P450 hydroxylase mutant and axially chiral allene alcohol synthesized by P450 hydroxylase mutant

Abstract

The invention discloses a P450 hydroxylase mutant (named as P450AHS) and application of the P450 hydroxylase mutant in biosynthesis of axially chiral allenol compounds, and relates to the technical field of synthesis of the axially chiral allenol compounds. The P450 hydroxylase mutant has a function of biologically catalyzing allene carbon-hydrogen bond hydroxylation to synthesize an axially chiral allene alcohol compound, has high catalytic activity, is environment-friendly, is simple and convenient to operate, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a P450 hydroxylase mutant and its synthetic axial chiral allenol. Background Technology

[0002] Cytochrome P450 hydroxylases (CYPs) are a superfamily of heme proteins whose reduced forms exhibit a characteristic absorption peak at 450 nm when bound to CO. As one of the largest protein superfamilies, genes encoding P450 hydroxylases have been discovered in animals, plants, fungi, bacteria, and protozoa. P450 hydroxylases can exert biocatalytic effects on substrates of various structural types under mild conditions, including hydroxylation, epoxidation, and dealkylation reactions. Currently, with the development of technologies such as directed evolution, an increasing number of P450 hydroxylases are being used as important biocatalysts in fields such as drug synthesis.

[0003] Axially chiral allenols are an important class of organic molecules with unique axial chiral structures. In these molecules, the substitutional genes at both ends of the allene double bond are sterically hindered and cannot rotate freely, forming a stable chiral axis. These compounds play an irreplaceable role in the synthesis of natural products (such as terpenes and alkaloids), drug development (such as intermediates for antiviral drugs), and asymmetric catalysis (such as chiral ligands). For example, axially chiral allenols can be stereoselectively transformed to construct complex molecules with multiple chiral centers, making them a key precursor in the synthesis of the anti-HIV drug deraviridine.

[0004] Currently, the synthesis methods for axially chiral allenols are mainly divided into two categories: chemical catalysis and biocatalysis. However, both methods suffer from drawbacks such as low yield, poor stereoselectivity, numerous byproducts, and harsh reaction conditions. For example, existing technology CN117568296A discloses a P450 hydroxylase mutant for the synthesis of R-haloalcohols. This mutant can be applied to catalyze the asymmetric hydroxylation of haloalkanes to synthesize R-configuration chiral haloalcohols. CN117568295A discloses the application of a class of P450 hydroxylase mutants in the synthesis of R-haloallyl alcohols. CN117568294A discloses a class of P450 hydroxylase mutants and their application in the synthesis of S-haloalcohols. However, there are no reports on the use of P450 hydroxylase mutants in the synthesis of axially chiral allenols. Therefore, obtaining highly efficient biocatalysts and corresponding biocatalytic synthesis methods remains a challenge and bottleneck in the development of biocatalytic preparation of axially chiral allenols. Summary of the Invention

[0005] The present invention aims to provide a P450 hydroxylase mutant and its synthesis of axial chiral allenols. The present invention solves the technical problems of "low efficiency, poor selectivity and narrow substrate range" in the synthesis of axial chiral allenols through a novel enzyme mutation design.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] The technical solution of this invention:

[0008] A P450 hydroxylase mutant, named P450AHS hydroxylase, has the amino acid sequence shown in SEQ ID No. 1.

[0009] A method for preparing axially chiral allenols, using alkyl and aryl substituted allenes as substrates and P450AHS hydroxylase as catalyst, involves a reaction under aqueous conditions, as shown in reaction formula I below:

[0010]

[0011] Wherein: Substituent R in reaction formula I 1 R 2 ,R 3 The compounds are methyl, ethyl, n-propyl, isopropyl, n-butyl, phenyl, 4-chlorophenyl, 4-fluorophenyl, 4-bromophenyl, 4-trifluoromethylphenyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 3-methoxyphenyl, 4-ethylphenyl, 4-tert-butylphenyl, 4-n-butoxyphenyl, 3,4-dimethoxyphenyl, 4-methoxyphenyl, 3,5-dimethoxyphenyl, 3,4,5-trimethoxyphenyl, 3-chloro-4-methoxyphenyl, 3-bromophenyl, 4-iodophenyl, 3-bromo-4-methoxyphenyl, 2-naphthyl, 4-cyanophenyl, and 2-furanyl.

[0012] This invention is implemented as follows:

[0013] In a first aspect, the present invention provides a P450 hydroxylase mutant, the amino acid sequence of which is shown in SEQ ID No. 1.

[0014] This invention relates to a P450AHS hydroxylase obtained through genetic modification using P450pyr enzyme as a template. The nucleotide sequence of P450AHS is shown in SEQ ID No. 2. This hydroxylase was constructed into genetically engineered bacteria and, after induction expression, the P450 hydroxylase P450AHS was obtained. Functional studies of this P450AHS hydroxylase revealed that it possesses the function of biosynthesizing axially chiral allenol compounds. Compared to existing methods for preparing axially chiral allenols, the biosynthesis of axially chiral allenols using the P450AHS hydroxylase provided in this invention has the technical advantages of high reaction rate and short reaction time (30 minutes). The P450AHS hydroxylase provided in this invention can be used to synthesize axially chiral allenols at room temperature, under normal pressure, and in an aqueous environment, with mild reaction conditions. Furthermore, the P450AHS hydroxylase provided in this invention does not require the use of transition metals, complex ligands, strong oxidants, or toxic reagents during the synthesis of axially chiral allenols. Therefore, the P450AHS hydroxylase provided in this invention has high catalytic activity, is environmentally friendly, and is easy to operate.

[0015] The aforementioned P450AHS hydroxylase contains heme units.

[0016] The amino acid sequence shown in SEQ ID No. 1 is as follows:

[0017] Mehtgqsaaatmpldsidvsipelfyndsvgeyfkrlrkddpvhycadsafgpywsitkyndimhvdtnhdi

[0018] fssdagyggfiiddgiqkggdggldlpnfiamdrprhdeqrkavspivapanlaalegtirervsktldglpvgeefdw

[0019] vdrvsieittqmlatlfdfpfeerrkltrwsdvttaapgggvveswdqrktellecaayfqvlwnervnkdpgndlisml

[0020] ahspatrnmtpeeylgnvlllivggndttrnsmtggvlalhknpdqfaklkanpalvetmvpeiirwqtplahmrrtai

[0021] adselggktirkgdkvvmwyysgnrddevidrpeefiidrprprqhlsfgfgihrcvgnrlaemqlrilweeiltrfsrie

[0022] vmaepervrsnfvrgyakmmvrvha.

[0023] Secondly, the present invention also provides an isolated nucleic acid molecule encoding the aforementioned P450 hydroxylase. The nucleotide sequence of the isolated nucleic acid molecule is shown in SEQ ID No. 2.

[0024] With the amino acid sequence of the P450AHS hydroxylase provided in this invention, those skilled in the art can easily obtain the nucleic acid sequence encoding the P450AHS hydroxylase based on the degeneracy of the codons, which is easily achieved by those skilled in the art.

[0025] For example, the isolated nucleic acid molecule has the nucleotide sequence shown in SEQ ID No. 2:

[0026]

[0027] Thirdly, the present invention also provides a carrier comprising the aforementioned nucleic acid molecules.

[0028] Vectors include, but are not limited to, bacterial expression vectors and fungal expression vectors; any vector capable of expressing or facilitating the transport of the aforementioned nucleic acid molecules is acceptable. This includes, but is not limited to, plasmids. Examples of bacterial expression vectors include E. coli expression vectors, Streptomyces expression vectors, and Mycobacterium expression vectors. Examples of fungal expression vectors include yeast vectors.

[0029] Fourthly, the present invention also provides a recombinant bacterium comprising the aforementioned vector.

[0030] In a preferred embodiment of the present invention, the recombinant bacteria are bacteria or fungi;

[0031] In a preferred embodiment of the present invention, the bacteria are Escherichia coli, Streptomyces or Mycobacterium.

[0032] Fungi, such as yeast.

[0033] In a preferred embodiment of the present invention, recombinant bacteria refers to at least one of resting cells of bacteria, live cells of recombinant bacteria, dead bacteria of recombinant bacteria, and cell fragments of recombinant bacteria.

[0034] The aforementioned recombinant bacteria (or genetically engineered bacteria) are prepared by transforming the recombinant expression vector into a host microorganism using conventional methods in the art. The host microorganism can be any conventional host microorganism in the art, as long as it satisfies the requirement that the recombinant expression vector can stably replicate on its own and that the P450AHS hydroxylase gene it carries can be effectively expressed. Preferably, it can be prepared by transforming the recombinant vector pET-28a(+)-P450 into Escherichia coli BL21(DE3).

[0035] Dead bacteria in recombinant bacteria include, but are not limited to, bacteria obtained after inactivation by means of heat, pressure, radiation, etc.

[0036] Cell debris refers to cell fragments obtained by altering cell membrane permeability through methods such as ultrasound, mechanical, chemical, and biological means, resulting in the leakage of cell contents.

[0037] Fifthly, the present invention also provides an immobilized enzyme, which is prepared by immobilizing the above-mentioned P450AHS hydroxylase using a carrier or a carrier-free method.

[0038] In a preferred embodiment of the present invention, the method of immobilization by carrier is selected from at least one of adsorption, cross-linking and embedding methods.

[0039] Adsorption methods include, but are not limited to, physical adsorption and ion adsorption to adsorb enzymes onto a carrier. Suitable adsorption carriers include: activated carbon, porous ceramics, diatomaceous earth, sawdust, porous glass, ion exchange resins, and plastics.

[0040] Cross-linking involves using bifunctional or multifunctional reagents to react with the reactive groups (such as amino, carboxyl, hydroxyl, thiol, imidazole, etc.) on the enzyme surface, thereby immobilizing the enzyme.

[0041] Immobilized enzymes can be obtained using microencapsulation or gel encapsulation methods. Microencapsulation involves encapsulating the enzyme in a semi-permeable polymer membrane to form microcapsules; gel encapsulation involves mixing the enzyme with a gel solution and then granulating it.

[0042] Sixthly, the present invention also provides an immobilized cell, which is prepared by immobilizing the above-mentioned bacteria or the above-mentioned cells.

[0043] In a preferred embodiment of the present invention, the method of cell immobilization via a carrier is selected from at least one of adsorption, covalent bonding, cross-linking, and microencapsulation.

[0044] Adsorption methods include, but are not limited to, physical adsorption and ion adsorption to adsorb cells onto a carrier. Suitable adsorption carriers include: activated carbon, porous ceramics, diatomaceous earth, wood chips, porous glass, ion exchange resins, and plastics.

[0045] Covalent bonding is a method that 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 and fix the cells.

[0046] Cross-linking involves using bifunctional or multifunctional reagents to react with reactive groups (such as amino, carboxyl, hydroxyl, thiol, imidazole, etc.) on the cell surface, thereby fixing the cells.

[0047] Immobilized cells can be obtained using microencapsulation or gel encapsulation methods. Microencapsulation involves encapsulating cells in a semi-permeable polymer membrane to form microcapsules; gel encapsulation involves mixing cells with a gel solution and then granulating the mixture.

[0048] In a preferred embodiment of the present invention, the immobilized cells are immobilized cells of live or dead bacteria.

[0049] In a seventh aspect, the present invention also provides the application of P450AHS hydroxylase, recombinant bacteria, recombinant cells, immobilized enzymes or immobilized cells in the biosynthesis of axially chiral allenol compounds.

[0050] P450 hydroxylase includes, but is not limited to, purified recombinant P450AHS hydroxylase protein and crude enzyme solution containing P450AHS hydroxylase.

[0051] Using the aforementioned P450AHS hydroxylase, recombinant bacteria, recombinant cells, immobilized enzymes, or immobilized cells, axially chiral allenol compounds can be biosynthesized efficiently, rapidly, and conveniently in ambient temperature, ambient pressure, and aqueous environments. The entire synthesis process is environmentally friendly and has broad application prospects.

[0052] In a preferred embodiment of the present invention, the substrate for the biosynthesis of axially chiral allenol compounds is an alkyl-substituted olefin.

[0053] Eighthly, the present invention also provides a method for biosynthesizing axially chiral allenol compounds, wherein the above-mentioned P450 hydroxylase, bacteria, cells, immobilized enzymes or immobilized cells are reacted with a substrate.

[0054] In a preferred embodiment of the present invention, the reaction temperature is 0-50°C; at the above reaction temperature, the biosynthesis efficiency of axially chiral allenol compounds is high.

[0055] In a preferred embodiment of the present invention, each 1-50 mM substrate is mixed with 1-25 mL of P450AHS hydroxylase, bacteria, cells, immobilized enzymes, or immobilized cells.

[0056] For example, each 1-20mM, 1-25mM, 30-40mM or 35-50mM substrate is mixed with 1-10mL, 5-25mL or 20-25mL of P450AHS hydroxylase, bacteria, cells, immobilized enzyme or immobilized cells.

[0057] In a preferred embodiment of the present invention, P450AHS hydroxylase, bacteria, cells, immobilized enzymes or immobilized cells are placed in a buffer solution, mixed with the substrate, and reacted on a shaker at 200-300 rpm.

[0058] In a preferred embodiment of the invention, the reaction product is extracted with an organic solvent, and the axially chiral allenol compound is obtained by column chromatography. In an optional embodiment, the organic solvent is selected from organic solvents readily compatible with the axially chiral allenol compound, such as esters, ethers, and alcohols. Esters include, for example, ethyl acetate. The purified axially chiral allenol compound is obtained by column chromatography.

[0059] In a preferred embodiment of the present invention, the buffer solution is a citric acid solution with a concentration of 50-200 mM and a pH of 6-9.

[0060] This invention provides a P450AHS hydroxylase capable of biosynthesizing axially chiral allenols. Compared to existing methods for preparing axially chiral allenols, using the P450AHS hydroxylase provided by this invention for the biosynthesis of axially chiral allenols offers advantages such as high reaction rate and short reaction time.

[0061] The P450AHS hydroxylase provided by this invention can be used to synthesize axially chiral allenols in a mild environment under normal temperature, normal pressure and aqueous phase conditions.

[0062] Furthermore, the P450AHS hydroxylase provided by this invention does not require the use of transition metals, complex ligands, strong oxidants, or toxic reagents when synthesizing axially chiral allenols. Therefore, the P450AHS hydroxylase provided by this invention has high catalytic activity, is environmentally friendly, easy to operate, and has broad application prospects. Attached Figure Description

[0063] Appendix Figure 1 The 1H and 1C NMR spectra of compound 2a are shown.

[0064] Appendix Figure 2 The NMR spectra of compound 2b are: 1H NMR and 1C NMR.

[0065] Appendix Figure 3 The NMR spectra of compound 2c are: 1H NMR and 1C NMR.

[0066] Appendix Figure 4 The images show the 1H and 1C NMR spectra of compound 2d.

[0067] Appendix Figure 5 The NMR spectra of compound 2e are the 1H and 1C NMR spectra.

[0068] Appendix Figure 6 The 1H and 1C NMR spectra of compound 2f are shown.

[0069] Appendix Figure 7 The 1H and 1C NMR spectra of compound 2g are shown.

[0070] Appendix Figure 8 The NMR spectra of compound 2h are 1H and 1C. Detailed Implementation

[0071] The following detailed description illustrates the specific implementation method:

[0072] Example 1

[0073] This embodiment provides a method for obtaining P450AHS hydroxylase, specifically including the following steps:

[0074] After activating the genetically engineered bacteria BL21(DE3) containing recombinant plasmids preserved in glycerol by streak plating, single colonies were picked and cultured in 5 ml of liquid LB medium containing the corresponding antibiotic at 37°C with shaking for 12-16 h. The next day, the colonies were transferred at a 1% inoculum to 100 mL of fresh TB liquid medium containing antibiotics and cultured at 37°C with shaking at 250 rpm until the OD600 reached 0.6 (2-3 h). IPTG was then added to a final concentration of 0.1 mM, and the culture was induced at 25°C with shaking at 200 rpm for 8-10 h. After induction, the bacterial cells were collected by centrifugation at 6000 rpm / min for 5 min and resuspended in citrate buffer.

[0075] Example 2

[0076]

[0077] The wet cells of the strain cultured in Example 1 were suspended at a cell concentration of 10 g / L in 5 mL of citrate buffer at pH 6.0. 4 mM of the substrate allylbenzene was added to the 5 mL reaction system. The mixture was reacted on a shaker at 30°C and 250 rpm for 0.5 h. Ethyl acetate was then added for extraction. Twenty flasks of the extracted organic phase were collected, dried with anhydrous sodium sulfate, centrifuged to remove sodium sulfate, and filtered. The solvent was removed by pressure evaporation, and the mixture was separated by silica gel column chromatography to obtain the axially chiral allenol compound. Product detection data: yield 53%, ee value 98% (Chiralcel OD-H, Hexane / i-PrOH = 90 / 10, flow rate 1.0 mL / min, λ = 254 nm, t(S) = 6.3 min (major), t(R) = 8.0 min (minor)). The specific rotation corresponds to the reported literature to determine the configuration,mp:79.6-81.5℃.1H NMR (400MHz, CDCl3) δ7.35-7.25(m,4H),7.25-7.17(m,1H),6.28(q,J=2.9Hz,1H),4.16(qd,J=13.1,3.0Hz,2H),1.86(d,J=2.9Hz,3H).13C NMR(100MHz, CDCl3)δ200.9,134.8,128.7,127.2,126.9,104.8,97.2,64.0,15.6.HRMS(ESI)m / z:calculated for C11H13O[M+H]+:161.0966,found:161.0972.

[0078] Example 3

[0079]

[0080] The wet cells of the strain cultured in Example 1 were suspended at a cell concentration of 10 g / L in 5 mL of citrate buffer at pH 6.0. 4 mM of the substrate allylbenzene was added to the 5 mL reaction system. The mixture was reacted on a shaker at 30°C and 250 rpm for 0.5 h. Ethyl acetate was then added for extraction. Twenty flasks of the extracted organic phase were collected, dried with anhydrous sodium sulfate, centrifuged to remove sodium sulfate, and filtered. The solvent was removed by pressure evaporation, and the mixture was separated by silica gel column chromatography to obtain the axially chiral allenol compound. Product detection data: yield 44%, ee value 98% (Chiralcel OD-H, Hexane / i-PrOH = 90 / 10, flow rate 1.0 mL / min, λ = 254 nm, t(S) = 5.6 min (major), t(R) = 6.1 min (minor)). 1HNMR(400MHz, CDCl3)δ7.29-7.19(m,1H),7.08-7.00(m,1H),7.03-6.95(m,1H),6.93-6.8 4(m,1H),6.21(q,J=2.9Hz,1H),4.25-4.00(m,2H),2.09(s,1H),1.84(d,J=2.9Hz,3H).13C NMR (100MHz, CDCl3) δ201.5, 163.2 (d, J = 243.8Hz), 137.5 (d, J = 7.6Hz), 130.1 (d, J = 8.4Hz), 122.6 (d, J = 2.7Hz),113.9(d,J=21.5Hz),113.3(d,J=22.0Hz),105.2,96.1,63.8,15.4.HRMS(ESI)m / z:calculated forC11H12FO[M+H]+:179.0872,found:179.0870.

[0081] Example 4

[0082]

[0083] The wet cells of the strain cultured in Example 1 were suspended at a cell concentration of 10 g / L in 5 mL of citrate buffer at pH 6.0. 4 mM of the substrate allylbenzene was added to the 5 mL reaction system. The mixture was reacted on a shaker at 30°C and 250 rpm for 0.5 h. Ethyl acetate was added for extraction, and 20 flasks of the extracted organic phase were collected. The phase was dried over anhydrous sodium sulfate, centrifuged to remove sodium sulfate, and filtered. The solvent was removed by pressure evaporation, and the mixture was separated by silica gel column chromatography to obtain the axially chiral allenol compound. Product detection data: yield 46%, ee value 99% (Chiralcel AD-H, Hexane / i-PrOH = 90 / 10, flow rate 1.0 mL / min, λ = 254 nm, t(S) = 6.0 min (major), t(R) = 6.4 min (minor)). mp:56.2-57.1℃.1H NMR(400MHz, CDCl3)δ7.26-7.20(m,2H),7.03-6.92(m,2H),6.23(q,J=3.0Hz,1H),4.19-4.08(m,2H),1.83(d,J=2.9Hz,3H).13C NMR (100MHz, CDCl3) δ200.6 (d, J = 2.4Hz), 162.1 (d, J = 244.7Hz), 130.8 (d, J = 3.3Hz), 128. 3(d,J=8.0Hz),115.7(d,J=21.6Hz),105.2,96.3,63.9,15.6.HRMS(ESI)m / z:calculated for C11H12FO[M+H]+:179.0872,found:179.0872.

[0084] Example 5

[0085]

[0086] The wet cells of the strain cultured in Example 1 were suspended at a cell concentration of 10 g / L in 5 mL of citrate buffer (pH 6.0). 4 mM of the substrate allylbenzene was added to the 5 mL reaction system. The mixture was reacted on a shaker at 30°C and 250 rpm for 0.5 h. Ethyl acetate was then added for extraction. Twenty flasks of the extracted organic phase were collected, dried with anhydrous sodium sulfate, centrifuged to remove sodium sulfate, and filtered. The solvent was removed by pressure evaporation, and the mixture was separated by silica gel column chromatography to obtain the axially chiral allenol compound. Product detection data: yield 24%, ee value 98% (Chiralcel OD-H, Hexane / i-PrOH = 90 / 10, flow rate 1.0 mL / min, λ = 254 nm, t(S) = 5.8 min (major), t(R) = 6.7 min (minor)). 1HNMR (400MHz, CDCl3) δ7.33-7.07(m,4H),6.24-6.13(m,1H),4.22-4.08(m,2H),1.94(s,1H),1.85(d,J=2.9Hz,3H).13C NMR(100MHz, CDCl3)δ201.4,137.0,134.6,129.9,127.1,126.6,125.1,105.4,96.0,63.8,15.4.HRMS(ESI)m / z:calculated for C11H11ClNaO[M+Na]+:217.0396,found:217.0394.

[0087] Example 6

[0088]

[0089] The wet cells of the strain cultured in Example 1 were suspended at a cell concentration of 10 g / L in 5 mL of citrate buffer (pH 6.0). 4 mM of the substrate allylbenzene was added to the 5 mL reaction system. The mixture was reacted on a shaker at 30°C and 250 rpm for 0.5 h. Ethyl acetate was then added for extraction. Twenty flasks of the extracted organic phase were collected, dried with anhydrous sodium sulfate, centrifuged to remove sodium sulfate, and filtered. The solvent was removed by pressure evaporation, and the mixture was separated by silica gel column chromatography to obtain the axially chiral allenol compound. Product detection data: yield 35%, ee value 99% (Chiralcel AD-H, Hexane / i-PrOH = 95 / 5, flow rate 1.0 mL / min, λ = 254 nm, t(S) = 9.4 min (major), t(R) = 10.3 min (minor)). mp:65.3-66.2℃.1H NMR(400MHz, CDCl3)δ7.29-7.22(m,2H),7.24-7.17(m,2H),6.27-6.18(m,1H),4.21-4.09(m,2H),1.85(d,J=2.9Hz,3H).13C NMR(100MHz, CDCl3)δ201.1,133.4,132.7,128.9,128.1,105.3,96.3,63.9,15.5.HRMS(ESI)m / z:calculated for C11H12ClO[M+H]+:195.0577,found:195.0571.

[0090] Example 7

[0091]

[0092] The wet cells of the strain cultured in Example 1 were suspended at a cell concentration of 10 g / L in 5 mL of citrate buffer at pH 6.0. 4 mM of the substrate allylbenzene was added to the 5 mL reaction system. The mixture was reacted on a shaker at 30°C and 250 rpm for 0.5 h. Ethyl acetate was then added for extraction. Twenty flasks of the extracted organic phase were collected, dried with anhydrous sodium sulfate, centrifuged to remove sodium sulfate, and filtered. The solvent was removed by pressure evaporation, and the mixture was separated by silica gel column chromatography to obtain the axially chiral allenol compound. Product detection data: yield 20%, ee value 96% (Chiralcel OD-H, Hexane / i-PrOH = 90 / 10, flow rate 1.0 mL / min, λ = 254 nm, t(S) = 11.4 min (major), t(R) = 12.9 min (minor)). mp:82.5-83.5℃.1H NMR (400MHz, CDCl3) δ7.63-7.50(m,2H),7.41-7.30(m,2H),6.25(s,1H),4.26-4.10(m,2H),1.86(s,3H),1.78(s,1H).1 3CNMR(100MHz,CDCl3)δ202.8,140.3,132.5,127.3,119.2,110.1,105.9,96.1,63.7,15.3.HRMS(ESI)m / z:calculated for C12H12NO[M+H]+:186.0919,found:186.0920.

[0093] Example 8

[0094]

[0095]

[0096] The wet cells of the strain cultured in Example 1 were suspended at a cell concentration of 10 g / L in 5 mL of citrate buffer at pH 6.0. 4 mM of the substrate allylbenzene was added to the 5 mL reaction system. The mixture was reacted on a shaker at 30°C and 250 rpm for 0.5 h. Ethyl acetate was then added for extraction. Twenty flasks of the extracted organic phase were collected, dried with anhydrous sodium sulfate, centrifuged to remove sodium sulfate, and filtered. The solvent was removed by pressure evaporation, and the mixture was separated by silica gel column chromatography to obtain the axially chiral allenol compound. Product detection data: yield 25%, ee value 94% (Chiralcel OD-H, Hexane / i-PrOH = 90 / 10, flow rate 1.0 mL / min, λ = 254 nm, t(S) = 5.3 min (major), t(R) = 5.4 min (minor)). mp:42.3-42.8℃.1H NMR (400MHz, CDCl3) δ7.56-7.51(m,2H),7.39-7.34(m,2H),6.31-6.23(m,1H),4.22-4.11(m,2H),1.95(s,1H),1.86(s,3H).13C NMR(100MHz, CDCl3)δ202.2,138.9,128.9(q,J=32.0Hz),127.0,125.6(q,J=4.0Hz),124.3(q,J=270Hz),105.5,96.0,63.8,15.3.HRMS(ESI)m / z:calculated forC12H12F3O[M+H]+:229.0840,found:229.0844.

[0097] Example 9

[0098]

[0099] The wet cells of the strain cultured in Example 1 were suspended at a cell concentration of 10 g / L in 5 mL of citrate buffer at pH 6.0. 4 mM of the substrate allylbenzene was added to the 5 mL reaction system, and the mixture was reacted on a shaker at 30°C and 250 rpm for 0.5 h. Ethyl acetate was then added for extraction, and 20 flasks of the extracted organic phase were collected. The phase was dried over anhydrous sodium sulfate, centrifuged to remove sodium sulfate, and filtered. The solvent was removed by pressure evaporation, and the mixture was separated by silica gel column chromatography to obtain the axially chiral allenol compound. Product detection data: yield 23%, ee value 98% (Chiralcel OD-H, Hexane / i-PrOH = 90 / 10, flow rate 1.0 mL / min, λ = 254 nm, t(S)-2h = 5.6 min (major), t(R)-2h = 7.6 min (minor)). 1HNMR (400MHz, CDCl3) δ7.22-7.17 (m, 2H), 7.12 (d, J = 7.8Hz, 2H), 6.29-6.23 (m,1H),4.14(qd,J=13.1,2.9Hz,2H),2.34(s,3H),1.85(d,J=2.9Hz,3H).13C NMR(100MHz, CDCl3)δ200.5,137.0,131.8,129.4,126.8,104.7,97.0,64.0,21.3,15.6.HRMS(ESI)m / z:calculated for C12H15O[M+H]+:175.1123,found:175.1126.

[0100] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A P450 hydroxylase mutant, characterized in that, Its amino acid sequence is shown in SEQ ID No.

1.

2. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encoding the P450 hydroxylase mutant has the nucleotide sequence shown in SEQ ID No.

2.

3. A carrier, characterized in that, It includes the nucleic acid molecule as described in claim 2.

4. A recombinant bacterium, characterized in that, The recombinant bacteria comprises the vector of claim 3, wherein the host cell of the recombinant bacteria is selected from bacteria or fungi.

5. An immobilized enzyme, characterized in that, The P450 hydroxylase mutant of claim 1 was prepared by immobilization using a vector or a vector-free method.

6. An immobilized cell, characterized in that, The recombinant bacteria described in claim 4 were fixed to obtain the bacteria.

7. The use of the P450 hydroxylase mutant of claim 1 (named P450AHS hydroxylase), the recombinant bacteria of claim 4, the recombinant cells of claim 5, or the immobilized enzyme of claim 6 in the biosynthesis of axially chiral allenol compounds.

8. A method for biosynthesizing axially chiral allenol compounds, characterized in that, The substrate was reacted using P450AHS hydroxylase, bacteria, immobilized enzymes, or immobilized cells.

9. The synthesis method according to claim 8, characterized in that, The substrate is an alkyl or aryl substituted allene.

10. A method for preparing an axially chiral allenol compound, characterized in that, Using alkyl and aryl substituted allenes as substrates and P450AHS hydroxylase as catalyst, the reaction is carried out under aqueous conditions, as shown in reaction formula I below: Wherein: Substituent R in reaction formula I 1 R 2 ,R 3 It is any one of methyl, ethyl, n-propyl, isopropyl, n-butyl, phenyl, 4-chlorophenyl, 4-fluorophenyl, 4-bromophenyl, 4-trifluoromethylphenyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 3-methoxyphenyl, 4-ethylphenyl, 4-tert-butylphenyl, 4-n-butoxyphenyl, 3,4-dimethoxyphenyl, 4-methoxyphenyl, 3,5-dimethoxyphenyl, 3,4,5-trimethoxyphenyl, 3-chloro-4-methoxyphenyl, 3-bromophenyl, 4-iodophenyl, 3-bromo-4-methoxyphenyl, 2-naphthyl, 4-cyanophenyl, and 2-furanyl.

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