A method for enzymatic synthesis of 1,2-hexanediol

Abstract

The application discloses an enzyme method for synthesizing 1,2-hexanediol, which comprises the following steps: S1, constructing a P450 monooxygenase strain, producing P450 monooxygenase from the P450 monooxygenase strain, and purifying the P450 monooxygenase; S2, generating 2-hydroxyhexanoic acid from n-hexanoic acid through P450 monooxygenase catalysis; and S3, generating 1,2-hexanediol from 2-hydroxyhexanoic acid through lipase catalysis in the presence of butanol. The application generates 2-hydroxyhexanoic acid from n-hexanoic acid through P450 monooxygenase catalysis, and generates 1,2-hexanediol from 2-hydroxyhexanoic acid through lipase catalysis in the presence of butanol. The application solves the use of toxic and harmful reagents in the existing chemical synthesis process, and has a good industrial application prospect, because the enzyme method for synthesizing 1,2-hexanediol provided by the application has a mild reaction environment, a simple and controllable process, and a green and environment-friendly production process.

Description

Technical Field

[0001] This invention belongs to the field of compound synthesis technology, and more specifically, relates to an enzymatic method for synthesizing 1,2-hexanediol. Background Technology

[0002] 1,2-Hexanediol is an organic compound with the chemical formula C6H2O. 14 O2 is a transparent, colorless to pale yellow liquid. 1,2-Hexanediol is miscible with a variety of organic compounds in any proportion, is non-corrosive, and is widely used in inkjet printer inks, cosmetics, and multi-purpose cleaning agents. Based on its application, 1,2-Hexanediol can be classified as pharmaceutical grade, chemical grade, and cosmetic grade. 1,2-Hexanediol possesses both hydrophobic and hydrophilic properties, and is commonly used in cosmetics and pharmaceuticals as a solvent and penetrant, while also exhibiting certain moisturizing and preservative effects.

[0003] The synthesis of 1,2-hexanediol generally begins with 1-hexene as a starting material, undergoing epoxidation with an oxidant and hydrolysis to produce 1,2-hexanediol. Alternatively, it can begin with hexanoic acid as a starting material, undergoing chlorination and hydrolysis to produce 2-hydroxyhexanoic acid, which is then reduced by a carboxyl group to generate 1,2-hexanediol. Current α-hydroxylation methods require two chemical steps and involve chlorine, which is listed in the "List of Hazardous Chemicals." If α-hydroxylation could be achieved in a single enzymatic step, production costs would be reduced, and the process would be more gentle, environmentally friendly, and less hazardous. Existing reports indicate that the enzymes used for α-hydroxylation lack high specificity, exhibiting hydroxylation at numerous other sites besides the α-position, or resulting in extremely low substrate and product concentrations, which are insufficient for industrial production. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides an enzymatic method for the synthesis of 1,2-hexanediol. The enzymatic method for synthesizing 1,2-hexanediol provided by this invention features a mild reaction environment, a simple and controllable process, and an environmentally friendly production process, demonstrating promising prospects for industrial application.

[0005] The objective of this invention and the technical problem it solves are achieved by the following technical solutions.

[0006] One aspect of the present invention provides a method for the enzymatic synthesis of 1,2-hexanediol, the method comprising the following steps:

[0007] S1: Construct a P450 monooxygenase strain, produce P450 monooxygenase from the P450 monooxygenase strain, and purify the P450 monooxygenase;

[0008] S2: The production of 2-hydroxyhexanoic acid from hexanoic acid is catalyzed by P450 monooxygenase;

[0009] S3: 1,2-Hexanediol is generated from 2-hydroxyhexanoic acid via lipase in the presence of butanol.

[0010] Furthermore, the method for constructing the P450 monooxygenase strain described in step S1 is as follows:

[0011] Using the Bacillus subtilis WB800N genome as a template, the ybdT fragment was amplified using primers ybdT-F and ybdT-R. The obtained fragment was recovered from the gel and ligated into the pBAD HisB vector, which was then transformed into Escherichia coli Fast-T1 competent cells. Positive clones were screened on LB plates containing ampicillin and verified by sequencing using primer PBAD-F.

[0012] The correctly sequenced plasmid was transferred into E. coli BW25113 competent cells to obtain the genetically engineered bacterium BW / pBAD-ybdT.

[0013] Further, the nucleotide sequence of primer ybdT-F is shown in SEQ ID NO:3, the nucleotide sequence of primer ybdT-R is shown in SEQ ID NO:4, and the nucleotide sequence of primer PBAD-F is shown in SEQ ID NO:5;

[0014] The PCR amplification system consisted of: 10 μL of 5×SF Buffer, 1 μL of dNTP Mix (10 mM each), 1 μL of Bacillus subtilis WB800N genome template, 2 μL each of primers (10 μM), 1 μL of Phanta Super-Fidelity DNA Polymerase, and 34 μL of distilled water, for a total volume of 50 μL.

[0015] The amplification conditions were as follows: 95°C pre-denaturation for 2 minutes, 1 cycle; 95°C denaturation for 10 seconds, 55°C annealing for 20 seconds, 72°C extension for 1.5 minutes, 30 cycles; 72°C extension for 10 minutes, 1 cycle.

[0016] Furthermore, the specific process of producing P450 monooxygenase from the P450 monooxygenase strain in step S1 is as follows:

[0017] The obtained genetically engineered bacteria BW / pBAD-ybdT were inoculated into 2YT liquid medium and cultured at 37℃ for 4-6 hours. Then, the resulting culture solution was inoculated into shake flasks at an inoculation rate of 1%-2% and cultured at 37℃ and 200 rpm for 2-3 hours. Then, enzyme production was induced with arabinose at a final concentration of 1wt%-2wt% at an induction temperature of 25℃-30℃, an induction speed of 220 rpm, and an induction time of 14-18 hours. After that, the cells were centrifuged and the cell products in the culture medium were collected.

[0018] The 2YT liquid culture medium consists of: 9-11 g / L of molecular-grade peptone, 4-6 g / L of molecular-grade yeast extract, 4-6 g / L of NaCl, 100 μg / mL of ampicillin, and a pH of 6.8-7.2.

[0019] Furthermore, the purification method for the P450 monooxygenase described in step S1 is as follows:

[0020] Add 20 mL of 1×PBS to the collected cell products, resuspend the cells at pH 7.4, sonicate to disrupt the cells, perform the operation on ice, disrupt for 1 second, cool for 3 seconds, for a total time of 30-45 minutes; centrifuge the lysis buffer at 10000 rpm for 30 minutes at 4°C to precipitate cell debris, collect the supernatant, filter through a 0.45 μm filter membrane, and then pass through Ni-NTA affinity chromatography to obtain the eluent;

[0021] The collected eluent was concentrated and desalted by ultrafiltration using a 10 kDa ultrafiltration tube, with 1×PBS added during the process. The mixture was centrifuged 5 times at 4000 rpm at pH 7.4 and 4°C. After the final concentration, no PBS was added to obtain the purified P450 monooxygenase ybdT.

[0022] Preferably, the above affinity chromatography process is as follows: A clarified sample containing a polyhistidine-tagged protein is loaded into the column at a flow rate of 0.5-1 mL / min, and the eluent is collected and passed through the column again; the column is washed with PBS at a flow rate of 1 mL / min to remove contaminating proteins, typically using 5 column volumes; the column is then washed with wash buffer containing 20 mM imidazole at a flow rate of 1 mL / min to remove contaminating proteins, typically using 8 column volumes; elution is then performed with 5-10 column volumes of elution buffer containing 250 mM imidazole at a flow rate of 0.5-1 mL / min, and the eluent is collected.

[0023] Furthermore, the P450 monooxygenase has the following characteristics:

[0024] (1) It has the amino acid sequence shown in SEQ ID NO:2;

[0025] (2) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids as described in (1), and which has the same or similar function as the amino acid sequence shown in (1); or

[0026] (3) A mutant polypeptide having at least 70%, at least 80%, or at least 90% sequence identity with the amino acid sequence of (1) or (2) and the fragment still having the activity of catalyzing the formation of 2-hydroxyhexanoic acid from hexanoic acid.

[0027] Furthermore, the specific process of generating 2-hydroxyhexanoic acid from hexanoic acid via P450 monooxygenase catalysis in step S2 is as follows:

[0028] Hexanoic acid, H2O2, pH 7.5 Tris-HCl, P450 monooxygenase, and ethanol were mixed and reacted at 25-37℃ for 12-24 h. Then, one-tenth of the HCl was added to terminate the reaction. 1 mL of sample was taken according to the sample:ethyl acetate volume ratio of 1:1 and 1 mL of ethyl acetate was added. After sonication for 10 min, the mixture was centrifuged at 10000 rpm for 10 min. 2-Hydroxyhexanoic acid was found in the supernatant.

[0029] Preferably, the supernatant is detected by gas chromatography, and the specific process is as follows:

[0030] Instrument: Shimadzu GC-2014

[0031] Column: Rtx-5, length 30m, inner diameter 0.32mm ID, film thickness 0.25μm, maximum temperature 350℃;

[0032] Injection volume: 1 μL, vaporization chamber temperature: 300 °C, split flow, carrier gas: N2, control mode: pressure

[0033] Pressure: 105.0 kPa, Total flow rate: 82.5 mL / min, Column flow rate: 3.79 mL / min; Linear velocity: 55.8 cm / sec; Purge flow rate: 3.0 mL / min; Split ratio: 20.0; Column temperature: 60℃; Equilibrium time: 2.0 min; Detector temperature: 320℃; End time: 31 min.

[0034] Further, the concentration of hexanoic acid is 0.02-0.3M, the initial concentration of H2O2 is 0.2-1.5mM, and then 0.2-1.5mM is added every 15 minutes. The concentration of Tris-HCl is 10-100mM, the concentration of P450 monooxygenase is 50-500μg / mL, the concentration of ethanol is 1%-5%, and the reaction volume is 2-10mL.

[0035] Furthermore, the specific process of catalyzing the production of 1,2-hexanediol from 2-hydroxyhexanoic acid by lipase in the presence of butanol in step S3 is as follows:

[0036] Mix 2-hydroxyhexanoic acid and butanol in a molar ratio of 1:1 to 1:5, then add 1% to 10% toluene and 0.1 to 0.5 g / L lipase (SIGMA, catalog number 89444), and react at 30°C and 250 rpm for 15 to 45 hours.

[0037] The expected effects and advantages of this invention are as follows:

[0038] This invention provides an enzymatic method for the synthesis of 1,2-hexanediol from hexanoic acid. First, a P450 monooxygenase catalyzes the formation of 2-hydroxyhexanoic acid from hexanoic acid. Then, a lipase catalyzes the formation of 1,2-hexanediol from 2-hydroxyhexanoic acid in the presence of butanol. The enzymatic synthesis of 1,2-hexanediol provided by this invention features a mild reaction environment, a simple and controllable process, and an environmentally friendly production process, demonstrating promising prospects for industrial application.

[0039] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below. Attached Figure Description

[0040] Figure 1 The results show the expression and purification of the P450 monooxygenase ybdT protein, with the last band representing the purified result.

[0041] Figure 2 This is a gas chromatogram of the detection peaks for n-hexanoic acid.

[0042] Figure 3 The image shows the gas phase detection peaks of 2-hydroxyhexanoic acid.

[0043] Figure 4 The image shows the gas phase peaks for 1,2-hexanediol. Detailed Implementation

[0044] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0045] Experimental methods not specified in detail in the following examples are generally performed under standard conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.

[0046] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0047] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0048] Example 1: Construction of strains of P450 monooxygenase

[0049] Using the genome of *Bacillus subtilis* WB800N (purchased from Beijing Bio-Labs Technology Co., Ltd.) as a template, the ybdT fragment was amplified using primer pairs ybdT-F and ybdT-R. The fragment was then recovered from the gel and ligated into the pBAD HisB vector using the Novizan ClonExpress II One Step Cloning Kit. This ligation was then used to transform *E. coli* Fast-T1 competent cells. Positive clones were screened on LB agar plates containing ampicillin and verified by sequencing using primer PBAD-F. The ybdT gene is the nucleotide sequence shown in SEQ ID NO:1 and the amino acid sequence is shown in SEQ ID NO:2. The full-length DNA is 1254 bp, with the start codon ATG and the stop codon TAA. This sequence has no introns, possesses a complete open reading frame, and encodes 418 amino acids.

[0050] The PCR amplification system consisted of: 10 μL of 5×SF Buffer, 1 μL of dNTP Mix (10 mM each), 1 μL of Bacillus subtilis WB800N genome template, 2 μL each of primers (10 μM), 1 μL of Phanta Super-Fidelity DNA Polymerase (Nanjing Novizan Biotechnology Co., Ltd., product catalog P501), and 34 μL of distilled water, for a total volume of 50 μL.

[0051] The amplification conditions were as follows: 95℃ pre-denaturation for 2 minutes (1 cycle); 95℃ denaturation for 10 seconds, 55℃ annealing for 20 seconds, 72℃ extension for 1.5 minutes (30 cycles); 72℃ extension for 10 minutes (1 cycle).

[0052] SEQ ID NO:1

[0053]

[0054]

[0055] SEQ ID NO:2

[0056]

[0057] The primer sequences used are as follows:

[0058] ybdT-F SEQ ID NO:3

[0059] 5'-TGACGATAAGGATCCGAGCTCGAGatgaatgagcagattccac-3'

[0060] ybdT-R SEQ ID NO:4

[0061] 5'-aaacagccaagcttcgaattctTAactttttcgtctgattcc-3'

[0062] PBAD-F SEQ ID NO:5

[0063] 5'-aggaggaattaaccatgggg-3'

[0064] The steps of E. coli Fast-T1 competent transformation are as follows:

[0065] (1) Take a tube of Fast-T1 competent cells stored at -80℃ containing 100 μL;

[0066] (2) Add the ligation product to the thawed competent cells, gently flicking them with your fingers during the process;

[0067] (3) Place the product of step (2) on ice for 20-30 minutes, then place it in a 42°C water bath for 45 seconds for heat shock. After the heat shock is completed, immediately remove it and place it back on ice to stand for 2-5 minutes.

[0068] (4) Add 800 μL of 2YT liquid culture medium and incubate for 40 min in a 37℃ shaking incubator (purchased from Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd., model ZWY-2102C) with a shaking frequency of 200 rpm;

[0069] (5) After centrifuging the transformed bacterial culture from step (4) at 4000 rpm for 2 min, spread it on a 2YT solid culture medium plate and incubate at 37℃ for 10-12 h.

[0070] (6) Select single colonies and place them into resistant liquid 2YT medium containing 100 μg / mL ampicillin. Incubate at 37°C for 4–6 h and then verify.

[0071] The correctly sequenced plasmid was transferred into E. coli BW25113 competent cells to obtain the genetically engineered bacterium BW / pBAD-ybdT.

[0072] The obtained genetically engineered bacteria were inoculated into 2YT liquid medium and cultured at 37℃ for 4-6 hours. Then, the resulting culture solution was inoculated into shake flasks at an inoculation rate of 1%-2% and cultured at 37℃ and 200 rpm for 2-3 hours. Enzyme production was induced with arabinose at a final concentration of 1%-2% (mass) at an induction temperature of 25℃-30℃, an induction speed of 220 rpm, and an induction time of 14-18 hours.

[0073] The steps for transforming E. coli BW25113 competent cells are as follows:

[0074] (1) Take a vial of E. coli BW25113 competent cells stored at -80℃ containing 50-200 μL and thaw it on ice for 20-30 min;

[0075] (2) Take 2-3 μL of the plasmid solution that has been verified to be correct and add it to the thawed competent cells. Gently flick the cells with your fingers during the addition process.

[0076] (3) Place the product of step (2) on ice for 20-30 minutes, then place it in a 42°C water bath for 90 seconds for heat shock. After the heat shock is completed, immediately remove it and place it back on ice to stand for 2-5 minutes.

[0077] (4) Add 800 μL of 2YT liquid culture medium and incubate at 37°C with a shaking frequency of 200 rpm (purchased from Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd., model ZWY-2102C) for 40 min; (5) Take 50 μL of the transformation bacterial solution from step (4) and spread it on a 2YT solid culture medium plate, and incubate at 37°C for 10-12 h;

[0078] (6) Select single colonies and incubate them in resistant liquid 2YT medium containing 100 μg / mL ampicillin at 37°C for 6–8 h;

[0079] (7) Take an equal volume of bacterial solution and an equal volume of sterile 80% (volume) glycerol solution, mix them and place them in a preservation tube, store them in a -80℃ refrigerator for later use.

[0080] Example 2: Production of P450 monooxygenase by P450 monooxygenase strain and purification of the enzyme.

[0081] The obtained genetically engineered bacteria BW / pBAD-ybdT were inoculated into 100 mL of 2YT medium (containing ampicillin 30). After culturing for 2 h, the OD was 0.6-0.8. After adding arabinose to a final concentration of 0.2%, the cells were induced at 30 °C for 16 h. The cells were then centrifuged at 4 °C and 6000 rpm for 10 min. Finally, 100 mL of the culture medium was collected.

[0082] Add 20 mL of 1×PBS to the collected cell products, pH 7.4, resuspend the cells, and sonicate to disrupt the cells. Perform the operation on ice, disrupting for 1 second, cooling for 3 seconds, for a total time of 30-45 minutes. Centrifuge the lysis buffer at 10,000 rpm for 30 minutes at 4°C to precipitate cell debris. Collect the supernatant, filter it through a 0.45 μm filter membrane, and then pass it through Ni-NTA affinity chromatography medium.

[0083] The affinity chromatography procedure is as follows: A clear sample containing polyhistidine-tagged proteins is loaded into the column at a flow rate of 0.5-1 mL / min. The eluent is collected and passed through the column again. The column is washed with PBS at a flow rate of 1 mL / min to remove contaminating proteins, typically using 5 column volumes. The column is then washed with wash buffer containing 20 mM imidazole at a flow rate of 1 mL / min to remove contaminating proteins, typically using 8 column volumes. Elution is then performed with 5-10 column volumes of elution buffer containing 250 mM imidazole at a flow rate of 0.5-1 mL / min, and the eluent is collected.

[0084] The collected eluent was concentrated and desalted by ultrafiltration using a 10 kDa ultrafiltration tube, with 1×PBS added during the process. The mixture was centrifuged five times at 4000 rpm at pH 7.4, 4°C, and the final concentration was performed without adding PBS to obtain the purified P450 monooxygenase ybdT. The electrophoresis results of this enzyme are shown below. Figure 1 As shown, the rightmost band represents the purified result.

[0085] The P450 monooxygenase ybdT protein gene obtained using the above method has the following characteristics:

[0086] (1) Has a nucleotide sequence as shown in SEQ ID NO:1;

[0087] (2) A nucleotide sequence obtained by substitution, deletion, or addition of one or more bases to the nucleotide sequence described in (1), and which has the same or similar function to the nucleotide sequence shown in (1); or

[0088] (3) A nucleotide sequence that is at least 80% homologous to the sequence to which (1) or (2) belongs.

[0089] The P450 monooxygenase obtained by the above method also has the following characteristics:

[0090] (1) It has the amino acid sequence shown in SEQ ID NO:2;

[0091] (2) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids as described in (1), and which has the same or similar function as the amino acid sequence shown in (1); or

[0092] (3) A mutant polypeptide having at least 70%, at least 80%, or at least 90% sequence identity with the amino acid sequence of (1) or (2), and the fragment still possessing the activity of catalyzing the conversion of hexanoic acid to 2-hydroxyhexanoic acid.

[0093] Example 3: Catalysis of n-hexanoic acid to 2-hydroxyhexanoic acid using P450 monooxygenase

[0094] Add 0.3M hexanoic acid (gas chromatographic assay) to a 100mL wide-mouth Erlenmeyer flask. Figure 3 As shown), 1.5 mM H2O2 (1.5 mM added every 15 min), 100 mM pH 7.5 Tris-HCl, P450 monooxygenase 100 μg / mL, and 12.5% ​​ethanol were reacted at 37℃ for 24 h. The reaction was then terminated by adding one-tenth of the total reaction volume of HCl. 1 mL of sample was added to 1 mL of ethyl acetate at a 1:1 sample-to-ethyl acetate ratio. After sonication for 10 min, the mixture was centrifuged at 10000 rpm for 10 min. The supernatant (containing 2-hydroxyhexanoic acid) was diluted appropriately and analyzed by gas chromatography (GC). The results are as follows. Figure 3 As shown.

[0095] The specific method for GC detection is as follows:

[0096] Instrument: Shimadzu GC-2014

[0097] Column: Rtx-5, length 30m, inner diameter 0.32mm ID, film thickness 0.25μm, maximum temperature 350℃;

[0098] Injection volume: 1 μL, vaporization chamber temperature: 300 °C, split flow, carrier gas: N2, control mode: pressure;

[0099] Pressure: 105.0 kPa, Total flow rate: 82.5 mL / min, Column flow rate: 3.79 mL / min; Linear velocity: 55.8 cm / sec; Purge flow rate: 3.0 mL / min; Split ratio: 20.0; Column temperature: 60℃; Equilibrium time: 2.0 min; Detector temperature: 320℃; End time: 31 min.

[0100] Detection results: 140 mM of 2-hydroxyhexanoic acid was generated in the reaction, with a conversion rate of 46%.

[0101] Example 4: Utilizing lipase to catalyze the production of 1,2-hexanediol from 2-hydroxyhexanoic acid.

[0102] 0.1M 2-hydroxyhexanoic acid and 0.3M butanol were added to 10% toluene, along with 0.1 g / L lipase (SIGMA, catalog number 89444). The mixture was reacted at 30°C and 250 rpm for 15 h, followed by the addition of 0.2 g / L lipase, and the reaction was continued at 30°C and 250 rpm for another 30 h. The reaction produced 1,2-hexanediol (as shown in the gas chromatogram). Figure 4 (As shown) 70mM, conversion rate 70%.

[0103] Currently, 1,2-hexanediol is synthesized entirely through chemical methods, such as patent CN 110272329 A. The specific process involves mixing hexene, acetonitrile, methanol, and potassium bicarbonate at room temperature, and then adding hydrogen peroxide dropwise at 50°C. After the hexene has largely reacted, it is distilled under normal pressure to obtain 1,2-epoxyhexane, with the remainder being the first product. This first product is then further distilled under reduced pressure to obtain 1,2-hexanediol. This reaction process requires a large amount of organic reagents. In contrast, this invention uses an enzymatic synthesis method, making the production process green and environmentally friendly. Patent WO2016135396 discloses the synthesis of α-haloacids via sodium bromide halogenation at 200°C, followed by reaction with a base to generate α-hydroxy acids. However, the concentration of the substrate hexanoic acid is only 0.03 mol, significantly lower than the concentration in this invention, making it difficult to meet the needs of industrial production.

[0104] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for the enzymatic synthesis of 1,2-hexanediol, characterized in that, The method includes the following steps: S1: Construct a P450 monooxygenase strain, produce P450 monooxygenase from the P450 monooxygenase strain, and purify the P450 monooxygenase; S2: The production of 2-hydroxyhexanoic acid from hexanoic acid is catalyzed by P450 monooxygenase; S3: The lipase catalyzes the formation of 1,2-hexanediol from 2-hydroxyhexanoic acid in the presence of butanol; The P450 monooxygenase is YbdT derived from Bacillus subtilis and has an amino acid sequence as shown in SEQ ID NO:2; the nucleotide sequence of YbdT is shown in SEQ ID NO:

1.

2. The method according to claim 1, characterized in that, The method for constructing the P450 monooxygenase strain in step S1 is as follows: Bacillus subtilis Bacillus subtilis WB800N Using the genome as a template, PCR amplification was performed using primers ybdT-F and ybdT-R to obtain the ybdT fragment. The obtained fragment was recovered from the gel and ligated into the pBAD HisB vector, which was then transformed into E. coli Fast-T1 competent cells. Positive clones were screened on LB plates containing ampicillin and verified by sequencing using primer PBAD-F. The correctly sequenced plasmid was transferred into E. coli BW25113 competent cells to obtain the genetically engineered strain BW / pBAD-ybdT. The nucleotide sequence of primer ybdT-F is shown in SEQ ID NO: 3; the nucleotide sequence of primer ybdT-R is shown in SEQ ID NO: 4; and the nucleotide sequence of primer PBAD-F is shown in SEQ ID NO:

5.

3. The method according to claim 2, characterized in that, The PCR amplification system consisted of: 10 μL of 5×SF Buffer, 1 μL of dNTP Mix (10 mM each), and template. Bacillus subtilis WB800N 1 μL of genome, 2 μL each of primers (10 μM), 1 μL of Phanta Super-Fidelity DNA Polymerase, and 34 μL of distilled water, for a total volume of 50 μL; The PCR amplification conditions were as follows: 95℃ pre-denaturation for 2 minutes, 1 cycle; 95℃ denaturation for 10 seconds, 55℃ annealing for 20 seconds, 72℃ extension for 1.5 minutes, 30 cycles; 72℃ extension for 10 minutes, 1 cycle.

4. The method according to claim 2, characterized in that, The specific process of producing P450 monooxygenase from the P450 monooxygenase strain in step S1 is as follows: The obtained genetically engineered bacteria BW / pBAD-ybdT were inoculated into 2YT liquid medium and cultured at 37℃ for 4-6 h. Then, the resulting culture solution was inoculated into shake flasks at an inoculation rate of 1%-2% and cultured at 37℃ and 200 rpm for 2-3 h. Enzyme production was induced with arabinose at a final concentration of 1wt%-2wt% at an induction temperature of 25℃-30℃, an induction speed of 220 rpm, and an induction time of 14-18 h. After that, the cells were centrifuged and the cell products in the culture medium were collected. The 2YT liquid culture medium consists of: 9-11 g / L of molecular-grade peptone, 4-6 g / L of molecular-grade yeast extract, 4-6 g / L of NaCl, 100 μg / mL of ampicillin, and a pH of 6.8-7.

2.

5. The method according to claim 4, characterized in that, The purification method for P450 monooxygenase in step S1 is as follows: Add 20 mL of 1×PBS to the collected cell products to resuspend the cells at pH 7.

4. Sonicate the cells to disrupt them on ice for 1 second followed by 3 seconds of cooling, for a total time of 30–45 minutes. Centrifuge the lysis buffer at 10,000 rpm for 30 minutes at 4 °C to precipitate cell debris. Collect the supernatant, filter it through a 0.45 μm filter membrane, and then pass it through Ni-NTA affinity chromatography to obtain the eluent. The collected eluent was concentrated and desalted by ultrafiltration using a 10 kDa ultrafiltration tube, with 1×PBS added during the process. The mixture was centrifuged 5 times at 4000 rpm at pH 7.4, 4℃. After the final concentration, no PBS was added to obtain the purified P450 monooxygenase ybdT.

6. The method according to claim 1, characterized in that, The specific process of converting n-hexanoic acid to 2-hydroxyhexanoic acid via P450 monooxygenase catalysis in step S2 is as follows: Hexanoic acid, H2O2, pH 7.5 Tris-HCl, P450 monooxygenase, and ethanol were mixed and reacted at 25-37℃ for 12-24 h. Then, HCl was added to one-tenth of the total reaction volume to terminate the reaction. 1 mL of sample was taken according to the sample:ethyl acetate volume ratio of 1:1 and 1 mL of ethyl acetate was added. After sonication for 10 min, the mixture was centrifuged at 10000 rpm for 10 min. 2-Hydroxyhexanoic acid was found in the supernatant.

7. The method according to claim 6, characterized in that, The concentration of hexanoic acid is 0.02-0.3 M, the initial concentration of H2O2 is 0.2-1.5 mM, and then it is added to 0.2-1.5 mM every 15 min. The concentration of Tris-HCl is 10-100 mM, the concentration of P450 monooxygenase is 50-500 μg / mL, the concentration of ethanol is 1%-5%, and the reaction volume is 2-10 mL.

8. The method according to claim 1, characterized in that, The specific process described in step S3, which involves the catalysis of 2-hydroxyhexanoic acid to 1,2-hexanediol by lipase in the presence of butanol, is as follows: Mix 2-hydroxyhexanoic acid and butanol in a molar ratio of 1:1 to 1:5, then add 1% to 10% toluene and 0.1 to 0.5 g / L lipase, and react at 30°C and 250 rpm for 15 to 45 hours.

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