A fatty acid decarboxylase P450BSβ mutant and its application in the preparation of γ-lactone

Abstract

The invention discloses a fatty acid decarboxylase P450BSβ mutant and its application in the preparation of γ-lactone, wherein the fatty acid decarboxylase P450BSβ mutant is obtained by mutation of the amino acid sequence of wild-type P450BSβ at positions 78, 85, 173 and 290, and can be directly oxidized lactonized to lactone by one step using hydrogen peroxide as a direct electron donor and fatty acid as a substrate. The catalytic substrate range is extended to fatty acids with a chain length of C6 to C18, fatty acids containing functional groups, and fatty acids containing aromatic (hetero) rings. The catalyst efficiency of the present invention is higher, the reaction is simpler, and the substrate range is wider, and it has good application prospects and industrial value in the preparation of γ-lactone.

Description

Technical Field

[0001] The invention relates to a fatty acid decarboxylase P450BSβ mutant and application thereof in the preparation of gamma-lactone, belonging to the field of proteins. Background Art

[0002] γ-lactone is a lactone with a five-membered ring structure formed by dehydration condensation of the hydroxyl group on the γ-carbon atom with carboxylic acid. It is one of the most abundant categories of lactones in nature. γ-lactone exists in the structure of many natural products, such as γ-decalactone, γ-butyrolactone and α-amino-γ-lactone, and γ-lactone exhibits excellent biological activity. For example, γ-decalactone has a peach aroma, γ-octalactone has a coconut aroma, and γ-caprolactone has a vanilla aroma. They are a class of natural peach flavors widely used in beverages, perfumes and pharmaceutical preparations. The world market volume of γ-lactone has reached millions of tons per year, with a value of US$300-6000 / kg, but it is on a downward trend due to the development of its production technology. In addition, some γ-lactone derivatives also have biological activities such as anti-tuberculosis (such as Micromolide) and insect pheromones. Therefore, the preparation of γ-lactone has received widespread attention.

[0003] Common methods for synthesizing simple lactones such as γ-lactone by chemical methods include cyclization, reduction and oxidation. Cyclization of γ-hydroxycarboxylic acid and its derivatives catalyzed by inorganic acids, sulfonic acids, p-toluenesulfonic acid and pyridine p-toluenesulfonate is one of the simplest and most efficient methods for synthesizing γ-lactone, but the corresponding γ-hydroxycarboxylic acid is still difficult to synthesize; it also requires the reaction of functional groups such as carbonyl at the γ position of the substrate to generate γ-hydroxycarboxylic acid, but such substrates are difficult to obtain in nature and require multi-step chemical synthesis; oxidation of substrates containing groups such as carbon-carbon double bonds at the γ position to generate γ-hydroxycarboxylic acid can also synthesize γ-lactone, but substrates containing double bonds and other groups at specific sites have similar chemical construction difficulties and economic values ​​as γ-hydroxycarboxylic acid. These methods either require numerous reaction steps and low yields, or require chiral auxiliaries synthesized by precious metals such as ruthenium and iridium, as well as environmentally unfriendly and harsh reaction conditions using chemical products. In recent years, biocatalysts have also been used in the synthesis of lactones, making it possible to use green and milder reagents and synthetic conditions. The existing biosynthetic methods are basically to construct engineered strains, and then use unsaturated fatty acids such as oleic acid as substrates to biosynthesize γ-lactones through fermentation. These methods still require a certain degree of activated substrates to synthesize γ-lactones. These substrates have the same economic value and industrial applicability as lactones. In addition, the generated γ-lactones have different carbon chain lengths and are difficult to separate and purify, and the product stereoisomer selectivity is not ideal.

[0004] Direct oxidation of carboxylic acids at the γ position to obtain γ-hydroxycarboxylic acids and subsequent cyclization to γ-lactones is an ideal, direct and economical preparation method. Previous studies have shown that the chemical reactivity and site selectivity of oxidative lactonization of aliphatic carboxylic acids using platinum and copper or peroxodisulfate as catalysts are poor. The efficiency of site-selective chemical synthesis guided by external directing groups is also low. In order to achieve the reaction without external directing groups, chemical catalysts have been developed that utilize the inherent bias of different C-H bonds in the substrate, such as Fe- and Mn-(PDP) catalysts, which can achieve the electron-rich and sterically hindered secondary and tertiary C (sp 3 )-H bond γ-lactonization, and changing the steric properties of these catalysts can achieve electron-poor but sterically less hindered primary C(sp 3 )-H bonds. Recent studies have also found that a ligand-controlled palladium catalyst can effectively γ-lactonize dicarboxylic acids.

[38] . Among these remarkable advances, substrates usually require a certain rigidity, substituent pattern or complementary function to achieve good selectivity or reactivity, and there are still problems in the control of flexible, linear substrates with multiple identical methylene groups and enantioselectivity. Therefore, asymmetric catalytic γ-CH lactonization of flexible fatty acids has not yet been achieved. Among the known enzymes, only P450MP from Methylobacterium populi can produce trace amounts of γ-hydroxy fatty acid byproducts while achieving the main reaction of β-oxidation of C12:0-C18:0 fatty acids. Therefore, there are no reports of enzymes that specifically and directly hydroxylate fatty acids at the γ position. Summary of the invention

[0005] In order to overcome the existing technical defects and solve the problems of low catalytic efficiency, low reaction activity, low site and enantioselectivity, and narrow substrate range of metal catalysts, the present invention provides a fatty acid decarboxylase P450BSβ mutant and its application in the preparation of γ-lactone. The fatty acid decarboxylase P450BSβ mutant provided by the present invention can convert fatty acids into γ-lactone using hydrogen peroxide, and the biocatalyst has the advantages of high reaction rate, wide substrate range, simple system, high synthesis efficiency, high regional and enantiomeric selectivity, and low cost.

[0006] The fatty acid decarboxylase P450BSβ mutant of the present invention has an amino acid sequence as shown in SEQ ID NO:1.

[0007] The nucleotide sequence encoding the fatty acid decarboxylase P450BSβ mutant is shown in SEQ ID NO:2.

[0008] The fatty acid decarboxylase P450BSβ mutant of the present invention is obtained by mutating the glycine at position 290 of the wild-type fatty acid decarboxylase P450BSβ into isoleucine, mutating the phenylalanine at position 173 into serine, mutating the glutamine at position 85 into phenylalanine, and mutating the leucine at position 78 into glycine, and specifically comprises the following steps:

[0009] Step 1: constructing a plasmid for expressing fatty acid decarboxylase P450BSβ: the nucleotide sequence of SEQ ID NO: 2 is digested and recombined into an expression vector;

[0010] Step 2: transforming the plasmid expressing fatty acid decarboxylase P450BSβ constructed in step 1 into Escherichia coli;

[0011] Step 3: Expression of fatty acid decarboxylase P450BSβ: The strain obtained in step 2 was cultured overnight in LB liquid medium, then added to TB liquid medium for expansion, and heme precursor δ-aminolevulinic acid and IPTG were added in the late logarithmic phase to induce expression, and the bacteria were collected by centrifugation;

[0012] Step 4: Preparation of cell lysate containing fatty acid decarboxylase P450BSβ: resuspend the bacterial cells obtained in step 3 with a buffer, lyse the cells by ultrasonication, and centrifuge to obtain a supernatant;

[0013] Step 5: Purification of fatty acid decarboxylase P450BSβ: Add the supernatant obtained in step 4 to a pre-treated nickel ion affinity column to allow the target protein with a His tag to bind to the nickel column, then remove impurities at a lower imidazole concentration, and finally elute the target protein with a buffer containing a high concentration of imidazole;

[0014] Step 6: The eluted target protein is dialyzed to remove imidazole, the protein is concentrated and lyophilized to prepare lyophilized powder, the target protein concentration is determined, and then stored at -80°C.

[0015] The application of the fatty acid decarboxylase P450BSβ mutant of the present invention is to use fatty acids with a chain length of C6 to C18 as substrates, hydrogen peroxide as an oxidant, and the fatty acid decarboxylase P450BSβ mutant as a catalyst to catalyze the dehydration condensation of the hydroxyl group on the γ-carbon atom of the fatty acid and the carboxylic acid to form γ-lactone.

[0016] In the above catalytic reaction, the reaction is carried out at room temperature, the reaction system is an aqueous buffer system, and Triton X-100 (0.625%) and 5% DMSO (v / v) are used as cosolvents.

[0017] The buffer composition is: 0.1M KPi, 0.3M KCl, pH 7.0.

[0018] The fatty acid is selected from natural saturated fatty acids or non-natural carboxylic acids with a chain length of C6 to C18.

[0019] Furthermore, the structure of the non-natural carboxylic acid contains one or more of terminal or internal olefins or alkynes, bromine, esters, amines, nitriles, oxygen-sensitive hydroxyl motifs, and cyclohexyl groups; the fatty acid is an aromatic or heteroaromatic fatty acid, and its structure contains one or more functional groups of N-, O-, S-heteroaromatic rings, thiazole, pyridine, quinoline, benzothiazole, furan, thiophene, oxazole, benzofuran, and benzothiophene.

[0020] Furthermore, the γ-lactone is an S-type γ-lactone.

[0021] The method of the present invention wherein the fatty acid decarboxylase P450BSβ mutant catalyzes the dehydration condensation of the hydroxyl group on the γ-carbon atom of the fatty acid with the carboxylic acid to form γ-lactone comprises the following steps:

[0022] The reaction system of γ-lactonization includes buffer A (0.1M KPi, 0.3M KCl, pH 7.0), fatty acid decarboxylation P450BSβ mutant protein (2μM), substrate (1mM), hydrogen peroxide (5mM×3, interval time=5min), Triton X-100 (0.625%), and 5% DMSO (v / v). Among them, Triton X-100 (0.625%) and 5% DMSO (v / v) are used as cosolvents. The reaction is reacted at room temperature for 1h in a final volume of 100mL; after the reaction is completed, HCl is used to terminate the reaction, 1mL of the reaction mixture is taken out, extracted, treated with TMSCHN2, and the product and unreacted substrate are quantitatively analyzed by GC, and the enantiomeric ratio is analyzed by chiral GC. The remaining reaction solution is extracted with ethyl acetate, dried with anhydrous MgSO4 and filtered. The filtrate is concentrated in vacuo and purified by silica gel column chromatography. All experiments are carried out in parallel three times to calculate the yield.

[0023] The fatty acid decarboxylase P450BSβ mutant of the invention catalyzes the production of gamma-lactone. The biocatalyst has the advantages of high reaction rate, wide substrate range, simple system, high synthesis efficiency, high regional and enantiomeric selectivity, low cost and the like.

[0024] The fatty acid decarboxylase P450BSβ mutant of the present invention is obtained by mutating the amino acid sequence of the wild type P450BSβ at positions 78, 85, 173 and 290. The fatty acid decarboxylase P450BSβ mutant has high reaction activity and directly uses cheap hydrogen peroxide to synthesize corresponding γ-lactone from natural saturated fatty acids with a chain length of C6 to C18 and non-natural carboxylic acid substrates containing one or more functional groups of terminal or internal olefins / alkynes, bromine, esters, amines, nitrites, oxygen-sensitive hydroxyl motifs, cyclohexyl, N-, O-, S-heteroaromatic rings, thiazole, pyridine, quinoline, benzothiazole, furan, thiophene, oxazole, benzofuran, and benzothiophene. With octanoic acid as the model substrate, the reaction rate (TOF) was as high as 364 / min and the turnover number (TON) was as high as 3278. Due to its high reaction activity, high selectivity and low cost, the fatty acid decarboxylase P450BSβ mutant has good industrial production prospects in the synthesis of γ-lactones.

[0025] The beneficial effects of the present invention are embodied in:

[0026] 1. The biocatalyst constructed by the present invention utilizes cheap hydrogen peroxide as a direct electron donor, and uses natural saturated fatty acids with a chain length of C6 to C18 and non-natural carboxylic acids containing one or more functional groups of terminal or internal olefins / alkynes, bromine, esters, amines, nitriles, oxygen-sensitive hydroxyl motifs, cyclohexyl, N-, O-, S-heteroaromatic rings, thiazole, pyridine, quinoline, benzothiazole, furan, thiophene, oxazole, benzofuran, and benzothiophene as substrates to synthesize the corresponding (S)-γ-lactones, and the catalytic substrate range is wider.

[0027] 2. Most P450 hydroxylases require expensive biological coenzyme NAD(P)H and additionally prepared redox chaperones to mediate electron transfer for hydroxylation reactions, so the reaction system is complex and costly. The novel enzyme fatty acid decarboxylase P450BSβ mutant only requires hydrogen peroxide as a direct redox donor and does not require chaperone proteins and expensive cofactors (such as NADPH), directly catalyzing the synthesis of fatty acids into γ-lactones, and the reaction system is simple and economical.

[0028] 3. The reaction process is mild and is carried out in an aqueous solution at room temperature. It has high synthesis efficiency, high regioselectivity and high enantioselectivity. DETAILED DESCRIPTION

[0029] The present invention is further described below. The following examples are only used to more clearly illustrate the technical solution of the present invention, and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, all of the following compounds and reagents were purchased from Sigma-Aldrich, EMD Millipore, Tokyo Chemical Industry, Macklin Biochemical, Bidepharm Biochemical and New England Biolabs.

[0030] Example 1: Construction of fatty acid decarboxylase P450BSβ gene expression vector

[0031] The fully synthesized wild P450 BSβ fragment (synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.) was double-digested with restriction endonucleases NcoI and XhoI (New England Biolabs) according to the instructions, and ligated to the expression vector pET28a double-digested by NcoI and XhoI using T4 ligase (New England Biolabs). The ligation product was transformed into Escherichia coli DH5α competent cells (Tiangen Biochemical Technology Co., Ltd.). The successfully transformed monoclonal colonies were picked from the solid LB medium plate containing 50 μg / ml kanamycin, and cultured overnight in LB liquid medium containing the same concentration of kanamycin at 37°C and a shaking speed of 220 rpm / min. The recombinant plasmid was extracted from the overnight culture solution using a plasmid extraction kit (Tiangen Biochemical Technology Co., Ltd.) according to the instructions, and the extracted plasmid was sent for sequencing identification (Suzhou Jinweizhi Biotechnology Co., Ltd.).

[0032] Example 2: Preparation of fatty acid decarboxylase P450BSβ mutant

[0033] 1. Construction and expression of the P450BSβ mutant library: Using the sequenced recombinant plasmid containing the wild-type P450BSβ gene as a template, overlapping saturation mutations were performed on the relevant amino acid residue sites of the wild-type P450BSβ. In each round, the obtained saturated mutant library was transformed into Escherichia coli Rosetta (DE3) and cultured in a 96-well deep-well plate at 37°C, with each well containing 300μLTB culture medium containing 50μg / mL kanamycin and 34μg / mL chloramphenicol. After shaking overnight at a shaker speed of 250rpm, 15μL of the overnight culture was transferred to a new deep-well plate, with each well containing 1.5mL TB (50μg / mL kanamycin and 34μg / mL chloramphenicol). When OD 600When it reaches 0.6, 0.5mM δ-aminolevulinic acid and 1mM IPTG are added to the culture medium for induction. After culturing for 16 hours at 25°C and a shaker speed of 220rpm, the cells are harvested by centrifugation at a speed of 5000rpm for 10min. The bacteria are resuspended with 0.5ml of bufferA (0.1M KPi, 0.3M KCl, pH 7.0) and then broken by ultrasound. After centrifugation at 5000rpm / min and 4°C for 30 minutes, the supernatant is obtained. Lysate, caprylic acid (1mM), H2O2 (2mM), and 5% DMSO (v / v) and 0.625% Triton X-100 (v / v) as cosolvents are added to the reaction to increase the solubility of the substrate in the reaction solution.

[0034] 2. Screening of fatty acid decarboxylase P450BSβ mutant plasmids: For screening, cells were resuspended in buffer A (0.1M KPi, 0.1M KCl, pH 7.0, 0.5mL per well) and disrupted by ultrasound. After centrifugation at 5000rpm / min and 4°C for 30 minutes, the supernatant was obtained. Lysis solution, octanoic acid (1mM), H2O2 (2mM), 0.625% Triton X-100 (v / v), and 5% DMSO (v / v) were added to the reaction as cosolvents to increase the solubility of the substrate in the reaction solution. The reaction was carried out in a sealed bottle at room temperature for 1 hour. Headspace gas chromatography was used to directly quantify 1-heptene. In order to quantify the unreacted substrate, 2- and 3-hydroxy products, and γ- and δ-lactones, the reaction was quenched with 75μL HCl (5N), shaken for 1 hour (for γ- and δ-lactonization), and extracted with ethyl acetate. The organic phase was dried over anhydrous MgSO4. The hydroxycarboxylic acids and substrates were derivatized to the corresponding methyl esters by mixing the dried organic phase with MeOH and then adding TMSCHN2. After incubation at 25°C for 1 h, the contents of methyl octanoate, methyl 2-hydroxy and 3-hydroxyoctanoate, and γ- and δ-octanolactone were determined by gas chromatography. Standards of octanoic acid, 1-heptene, 2- and 3-hydroxyoctanoic acids, γ- and δ-octanolactone were treated in the same manner and served as references. The catalytic performance of the variants with higher C-γ selectivity than the parent was tested in triplicate and the average of the three experiments was taken.

[0035] Example 3: Large-scale expression and purification of P450BSβ mutant protein

[0036] After pre-culture, the cells containing the recombinant plasmid were transferred to TB medium containing 50 μg / mL kanamycin and 34 μg / mL chloramphenicol. The culture was carried out at 37°C and a shaking speed of 220 rpm / min. When OD 600When the p-value reached 0.6, 0.5 mM of precursor ALA (δ-aminolevulinic acid) and 1 mM IPTG were added for induction. The cells were cultured at 25°C with a shaker speed of 220 rpm for 16 h, and the cells were harvested by centrifugation at a centrifugal speed of 5000 rpm for 10 min. The cells were resuspended in buffer A and lysed by ultrasonic disruption. The supernatant cell lysate was obtained by centrifugation at 4°C with a centrifugal speed of 10000 rpm for 45 min, and loaded onto a nickel ion affinity column. The column was washed with buffer A containing 35 mM imidazole to remove impurities, and the P450BSβ mutant protein was detached from the column with a buffer containing 250 mM imidazole. Buffer A was dialyzed to remove imidazole, and the purified protein was concentrated and stored at -80°C. The protein concentration was determined by the P450-CO spectrometry developed by Omura and Sato.

[0037] Example 4: Identification and analysis of fatty acid deacidase P450BSβ mutant products

[0038] GC: The mutagenesis library was screened by gas chromatography on an Agilent 7890A gas chromatograph system with FID detector, column: DB-WAX (30 m × 0.25 mm, 0.25 μm membrane), N2 as carrier gas. Injection temperature: 250 °C, splitless mode. Detector temperature: 300 °C. Program: 40 °C for 1 min, 10 °C for 1 min to 250 °C for 15 min, for a total of 40 min.

[0039] GC-MS: After the reaction of natural saturated fatty acids with a chain length of C6 to C18 and non-natural carboxylic acids containing one or more functional groups of terminal or internal olefins / alkynes, bromine, esters, amines, nitriles, oxygen-sensitive hydroxyl motifs, cyclohexyl, N-, O-, S-heteroaromatic rings, thiazole, pyridine, quinoline, benzothiazole, furan, thiophene, oxazole, benzofuran, and benzothiophene as substrates, the reaction was terminated with 5N HCl and extracted with an equal volume of ethyl acetate. Samples were taken and the corresponding γ-lactones were quantified by gas chromatography-mass spectrometry. Chromatographic column: TG-5MS (30m×0.25mm, 0.25μm membrane). Injection temperature: 250℃, splitless mode. Program: 60℃ for 1 minute, 20℃min -1 To 320°C, 14 minutes.

[0040] GC: Chiral gas chromatography, on an Agilent 7890A gas chromatography system, using a FID detector, chromatographic column: CP-Chirasil-Dex CB (25m×0.25mm, 0.25μm membrane), N2 as carrier gas, injection temperature: 250℃, split mode, split ratio 100. Detector temperature: 275℃. Program: 100℃ for 1 minute, 2℃ for 1 minute to 180℃ for 4 minutes, for a total of 45 minutes.

[0041] HPLC: On a high performance liquid chromatograph (Agilent 1260 Infinity), the column was CHIRALPAK IA (Daicel, 4.6×250 mm, 5 μm column), the mobile phase was hexane and isopropanol (isopropanol: hexane: = 1:99 to 15:85), the flow rate was 1.0 mL / min, and the detection wavelength was 210 nm. According to the published procedure, the purified γ-lactone prepared by the enzymatic reaction and its racemic standard were converted into the corresponding benzamide.

[0042] NMR: recorded on an Agilent DD2400 MHz spectrometer or a BrukerAvance 600 MHz instrument 1 H- and 13 C-NMR spectrum.

[0043] HEMS: High resolution mass spectrometry (HRMS) was performed on an AB 5800 MALDI TOF / TOF and is presented as m / z.

[0044] Example 5: Fatty acid decarboxylase P450BSβ mutant catalyzes the production of γ-lactone

[0045] The γ-lactonization reaction system includes buffer A (0.1M KPi, 0.3M KCl, pH 7.0), purified P450BSβ mutant (5 or 7.5μM), substrate (5mM), Triton X-100 (0.625% v / v) and 5% DMSO (v / v) as cosolvents, H2O2 (5mM×3, interval time=5min). The reaction is reacted at room temperature for 1h in a final volume of 100mL. After quenching the reaction with HCl (5N), the pH is 1.0-2.0, and the reaction solution is neutralized with ammonium hydroxide to pH=7.0. 1mL of the reaction mixture is taken out, extracted, treated with TMSCHN2, and the product and unreacted substrate are quantitatively analyzed by GC, and the enantiomeric ratio is analyzed by chiral GC. The remaining reaction solution is extracted with ethyl acetate, dried over anhydrous MgSO4, and filtered. The filtrate is concentrated in vacuo and purified by silica gel column chromatography to obtain γ-lactone. All experiments were carried out three times in parallel to calculate the yield, and the results are shown in the following table:

[0046]

[0047] Example of γ-lactone catalysis by P450BSβ-L78G / Q85F / F173S / G290I

[0048] The biocatalyst constructed by the present invention uses cheap hydrogen peroxide as a direct electron donor, and uses natural saturated fatty acids with a chain length of C6 to C18 and non-natural carboxylic acids containing one or more functional groups of terminal or internal olefins / alkynes, bromine, esters, amines, nitriles, oxygen-sensitive hydroxyl motifs, cyclohexyl, N-, O-, S-heteroaromatic rings, thiazole, pyridine, quinoline, benzothiazole, furan, thiophene, oxazole, benzofuran, and benzothiophene as substrates to synthesize the corresponding (S)-γ-lactone in one step. The method has the following advantages: 1) the novel fatty acid deacidase P450BSβ mutant has a wider range of catalytic substrates, and can catalyze not only natural straight-chain saturated fatty acids, but also non-natural carboxylic acids. 2) the novel fatty acid deacidase P450BSβ mutant catalyzes the synthesis of γ-lactone from carboxylic acids without the need for NAD(P)H redox chaperones and auxiliary factors, and the reaction system is simple and inexpensive. 3) The reaction process is mild and is carried out in an aqueous solution at room temperature. It has high reactivity, high regioselectivity and enantioselectivity, and mild reaction conditions. It has good industrial production prospects in the synthesis of γ-lactone.

[0049] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A fatty acid decarboxylase P450BSβ mutant, characterized in that Prepared by a method comprising the following steps: Step 1: Construction of plasmid expressing fatty acid decarboxylase P450BSβ The nucleotide sequence shown in SEQ ID NO: 2 was digested and recombined into the expression vector pET28a; Step 2: transforming the plasmid expressing fatty acid decarboxylase P450BSβ constructed in step 1 into Escherichia coli; Step 3: Expression of fatty acid decarboxylase P450BSβ The strain obtained in step 2 was cultured overnight in LB liquid medium, then added to TB liquid medium for expansion, and after adding heme precursor δ-aminolevulinic acid and IPTG to induce expression in the late logarithmic phase, the bacteria were collected by centrifugation; Step 4: Preparation of cell lysate containing fatty acid decarboxylase P450BSβ Resuspend the cells obtained in step 3 with a buffer, lyse the cells by ultrasonication, and centrifuge to obtain a supernatant; Step 5: Purification of fatty acid decarboxylase P450BSβ The supernatant obtained in step 4 is added to a pre-treated nickel ion affinity column to allow the target protein with the His tag to bind to the nickel column, and then the impurities are removed at a lower imidazole concentration, and finally the target protein is eluted with a buffer containing a high concentration of imidazole; Step 6: The eluted target protein is dialyzed to remove imidazole, the protein is concentrated and lyophilized to prepare lyophilized powder, the target protein concentration is determined, and then stored at -80°C.

2. The use of the fatty acid decarboxylase P450BSβ mutant according to claim 1, characterized in that: Using fatty acids with chain lengths of C6 to C18 as substrates, hydrogen peroxide as an oxidant, and a fatty acid decarboxylase P450BSβ mutant as a catalyst, the hydroxyl group on the γ-carbon atom of the fatty acid is catalyzed to dehydrate and condense with the carboxylic acid to form γ-lactone.

3. The use according to claim 2, characterized in that: During the catalytic reaction, the reaction is carried out at room temperature, the reaction system is an aqueous buffer system, and Triton X-100 and DMSO are used as cosolvents.

4. The use according to claim 3, characterized in that: The buffer composition is: 0.1 M KPi, 0.3 M KCl, pH 7.

0.

5. The use according to claim 2, characterized in that: The fatty acid is selected from natural saturated fatty acids or non-natural carboxylic acids with a chain length of C6 to C18.

6. The use according to claim 5, characterized in that: The structure of the non-natural carboxylic acid contains one or more of terminal or internal olefins or alkynes, bromine, esters, amines, nitriles, oxygen-sensitive hydroxyl motifs, and cyclohexyl groups; the fatty acid is an aromatic or heteroaromatic fatty acid, and its structure contains one or more functional groups of N-, O-, S-heteroaromatic rings, thiazole, pyridine, quinoline, benzothiazole, furan, thiophene, oxazole, benzofuran, and benzothiophene.

7. The use according to claim 2, characterized in that: The γ-lactone is an S-type γ-lactone.

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