Method for green synthesis of chiral o-triol
By constructing a three-enzyme cascade catalytic reaction of P450 monooxygenase and epoxy hydrolase, the problems of insufficient use of strong oxidants and transition metal catalysis in the existing chiral ortho-triol compound synthesis technology are solved, and high efficiency is achieved. Environmentally friendly biosynthesis method.
Patent Information
- Application Number
- CN202411386643.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-30
- Publication Date
- 2025-02-14
AI Technical Summary
The existing chiral ortho-triol compound synthesis technology has insufficient use of strong oxidants, transition metal catalysis, poor stereoselectivity, many by-products, and harsh reaction conditions. There is a lack of syntheses with high activity and high stereoselectivity, resulting in catalytic efficiency. Low.
Construct the three-cascade catalytic reaction of P450 monooxygenase P450DA-M1, P450PL-M1 and epoxy hydrolase LEH. These enzymes are used to catalyze allylbenzene under aqueous conditions to achieve chiral ortho-triol compounds biosynthesis.
A high reaction rate, no need for intermediate separation, short time, mild reaction conditions, no need for transition metals or toxic reagents, has high catalytic activity and environmentally friendly biosynthesis method.
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Figure CN119432781A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of synthesis of chiral vicinal triol compounds, and in particular to the construction of a three-enzyme cascade catalytic reaction of a P450 monooxygenase (named P450DA-M1), a P450 monooxygenase (named P450PL-M1) and an epoxide hydrolase (named LEH), and the application thereof in the biosynthesis of chiral vicinal triol compounds. Background Art
[0002] Chiral vicinal triols are a very important class of compounds that can be used as drug intermediates and important catalyst intermediates, and are widely used in organic synthesis and drug synthesis. The existing synthesis technology of chiral vicinal triols still has shortcomings such as the use of strong oxidants, transition metal catalysis, poor stereoselectivity, many by-products, and harsh reaction conditions. The high efficiency and high selectivity of bioenzymes make biocatalysis technology expected to develop into a new way for industrial green preparation of chiral vicinal triols. However, existing studies have shown that chiral vicinal triol synthases with high activity and high stereoselectivity are still very scarce. Therefore, obtaining bioenzymes with high catalytic efficiency and corresponding biocatalytic synthesis methods is still a difficulty in the development of biocatalytic preparation of chiral vicinal triols. Summary of the invention
[0003] The purpose of the present invention is to provide a construction of a three-enzyme cascade catalytic reaction of P450 monooxygenase (named P450DA-M1), P450 monooxygenase (named P450PL-M1) and epoxide hydrolase (named LEH) and its application in the biosynthesis of chiral vicinal triol compounds to solve the above technical problems.
[0004] The technical solution of the present invention:
[0005] A method for preparing a chiral vicinal triol compound is characterized in that allylbenzene is used as a substrate, P450DA-M1, P450PL-M1 monooxygenase and epoxide hydrolase LEH are used as catalysts, and the reaction is carried out under aqueous conditions, and the reaction formula is as follows:
[0006] in:
[0007] R is methyl, ethyl, fluorine, chlorine, bromine, n-propyl, isopropyl, n-butyl, phenyl, methylphenyl, methoxyphenyl, bromophenyl, chlorophenyl, or fluorophenyl. The present invention is achieved in that:
[0008] In the first aspect, the present invention provides a P450 monooxygenase P450DA-M1, whose amino acid sequence is shown in SEQ ID No.1; the present invention provides a P450 monooxygenase P450PL-M1, whose amino acid sequence is shown in SEQ ID No.3; the present invention provides an epoxide hydrolase LEH, whose amino acid sequence is shown in SEQ ID No.5.
[0009] The P450 monooxygenase P450DA-M 1, P450PL-M 1 and epoxide hydrolase LEH genes of the present invention are constructed into genetic engineering bacteria, and induced to express to obtain P450 monooxygenase P450DA-M 1, P450PL-M 1 and epoxide hydrolase LEH. It is found that the P450 monooxygenase P450DA-M 1, P450PL-M 1 and hydrolase LEH have the function of synthesizing chiral vicinal triol compounds by three-enzyme cascade reaction. Compared with the existing preparation method of chiral vicinal triol compounds, the three-enzyme cascade reaction provided by the present invention is used to biosynthesize chiral vicinal triol compounds, which has the technical advantages of high reaction rate, no need for intermediate separation and short time consumption; when synthesizing chiral vicinal triol compounds, the three-enzyme cascade of monooxygenase P450DA-M1, P450PL-M1 and hydrolase LEH provided by the present invention can be carried out at normal temperature, normal pressure and aqueous phase environment, and the reaction conditions are mild; in addition, when synthesizing chiral vicinal triol compounds, the two-enzyme cascade of monooxygenase P450DA-M1, P450PL-M1 and epoxide hydrolase LEH provided by the present invention does not need to use transition metals or toxic reagents, so the two-enzyme cascade of monooxygenase P450DA-M1, P450PL-M1 and epoxide hydrolase LEH provided by the present invention has high catalytic activity, is environmentally friendly and easy to operate.
[0010] The amino acid sequence of monooxygenase P450DA-M1 shown in SEQ ID No. 1 is as follows:
[0011] In a second aspect, the present invention further provides an isolated nucleic acid molecule encoding the above-mentioned monooxygenase P450DA-M1, wherein the isolated nucleic acid molecule has a nucleotide sequence as shown in SEQ ID No. 2:
[0012] In the third aspect, the amino acid sequence of monooxygenase P450PL-M1 shown in SEQ ID No.3 is as follows:
[0013] The present invention also provides an isolated nucleic acid molecule encoding the above-mentioned monooxygenase P450PL-M1, and the isolated nucleic acid molecule has the nucleotide sequence shown in SEQ ID No. 4 as follows:
[0014] In a fourth aspect, the amino acid sequence of epoxide hydrolase LEH shown in SEQ ID No.5 is as follows:
[0015] The present invention also provides an isolated nucleic acid molecule encoding the above-mentioned epoxide hydrolase LEH, and the isolated nucleic acid molecule has the nucleotide sequence shown in SEQ ID No.6 as follows:
[0016] In a fifth aspect, the present invention also provides a vector comprising the above-mentioned nucleic acid molecule.
[0017] The vector includes but is not limited to bacterial expression vectors, fungal expression vectors, etc., as long as the vector can achieve the expression of the above-mentioned nucleic acid molecules and the shuttle of the above-mentioned nucleic acid molecules. Including but not limited to plasmids, etc. Bacterial expression vectors such as Escherichia coli expression vectors, Streptomyces or Mycobacterium expression vectors. Fungal expression vectors such as yeast vectors.
[0018] In a sixth aspect, the present invention also provides a recombinant bacterium, which comprises the above-mentioned vector.
[0019] In a preferred embodiment of the present invention, the recombinant bacteria is a bacterium or a fungus; in a preferred embodiment of the present invention, the bacteria is Escherichia coli, Streptomyces or Mycobacterium.
[0020] Fungi such as yeast.
[0021] In a preferred embodiment of the present invention, the recombinant bacteria refers to at least one of resting cells of bacteria, living cells of recombinant bacteria, dead cells of recombinant bacteria, and cell fragments of recombinant bacteria.
[0022] The above-mentioned recombinant bacteria (or genetically engineered bacteria) are obtained by transforming a recombinant expression vector into a host microorganism by conventional methods in the art; the host microorganism can be various conventional host microorganisms in the art, as long as the recombinant expression vector can stably replicate itself and the P450 monooxygenase and epoxide hydrolase genes carried by it can be effectively expressed; preferably, it can be obtained by the following method: transforming the recombinant vector pET-28a(+)-P450 into Escherichia coli BL21(DE3).
[0023] Dormant cells, also known as resting cells, are a special cell state. In this state, cells do not grow and reproduce, but still contain various enzyme systems and have oxidation and fermentation capabilities. Under suitable conditions, dormant cells can resume growth. The characteristics of dormant cells include: a. Cells maintain growth potential: Although in a dormant state, these cells can re-enter the cell cycle and restore their proliferation ability when given appropriate stimulation. b. Strong specificity: Dormant cells have strong specificity in the reaction and can improve the substrate conversion rate. c. Not easy to be contaminated by foreign bacteria: Due to its characteristics, dormant cells can reduce the inhibition of bacterial growth and enzyme synthesis by products during use.
[0024] In a seventh aspect, the present invention further provides an immobilized cell, which is obtained by immobilizing the above-mentioned bacteria or the above-mentioned cells.
[0025] In a preferred embodiment of the present invention, the method of fixing cells via a carrier is selected from at least one of an adsorption method, a covalent binding method, a cross-linking method and a microencapsulation method.
[0026] Adsorption methods include but are not limited to physical adsorption and ion adsorption to adsorb cells onto a carrier. The carriers that can be used for adsorption are: diatomaceous earth, wood chips, porous glass, activated carbon, porous ceramics, ion exchange resins and plastics.
[0027] The covalent binding method uses reactive groups on the cell surface (such as amino, carboxyl, hydroxyl, thiol, imidazole, etc.) to react with activated inorganic or organic carriers to form covalent bonds to fix the cells.
[0028] The cross-linking method uses bifunctional or multifunctional reagents to react with reactive groups on the surface of cells (such as amino, carboxyl, hydroxyl, sulfhydryl, imidazole, etc.), thereby fixing the cells.
[0029] The embedding method can use microencapsulation method or gel embedding method to obtain immobilized cells.
[0030] The microencapsulation method, for example, uses a semipermeable polymer film to wrap cells to form microcapsules; the gel encapsulation method is to mix cells with a gel solution and granulate them. In a preferred embodiment of the present invention, the immobilized cells are immobilized cells of living bacteria or dead bacteria.
[0031] In an eighth aspect, the present invention also provides the use of P450 monooxygenase and epoxide hydrolase, recombinant bacteria, recombinant cells, immobilized enzymes or immobilized cells in the biosynthesis of chiral vicinal triol compounds.
[0032] P450 monooxygenase and epoxide hydrolase include but are not limited to purified recombinant P450 monooxygenase and epoxide hydrolase proteins, and crude enzyme solutions containing P450 monooxygenase and epoxide hydrolase.
[0033] The above-mentioned P450 monooxygenase and epoxide hydrolase, recombinant bacteria, recombinant cells, immobilized enzymes or immobilized cells can efficiently, quickly and simply realize the biosynthesis of chiral vicinal triol compounds at room temperature, normal pressure and in an aqueous environment. The entire synthesis process is environmentally friendly and has broad application prospects.
[0034] In a preferred embodiment of the present invention, the substrate for the biosynthesis of chiral vicinal triol compounds is an allylbenzene compound.
[0035] In a ninth aspect, the present invention also provides a method for biosynthesizing chiral vicinal triol compounds, using the above-mentioned P450 monooxygenase and epoxide hydrolase, bacteria, cells, immobilized enzymes or immobilized cells to react with substrates.
[0036] In a preferred embodiment of the present invention, the reaction temperature is 0-50° C.; at the above reaction temperature, the biosynthesis efficiency of chiral vicinal triol compounds is relatively high.
[0037] In a preferred embodiment of the present invention, 10-50 mM of substrate is mixed with 5-25 mL of P450 monooxygenase and epoxide hydrolase, bacteria, cells, immobilized enzyme or immobilized cells.
[0038] For example, 10-20 mM, 10-25 mM, 30-40 mM or 35-50 mM of substrate is mixed with 5-10 mL, 15-25 mL or 20-25 mL of P450 monooxygenase and epoxide hydrolase, bacteria, cells, immobilized enzyme or immobilized cells.
[0039] In a preferred embodiment of the present invention, P450 monooxygenase and epoxide hydrolase, bacteria, cells, immobilized enzyme or immobilized cells are placed in a buffer, mixed with a substrate and reacted on a shaker at 200-250 rpm.
[0040] In a preferred embodiment of the present invention, the reaction product is extracted with an organic solvent, and the chiral vicinal triol compound is obtained by column chromatography. In an optional embodiment, the organic solvent is selected from an organic solvent that is easily compatible with the chiral vicinal triol compound, such as ethyl acetate. The chiral vicinal triol compound is purified by column chromatography.
[0041] In a preferred embodiment of the present invention, the buffer is a sodium phosphate buffer with a concentration of 50-200 mM and a pH of 6-9.
[0042] The beneficial effects of adopting the above technical solution are:
[0043] The invention provides a construction of a cascade catalytic reaction of P450 monooxygenase P450DA-M1, P450PL-M1 and epoxide hydrolase LEH and has the function of biosynthesizing chiral vicinal triol compounds. Compared with the existing preparation method of chiral vicinal triol compounds, the cascade reaction of P450 monooxygenase P450DA-M1, P450PL-M1 and epoxide hydrolase LEH provided by the invention is used to biosynthesize chiral vicinal triol compounds, which has the technical advantages of high reaction rate and short time consumption.
[0044] When synthesizing chiral vicinal triol compounds, the cascade reaction of P450 monooxygenase P450DA-M1, P450PL-M1 and epoxide hydrolase LEH provided by the invention can be carried out at normal temperature, normal pressure and in an aqueous phase environment, and the reaction conditions are mild.
[0045] In addition, the cascade reaction of P450 monooxygenase P450DA-M1, P450PL-M1 and epoxide hydrolase LEH provided by the present invention does not require the use of metal catalysts or toxic reagents when synthesizing chiral vicinal triol compounds. Therefore, the cascade reaction of P450 monooxygenase P450DA-M1, P450PL-M1 and epoxide hydrolase LEH provided by the present invention has high catalytic activity, is environmentally friendly, is easy to operate, and has broad application prospects. DETAILED DESCRIPTION
[0046] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.
[0047] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.
[0048] The features and performance of the present invention are further described in detail below in conjunction with the embodiments. Example 1
[0049] This embodiment provides a method for obtaining P450 monooxygenase P450DA-M1, which specifically comprises the following steps:
[0050] After the genetically engineered bacteria BL21 (DE3) containing the recombinant plasmid stored in glycerol was activated by streaking a plate, a single colony was picked and placed in 5 ml of liquid LB medium containing the corresponding antibiotics, and cultured at 37°C with shaking for 12 h. The next day, the colony was transferred to 100 mL of fresh LB liquid medium containing antibiotics at a 1% inoculum and cultured at 37°C with shaking at 250 rpm until the OD 600 The concentration of IPTG was 0.6 (about 3 hours), and IPTG was added to a final concentration of 0.2 mM, and the culture was induced at 25°C and 160 rpm for 8 hours. After the induction, the cells were collected by centrifugation at 5000 rpm / min for 5 minutes, and the cells were resuspended in PBS buffer to obtain the monooxygenase P450DA-M1 catalyst. Example 2
[0051] This embodiment provides a method for obtaining P450 monooxygenase P450PL-M1, which specifically comprises the following steps:
[0052] After the genetically engineered bacteria BL21 (DE3) containing the recombinant plasmid stored in glycerol was activated by streaking a plate, a single colony was picked and placed in 5 ml of liquid LB medium containing the corresponding antibiotics, and cultured at 37°C with shaking for 12 h. The next day, the colony was transferred to 100 mL of fresh LB liquid medium containing antibiotics at a 1% inoculum and cultured at 37°C with shaking at 250 rpm until the OD 600 The concentration of IPTG was 0.6 (about 3 hours), and IPTG was added to a final concentration of 0.2 mM, and the culture was induced at 25°C and 160 rpm for 8 hours. After the induction, the cells were collected by centrifugation at 5000 rpm / min for 5 minutes, and the cells were resuspended in PBS buffer to obtain the monooxygenase P450PL-M1 catalyst. Example 3
[0053] This embodiment provides a method for obtaining epoxide hydrolase LEH, which specifically comprises the following steps:
[0054] After the genetically engineered bacteria BL21 (DE3) containing the recombinant plasmid stored in glycerol was activated by streaking a plate, a single colony was picked and placed in 5 ml of liquid LB medium containing the corresponding antibiotics, and cultured at 37°C with shaking for 12 h. The next day, the colony was transferred to 100 mL of fresh LB liquid medium containing antibiotics at a 1% inoculum and cultured at 37°C with shaking at 250 rpm until the OD 600 The concentration of IPTG was 0.6 (about 3 hours), and IPTG was added to a final concentration of 0.2 mM, and the culture was induced at 20°C and 160 rpm for 12 hours. After the induction, the cells were collected by centrifugation at 5000 rpm / min for 5 minutes, and the cells were resuspended in PBS buffer to obtain the epoxide hydrolase LEH catalyst. Example 4
[0055] The wet cells of the cultured strains in Example 1, Example 2 and Example 3 were suspended in 5 mL of PBS buffer with a pH of 8.0 at a cell concentration of 20 g / L. The substrate allylbenzene with a concentration of 20 mM was added to the above 5 mL reaction system, and the mixture was placed in a shaker at 20°C and 250 rpm for 24 hours. Ethyl acetate was added for extraction, and 10 bottles of organic phases after extraction were collected, dried over anhydrous sodium sulfate, centrifuged to remove sodium sulfate and filtered. The solvent was evaporated under pressure, and the chiral triol compound was obtained by silica gel column chromatography. Product detection data: yield was 38%, ee value was 98%, (Chiral IG, hexane / i-PrOH=78 / 28, flow rate=0.3 mL / min, λ=210 nm, retention time: t (2R,3S)-8a =19.620min,t (2S,3R)-8a =20.237min,t (2S,3S)-8a =21.097min,t (2R,3R)-8a =22.781min),[α] D 25 =-29.8 (c = 0.85, MeOH). 1 H NMR (400MHz, D2O): δ7.30-7.14(m,5H),4.49(d,J=6.4Hz,1H),3.73-3.64(m,1H),3.33(dd,1H),3.22(dd,1H). 13 C NMR (101MHz, D2O): δ140.3,128.6,128.5,128.1,127.1,126.6,75.3,74.1,62.3.HRMS(ESI-TOF)Calcd.for C9H 12 O3Na[M+Na] + :191.0684; found:191.0690.
[0056] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A P450 monooxygenase and epoxide hydrolase, characterized in that: The amino acid sequence of P450DA-M1 is shown in SEQ ID No. 1; The amino acid sequence of P450PL-M1 is shown in SEQ ID No. 3; The amino acid sequence of LEH is shown in SEQ ID No.
5.
2. An isolated nucleic acid molecule, characterized in that: It encodes the P450 monooxygenase and epoxide hydrolase described in claim 1.
3. A carrier, characterized in that: It comprises the nucleic acid molecule according to claim 2.
4. A recombinant bacterium, characterized in that: It comprises the vector according to claim 3; The recombinant bacteria are bacteria or fungi; The bacteria is Escherichia coli, Streptomyces or Mycobacterium; The recombinant bacteria refers to at least one of resting cells of the recombinant bacteria, living cells of the recombinant bacteria, dead cells of the recombinant bacteria, and cell fragments of the recombinant bacteria.
5. A recombinant cell, characterized in that: It comprises the vector according to claim 3, and the recombinant cell is an insect cell.
6. An immobilized cell, characterized in that: The recombinant bacteria according to claim 4 or the recombinant cells according to claim 5 are fixed; The method of cell fixation via the carrier is selected from at least one of an adsorption method, a covalent binding method, a cross-linking method, and a microencapsulation method; The immobilized cells are live or dead bacteria.
7. Use of the P450 monooxygenase and epoxide hydrolase according to claim 1, the recombinant bacteria according to claim 4, the recombinant cell according to claim 5, and the immobilized enzyme according to claim 6 in the biosynthesis of chiral vicinal triol compounds.
8. The use according to claim 7, characterized in that: The substrate for the biosynthesis of the chiral vicinal triol compound is allylbenzene.
9. A method for biosynthesizing chiral vicinal triol compounds, characterized in that: Using the P450 monooxygenase and epoxide hydrolase of claim 1, the recombinant bacteria of claim 4, the recombinant cell of claim 5, the immobilized enzyme of claim 6 to react with a substrate; The reaction temperature is 0-50°C; Mixing 10-50 mM of the substrate with 5-25 mL of the P450 monooxygenase and epoxide hydrolase, the bacteria, the cells, the immobilized enzyme or the immobilized cells; The P450 monooxygenase and epoxide hydrolase, the recombinant bacteria, the recombinant cells, the immobilized enzyme or the immobilized cells are placed in a buffer, mixed with the substrate, and reacted on a shaker at 200-250 rpm; The reaction product is extracted with an organic solvent, and a chiral vicinal triol compound is obtained by column chromatography analysis; The buffer is a sodium phosphate buffer with a concentration of 50-200 mM and a pH of 6-9.
10. A method for preparing a chiral vicinal triol compound, characterized in that: It uses allylbenzene as substrate, P450DA-M1, P450PL-M1 monooxygenase and epoxide hydrolase LEH as catalysts, and reacts under aqueous conditions. The reaction formula is as follows: in: R is methyl, ethyl, fluorine, chlorine, bromine, n-propyl, isopropyl, n-butyl, phenyl, methylphenyl, methoxyphenyl, bromophenyl, chlorophenyl, or fluorophenyl.