Cytochrome P450 monooxygenase and application thereof in synthesis of chiral tertiary alcohol
通过优化细胞色素P450单加氧酶的核苷酸和氨基酸序列,利用其催化外消旋底物形成手性叔醇,解决了现有技术中酶活力低和产物浓度不足的问题,实现了高效 , synthesis of chiral tertiary alcohols with high purity, suitable for industrial production.
Patent Information
- Application Number
- CN202411240920.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-05
- Publication Date
- 2024-12-13
AI Technical Summary
In the prior art, the enzyme activity of biocatalytic preparation of chiral tertiary alcohols is relatively low, the steps are complicated and the product concentration is insufficient, making it difficult to meet the needs of industrial production.
Provide a cytochrome P450 monooxygenase and its application in the synthesis of chiral tertiary alcohols. By using this enzyme in buffer, the formation of chiral tertiary alcohols is catalyzed by using the enzyme in the buffer, and the recombinant cell-integrated and crude enzymes are used. nucleotide and amino acid sequences are optimized to improve catalytic efficiency.
It achieves a reaction yield of up to 45% and a product optical purity up to 99%ee. It has extremely high three-dimensional and location selectivity, is suitable for industrial production, reduces the complexity of by-product generation and operation, and is suitable for large-scale applications.
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Figure CN119120399A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of enzyme engineering, and more specifically to a cytochrome P450 monooxygenase and application thereof in synthesizing chiral tertiary alcohols. Background Art
[0002] Chiral tertiary alcohols are an important class of compounds that are widely present in a variety of drugs and bioactive natural products. They are also versatile building blocks in synthesis. Many chemists and pharmaceutical workers have reported their synthetic methods and strategies, usually through the kinetic resolution of racemic tertiary alcohols and asymmetric nucleophilic addition to ketones to prepare chiral tertiary alcohols. In addition, they can also be synthesized by direct upgrading of secondary alcohols. However, all these methods require pre-oxidation on the corresponding CH bond to form alcohol or ketone precursors. Therefore, for substrates without pre-oxygen functionalization of the target CH bond, direct CH hydroxylation is an ideal and straightforward strategy to construct chiral tertiary alcohols. CH bonds are very common, with high bond energy and low polarity, which make CH bonds highly inert. Breaking CH bonds under mild conditions is one of the most challenging reactions in organic synthesis, so the synthesis of such chiral tertiary alcohols has not been fully developed. In recent years, highly selective enzymatic hydroxylation has shown promise in this field and has become a promising solution.
[0003] Biocatalysts are usually highly chemically, regio- and stereo-selective, and can carry out non-toxic catalytic reactions under mild conditions. Oxidase and reductase perform well in asymmetric catalysis and can generate primary and secondary alcohols. However, due to the tertiary carbon structure of tertiary alcohols, ketoreductases cannot synthesize chiral tertiary alcohols. P450 enzymes with excellent CH bond hydroxylation performance provide a feasible synthesis scheme for chiral tertiary alcohols. Cytochrome P450 monooxygenase (P450) is a superfamily of heme-containing enzymes that play an important role in the biosynthesis of secondary metabolites or the metabolism of xenobiotics in organisms. P450 usually acts as a monooxygenase, using molecular oxygen as a more environmentally friendly oxidant at normal pressure, and shows amazing regio- and stereo-selectivity in the catalytic process. P450 enzymes are the most widely studied oxygenases, and it has been reported that P450 enzymes can catalyze more than 40 different types of reactions.
[0004] At present, the literature on the synthesis of tertiary alcohols by P450 enzymes is mainly limited to some natural product gene synthesis clusters with single-structure substrates, such as 25-hydroxyvitamin D3 and mycinamicin V, while the hydroxylation of racemic tertiary carbon substrates has not been widely studied. To achieve this reaction, it is necessary to overcome the steric hindrance caused by the three non-hydrogen atom groups of the tertiary carbon and achieve single-structure kinetic resolution within the racemic substrate. Zhang Ruyue et al. reported a method for synthesizing chiral tertiary alcohols through CH hydroxylation using P450PL2 and its mutants. However, this method is limited to laboratory scale, and there are problems such as low hydroxylase activity and low product concentration, which is not suitable for industrial production of chiral tertiary alcohols.
[0005] Therefore, it is of great significance to screen cytochrome P450 monooxygenases with high activity and the ability to obtain high product concentrations in a short time to meet the needs of industrial production of chiral tertiary alcohols. Summary of the invention
[0006] The purpose of the present invention is to provide a cytochrome P450 monooxygenase and its application in the synthesis of chiral tertiary alcohols, so as to solve the problems of low enzyme activity, complicated steps and insufficient product concentration in the biocatalytic reaction of preparing chiral tertiary alcohols in the prior art.
[0007] The above technical purpose of the present invention is achieved through the following technical scheme: a cytochrome P450 monooxygenase, the nucleotide sequence of the cytochrome P450 monooxygenase is shown in SEQ ID NO.1, and the amino acid sequence of the cytochrome P450 monooxygenase is shown in SEQ ID NO.2.
[0008] Another object of the present invention is to provide a use of the above-mentioned cytochrome P450 monooxygenase in the synthesis of chiral tertiary alcohols, specifically comprising: in a buffer, catalyzing a racemic substrate to form a chiral tertiary alcohol under the catalysis of the cytochrome P450 monooxygenase.
[0009] Furthermore, the buffer solution is a phosphate buffer solution with a phosphate concentration of 0.05 mol / L.
[0010] Furthermore, the concentration of the racemic substrate is 1 to 50 mmol / L.
[0011] Furthermore, the dosage of the cytochrome P450 monooxygenase is 1-5 kU / L.
[0012] Furthermore, the cytochrome P450 monooxygenase is added in the form of recombinant whole cells, crude enzyme solution or lyophilized cells containing cytochrome P450 monooxygenase.
[0013] Another object of the present invention is to provide a protein recombinant bacterial cell containing the above cytochrome P450 monooxygenase.
[0014] Another object of the present invention is to provide a vector carrying the gene encoding the above cytochrome P450 monooxygenase.
[0015] Another object of the present invention is to provide a recombinant bacterium expressing the above cytochrome P450 monooxygenase.
[0016] By adopting the above technical solution, the present invention adopts the technical solution provided by the present invention, and cytochrome P450 monooxygenase is used as a catalyst, and the yield of the reaction can reach up to 45%, which means that most of the substrate can be successfully converted into the target product during the reaction, and the optical purity of the product can reach up to 99% ee (enantiomeric excess), which indicates that the product is almost entirely in one chiral form, has extremely high stereoselectivity, and is particularly important for the preparation of chiral drugs and other chiral compounds;
[0017] The reaction of the present invention not only has high stereoselectivity, but also has excellent regioselectivity, ensuring that the reaction occurs at the target location rather than producing by-products, which is particularly important for the synthesis of complex molecules. The substrate concentration of the present invention is high, which means that the reaction can be carried out at a higher concentration, thereby improving the reaction efficiency, reducing the amount of solvent used, and reducing the cost. The reaction conditions of the present invention are mild, which not only helps to protect the structural integrity of the substrate and the product, but also reduces energy consumption and improves the safety and operability of the reaction.
[0018] The catalytic process of the present invention is environmentally friendly, does not produce harmful by-products, conforms to the principles of green chemistry, helps to reduce environmental pollution, is easy to operate, and the reaction system is easy to control and adjust, suitable for large-scale industrial production, reducing the need for complex equipment and operations, so that the cytochrome P450 monooxygenase and its gene described in the present invention have broad industrial application prospects and can be used to prepare high-value-added chiral compounds such as drug intermediates, pesticides and other fine chemicals. Due to its high efficiency, safety and environmental protection, the technology is expected to be widely promoted and applied in the industry.
[0019] In summary, the present invention has the following beneficial effects:
[0020] 1. The cytochrome P450 monooxygenase provided by the present invention exhibits excellent catalytic efficiency in the application of preparing chiral tertiary alcohols from racemic tertiary carbon substrates, and the reaction yield can be as high as 45%. The high yield means that more target products can be obtained under the same reaction conditions, thereby improving production efficiency and reducing production costs; the optical purity of the product of the present invention can be as high as 99%ee, indicating that the catalytic system of the present invention has extremely high stereoselectivity, and chiral tertiary alcohols with high optical purity have important applications in the fields of medicine and fine chemicals, and can significantly improve the purity of the product and reduce by-products;
[0021] 2. The present invention exhibits good stereo and regio selectivity during the reaction process, ensuring the uniqueness and purity of the product structure, and the high selectivity reduces the generation of by-products, simplifying the subsequent separation and purification steps; the reaction conditions of the present invention are mild, the conditions are easy to control, suitable for industrial production, and have low requirements on equipment and operators, which is conducive to large-scale application and promotion;
[0022] 3. The cytochrome P450 monooxygenase provided by the present invention can be added in the form of recombinant whole cells, crude enzyme solution or lyophilized cells, which is simple to operate, easy to store and transport, and reduces the complexity and cost of the production process; the cytochrome P450 monooxygenase and its gene described in the present invention have good industrial application development prospects and can significantly improve the industrial production level of chiral tertiary alcohols. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 The synthetic pathway diagram of the chiral tertiary alcohol product catalyzed by cytochrome P450 monooxygenase in Example 1 of the present invention is shown.
[0024] Figure 2 This is a liquid phase analysis spectrum of chiral (R)-2-phenyl-1,2-propanediol prepared by the reaction in Example 3 of the present invention.
[0025] Figure 3 This is a liquid phase analysis spectrum of the chiral (S)-3-hydroxy-3-phenylbutyronitrile prepared by the reaction in Example 4 of the present invention.
[0026] Figure 4 This is a liquid phase analysis spectrum of chiral (R)-1-chloro-2-phenyl-2-propanol prepared by the reaction in Example 5 of the present invention. DETAILED DESCRIPTION
[0027] The following is combined with Figure 1-4The present invention is further described in detail by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are selected according to conventional methods and conditions, or according to the product specifications. The room temperature is the conventional room temperature in the art, and the room temperature range is 20 to 40°C.
[0028] Example 1: Preparation of a cytochrome P450 monooxygenase recombinant plasmid and recombinant expression transformant
[0029] In order to optimize the amino acid sequence described in SEQ ID NO.2 and make it more suitable for expression in E. coli, we optimized the nucleotide sequence according to the codon preference of E. coli to obtain the optimized gene nucleotide sequence SEQ ID NO.1. The optimized gene SEQ ID NO.1 was artificially synthesized and connected to the E. coli expression vector pET28a to construct the recombinant expression plasmid pET28a-P450. The specific steps are as follows:
[0030] Gene optimization and synthesis: The amino acid sequence described in SEQ ID NO.2 was optimized according to the codon preference of Escherichia coli to obtain the nucleotide sequence SEQ ID NO.1, and the optimized gene SEQ ID NO.1 was synthesized by a whole gene artificial synthesis method;
[0031] Plasmid construction: The synthesized SEQ ID NO.1 gene fragment was inserted into the E. coli expression vector pET28a to obtain the recombinant expression plasmid pET28a-P450;
[0032] Transformation and screening: The constructed recombinant expression plasmid pET28a-P450 was transformed into Escherichia coli BL21 (DE3) competent cells, and the transformation solution was spread on LB plates containing kanamycin, and incubated inverted at 37°C overnight to screen out resistant colonies;
[0033] Clone verification: The selected colonies were preliminarily screened by colony PCR, and positive clones were selected. The positive clones were verified by gene sequencing to confirm the correct insertion and expression of the recombinant plasmid pET28a-P450 in E. coli BL21 (DE3).
[0034] Example 2: Expression of cytochrome P450 monooxygenase
[0035] The recombinant E. coli BL21 (DE3) / pET28a-P450 obtained in Example 1 was cultured and induced for expression according to the following steps:
[0036] Seed culture: The recombinant E. coli was inoculated into LB medium containing 50 μg / mL kanamycin. The LB medium composition was: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.0. The culture was shaken overnight at 37°C and 250 rpm to allow the bacterial liquid to grow fully.
[0037] Expanded culture: On the second day, inoculate the overnight cultured seed solution into a 500 mL Erlenmeyer flask containing 100 mL LB medium at a 1% (v / v) inoculation volume. The LB medium also contains 50 μg / mL kanamycin. Continue shaking culture at 37°C and 250 rpm until the OD600 value of the culture solution reaches about 0.6, which usually takes about 2-3 hours.
[0038] Induced expression: When the OD600 value reached 0.6, isopropyl-β-D-thiogalactoside (IPTG) was added to the culture medium at a final concentration of 0.1 mmol / L as an inducer to induce the expression of P450 monooxygenase. The induced culture system was shaken and cultured at 25°C and 250 rpm for 12 hours to fully express the recombinant protein.
[0039] Bacteria collection: After 12 hours, the culture medium was collected by centrifugation in a centrifuge. The centrifugation conditions were set at 4°C, 5000 rpm, and the centrifugation time was 10 minutes. After centrifugation, the supernatant was discarded and the precipitated recombinant bacterial cells were collected. These cells contained the expressed P450 monooxygenase protein.
[0040] Storage and application: The collected recombinant bacterial cells can be directly used in subsequent biocatalytic reactions, or further processed as needed, such as freeze-drying or preparing crude enzyme solution;
[0041] Through the above steps, recombinant E. coli cells containing P450 monooxygenase can be successfully cultured and induced to express, providing an efficient catalyst source for the biocatalytic synthesis of chiral tertiary alcohols. The synthesis pathway of chiral tertiary alcohol products catalyzed by cytochrome P450 monooxygenase is shown in the attached figure. Figure 1 shown.
[0042] Example 3: P450 monooxygenase recombinant cells catalyze the synthesis of chiral (R)-2-phenyl-1,2-propanediol from 2-phenyl-1-propanol. The synthesis pathway is shown in Formula 1 below:
[0043] Formula 1:
[0044] In a 500mL Erlenmeyer flask, add 100mL of phosphate buffer. The concentration of the buffer is 50mM, and the pH is adjusted to 8.5 to ensure a suitable reaction environment. Recombinant cells are added to the buffer with a cell dry weight concentration (cdw) of 20g / L. These recombinant cells contain expressed P450 monooxygenases that can catalyze the target reaction; 2-phenyl-1-propanol is added to the Erlenmeyer flask at a final concentration of 50mM as a reaction substrate. Ensure that the substrate is evenly distributed in the buffer, place the Erlenmeyer flask on a constant temperature shaker at 30°C, and set the shaking speed to 250rpm. The reaction is stirred under this condition to ensure sufficient oxygen supply and uniform mixing of the reactants. The reaction duration is set to 24 hours to ensure that the substrate is fully converted into the target product;
[0045] After the reaction is completed, the reaction solution is taken out and centrifuged in a centrifuge, and the centrifugation conditions are set to 4°C, 8000rpm, and the centrifugation time is 10 minutes to precipitate and remove the cell bodies; after centrifugation, the precipitated bacteria are discarded, and the supernatant is collected to prepare for subsequent product extraction; the separated supernatant is extracted with 100mL of ethyl acetate, and each extraction is fully shaken and mixed, and then allowed to stand for stratification, and extraction is performed three times to ensure sufficient extraction of the product, and the organic phases obtained from each extraction are separated and combined into a container; the combined organic phases are dried with anhydrous sodium sulfate to absorb the moisture in the organic phase, and allowed to stand for a period of time to ensure sufficient drying. After drying, the organic phases are filtered through filter paper to remove the anhydrous sodium sulfate in the organic phase, and the dried filtrate is collected;
[0046] The filtrate obtained by filtration was concentrated by vacuum distillation. The purpose of vacuum distillation was to remove the ethyl acetate solvent and concentrate the target product. The temperature and pressure needed to be controlled during the concentration process to prevent the degradation of the target product. Finally, the target product (R)-2-phenyl-1,2-propanediol was obtained by vacuum distillation. The yield of the product was 47%, and the optical purity (enantiomeric excess, ee value) was 96%, indicating that the product had a high optical purity. The purity and composition of the chiral (R)-2-phenyl-1,2-propanediol prepared by the reaction were verified by liquid chromatography (HPLC). The liquid phase analysis spectrum is shown in the attached figure. Figure 2 As shown, liquid chromatography analysis can determine the purity and optical purity of the product and ensure the accuracy of the experimental results.
[0047] Example 4: P450 monooxygenase recombinant cells catalyze the synthesis of chiral (S)-3-hydroxy-3-phenylbutyronitrile from 3-phenylbutyronitrile. The synthesis pathway is shown in Formula 2 below:
[0048] Formula 2:
[0049] In a 500mL Erlenmeyer flask, add 100mL of phosphate buffer. The concentration of the buffer is 50mM, and the pH value is adjusted to 8.5 to ensure a suitable reaction environment. Recombinant cells are added to the buffer with a cell dry weight concentration (cdw) of 20g / L. These recombinant cells contain expressed P450 monooxygenases that can catalyze the target reaction. Add 3-phenylbutyronitrile to the Erlenmeyer flask at a final concentration of 50mM as a reaction substrate to ensure that the substrate is evenly distributed in the buffer. Place the Erlenmeyer flask on a constant temperature shaker at 30°C and set the shaking speed to 250rpm. Stir the reaction under these conditions to ensure sufficient oxygen supply and uniform mixing of the reactants. The reaction duration is set to 24 hours to ensure that the substrate is fully converted into the target product.
[0050] After the reaction is completed, the reaction solution is taken out and centrifuged in a centrifuge. The centrifugation conditions are set to 4°C and 8000rpm for 10 minutes to precipitate and remove the cell bodies. After centrifugation, the precipitated bacteria are discarded, and the supernatant is collected to prepare for subsequent product extraction; the separated supernatant is extracted with 100mL of ethyl acetate, and each extraction is fully shaken and mixed and then allowed to stand for stratification. Three extractions are performed to ensure sufficient extraction of the product, and the organic phases obtained from each extraction are separated and combined into a container; the combined organic phases are dried with anhydrous sodium sulfate to absorb the moisture in the organic phase, and allowed to stand for a period of time to ensure sufficient drying. After drying, the organic phases are filtered through filter paper to remove the anhydrous sodium sulfate in the organic phase, and the dried filtrate is collected;
[0051] The filtrate obtained by filtration is concentrated by vacuum distillation. The purpose of the vacuum distillation is to remove the ethyl acetate solvent and concentrate the target product. The temperature and pressure need to be controlled during the concentration process to prevent the degradation of the target product. Finally, the target product (S)-3-hydroxy-3-phenylbutyronitrile is obtained by vacuum distillation. The yield of the product is 46%, and the optical purity (enantiomeric excess, ee value) is 99%, indicating that the product has extremely high optical purity. The purity and composition of the chiral (S)-3-hydroxy-3-phenylbutyronitrile prepared by the reaction are verified by liquid chromatography (HPLC) analysis. The liquid phase analysis spectrum is shown in the attached figure. Figure 3 As shown, liquid chromatography analysis can determine the purity and optical purity of the product and ensure the accuracy of the experimental results.
[0052] Example 5: P450 monooxygenase recombinant cells catalyze (1-chloro-2-propyl)benzene to synthesize chiral (R)-1-chloro-2-phenyl-2-propanol. The synthesis pathway is shown in Formula 3 below:
[0053] Formula 3:
[0054] Reaction preparation: In a 500 mL Erlenmeyer flask, add 100 mL of phosphate buffer with a concentration of 50 mM and a pH value adjusted to 8.5 to ensure a suitable reaction environment. Add recombinant cells with a cell dry weight concentration (cdw) of 20 g / L to the buffer. These recombinant cells contain expressed P450 monooxygenases that can catalyze the target reaction.
[0055] Substrate addition: Add (1-chloro-2-propyl)benzene to a final concentration of 50 mM as the reaction substrate to the conical flask, ensuring that the substrate is evenly distributed in the buffer;
[0056] Reaction condition setting: Place the conical flask on a constant temperature shaker at 30°C and set the shaking speed to 250 rpm. Stir the reaction under this condition to ensure sufficient oxygen supply and uniform mixing of the reactants. The reaction duration is set to 24 hours to ensure that the substrate is fully converted into the target product.
[0057] Reaction termination and separation: After the reaction is completed, the reaction solution is taken out and centrifuged in a centrifuge. The centrifugation conditions are set at 4°C and 8000 rpm for 10 minutes to precipitate and remove the cell bodies. After centrifugation, the precipitated cells are discarded and the supernatant is collected for subsequent product extraction.
[0058] Product extraction: The separated supernatant was extracted with 100 mL of ethyl acetate. After each extraction, the mixture was fully shaken and mixed, and then allowed to stand for stratification. The extraction was performed three times to ensure that the product was fully extracted. The organic phases obtained from each extraction were separated and combined into one container;
[0059] Treatment of organic phase: The combined organic phase is dried with anhydrous sodium sulfate to absorb the water in the organic phase, and is allowed to stand for a period of time to ensure sufficient drying. After drying, the organic phase is filtered through filter paper to remove the anhydrous sodium sulfate in the organic phase, and the dried filtrate is collected;
[0060] Product concentration: The filtrate obtained by filtration is concentrated by vacuum distillation. The purpose of vacuum distillation is to remove the ethyl acetate solvent and concentrate the target product. The temperature and pressure need to be controlled during the concentration process to prevent degradation of the target product;
[0061] Obtaining the target product: Finally, the target product (R)-1-chloro-2-phenyl-2-propanol was obtained by reduced pressure distillation. The yield of the product was 44% and the optical purity (enantiomeric excess, ee value) was 94%, indicating that the product had a high optical purity;
[0062] Analysis and verification: The purity and composition of the chiral (R)-1-chloro-2-phenyl-2-propanol prepared by the reaction were verified by liquid chromatography (HPLC). The liquid phase analysis spectrum is shown in the attached figure. Figure 4As shown, liquid chromatography analysis can determine the purity and optical purity of the product and ensure the accuracy of the experimental results.
[0063] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.
Claims
1. A cytochrome P450 monooxygenase, characterized in that: The nucleotide sequence of the cytochrome P450 monooxygenase is shown in SEQ ID NO.1, and the amino acid sequence of the cytochrome P450 monooxygenase is shown in SEQ ID NO.
2.
2. The use of the cytochrome P450 monooxygenase according to claim 1 in the synthesis of chiral tertiary alcohols, characterized in that: include: In a buffer, the racemic substrate is catalyzed to form a chiral tertiary alcohol under the catalysis of the cytochrome P450 monooxygenase.
3. The use according to claim 2, characterized in that: The buffer solution is a phosphate buffer solution with a phosphate concentration of 0.05 mol / L.
4. The use according to claim 2, characterized in that: The concentration of the racemic substrate is 1-50 mmol / L.
5. The use according to claim 2, characterized in that: The dosage of the cytochrome P450 monooxygenase is 1-5 kU / L.
6. The application according to claim 2, characterized in that: The added form of the cytochrome P450 monooxygenase is recombinant whole cells, crude enzyme solution or lyophilized cells containing cytochrome P450 monooxygenase.
7. A protein recombinant bacterial cell containing the cytochrome P450 monooxygenase according to claim 1.
8. A vector carrying the gene encoding cytochrome P450 monooxygenase as claimed in claim 1.
9. A recombinant bacterium expressing the cytochrome P450 monooxygenase according to claim 1.