首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 218 毫秒
1.
Epoxygenases are attractive enzymes for synthesizing important chemical synthons. Directed evolution of epoxygenase properties to production demands have been limited until recently by a lack of screening systems. The previously reported p-nitrothiophenolate (pNTP) screening system was validated through improving styrene epoxidation activity of P450 BM-3 from Bacillus megaterium. Unlike the catalytically self-sufficient P450 BM-3, most epoxygenases are multi-component systems and often significantly less active. We improved the pNTP screening system for a two-component epoxygenase, styrene monooxygenase StyAB from Pseudomonas species, by enhancing the sensitivity of the pNTP assay from 400 to 140 μM and reducing styrene evaporation from 72 to 52%. These improvements were achieved using methylated β-cyclodextrins (mβ-CD) as inclusion host for styrene. Incorporation of mβ-CD increases styrene availability over the assay period and thus enables screening for improved mutants. The pNTP screening procedure for StyAB was subsequently verified in 96-well microtiter plate screens by gas chromatography analysis of styrene conversions.  相似文献   

2.
Cytochrome P450 BM-3 from Bacillus megaterium catalyzes the subterminal hydroxylation of medium- and long-chain fatty acids at the positions omega-1, omega-2, and omega-3. A rapid and continuous spectrophotometric activity assay for cytochrome P450 BM-3 based on the conversion of p-nitrophenoxycarboxylic acids (pNCA) to omega-oxycarboxylic acids and the chromophore p-nitrophenolate was developed. In contrast to the commonly used activity assays for this enzyme, relying on the consumption of oxygen or NADPH or the use of 14C-labeled carboxylic acids, the pNCA assay can even be used with crude extracts of the recombinant enzyme from lysed Escherichia coli cells. The kinetics of p-nitrophenolate formation are directly measured at a wavelength of 410 nm using a spectrophotometer or microtiter plate reader. Sensitivity of the assay is greatly enhanced if p-nitrophenoxydodecanoic or p-nitrophenoxypentadecanoic acid are used with the F87A mutant instead of the wild-type P450 BM-3 enzyme.  相似文献   

3.
催化吲哚生成靛蓝的细胞色素P450BM-3 定向进化研究   总被引:6,自引:0,他引:6  
以催化吲哚产生的靛蓝在 630 nm 处具有特殊的吸收峰为高通量筛选指标,将来源于 Bacillus megaterium 的细胞色素 P450BM-3 单加氧酶的基因序列用易错聚合酶链式反应进行定向进化,通过多轮突变,在原有的能产靛蓝的高活力突变酶的基础上成功获得了三个高于亲本酶的突变酶,突变酶的酶活分别是亲本酶的 6.6 倍 (hml001) , 9.6 倍 (hml002) 和 5.3 倍 (hml003) ,并对突变酶的动力学参数进行了分析 . 突变酶 DNA 测序的结果表明, hml001 含有一个有义氨基酸置换 I39V , hml002 含有三个有义氨基酸置换 D168N , A225V , K440N , hml003 含有一个有义氨基酸置换 E435D ,这些突变位点有些远离底物结合部位,有些位于底物结合部位 .  相似文献   

4.
以来自于巨大芽孢杆菌的细胞色素P450BM-3为研究对象,采用随机突变和饱和定点突变定向进化技术对P450BM-3进行改造,通过突变体催化靛蓝显色的特性采用活性琼脂平板分析和96微孔板相结合的高通量筛选成功获得了几个具有更高催化性能的突变体。  相似文献   

5.
Cytochrome P450 BM-3 from Bacillus megaterium is a fatty acid hydroxylase exhibiting selectivity for long-chain substrates (12–20 carbons). Replacement of Phe87 in P450 BM-3 by Val (F87V) greatly increased its activity towards a variety of aromatic and phenolic compounds. The apparent initial reaction rates of F87V as to benzothiophene, indan, 2,6-dichlorophenol, and 2-(benzyloxy)phenol were 227, 204, 129, and 385 nmol min–1 nmol–1 P450, which are 220-, 66-, 99-, and 963-fold those of the wild type, respectively. These results indicate that Phe87 plays a critical role in the control of the substrate specificity of P450 BM-3. Furthermore, F87V catalyzed regioselective hydroxylation at the para position of various phenolic compounds. In particular, F87V showed high activity as to the hydroxylation of 2-(benzyloxy)phenol to 2-(benzyloxy)hydroquinone. With F87V as the catalyst, 0.71 mg ml–1 2-(benzyloxy)hydroquinone was produced from 1.0 mg ml–1 2-(benzyloxy)phenol in 4 h, with a molar yield of 66%.  相似文献   

6.
Rational evolution of a medium chain-specific cytochrome P-450 BM-3 variant   总被引:3,自引:0,他引:3  
The single mutant F87A of cytochrome P-450 BM-3 from Bacillus megaterium was engineered by rational evolution to achieve improved hydroxylation activity for medium chain length substrates (C8-C10). Rational evolution combines rational design and directed evolution to overcome the drawbacks of these methods when applied individually. Based on the X-ray structure of the enzyme, eight mutation sites (P25, V26, R47, Y51, S72, A74, L188, and M354) were identified by modeling. Sublibraries created by site-specific randomization mutagenesis of each single site were screened using a spectroscopic assay based on omega-p-nitrophenoxycarboxylic acids (pNCA). The mutants showing activity for shorter chain length substrates were combined, and these combi-libraries were screened again for mutants with even better catalytic properties. Using this approach, a P-450 BM-3 variant with five mutations (V26T, R47F, A74G, L188K, and F87A) that efficiently hydrolyzes 8-pNCA was obtained. The catalytic efficiency of this mutant towards omega-p-nitrophenoxydecanoic acid (10-pNCA) and omega-p-nitrophenoxydodecanoic acid (12-pNCA) is comparable to that of the wild-type P-450 BM-3.  相似文献   

7.
Replacement of phenylalanine 87 with alanine or glycine (mutant F87A or F87G) greatly increased the H2O2-supported substrate hydroxylation activity of cytochrome P450BM-3, whose original H2O2-supported activity is hardly detectable. On the other hand, replacement of phenylalanine 87 with valine (mutant F87V) did not. In the oxidation of p-nitrophenoxydodecanoic acid (12-pNCA), the turnover numbers of the mutant F87A in the presence of NADPH and O2, or H2O2 were 493 and 162 nmol/min/nmol, respectively. The H2O2-supported F87A hydroxylation activity was further confirmed with free fatty acids as substrates. Moreover, the stability of F87A in H2O2 solutions also largely increased. The order of the stability of the wild type (WT), F87A, and their substrate (12-pNCA)-binding complexes in H2O2 solutions listed from high to low was F87A, WT, F87A substrate-binding complex, and WT substrate-binding complex. We propose that the free space size in the vicinity of the heme iron significantly influences P450BM-3 H2O2-supported activity and H2O2 inactivation.  相似文献   

8.
The substrate oxidation rates of P450(BM-3) are unparalleled in the cytochrome P450 (CYP) superfamily of enzymes. Furthermore, the bacterial enzyme, originating from Bacillus megaterium, has been used repeatedly as a model to study the metabolism of mammalian P450s. A specific example is presented where studying P450(BM-3) substrate dynamics can define important enzyme-substrate characteristics, which may be useful in modeling omega-hydroxylation seen in mammalian P450s. In addition, if the reactive species responsible for metabolism can be controlled to produce specific products this enzyme could be a useful biocatalyst. Based on crystal structures and the fact that the P450(BM-3) F87A mutant produces a large isotope in contrast to the native enzyme, we propose that phenylalanine 87 is responsible for hindering substrate access to the active oxygen species for nonnative substrates. Using kinetic isotopes and two aromatic substrates, p-xylene and 4,4'-dimethylbiphenyl, the role phenylalanine 87 plays in active-site dynamics is characterized. The intrinsic KIE is 7.3 +/- 2 for wtP450(BM-3) metabolism of p-xylene. In addition, stoichiometry differences were measured with the native and mutant enzyme and 4,4'-dimethylbiphenyl. The results show a more highly coupled substrate/NADPH ratio in the mutant than in the wtP450(BM-3).  相似文献   

9.
Cytochrome P450 BM-3 from Bacillus megaterium is an extensively studied enzyme for industrial applications. A major focus of current protein engineering research is directed to improving the catalytic performance of P450 BM-3 toward nonnatural substrates of industrial importance in the presence of organic solvents or cosolvents. For the latter reason, it is important to study the effect of organic cosolvent molecules on the structure and dynamics of the enzyme, in particular, the effect of cosolvent molecules on the active site's structure and dynamics. In this paper, we have studied, using molecular dynamics (MD) simulations, the F87A mutant of P450 BM-3 in the presence of DMSO as cosolvent, to understand the role of the F87A substitution for its catalytic activity. This mutant exhibits an altered regioselectivity and substrate specificity compared with wild-type; however, it has lower tolerance toward DMSO. The simulation results offer an explanation for the DMSO sensitivity of the F87A mutant. Our simulation results show that the F87 side chain prevents the disturbance of the water molecule bound to the heme iron by DMSO molecules. The absence of the phenyl ring in F87A mutant promotes interactions of the DMSO molecule with the heme iron resulting in water displacement by DMSO at the catalytic heme center.  相似文献   

10.
16,17-Epoxysterol plays an important role in pharmaceutical steroid synthesis. To investigate the potential application of cytochrome P450 for epoxysterol synthesis, an approach to the epoxidation of 16,17-epoxysterol, based on directed evolution of cytochrome P450 BM-3, was developed. This comprised random gene mutagenesis for optimizing the activity of P450 BM-3 for epoxidation of hydrophobic sterol, followed by the 7-ethoxycoumarin de-ethylation assay for general enzyme activity detection and the modified picric acid assay for epoxidation activity screening. By the two-step screening, one mutant from 792 clones showed specific substrate activity of converting progesterone to 16,17-epoxysterol, which validated the possibility to evolve the cytochrome P450 for the synthesis of steroidal epoxides.  相似文献   

11.
Cytochrome P450 BM-3 (CYP102) catalyzes the subterminal hydroxylation of fatty acids with a chain length of 12–22 carbons. The paper focuses on the regioselectivity and substrate specificity of the purified wild-type enzyme and five mutated variants towards caprylic, capric, and lauric acid. The enzymes were obtained by random mutagenic fine-tuning of the mutant F87A(LARV). F87A(LARV) was selected as the best enzyme variant in a previous study in which the single mutant F87A was subjected to rational evolution to achieve hydroxylation activity for short chain length substrates using a p-nitrophenolate-based spectrophotometric assay.

The best mutants, F87V(LAR) and F87V(LARV), show a higher catalytic activity towards ω-(p-nitrophenoxy)decanoic acid (10-p-NCA) than F87A(LARV). In addition, they proved capable of hydroxylating ω-(p-nitrophenoxy)octanoic acid (8-p-NCA) which the wild-type enzyme is unable to do. Both variants catalyzed hydroxylation of capric acid, which is not a substrate for the wild-type, with a conversion rate of up to 57%. The chain length specificity of the mutants in fatty acid hydroxylation processes shows a good correlation with their activity towards p-NCA pseudosubstrates. The p-NCA assay therefore, allows high-throughput screening of large mutant libraries for the identification of enzyme variants with the desired catalytic activity towards fatty acids as the natural substrates.  相似文献   


12.
Electro enzymatic processes offer novel opportunities in catalysis by combining advantages of enzyme catalysis and electrochemistry. An efficient electrochemical cofactor substitution system can help to overcome economical hurdles for the technical use of cofactor dependent enzymes. The in vitro biocatalysis with P450 BM-3 was investigated aiming for the substitution of the expensive natural cofactor NADPH by electrochemistry as “electron source”. An electrochemical 24-well microtiter plate (eMTP) was developed, which can be employed in a standard microtiter plate reader and enables parallelized electrochemical experiments in combination with simultaneous optical measurements. The eMTP was applied to screen a P450 monooxygenase BM-3 mutein library and determine the behavior of P450 BM-3 muteins in an electrochemically driven surrogate assay with the mediator cobalt sepulchrate. Besides determining reaction rates also the influence of single reaction parameters e.g. applied potential, enzyme and mediator concentration were measured. Additionally the developed eMTP based screening system allows a fast development of an electro enzymatic process.  相似文献   

13.
在体外,利用野生型CYP450BM-3对瓦伦西亚烯进行催化,酶-底物复合物催化NADPH氧化的速率为31±1.0 nmol(nmol P450)-1min-1,但催化产物中没有检测到圆柚酮的生成。突变体R47L/Y51F/F87A与底物复合物催化NADPH氧化的速率高于野生型,为79±6.5 nmol(nmol P450)-1min-1,并在催化产物中检测到圆柚酮的生成,但其产物选择性较差,圆柚酮的含量仅占总产物的6.8%。与此同时,检测了另一个突变体A74G/F87V/L188Q对瓦伦西亚烯的催化效果,发现其与底物复合物对NADPH的氧化速率与突变体R47L/Y51F/F87A相当,但产物中圆柚酮的比率更高,达8.0%。  相似文献   

14.
Cytochrome P450 BM-3, a self-sufficient P450 enzyme from Bacillus megaterium that catalyzes the subterminal hydroxylation of long-chain fatty acids, has been engineered into a catalyst for the oxidation of polycyclic aromatic hydrocarbons. The activities of a triplet mutant (A74G/F87V/L188Q) towards naphthalene, fluorene, acenaphthene, acenaphthylene, and 9-methylanthracene were 160, 53, 109, 287, and 22/min, respectively. Compared with the activities of the wild type towards these polycyclic aromatic hydrocarbons, those of the mutant were improved by up to 4 orders of magnitude. The coupling efficiencies of the mutant towards naphthalene, fluorene, acenaphthene, acenaphthylene, and 9-methylanthracene were 11, 26, 5.4, 15, and 3.2%, respectively, which were also improved several to hundreds fold. The high activities of the mutant towards polycyclic aromatic hydrocarbons indicate the potential of engineering P450 BM-3 for the biodegradation of these compounds in the environment.  相似文献   

15.
Cytochrome P450 BM-3, a self-sufficient P450 enzyme from Bacillus megaterium that catalyzes the subterminal hydroxylation of long-chain fatty acids, has been engineered into a catalyst for the oxidation of polycyclic aromatic hydrocarbons. The activities of a triplet mutant (A74G/F87V/L188Q) towards naphthalene, fluorene, acenaphthene, acenaphthylene, and 9-methylanthracene were 160, 53, 109, 287, and 22/min, respectively. Compared with the activities of the wild type towards these polycyclic aromatic hydrocarbons, those of the mutant were improved by up to 4 orders of magnitude. The coupling efficiencies of the mutant towards naphthalene, fluorene, acenaphthene, acenaphthylene, and 9-methylanthracene were 11, 26, 5.4, 15, and 3.2%, respectively, which were also improved several to hundreds fold. The high activities of the mutant towards polycyclic aromatic hydrocarbons indicate the potential of engineering P450 BM-3 for the biodegradation of these compounds in the environment.  相似文献   

16.
Highly miniaturized P450 screening assays designed to enable facile analysis of P450 drug interactions in a 1536-well plate format with the principal human cytochrome P450 enzymes (CYP3A4, 2D6, 2C9, 2C19, and 1A2) and Vivid fluorogenic substrates were developed. The detailed characterization of the assays included stability, homogeneity, and reproducibility of the recombinant P450 enzymes and the kinetic parameters of their reactions with Vivid fluorogenic substrates, with a focus on the specific characteristics of each component that enable screening in a low-volume 1536-well plate assay format. The screening assays were applied for the assessment of individual cytochrome P450 inhibition profiles with a panel of selected assay modifiers, including isozyme-specific substrates and inhibitors. IC(50) values obtained for the modifiers in 96- and 1536-well plate formats were similar and comparable with values obtained in assays with conventional substrates. An overall examination of the 1536-well assay statistics, such as signal-to-background ratio and Z' factor, demonstrated that these assays are a robust, successful, and reliable tool to screen for cytochrome P450 metabolism and inhibition in an ultra-high-throughput screening format.  相似文献   

17.
18.
Cytochrome P450 BM-3 from Bacillus megaterium catalyzed NADPH-supported indole hydroxylation under alkaline conditions with homotropic cooperativity toward indole. The activity was also found with the support of H2O2, tert-butyl hydroperoxide (tBuOOH), or cumene hydroperoxide (CuOOH). Enhanced activity and heterotropic cooperativity were observed in CuOOH-supported hydroxylation, and both the Hill coefficient and substrate concentration required for half-maximal activity in the CuOOH-supported reaction were much lower than those in the H2O2-, tBuOOH-, or NADPH-supported reactions. CuOOH greatly enhanced NADPH consumption and indole hydroxylation in the NADPH-supported reaction. However, when CuOOH was replaced by tBuOOH or H2O2, heterotropic cooperativity was not observed. Spectral studies also confirmed that CuOOH stimulated indole binding to P450 BM-3. Interestingly, a mutant enzyme with enhanced indole-hydroxylation activity, F87V (Phe87 was replaced by Val), lost homotropic cooperativity towards indole and heterotropic cooperativity towards CuOOH, indicating that the active-site structure affects the cooperativities.  相似文献   

19.
20.
Wild-type cytochrome P450 monooxygenase from Bacillus megaterium (P450 BM-3) has a low hydroxylation activity for β-ionone (<1 min−1). Substitution of phenylalanine by valine at position 87 led to a more than 100-fold increase in β-ionone hydroxylation activity (115 min−1). Enzyme activity could be further increased by both site-directed and random mutagenesis. The mutant R47L Y51F F87V, designed by site-directed mutagenesis, and the mutant A74E F87V P386S, obtained after two rounds of error-prone polymerase chain reaction, exhibited an increase in activity of up to 300-fold compared to the wild-type enzyme. The triple mutant R47 LY51F F87V exhibited moderate enantioselectivity, forming (R)-4-hydroxy-β-ionone with an optical purity of 39%. All mutants regioselectively converted β-ionone into 4-hydroxy-β-ionone. The regioselectivity is determined amongst others by the absolute configuration of the substrate.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号