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1.
Enhancement of 7-methoxyresorufin O-demethylation activity of human cytochrome P450 1A2 by molecular breeding 总被引:2,自引:0,他引:2
Alkylresorufins are model substrates for cytochrome P450 (P450) 1A2. The ability of human P450 1A2 to catalyze 7-methoxyresorufin O-demethylation was improved by screening of random mutant libraries (expressed in Escherichia coli) on the basis of 7-methoxyresorufin O-demethylation. After three rounds of mutagenesis and screening, the triple mutant E163K/V193M/K170Q yielded a kcat > five times faster than wild type P450 1A2 in steady-state kinetic analysis using either isolated membrane fractions or purified, reconstituted enzymes. The enhanced catalytic activity was not attributed to changes in substrate affinity. The kinetic hydrogen isotope effect of the triple mutant did not change from wild type enzyme and suggests that C-H bond cleavage is rate-limiting in both enzymes. Homology modeling, based on an X-ray structure of rabbit P450 2C5, suggests that the locations of mutated residues are not close to the substrate binding site and therefore that structural elements outside of this site play roles in changing the catalytic activity. This approach has potential value in understanding P450 1A2 and generating engineered enzymes with enhanced catalytic activity. 相似文献
2.
Gillam EM 《Archives of biochemistry and biophysics》2007,464(2):176-186
Cytochrome P450 enzymes are amongst the most versatile enzymatic catalysts known. The ability to introduce a single atom of oxygen into an organic substrate has led to the diversification and exploitation of these enzymes throughout nature. Nowhere is this versatility more apparent than in the mammalian liver, where P450 monooxygenases catalyze the metabolic clearance of innumerate drugs and other environmental chemicals. In addition to the aromatic and aliphatic hydroxylations, N- and O-dealkylations, and heteroatom oxidations that are common in drug metabolism, many more unusual reactions catalyzed by P450s have been discovered, including reductions, group transfers and other biotransformations not typically associated with monooxygenases. A research area that shows great potential for development over the next few decades is the directed evolution of P450s as biocatalysts. Mammalian xenobiotic-metabolizing P450s are especially well suited to such protein engineering due to their ability to interact with relatively wide ranges of substrates with marked differences in structure and physicochemical properties. Typical characteristics, such as the low turnover rates and poor coupling seen during the metabolism of xenobiotics, as well as the enzyme specificity towards particular substrates and reactions, can be improved by directed evolution. This mini-review will cover the fundamental enabling technologies required to successfully engineer P450s, examine the work done to date on the directed evolution of mammalian forms, and provide a perspective on what will be required for the successful implementation of engineered enzymes. 相似文献
3.
Fluorescence-based assays for the cytochrome P450 BM3 monooxygenase from Bacillus megaterium address an attractive biotechnological challenge by facilitating enzyme engineering and the identification of potential substrates of this highly promising biocatalyst. In the current study, we used the scarcity of corresponding screening systems as an opportunity to evaluate a novel and continuous high-throughput assay for this unique enzyme. A set of nine catalytically diverse P450 BM3 variants was constructed and tested toward the native substrate-inspired fluorogenic substrate 12-(4-trifluoromethylcoumarin-7-yloxy)dodecanoic acid. Particularly high enzyme-mediated O-dealkylation yielding the fluorescent product 7-hydroxy-4-trifluoromethylcoumarin was observed with mutants containing the F87V substitution, with A74G/F87V showing the highest catalytic efficiency (0.458 min−1 μM−1). To simplify the assay procedure and show its versatility, different modes of application were successfully demonstrated, including (i) the direct use of NADPH or its oxidized form NADP+ along with diverse NADPH recycling systems for electron supply, (ii) the use of cell-free lysates and whole-cell preparations as the biocatalyst source, and (iii) its use for competitive inhibition screens to identify or characterize substrates and inhibitors. A detailed comparison with known, fluorescence-based P450 BM3 assays finally emphasizes the relevance of our contribution to the ongoing research. 相似文献
4.
Sequence-based screening for self-sufficient P450 monooxygenase from a metagenome library 总被引:2,自引:0,他引:2
Kim BS Kim SY Park J Park W Hwang KY Yoon YJ Oh WK Kim BY Ahn JS 《Journal of applied microbiology》2007,102(5):1392-1400
AIMS: Cytochrome P450 monooxygenases (CYPs) are useful catalysts for oxidation reactions. Self-sufficient CYPs harbour a reductive domain covalently connected to a P450 domain and are known for their robust catalytic activity with great potential as biocatalysts. In an effort to expand genetic sources of self-sufficient CYPs, we devised a sequence-based screening system to identify them in a soil metagenome. METHODS AND RESULTS: We constructed a soil metagenome library and performed sequence-based screening for self-sufficient CYP genes. A new CYP gene, syk181, was identified from the metagenome library. Phylogenetic analysis revealed that SYK181 formed a distinct phylogenic line with 46% amino-acid-sequence identity to CYP102A1 which has been extensively studied as a fatty acid hydroxylase. The heterologously expressed SYK181 showed significant hydroxylase activity towards naphthalene and phenanthrene as well as towards fatty acids. CONCLUSIONS: Sequence-based screening of metagenome libraries is expected to be a useful approach for searching self-sufficient CYP genes. The translated product of syk181 shows self-sufficient hydroxylase activity towards fatty acids and aromatic compounds. SIGNIFICANCE AND IMPACT OF THE STUDY: SYK181 is the first self-sufficient CYP obtained directly from a metagenome library. The genetic and biochemical information on SYK181 are expected to be helpful for engineering self-sufficient CYPs with broader catalytic activities towards various substrates, which would be useful for bioconversion of natural products and biodegradation of organic chemicals. 相似文献
5.
The use of cytochrome P450 (P450 or CYP) enzymes as biocatalysts for the production of fine chemicals, including pharmaceuticals, has been of increasing interest, primarily owing to their catalytic diversity and broad substrate range. CYP102A1 (P450 BM3) from Bacillus megaterium integrates an entire monooxygenase system into one polypeptide and represents an appropriate prokaryotic model for industrial applications of mammalian P450 activities. CYP102A1 not only exhibits the highest catalytic activity ever detected in a P450 monooxygenase but also provides a potentially versatile biocatalyst for the production of human P450 metabolites. CYP102A1 can be further engineered to be a drug-metabolizing enzyme, making it a promising candidate to use as a biocatalyst in drug discovery and synthesis. 相似文献
6.
Cytochrome P450 (P450) open reading frames (ORFs) identified in genome sequences of Bacillus species are potential resources for new oxidation biocatalysts. Phylogenetic analysis of 29 Bacillus P450 ORFs revealed that the P450s consist of a limited number of P450 families, CYP102, CYP106, CYP107, CYP109, CYP134, CYP152, and CYP197. Previously, we identified the catalytic activities of three P450s of Bacillus subtilis towards steroids by rapid substrate screening using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR/MS). Here, we further applied this method to evaluate the activity of Bacillus cereus P450s towards steroids. Five P450 genes were cloned from B. cereus ATCC 10987 based on its genomic sequence and were expressed in Escherichia coli. These P450s were reacted with a mixture of 30 compounds that mainly included steroids, and the reaction mixtures were analyzed using FT-ICR/MS. We found that BCE_2659 (CYP106) catalyzed the monooxygenation of methyltestosterone, progesterone, 11-ketoprogesterone, medroxyprogesterone acetate, and chlormadinone acetate. BCE_2654 (CYP107) monooxygenated testosterone enanthate, and BCE_3250 (CYP109) monooxygenated testosterone and compactin. Based on the phylogenetic relationship and the known substrate specificities including ones identified in this study, we discuss the catalytic potential of Bacillus P450s towards steroids. 相似文献
7.
Sun L Chen CS Waxman DJ Liu H Halpert JR Kumar S 《Archives of biochemistry and biophysics》2007,458(2):167-174
Based on recent directed evolution of P450 2B1, six P450 2B11 mutants at three positions were created in an N-terminal modified construct termed P450 2B11dH and characterized for enzyme catalysis using five substrates. Mutant I209A demonstrated a 3.2-fold enhanced k(cat)/K(m) for 7-ethoxy-4-trifluoromethylcourmarin O-deethylation, largely due to a dramatic decrease in K(m) (0.72 microM vs. 18 microM). I209A also demonstrated enhanced selectivity for testosterone 16beta-hydroxylation over 16alpha-hydroxylation. In contrast, V183L showed a 4-fold increased k(cat) for 7-benzyloxyresorufin debenzylation and a 4.7-fold increased k(cat)/K(m) for testosterone 16alpha-hydroxylation. V183L also displayed a 1.7-fold higher k(cat)/K(m) than P450 2B11dH with the anti-cancer prodrugs cyclophosphamide and ifosfamide, resulting from a approximately 4-fold decrease in K(m). Introduction of the V183L mutation into full-length P450 2B11 did not enhance the k(cat)/K(m). Overall, the re-engineered P450 2B11dH enzymes exhibited enhanced catalytic efficiency with several substrates including the anti-cancer prodrugs. 相似文献
8.
P450 enzymes are of great interest for drug metabolism and as potential biocatalysts. Like most P450s, purified CYP3A4 is normally handled and stored in solution because lyophilization greatly reduces its activity. We show here that colyophilization of this enzyme with sucrose or trehalose, but not mannitol, crown ethers or cyclodextrins, allow recovery of full enzymatic activity after rehydration. Sorbitol was almost as efficient, with 85% retention of the original activity. We also show that similar protection is observed through colyophilization of CYP2D6 with trehalose. This procedure should greatly facilitate handling, storage, or use of these enzymes in anhydrous media. 相似文献
9.
Kurt L. Harris Raine E.S. Thomson Yosephine Gumulya Gabriel Foley Saskya E. Carrera-Pacheco Parnayan Syed Tomasz Janosik Ann-Sofie Sandinge Shalini Andersson Ulrik Jurva Mikael Bodn Elizabeth M.J. Gillam 《Molecular biology and evolution》2022,39(6)
The cytochrome P450 family 1 enzymes (CYP1s) are a diverse family of hemoprotein monooxygenases, which metabolize many xenobiotics including numerous environmental carcinogens. However, their historical function and evolution remain largely unstudied. Here we investigate CYP1 evolution via the reconstruction and characterization of the vertebrate CYP1 ancestors. Younger ancestors and extant forms generally demonstrated higher activity toward typical CYP1 xenobiotic and steroid substrates than older ancestors, suggesting significant diversification away from the original CYP1 function. Caffeine metabolism appears to be a recently evolved trait of the CYP1A subfamily, observed in the mammalian CYP1A lineage, and may parallel the recent evolution of caffeine synthesis in multiple separate plant species. Likewise, the aryl hydrocarbon receptor agonist, 6-formylindolo[3,2-b]carbazole (FICZ) was metabolized to a greater extent by certain younger ancestors and extant forms, suggesting that activity toward FICZ increased in specific CYP1 evolutionary branches, a process that may have occurred in parallel to the exploitation of land where UV-exposure was higher than in aquatic environments. As observed with previous reconstructions of P450 enzymes, thermostability correlated with evolutionary age; the oldest ancestor was up to 35 °C more thermostable than the extant forms, with a 10T50 (temperature at which 50% of the hemoprotein remains intact after 10 min) of 71 °C. This robustness may have facilitated evolutionary diversification of the CYP1s by buffering the destabilizing effects of mutations that conferred novel functions, a phenomenon which may also be useful in exploiting the catalytic versatility of these ancestral enzymes for commercial application as biocatalysts. 相似文献
10.
旨在对鸡细胞色素P450 1A5(CYP1A5)蛋白进行体外功能研究,采用大肠杆菌系统进行CYP1A5的异源表达。以鸡的cDNA为模板,扩增出CYP1A5基因,将该基因的N端编码区进行修饰,并连接到pCW载体中构建His-CYP1A5,经IPTG诱导在大肠杆菌中表达。经CO-差示光谱检测,所获得的His-CYP1A5具有典型的P450吸收峰。该蛋白与细胞色素P450还原酶(CPR)进行体外重组,构成的重组酶系表现出乙氧基试卤灵-O-脱乙基酶活性。结果表明,所采用的表达策略可以成功产生出具有催化活性的鸡细胞色素P450 1A5(CYP1A5)蛋白。 相似文献
11.
Heme-iron oxygenases: powerful industrial biocatalysts? 总被引:1,自引:0,他引:1
Are cytochrome P450 enzymes powerful industrial biocatalysts? Next to market demands, well-defined enzyme functionalities and process parameters allow generalizations on the basis of process windows. These can provide useful guidelines for the design of improved biocatalysts. Oxygenase-catalyzed reactions are of special interest for selective C-H bond oxidation. The versatile class of cytochrome P450 mono-oxygenases attracts particular attention, and impressive advances have been achieved with respect to mechanistic insight, enzyme activity, stability, and specificity. Recent major achievements include significant increases in productivities, yields, and rates of catalytic turnover as well as modification of substrate specificity and efficient multistep reactions in whole-cell biocatalysts. For some biocatalysts, these parameters are already of an industrially useful magnitude. 相似文献
12.
Pravastatin, an important cholesterol lowering drug, is currently produced by hydroxylation of mevastatin (ML-236B) with Streptomyces carbophilus, in which the enzyme P450sca-2 plays a key role. Little information on the recombinant expression of this enzyme is available. As it is of industrial interest to develop an alternative simplified enzymatic process for pravastatin, as a first step, further study on the heterologous expression of this enzyme is warranted. We report here, for the first time, the purification, and characterization of P450sca-2 expressed in Escherichia coli. A synthetic gene encoding P450sca-2 was designed to suit the standard codon usage of E. coli. Expression of P450sca-2 in E. coli under optimized conditions yielded about 100 nmol purified active P450sca-2 per liter. Directed evolution was further carried out to improve the soluble expression level. In the absence of a facile and sensitive assay, green fluorescent protein (GFP) was used as a reporter to enable high-throughput screening. After three rounds of evolution by error-prone PCR and DNA shuffling, six almost totally soluble mutants were obtained, with the soluble expression levels dramatically improved by about 30-fold. For six most frequently occurring mutations, the corresponding single mutants were created to dissect the effects of these mutations. A single mutation, P159A, was found to be responsible for most of the enhanced solubility observed in the six mutants, and the corresponding single mutant also retained the hydroxylation activity. Our study provides a foundation for future work on improving functional expression of P450sca-2 in E. coli. 相似文献
13.
Chrispian W. Theron Michel Labuschagné Jacobus Albertyn Martha S. Smit 《Microbial biotechnology》2019,12(6):1126-1138
Cytochrome P450 monooxygenases (P450) are enzymes with high potential as biocatalysts for industrial applications. Their large-scale applications are, however, limited by instability and requirement for coproteins and/or expensive cofactors. These problems are largely overcome when whole cells are used as biocatalysts. We previously screened various yeast species heterologously expressing self-sufficient P450s for their potential as whole-cell biocatalysts. Most P450s are, however, not self-sufficient and consist of two or three protein component systems. Therefore, in the present study, we screened different yeast species for coexpression of P450 and P450-reductase (CPR) partners, using CYP53B1 from Rhodotorula minuta as an exemplary P450. The abilities of three different coexpressed CPR partners to support P450 activity were investigated, two from basidiomycetous origin and one from an ascomycete. The various P450-CPR combinations were cloned into strains of Saccharomyces cerevisiae, Kluyveromyces marxianus, Hansenula polymorpha, Yarrowia lipolytica and Arxula adeninivorans, using a broad-range yeast expression vector. The results obtained supported the previous finding that recombinant A. adeninivorans strains perform excellently as whole-cell biocatalysts. This study also demonstrated for the first time the P450 reductase activity of the CPRs from R. minuta and U. maydis. A very interesting observation was the variation in the supportive activity provided by the different reductase partners tested and demonstrated better P450 activity enhancement by a heterologous CPR compared to its natural partner CPR. This study highlights reductase selection as a critical variable for consideration in the pursuit of optimal P450-based catalytic systems. The usefulness of A. adeninivorans as both a host for recombinant P450s and whole-cell biocatalyst was emphasized, supporting earlier findings. 相似文献
14.
Cytochrome P450 enzymes (P450s) are able to regioselectively and stereoselectively introduce oxygen into organic compounds
under mild reaction conditions. These monooxygenases in particular easily catalyze the insertion of oxygen into less reactive
carbon–hydrogen bonds. Hence, P450s are of considerable interest as oxidation biocatalysts. To date, although several P450s
have been discovered through screening of microorganisms and have been further genetically engineered, the substrate range
of these biocatalysts is still limited to fulfill the requirements for a large number of oxidation processes. On the other
hand, the recent rapid expansion in the number of reported microbial genome sequences has revealed the presence of an unexpectedly
vast number of P450 genes. This large pool of naturally evolved P450s has attracted much attention as a resource for new oxidation
biocatalysts. In this review, we focus on aspects of the genome mining approach that are relevant for the discovery of novel
P450 biocatalysts. This approach opens up possibilities for exploitation of the catalytic potential of P450s for the preparation
of a large choice of oxidation biocatalysts with a variety of substrate specificities. 相似文献
15.
An artificial self‐sufficient cytochrome P450 directly nitrates fluorinated tryptophan analogs with a different regio‐selectivity
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Ran Zuo Yi Zhang Jose C. Huguet‐Tapia Mishal Mehta Evelina Dedic Steven D. Bruner Rosemary Loria Yousong Ding 《Biotechnology journal》2016,11(5):624-632
Aromatic nitration is an immensely important industrial process to produce chemicals for a variety of applications, but it often suffers from multiple unsolved challenges. Enzymes as biocatalysts have been increasingly used for organic chemistry synthesis due to their high selectivity and environmental friendliness, but nitration has benefited minimally from the development of biocatalysis. In this work, we aimed to develop TxtE as practical biocatalysts for aromatic nitration. TxtE is a unique class I cytochrome P450 enzyme that nitrates the indole of l ‐tryptophan. To develop cost‐efficient nitration processes, we fused TxtE with the reductase domains of CYP102A1 (P450BM3) and of P450RhF to create class III self‐sufficient biocatalysts. The best engineered fusion protein was comparable with wild type TxtE in terms of nitration performance and other key biochemical properties. To demonstrate the application potential of the fusion enzyme, we nitrated 4‐F‐dl ‐tryptophan and 5‐F‐l ‐tryptophan in large scale enzymatic reactions. Tandem MS/MS and NMR analyses of isolated products revealed altered nitration sites. To our knowledge, these studies represent the first practice in developing biological nitration approaches and lay a solid basis to the use of TxtE‐based biocatalysts for the production of valuable nitroaromatics. 相似文献
16.
Yohei Iizaka Ryusei Arai Akari Takahashi Mikino Ito Miho Sakai Atsushi Fukumoto David H Sherman Yojiro Anzai 《Journal of industrial microbiology & biotechnology》2022,49(1)
MycG is a multifunctional P450 monooxygenase that catalyzes sequential hydroxylation and epoxidation or a single epoxidation in mycinamicin biosynthesis. In the mycinamicin-producing strain Micromonospora griseorubida A11725, very low-level accumulation of mycinamicin V generated by the initial C-14 allylic hydroxylation of MycG is observed due to its subsequent epoxidation to generate mycinamicin II, the terminal metabolite in this pathway. Herein, we investigated whether MycG can be engineered for production of the mycinamicin II intermediate as the predominant metabolite. Thus, mycG was subject to random mutagenesis and screening was conducted in Escherichia coli whole-cell assays. This enabled efficient identification of amino acid residues involved in reaction profile alterations, which included MycG R111Q/V358L, W44R, and V135G/E355K with enhanced monohydroxylation to accumulate mycinamicin V. The MycG V135G/E355K mutant generated 40-fold higher levels of mycinamicin V compared to wild-type M. griseorubida A11725. In addition, the E355K mutation showed improved ability to catalyze sequential hydroxylation and epoxidation with minimal mono-epoxidation product mycinamicin I compared to the wild-type enzyme. These approaches demonstrate the ability to selectively coordinate the catalytic activity of multifunctional P450s and efficiently produce the desired compounds. 相似文献
17.
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
18.
Characterization of xenobiotic metabolizing cytochrome P450s (P450s) was carried out in rat brain microsomes using the specific substrates, 7-pentoxy- and 7-ethoxyresorufin (PR and ER), metabolized in the liver by P450 2B1/2B2 and 1A1/1A2 respectively and 7-benzyloxyresorufin (BR), a substrate for both the isoenzymes. Brain microsomes catalysed the O-dealkylation of PR, BR and ER in the presence of NADPH. The ability to dealkylate alkoxyresorufins varied in different regions of the brain. Microsomes from the olfactory lobes exhibited maximum pentoxyresorufin-O-dealkylase (PROD), benzyloxyresorufin-O-dealkylase (BROD) and ethoxyresorufin-O-dealkylase (EROD) activities. The dealkylation was found to be inducer selective. While pretreatment with phenobarbital (PB; 80 mg/kg; i.p. × 5 days) resulted in significant induction in PROD (3-4 fold) and BROD (4-5 fold) activities, 3-methylcholanthrene (MC; 30 mg/kg; i.p. × 5 days) had no effect on the activity of PROD and only a slight effect on that of BROD (1.4 fold). MC pretreatment significantly induced the activity of EROD (3 fold) while PB had no effect on it. Kinetic studies have shown that this increase in the activities following pretreatment with P450 inducers was associated with a significant increase in the velocity of the reaction (Vmax) of O-dealkylation. In vitro studies using organic inhibitors and antibodies have further provided evidence that the O-dealkylation of alkoxyresorufins is isoenzyme specific. While in vitro addition of a-naphthoflavone (ANF), an inhibitor of P450 1A1/1A2 catalysed reactions and antibody for hepatic P450 1A1/1A2 isoenzymes produced a concentration-dependent inhibition of EROD activity, metyrapone, an inhibitor of P450 2B1/2B2 and antibody for hepatic P450 2B1/2B2 significantly inhibited the activity of PROD and BROD in vitro. The data suggest that, as in the case of liver, dealkylation of alkoxyresorufins can be used as a biochemical tool to characterise the xenobiotic metabolising P450s and substrate selectivity of P450 isoenzymes in rat brain microsomes. 相似文献
19.
Kumar S 《Journal of biomolecular screening》2007,12(5):677-682
The author sought to develop a high-throughput activity screening assay to carry out rapid kinetic analysis, inhibitor screening, and directed evolution of cytochrome P450 2C enzymes. Initially, of the 9 fluorescent substrates and 10 P450 2C enzymes tested, several P450 2C enzymes showed > 1 nmol/min/nmol P450 activity in cumene hydroperoxide (CuOOH)-supported reaction with a laser dye, 7-dimethylamino-4-trifluoromethylcoumarin (C152). A high-throughput steady-state kinetic analysis of the human P450 2C8, 2C9, and 2C19 showed 1) k(cat) = 3 to 6 min(-1), 2) K(m, CuOOH) = 100 to 200 microM, and 3) S(50, C152) = 10 to 20 microM in the CuOOH system. In addition, P450 2C9 and 2C19 showed a very high k(ca)t (27 and 38 min(-1), respectively) in the nicotinamide adenine dinucleotide phosphate (NADPH)-supported reaction. Subsequently, when mammalian P450s from the other subfamilies were tested, P450 2B1dH, 2B4dH, 2B5dH, 3A4, and 3A5 exhibited a significant activity in both CuOOH and NADPH systems. Furthermore, a high-throughput activity screening assay using whole-cell suspensions of the human P450 2C8, 2C9, and 2C19 was optimized. Overall, the data suggested that C152 can be used as a model substrate for mammalian P450s in CuOOH-supported reaction to perform rapid kinetic analysis, inhibitor screening, and directed evolution. 相似文献
20.
Uno T Nakao A Masuda S Taniguchi Y Kanamaru K Yamagata H Nakamura M Imaishi H Oono K 《Journal of industrial microbiology & biotechnology》2006,33(12):1043-1050
We developed a system for bioconverting diverse compounds using P450s produced in Escherichia coli. Vectors for the expressing various P450 cDNAs quickly and easily in E. coli were developed by using several restriction enzyme sites. Three types of P450 (2C2, 2C29, and 2D22) were produced using these plasmids. Substrates were directly added to the incubation medium and metabolized. To obtain pure product from the medium, we first tried production of P450 in synthetic medium. The amount of another P450 2C43 produced in the synthetic medium was similar to the amount produced in Luria broth (LB) medium. Next, estradiol, a steroid, was added as a substrate, incubated, and the metabolite was extracted and analyzed by high-performance liquid chromatography. The metabolite extracted from synthetic medium was purer than that obtained from LB medium. Three P450s (2C29, 2C2, and 2A4) metabolized testosterone at different positions. P450 2C29 metabolized 7-ethoxycoumarin, androstendione, and dehydroepiandrosterone in this medium. P450s produced in the synthetic medium may be useful for producing various modified compounds for high-throughput screening. 相似文献