Biocatalysis method for synthesizing chiral vicinal diol
The synthesis of chiral ortho-diol compounds under aqueous phase conditions through the cascade catalytic reaction of P450 monooxygenase and epoxy hydrolase has solved the problems of harsh reaction conditions and poor stereoselectivity in the prior art, and achieved high efficiency and environmental protection biosynthesis process.
Patent Information
- Application Number
- CN202411386645.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-30
- Publication Date
- 2024-12-13
AI Technical Summary
The existing chiral o-diol compound synthesis technology has problems such as the use of transition metal catalysis, many by-products, poor stereoselectivity, and harsh reaction conditions. There is a lack of synthesizer enzymes for chiral o-diol compounds with high activity and high regio-selectivity. , it is difficult to achieve efficient biocatalytic preparation.
Using the cascaded catalytic reaction of P450 monooxygenase (P450DA-M1) and epoxy hydrolase (SpEH) is constructed through genetic engineering and catalyzed under aqueous phase conditions. Allylbenzene is used as substrate to achieve chiral orthogonal The biosynthesis of diol compounds avoids the use of transition metals and toxic agents.
It realizes the efficient synthesis of chiral ortho-diol compounds in room temperature, normal pressure and aqueous environment. It has high reaction rate, short time consumption, high catalytic activity and environmentally friendly advantages, and is easy to operate.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of synthesis of chiral vicinal diol compounds, and in particular to the construction of a cascade catalytic reaction of a P450 monooxygenase (named P450DA-M1) and an epoxide hydrolase (named SpEH) and its application in the biosynthesis of chiral vicinal diol compounds. Background Art
[0002] Chiral vicinal diol compounds are a very important class of compounds that can be used as drug intermediates and catalyst intermediates and are widely used in organic synthesis and drug synthesis. The existing synthesis technology of chiral vicinal diol compounds still has shortcomings such as the use of transition metal catalysis, many by-products, poor stereoselectivity, and harsh reaction conditions. The high efficiency and high selectivity of bioenzymes make biocatalysis technology expected to develop into a new way for the industrial green preparation of chiral vicinal diol compounds. However, existing studies have shown that chiral vicinal diol compound synthases with high activity and high regioselectivity 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 diol compounds. Summary of the invention
[0003] The purpose of the present invention is to provide a construction of a cascade catalytic reaction of a P450 monooxygenase (named P450DA-M1) and an epoxide hydrolase (named SpEH) and its application in the biosynthesis of chiral vicinal diol compounds to solve the above technical problems.
[0004] The technical solution of the present invention:
[0005] A method for preparing a chiral vicinal diol compound, characterized in that allylbenzene is used as a substrate, P450DA-M1 monooxygenase and epoxide hydrolase SpEH are used as catalysts, a double enzyme cascade reaction is constructed, and the reaction is carried out under aqueous conditions, and the reaction formula is as follows:
[0006] in:
[0007] R is methyl, ethyl, n-propyl, isopropyl, n-butyl, phenyl, methylphenyl, methoxyphenyl, bromophenyl, chlorophenyl, fluorophenyl, 2-furyl, or 2-thienyl.
[0008] The present invention is achieved in that:
[0009] In a 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 an epoxide hydrolase SpEH, whose amino acid sequence is shown in SEQ ID No.3.
[0010] The P450 monooxygenase P450DA-M1 and epoxide hydrolase SpEH genes of the present invention are constructed into genetically engineered bacteria, and induced to express to obtain the P450 monooxygenase and epoxide hydrolase. The function of the P450 monooxygenase and hydrolase cascade reaction to synthesize chiral vicinal diol compounds is found. Compared with the existing preparation method of chiral vicinal diol compounds, the use of the dual enzyme cascade reaction provided by the present invention to biosynthesize chiral vicinal diol compounds has the technical advantages of high reaction rate, no need for intermediate separation and short time consumption; when synthesizing chiral vicinal diol compounds, the monooxygenase P450DA-M1 and epoxide hydrolase SpEH dual enzyme cascade provided by the present invention can be carried out at room temperature, normal pressure and aqueous environment, and the reaction conditions are mild; in addition, when synthesizing chiral vicinal diol compounds, the monooxygenase P450DA-M1 and epoxide hydrolase SpEH dual enzyme cascade provided by the present invention does not need to use transition metals or toxic reagents, therefore, the monooxygenase P450DA-M1 and epoxide hydrolase SpEH dual enzyme cascade provided by the present invention has high catalytic activity, is environmentally friendly, and is easy to operate.
[0011] The above monooxygenases contain a heme unit.
[0012] The amino acid sequence of monooxygenase P450DA-M1 shown in SEQ ID No. 1 is as follows:
[0013] 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:
[0014] In the third aspect, the amino acid sequence of the epoxide hydrolase SpEH shown in SEQ ID No.3 is as follows:
[0015] In a fourth aspect, the present invention further provides an isolated nucleic acid molecule encoding the above-mentioned epoxide hydrolase SpEH, and the isolated nucleic acid molecule has the nucleotide sequence shown in SEQ ID No. 4 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 are bacteria or fungi;
[0020] In a preferred embodiment of the present invention, the bacteria are Escherichia coli, Streptomyces or Mycobacterium.
[0021] Fungi such as yeast.
[0022] 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.
[0023] The above-mentioned recombinant bacteria (or genetically engineered bacteria) are prepared by transforming a recombinant expression vector into a host microorganism by a conventional method 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 can be effectively expressed; preferably, it can be prepared by the following method: transforming the recombinant vectors pET-28a(+)-P450 and pET-22a(+)-SpEH into Escherichia coli BL21(DE3).
[0024] 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.
[0025] 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.
[0026] 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.
[0027] 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.
[0028] 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.
[0029] 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.
[0030] The embedding method can be used to obtain fixed cells by microencapsulation or gel embedding. The microencapsulation method uses a semipermeable polymer film to encapsulate the cells to form microcapsules; the gel embedding method is to mix the cells with a gel solution and granulate them.
[0031] In a preferred embodiment of the present invention, the immobilized cells are immobilized cells of living bacteria or dead bacteria.
[0032] 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 diol compounds.
[0033] The P450 monooxygenase includes, but is not limited to, purified recombinant P450 monooxygenase protein and crude enzyme solution containing P450 monooxygenase.
[0034] 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 diol compounds at room temperature, normal pressure and in an aqueous environment. The entire synthesis process is environmentally friendly and has broad application prospects.
[0035] In a preferred embodiment of the present invention, the substrate for the biosynthesis of chiral vicinal diol compounds is an allylbenzene compound.
[0036] In a ninth aspect, the present invention also provides a method for biosynthesizing chiral vicinal diol compounds, using the above-mentioned P450 monooxygenase and epoxide hydrolase, bacteria, cells, immobilized enzymes or immobilized cells to react with substrates.
[0037] In a preferred embodiment of the present invention, the reaction temperature is 5-50° C.; at the above reaction temperature, the biosynthesis efficiency of chiral vicinal diol compounds is relatively high.
[0038] 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.
[0039] 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.
[0040] 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.
[0041] In a preferred embodiment of the present invention, the reaction product is extracted with an organic solvent, and the chiral vicinal diol compound is obtained by column chromatography. In an optional embodiment, the organic solvent is selected from organic solvents that are easily compatible with the chiral vicinal diol compound, such as esters. Esters are such as ethyl acetate. Column chromatography is performed to obtain the purified chiral vicinal diol compound.
[0042] 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.
[0043] The beneficial effects of adopting the above technical solution are:
[0044] The present invention provides a construction of a cascade catalytic reaction of a P450 monooxygenase (named P450DA-M1) and an epoxide hydrolase (named SpEH) and has the function of biosynthesizing chiral vicinal diol compounds. Compared with the existing preparation method of chiral vicinal diol compounds, the use of the cascade reaction of the P450 monooxygenase and the epoxide hydrolase provided by the present invention to biosynthesize chiral vicinal diol compounds has the technical advantages of high reaction rate and short time consumption.
[0045] When synthesizing chiral vicinal diol compounds, the cascade reaction of P450 monooxygenase and epoxide hydrolase 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.
[0046] In addition, the P450 monooxygenase and epoxide hydrolase cascade reaction provided by the present invention does not require the use of metal catalysts or toxic reagents when synthesizing chiral vicinal diol compounds. Therefore, the P450 monooxygenase and epoxide hydrolase cascade reaction provided by the present invention has high catalytic activity, is environmentally friendly, is easy to operate, and has broad application prospects. DETAILED DESCRIPTION
[0047] 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.
[0048] 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.
[0049] The features and performance of the present invention are further described in detail below in conjunction with the embodiments. Example 1
[0050] This embodiment provides a method for obtaining P450 monooxygenase P450DA-M1, which specifically comprises the following steps:
[0051] After the genetically engineered bacteria BL21 (DE3) containing the recombinant plasmid P450DA-M1 stored in glycerol were 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
[0052] This embodiment provides a method for obtaining epoxide hydrolase SpEH, which specifically comprises the following steps:
[0053] After the genetically engineered bacteria BL21 (DE3) containing the recombinant plasmid SpEH stored in glycerol were 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 for 12 h with shaking. 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 at 250 rpm with shaking until the OD 600The 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 SpEH catalyst. Example 3
[0054] The wet cells of the strains cultured in Example 1 and Example 2 were suspended in 5 mL of PBS buffer with a pH of 8.0 at a cell concentration of 20 g / L. A 10 mM concentration of substrate allylbenzene 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 then added for extraction. 20 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 vicinal diol compound was obtained by silica gel column chromatography. Product detection data: yield was 39%, ee value was 90%, (Chiral AS-H, hexane / i-PrOH = 90 / 10, flow rate = 1.0 mL / min, λ = 210 nm, retention time: t (R)-6a =12.201min,t (S)-6a =14.797min),[α] D 25 =47.8(c=0.525,CH 2 Cl 2 ). 1 H NMR (400 MHz, DMSO-D 6 ): δ7.30-7.09(m,5H),4.54(t,J=6.0Hz,2H),3.64-3.53(m,1H),3.29-3.18(m,2H),2.73(dd,J=13.6,4.8Hz,1H),2.51(d,J=7.9Hz,1H). 13 C NMR (100 MHz, DMSO-D 6 ):δ140.1,129.8,128.4,126.1,72.9,65.7,39.9.HRMS(ES I-TOF)Cal cd.for C 9 H 12 O 2 Na[M+Na] + :175.0735; found:175.0741.
[0055] 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 P450 monooxygenase is shown in SEQ ID No.1; the amino acid sequence of epoxide hydrolase is shown in SEQ ID No.
3.
2. An isolated nucleic acid molecule, characterized in that It encodes the P450 monooxygenase P450DA-M1 and epoxide hydrolase SpEH 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 diol compounds.
8. The use according to claim 7, characterized in that: The substrate for the biosynthesis of the chiral vicinal diol compound is an allylbenzene molecule.
9. A method for biosynthesizing chiral vicinal diol 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 5-50°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 diol 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 diol compound, characterized in that: Allylbenzene was used as substrate, P450DA-M1 monooxygenase and epoxide hydrolase SpEH were used as catalysts, and a dual enzyme cascade reaction system was constructed to react under aqueous conditions. The reaction formula is as follows: in: R is fluorine, chlorine, bromine, methyl, ethyl, n-propyl, isopropyl, n-butyl, phenyl, methylphenyl, methoxyphenyl, bromophenyl, chlorophenyl, fluorophenyl, 2-furyl, or 2-thienyl.