【作者】 李红梅；

【导师】 梅乐和；

【作者基本信息】 浙江大学 ， 生物化工， 2006， 博士

【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Mn²⁺浓度，确定0.05mmol/L的Mn²⁺浓度是进行体外诱变的最适浓度，同时发现Mn²⁺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。更多还原

【Abstract】 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. Mn²⁺ concentration as a gene mutagenesis in vitro was optimized and 0.05 mmol/L Mn²⁺ 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 PCRrandom 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 explanationsfor 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.更多还原