Tuck S W, Arnold F H, Schwaneberg U. Laboratory Evolution of CytochromeP450 BM-3 Monooxygenase for Organic Cosolvents. Biotechnol. Bioeng.,2004, 85:351-358
89. Otey C R, Bandara G, Lalonde J, Takahashi K, Arnold F H. Preparation of human metabolites of propranolol using laboratory-evolved bacterial cytochromes P450. Bioteehnol. Bioeng., 2006, 93:494-499
90. Kubo T, Peters M W, Peter M, Arnold F H. Enantioselective Epoxidation of Terminal Alkenes to (R)- and (S)-Epoxidesby Engineered Cytochromes P450 BM-3. Chem. Eur. J., 2006, 12: 1216-1220
91．姚昕，秦文，齐春梅等，花生四烯酸的生理活性及其应用，粮油加工与食品机械．2004，5：57-59
92. Capdevila J H, Wei S, Helvig C, Falck J R, Belosludtsev Y, Truan G, Graham-Lorence S E, Peterson J A. The Highly Stereoselective Oxidation of Polyunsaturated Fatty Acids by Cytochrome P450 BM-3. J. Biol. Chem., 1996, 271:22663-22671
93. Falck J R, Reddy Y K, Haines D C, Roman RJ. Practical, Enantiospecific Syntheses of 14, 15-EET and Leukotoxin B (Vernolic Acid). Tetrahedron. Lett., 2001, 25:4131-4133
94. Harford-Cross C F, Carmichael A B, Allan F K, England PA, Rouch DA, Wong LL. Protein Engineering of Cytochrome P450cam (CYP101) for the Oxidation of Polycyclic Aromatic Hydrocarbons. Protein. Eng., 2000, 13:121-128
95. Joo H, Lin Z, Arnold F H. Laboratory Evolution of Peroxide Mediated Cytochrome P450 Hydroxylation. Nature, 1999, 339:670-673
96. Shou M, Gonzalez F J, Gelboin H V. Stereoselective Epoxidation and Hydration at the K-region of Polycyclic Aromatic Hydrocarbons by cDNA-expressed Cytochromes P450 1Al, lA2, and Epoxide Hydrolase. Biochemistry, 1996, 35:15807-15813
97．潘湘波，梁静娟，刘雄民，熊德元，李飘英．15-羟基十五烷酸的脂肪酶催化合成环十五内酯，应用化学，2004，21：850-852
98. Strobel J, Groger D. Biochemie und Physioloie der Panzen. 1989,184:321
99. Koehler C S W. Synthetic dyes and the German chemical industry. Today's Chemist at Work, 1999, 8: 85-91.
100．化学史编写组．化学发展简史．北京：科学出版社，1980：185．
101. Abbott D. The Biographical Dictionary of Scientists-Chemists. Frome &Lond: Batler &Tanner Ltd., 1983:13～14.
102. Murdock D, Ensley B D, Serdar C, and Thalen M. Construction of metabolic operons catalyzing the de novo biosynthesis of indigo in Eschedchia coli. Bio.Teehnology, 1993, 11：381-386
103．吴祺，拜耳与合成靛蓝．化学通报，2001，8：527-528
104．任吉远，王文涛，李红．近期靛蓝生产工艺研究进展．燃料染料工业，1997，4：22-24
105. Hill R R, Illing N, Kirby R, Woods DR. Cloning and expression of Rhodococcus genes encoding pigment production in Escherichia coli. Journal of General Microbiology, 1989, 135:1507-1520
106. Eyal J, Spencer, Michael G. Natural blue pigment. United States Patent, 5077201, 1991
107. Hart T, Kirby P, David R. Structure of a Rhodococcus gene encoding pigment production in Escherichia coli. Journal of General Microbiology, 1990, 136:1357-1363
108. Hart T, Woods D R. Construction of an insertional-inactivation cloning vector for Eschedchia coli using a Rhodococcus gene for indigo production. Journal of General Microbiology, 1992, 138:205-214
109. Drewlo S, Christian O. Bramer, Mohamed Madkour, Frank Mayer, and Alexander Steinbüchel. Cloning and expression of a Ralstonia eutropha HF39 gene mediating indigo formation in Escherichia coli. Applied and Environmental Microbiolog, 2001, 67:1964-1969
110. Elizabeth M. J, Anna M A, Lisa M N, Kim D, Mundkowski R G, Alexander A. Volkov, Arnold F H, Soucek P, DeVoss J J, Peter F. Guengerich. Formation of indigo by recombinant mammalian cytochrome P450. Bioehem. Biophys. Res. Commun., 1999, 265:469-472
111. Gillam EM, Notley LM, Cai H, De Voss JJ, Guengerich FP. Oxidation of indole by cytochrome P450 enzymes. Biochemistry, 2000, 39:13817-13824
112. Katsunori N, Martha V M, Peter G. Random mutagenesis of human cytochrome P450 2A6 and screening with indole oxidation products. Arch. Biochem. Biophys., 2001, 395:25-31
113. Li Q S, Schwaneberg U, Schmid R D, Urlacher V B. Directed evolution of the fatty-acid hydroxylase P450 BM-3 into an indole-hydroxylating catalyst. Chemistry-a European Journal, 2000, 6:1531-1536
114. Miller V P, Fruetel J A, Ortiz de Montellano P R. Cytochrome P450cam-catalyzed oxidation of a hypersensitive radical probe. Arch. Biochem. Biophys., 1992, 298:697-702
115. Martin P. M, Vytautas R, Marcia J h, Vincent L V. Improving the Cytochrome P450 Enzyme System for Electrode-Driven Biocatalysis of Styrene Epoxidation. Biotechnol. Prog., 2000, 16:610-616
116. Nickerson D P, Harford-Cross C F, Fulcher S R, Wong L L.The catalytic activity of cytochrome P450cam towards styrene oxidation is increased by site-specific mutagenesis. FEBS Letters, 1997,405:153-156
117. Farinas E T, Alcalde M, Arnold F H. Alkene epoxidation catalyzed by cytochrome P450 BM-3 139-3. Tetrahedron, 2004,605:25-528
118. Soumillion P, Fastrez J. Novel concepts for selection of catalytic activity. Curr. Opin. Biotech, 2001,12: 387-394
1. Li Q S, Schwaneberg U, Fischer P, Schmid RD. Directed evolution of the fatty-acid hydroxylase P450 BM-3 into an indole-hydroxylating catalyst. Chem., 2000, 6(9):1531-1536
2．J．萨姆布鲁克，E．F．弗里奇，T．曼尼阿蒂斯，Molecular Cloning：A laboratory Manual(分子克隆实验指南)，科学出版社，北京，1999
3. Vandeyar M A, Weiner M P, Hutton C J, Batt C A. A simple andrapid method for the selection of oligodeoxynucleotide-directedmutants. Gene., 1988, 65:129-133
4. Budde M, Maurer S C, Schmid R D, Urlacher V B. Cloning, expression and characterization of CYP102A2, a self-sufficient P450 monooxygenase from Bacillus subtility. Appl. Microbiol. Bioteehnol., 2004, 66(2):180-186
5．汪家政，范明．蛋白质技术手册(第一版)．科学出版社，2001
6. Omura T, Sato R. The carbon monoxide-binding pigment of liver microsomers. J. biol. Che., 1964, 239(7): 2370～2378
7. Li Q-S, Schwaneberg U, Fischer M, Schmitt J, Pleiss J, Lutz-Wahl S, Schmid R D. Rational evolution of a medium chain-specific cytochrome P-450 BM-3. Biochimica Biophysica Acta, 2001, 1545:114-121
8. Maurer S, Schmid R D, Urlacher V B. Immobilization of P450 BM-3 and an NADP+ Cofactor Recycling System: Towards a Technical Appication of Heine-containing Monooxygenases i Fine Chemical Synthesis. Adv. Synth. Coral., 2003, 345:802-810
9．pET系列操作手册(第十版，中译本)，Novagen Inc．
10. Gorsky L D, Koop D R, Coon M J. On the stoichiometry ofthe oxidase and monooxygenase reactions catalyzed by liver microsomal cytochrome P-450. Products of oxygen reduction. J. Biol. Chem., 1984, 259:6812-6817
11. Leung D W, Chen E, Goeddel, DV. A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique, 1989, 1:11-15
12．吴乃虎．基因工程原理：上册，北京：科学出版社，1999
13. Moore J C, Jin H M, Kuchner O, Arnold FH. Strategies for the in vitro evolution of protein function: enzyme evolution by random recombination of improved sequences. J. Mol. Biol., 1997, 272:336--347
14. Arnold F H. When blind is better: protein design by evolution. Nat. Bioteehnol., 1998, 16:617-618
15. Arnold F H. Enzyme engineering reaches the boiling point. Proc. Natl. Aead. Sei. USA., 1998, 95:2035-2036
16. Kocks C, Rajewsky K. Step wise intraclonal maturation of antibody affinity through somatic hypermutation. Proc. Natl. Aead. Sci. USA., 1988,85:8206-8210
17. Hanczyc M M, Dorit R L. Replicability and recurrence in the experimental evolution of a group Ⅰ ribozyme. Mol. Biol. Evol., 2000, 17:1050-1060
18. Miyazaki K, Arnold F H. Exploring non-natural evolutionary pathways by saturation mutagenesis: rapid improvement of protein function. J. Mol. Evol., 1999, 49(6): 716-736
19. Kast P, Hilvert D. 3D structural information as a guide to protein engineering using genetic selection. Curt. Opin. Struet. Biol., 1997, 7:470-479
20. Stemmol-Ler W P C, Minshull J. DNA shuffling of subogenomic sequences of subtilisin. Nat. Struet. Biol., 1999,17:893-896
21．陈曾樊等．生物化学实验，中国科技大学出版社，1994
22. Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Biotechnology, 1992, 24:104-108.
23．沈同，王镜岩．《生物化学》(第三版)．高等教育出版社，北京，2000
24. Gillam EM, Notley LM, Cai H, De Voss JJ, Guengerich FP. Oxidation of indole by cytochrome P450 enzymes. Biochemistry, 2000, 39:13817-13824
1. Vandeyar M A, Weiner M P, Hutton C J, Batt CA. A simple and rapid method for the selection of oligodeoxy-nucleotide-directed mutants. Gene., 1988, 65: 129-133
2. Miyazaki K, Arnold F H. Exploring nonnatural evolutionary pathways by saturation mutagenesis: rapid improvement of protein function. J. Mol. Evol., 1999, 49(6): 716-736
3. Appel D, Lutz-Wahl S, Fischer P, Schwaneberg U, Schmid R D. A P450 BM-3 mutant hydroxylates alkanes, cycloalkanes, arenes and heteroarenes. J. Biotechnol., 2001, 88:167-171
1. Urlacher V B, Schmid R D. Protein Engineering of the Cytochrome P450 Monooxygenase from Bacillus megaterium. Methods Enzymology, 2004, 388: 208-224.
2. Budde M, Maurer S C, Schmid R D, Urlacher V B. Cloning, expressionand characterisation of CYPI02A2, a self-sufficient P450 monooxygenase from Bacillus subtility. Appli. Mierobiol. Bioteehaol., 2004, 66:180-186
3. Narhi I Q, Fuko A J. Characterization of a catalytically self-sufficient 119,000 Dalton cythrome P450 monoxygenase induced by harbiturates in Bacillus megaserium. J. Biol. Chemmol., 1989, 264:10987-10995
4. Li H, Poulos T L. The structure of the cytoehrome P450 BM-3 haem domain complexed with the fatty acid substrate, palmitoleicacid. Nat. Struet. Biol., 1997, 4:140-146
5．冷欣夫，邱星辉．细胞色素P450酶系的结构功能和应用前景．科学出版社，北京，2001
6．沈同，王镜岩．《生物化学》(第三版)．高等教育出版社，北京，2000
7. Appel D, Lutz-Wahl S, Fischer P, Schwaneberg U, Schmid R D. A P450 BM-3 mutant hydroxylates alkanes, cycloalkanes, arenes and heteroarenes. J.Bioteehnol., 2001, 88:167-171
8. Hart T, Woods D R. Construction of an insertional-inactivation cloning vector for Escherichia coli using a Rhodococcus gene for indigo production. J. Gen. Mierobiol., 1992, 138:205-209
9. L Q-S, Ulrich S, Peter F, Fischer P, Schmid RD. Directed evolution of the fatty-acid hydroxylase P450 BM-3 into an indole- hydroxylating catalyst, J. Chem. Eur., 2000, 6: 1531-1536
10．夏伟．细胞色素P450的研究进展．国外医学卫生学分册，2000，27：41-47
11. Gorsky LD, Koop D R, Coon M J. On the stoichiometry of the oxidase and monooxygenase reactions catalyzed by liver microsomal cytochrome P 450 Products of oxygen reduction. J. Biol. Chem., 1984, 259:6812-6817
1. Li Q S, Schwaneberg U, Fischer P, Schmid R D. Directed evolution of the fatty-acid hydroxylase P450 BM-3 into an indole-hydroxylating catalyst. Chem., 2000, 6:1531-1536
2. Li Q S, Ogawa J, Schmid R D, Shimizu S. Engineering Cytochrome P450 BM-3 for Oxidation of Polycyclic Aromatic Hydrocarbons. Appl. Environ. Microbiol., 2001, 67:5735-5739
3. Li Q S, Ogawa J, Schmid R D, Shimizu S. Residue size at position 87 of cytochrome P450 BM-3 determines its stereoselectivity in propylbenzene and 3-chlorostyrene oxidation. FEBS. Lett., 2001, 508: 249-252
4. Farinas E T, Alcalde M, Arnold F H. Alkene epoxidation catalyzed by cytochrome P450 BM-3 139-3. Tetrahedron., 2004, 605:25-528
5. Richard A, Friesner. Computational Methods for Protein Folding: Advances in Chemical Physics. John Wiley & Sons, Inc. ISBNs: 0-471-20955-4 (Hardback); 0-471-22442-1 (Electronic). 2002, 120
6. Thompson JD, Higgins DG, Gibson TJ.CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res., 1994,22:4673-80
7. Peitsch MC. Protein modeling, Bio.Technology., 1995, 13: 658-660
8. Guex N, Peitsch MC SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modeling. Electrophoresis. 1997,18: 2714-2723
9. Schwede T, Kopp J, Guex N. Peitsch MCSWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res., 2003, 31: 3381-3385
10. Van Gunsteren W F, Billeter S R, Eising A A. In Biomolecular simulation: the GROMOS96 manual and user guide. Vdf Hochschulverlag ETHZ. 1996
11. Elizabeth M. J, Anna M A, Lisa M N, Kim D, Mundkowski R G, Alexander A. Volkov, Arnold F H, Soucek P, DeVoss J J, Guengerich P F. Formation of indigo by recombinant mammalian cytochrome P450. Biochem. Biophys. Res. Commun., 1999,265: 469-472
12. Gillam EM, Notley LM, Cai H, De Voss JJ, Guengerich FP. Oxidation of indole by cytochrome P450 enzymes. Biochemistry, 2000,39: 13817-13824
13. Katsunori N, Martha V M, Peter G. Random mutagenesis of human cytochrome P450 2A6 and screening with indole oxidation products. Arch. Bioehem. Biophys., 2001, 395:25-31
14．季海涛，张万年，周有骏．细胞色素P450超家族4种蛋白质晶体结构比较研究．生物化学与生物物理学报，1998，30：420-428
15. Li H, Poulos T L. The structure of the cytochrome P450 BM-3 haem domain complexed with the fatty acid substrate, palmitoleic acid. Nat. Struct. Biol., 1997, 4(2):140-146
16. Ravichandran K G, Sekhar S, Boddupalli S, Hasemann C A, Peterson J A, Deisenhofer J. Crystal structure of hemoprotein domain of P450BM-3, a prototype for microsomal P450's. Science, 1993,261:731-736
17. Modi S, Sutcliffe M J, Primrose WU, L.Y. lian, Roberts G C K, Nat.Struct.Biol., 1996, 3:414-417
18. Wong S, Machajewski T D, Mak C C, Wong C. Directed evolution of D-2-keto-3 deoxy-6-phosphogluconate aldolase to new variants for the efficient synthesis of D- and L-sugars. Chem. Biol., 2000, 7:873-883
19. Carmichael A B, Wong L L. Protein engineering of Bacillus megaterium CYP102. The oxidation of polycyclic aromatic hydrocarbons. Eur. J. Biochem., 2001 268:3117-3125
20. Cowart L A, Falck J R, Capdevila J H. Structural determinants of active site binding affinity and metabolism by cytochrome P450 BM-3. Arch. Biochem. Biophys., 2001, 387:117-124
21. Graham-Lorence S, Truan G, Peterson J A, Falck JR, Wei S, Helvig C, Capdevila JH . An active site substitution, F87V, converts cytochrome P450 BM-3 into a regio- and stereoselective (14S,15R)-arachidonic acid epoxygenase.J. Biol. Chem., 1997,272: 1127-1135
22. Urlacher V B, Makhsumkhanov A, Schmid R D, Biotransformation of ionones by engineered cytochrome P450 BM-3. Appl. Microb.Biotechnol., 2005, 70:53-59
1．邢其毅、徐瑞秋，周政 裴伟伟．基础有机化学上册(第二版)，高等教育出版社，北京，2000
2．徐成华，吕绍洁，邱发礼．苯乙烯环氧化制环氧化苯乙烷的研究．石油与天然气化工，1998，27(2)：72-77
3．沈同，王镜岩．生物化学上册(第二版)，高等教育出版社，北京，1991
4. Schwaneberg U. Schmitt J A, Schmid R D. P450 in biotechnology: zinc driven w-hydroxylation of p-nitrophenoxydodecanoic acid using P450 BM-3 F87A as a catalyst. J. Biotechnol., 2000, 84:249-257
5. Seng Wong T, Arnold FH, Schwaneberg U. Laboratory evolution of cytochrome p450 BM-3 monooxygenase for organic cosolvents. Biotechnol. Bioeng., 2004,85:351-358
6. Schwaneberg U, Schmidt-Dannert C, Schmid R D. A continuous spectrophotometric assay for P450 BM-3, a fatty acid hydroxylating enzyme, and its mutant F87A. Anal. Biochem., 1999, 269:359-366
7．冷欣夫，邱星辉 细胞色素P450酶系的结构、功能与应用前景，科学出版社，北京，2001