A baeyer-villiger monooxygenase mutant and its use in the preparation of 9-hydroxynonanoic acid

Abstract

The present application relates to a kind of Baeyer-Villiger monooxygenase mutant and its application in 9-hydroxynonanoic acid preparation.The specific application provides a kind of Baeyer-Villiger monooxygenase PpBVMO mutant derived from Pseudomonas putida with significantly improved catalytic activity and significantly improved substrate inhibition, its encoding nucleic acid, recombinant expression vector containing the nucleic acid sequence and recombinant expression transformant, and the application of using the recombinant Baeyer-Villiger monooxygenase mutant or recombinant expression transformant as catalyst to catalyze synthesis 9-hydroxynonanoic acid.Compared with prior art, the Baeyer-Villiger monooxygenase of the present application has the advantages of high activity, weak substrate inhibition, mild reaction condition, green and environmentally friendly process, simple operation, easy to scale up, etc., and has good application prospect in the production of 9-hydroxynonanoic acid.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and specifically relates to a Baeyer-Villiger monooxygenase mutant, a nucleic acid encoding the Baeyer-Villiger monooxygenase mutant, a recombinant expression vector and a recombinant expression transformant containing the nucleic acid, a method for preparing the recombinant Baeyer-Villiger monooxygenase mutant, and an application of the recombinant Baeyer-Villiger monooxygenase mutant in the preparation of 9-hydroxynonanoic acid. Background Art

[0002] Plant oils and fats are a vital component of biomass resources and have become a crucial renewable raw material in the chemical industry, widely used in surfactants, cosmetics, and lubricants. For example, as early as 1864, the industrial production of oilcloth, primarily made from linseed oil, was established in London, England. This oilcloth is more durable and environmentally friendly than polyvinyl chloride (PVC).

[0003] With the advancement of deep-processing technology for vegetable oils and fats, their utilization has expanded to the synthesis of high-value-added functional chemicals, such as medium-chain ω-hydroxy fatty acids and α,ω-dicarboxylic acids. As important functional chemicals, the presence of reactive groups in these molecules allows them to be used not only as surfactants, lubricants, adhesives, and rust inhibitors, but also in the synthesis of high-performance polymers such as polyesters and polyamides. 1,9-Azelaic acid, among others, is widely used in the production of polyamides and polyesters, pharmaceuticals, plasticizers, lubricants, and hydraulic fluids, and its synthesis pathway has garnered considerable attention.

[0004] Currently, the production of 9-hydroxynonanoic acid, an intermediate of 1,9-nonanedioic acid, mainly involves chemical synthesis and enzymatic hydroxylation of fatty acids. Traditional chemical methods for preparing medium-chain ω-hydroxy fatty acids generally require high temperatures, strong acids, or toxic oxidants and strong reducing agents. These methods include the following: High-purity monomeric ω-hydroxy fatty acids with a carbon chain length greater than 6 are usually prepared by saponification of their corresponding lactones or ω-hydroxy carboxylates, followed by sulfuric acid acidification and freeze-drying ( Org. Process. Res. Dev. 2006, 10, 481); prepared by cleavage of alkali and castor oil at high temperature and under strong base catalysis. The enzymatic terminal hydroxylation of fatty acids is limited by the specificity of the cytochrome P450 enzyme system for substrate action and low catalytic activity ( Chem. Commun. (Camb). 2012, 48, 5115); a multi-step enzymatic cascade preparation starting from cyclohexanol. The substrate cyclohexanol is mainly prepared by phenol hydrogenation or cyclohexane oxidation. The former requires high temperature and high pressure conditions, while the latter requires a complex reaction pathway and is accompanied by the generation of multiple by-products.

[0005] Starting from vegetable oils, the biocleavage pathway of unsaturated fatty acids involving Baeyer-Villiger monooxygenase (BVMO) can efficiently produce 9-hydroxynonanoic acid, an intermediate of 1,9-nonanedioic acid, which has the advantages of mild reaction conditions, wide and renewable raw materials, etc. compared with traditional chemical synthesis and enzymatic hydroxylation of fatty acids, and is an important direction of green chemistry development.

[0006] Currently, the most important rate-limiting enzyme in the synthesis of 9-hydroxynonanoic acid is Baeyer-Villiger monooxygenase. Baeyer-Villiger oxidation reaction refers to the reaction that a ketone compound introduces an oxygen atom between the carbonyl group and its adjacent hydrocarbon group under the oxidation of peroxide (such as peroxycarboxylic acid, hydrogen peroxide, etc.), thereby generating the corresponding ester or lactone. Specifically, NADPH is first combined with the enzyme molecule containing FAD, FAD is reduced, and at the same time, NADPH undergoes a conformational change to better stabilize the reduced FAD. Molecular oxygen reacts with the reduced FAD to form a peroxidized intermediate. Next, the substrate is loosely bound to the pocket of the enzyme molecule and triggers the recognition of the flexible loop to the substrate, so that the enzyme molecule adopts a more tightly bound conformation. After the substrate is accepted by the enzyme molecule, the substrate will further react to form a Kiegiel intermediate, which further undergoes rearrangement reaction, thereby releasing the product, and releasing water molecules and NADPH. + ( Sci. Rep. 2018, 8, 11). Baeyer-Villiger monooxygenase catalyzes 10-carbonyloctadecanoic acid to convert into nonanoic acid-9-carboxylnonyl ester by inserting an oxygen atom on the side with high substituents, and then forms 9-hydroxynonanoic acid and n-nonanoic acid by alkaline hydrolysis. Therefore, the molecular modification to improve the catalytic performance of Baeyer-Villiger monooxygenase in the synthesis of 9-hydroxynonanoic acid has attracted widespread attention from researchers, among which the most studied is the BVMO from Pseudomonas putida KT2400. Researchers found that cysteine at position 302 of the BVMO is a key amino acid site affecting its stability, and the mutant obtained by mutating it to leucine has enhanced oxidation stability and thermal stability (Biotechnol. Lett. 2016, 6, 9), which lays the foundation for the efficient synthesis of 9-hydroxynonanoic acid. Pseudomonas putida Pp Pp Sci. Rep. Pseudomonans putida Pp Metab. Eng. ​​​​​​2019,54, 137). The researchers obtained Pp The three-dimensional structure of BVMO was investigated, and molecular modifications were performed based on this to obtain mutants with improved oxidative and thermal stability. In addition, the stability of the exogenous plasmid in the host Escherichia coli was improved through a stable and regulatable plasmid (STAPL) system, and the expression level of BVMO was optimized. On this basis, fatty acid double bond hydratase, long-chain secondary alcohol dehydrogenase, and the whole cell catalyst of the modified PpBVMO were recombinantly expressed, and the yield of C9 chemicals from oleic acid reached 6 mmol / g dry cell, which was about 2.4 times higher than before the modification ( ACS. Synth. Biol. 2019, 8, 1055).

[0007] In summary, in the research on the synthesis of 9-hydroxynonanoic acid from oleic acid, the monooxygenase from Pseudomonas putida is currently widely used in this biotransformation pathway. Pp Researchers have used strategies such as coupling esterases and molecular engineering of the component enzymes to reduce catalyst usage and improve overall conversion efficiency. However, Baeyer-Villiger monooxygenases still suffer from common drawbacks such as severe substrate inhibition and poor catalytic activity. These drawbacks inevitably lead to low concentrations of the target product in the reaction system, significantly complicating product isolation and increasing separation costs, thus significantly hindering their industrial application. Therefore, to enable large-scale biocatalytic production of 9-hydroxynonanoic acid, the development of highly active Baeyer-Villiger monooxygenases with minimal substrate inhibition is crucial. Summary of the Invention

[0008] To address the shortcomings of existing Baeyer-Villiger monooxygenase (BVMO), such as severe substrate inhibition and poor catalytic activity, the present invention provides a Baeyer-Villiger monooxygenase mutant with high catalytic activity, high thermal stability, and good substrate tolerance, a nucleic acid encoding the Baeyer-Villiger monooxygenase mutant, a recombinant expression vector and a recombinant expression transformant containing the Baeyer-Villiger monooxygenase mutant gene, a method for preparing the recombinant Baeyer-Villiger monooxygenase mutant catalyst, and a method for synthesizing 9-hydroxynonanoic acid using the recombinant Baeyer-Villiger monooxygenase mutant.

[0009] The purpose of the present invention can be achieved by the following technical solutions:

[0010] One of the technical solutions adopted in the present invention:

[0011] The present invention provides a Baeyer-Villiger monooxygenase mutant, which is a derivative protein obtained by replacing one or more amino acid residues in the amino acid sequence shown in SEQ ID No. 2, and the Baeyer-Villiger monooxygenase mutant has a higher Pp BVMO M0 Higher enzyme activity, Pp BVMO M0 The amino acid sequence is shown in SEQ ID No. 2.

[0012] The present invention is based on the E6- Pp BVMO C302L / M340L ( Metab. Eng. 2019, 54, 137) and laboratory-constructed co-expression Gs BVMO C308L Starting from the two monooxygenases, the soluble expression, crude enzyme conversion rate and pure enzyme specific activity of the two enzymes were comprehensively compared. The results showed that the enzymes from Bacillus putida Pseudomonas putida Baeyer-Villiger monooxygenase mutant E6 of KT2400 Pp BVMO C302L / M340L It has high catalytic activity. The amino acid sequence of the enzyme is shown in SEQ ID No. 2, hereinafter referred to as Pp BVMO M0 , whose nucleotide sequence is shown in SEQ ID No. 1.

[0013] On this basis, site-directed saturation mutagenesis was used to Pp BVMO M0 Perform directed evolution. Use AlphaFold according to Pp BVMO M0 The sequence was modeled and the model structure was molecularly docked with the substrate 10-carbonyloctadecanoic acid to determine the site where the enzyme binds to the substrate. Key sites were selected for modification and screening to obtain a group of mutants with improved activity. Specifically, the sequence of the Baeyer-Villiger monooxygenase mutant is shown below:

[0014] (1) replacing leucine at position 88 of the amino acid sequence shown in SEQ ID No. 2 with isoleucine;

[0015] (2) replacing glutamine at position 229 of the amino acid sequence shown in SEQ ID No. 2 with methionine;

[0016] (3) replacing glutamine at position 229 of the amino acid sequence shown in SEQ ID No. 2 with isoleucine;

[0017] (4) replacing glutamine at position 229 of the amino acid sequence shown in SEQ ID No. 2 with threonine;

[0018] (5) replacing glutamine at position 229 of the amino acid sequence shown in SEQ ID No. 2 with cysteine;

[0019] (6) replacing serine at position 309 of the amino acid sequence shown in SEQ ID No. 2 with arginine;

[0020] (7) replacing serine at position 309 of the amino acid sequence shown in SEQ ID No. 2 with valine;

[0021] (8) replacing serine at position 403 of the amino acid sequence shown in SEQ ID No. 2 with cysteine;

[0022] (9) replacing the serine at position 403 of the amino acid sequence shown in SEQ ID No. 2 with threonine;

[0023] (10) The glutamine at position 229 of the amino acid sequence shown in SEQ ID No. 2 is replaced by threonine, and the serine at position 309 is replaced by arginine;

[0024] (11) The leucine at position 88 of the amino acid sequence shown in SEQ ID No. 2 is replaced by isoleucine, and the glutamine at position 229 is replaced by threonine;

[0025] (12) replacing the leucine at position 88 of the amino acid sequence shown in SEQ ID No. 2 with isoleucine, and replacing the serine at position 309 with arginine;

[0026] (13) The amino acid sequence shown in SEQ ID No. 2 is substituted with isoleucine at position 88, glutamine at position 229 with threonine, and serine at position 309 with arginine.

[0027] The second technical solution adopted by the present invention is:

[0028] Provided is a nucleic acid encoding the Baeyer-Villiger monooxygenase mutant as described in technical solution 1.

[0029] The method for preparing the nucleic acid of the present invention is a conventional method in the art, and the method preferably comprises:

[0030] A DNA molecule encoding a Baeyer-Villiger monooxygenase mutant is obtained by gene cloning technology; or a DNA molecule encoding a Baeyer-Villiger monooxygenase mutant is obtained by artificial sequence total synthesis.

[0031] The method of obtaining DNA encoding Baeyer-Villiger monooxygenase mutant by gene cloning technology of the present invention is as follows:

[0032] Forward primer 5'- ATGGAATTC GAATTCATGGAAAGAAGAAG-3' (SEQ ID No. 3),

[0033] Reverse primer 5'- TTGAAGCTT AAGCTTTTAGTGGTGGTGG-3' (SEQ ID No. 4),

[0034] The DNA sequence of the Baeyer-Villiger monooxygenase mutant in the technical solution 1 is amplified using polymerase chain reaction technology.

[0035] The present invention provides the following feasible gene amplification methods:

[0036] PCR system (50 μL): 2 × Prime Star Mix 20 μL, template plasmid approximately 100 ng, upstream and downstream primers 1.5 μL each, and ddH2O to 50 μL.

[0037] PCR reaction procedure: (1) pre-denaturation at 95°C for 3 min; (2) denaturation at 98°C for 10 s; (3) annealing at 60°C for 15 s; (4) extension at 72°C for 1.5 min; (5) steps (2) to (4) were repeated for 30 cycles, with a final extension at 72°C for 10 min and storage at 4°C.

[0038] The third technical solution adopted by the present invention is:

[0039] A recombinant expression vector comprising the nucleic acid sequence of the Baeyer-Villiger monooxygenase mutant described herein is provided. This vector can be constructed by ligating the nucleic acid sequence of the Baeyer-Villiger monooxygenase mutant gene described herein to various suitable vectors using conventional methods in the art. The vector can be any conventional vector in the art, preferably a plasmid, more preferably the plasmid pMAL-c5x. The Baeyer-Villiger monooxygenase mutant gene can be operably linked downstream of a regulatory sequence suitable for expression in the selected vector to achieve constitutive or inducible expression of the Baeyer-Villiger monooxygenase mutant.

[0040] Preferably, the recombinant expression vector of the present invention can be prepared by the following method: the gene sequence DNA fragment of the Baeyer-Villiger monooxygenase mutant obtained by PCR amplification is cleaved with restriction endonucleases Eco RI and Hind III double enzyme digestion, and the empty plasmid pMAL-c5x was also digested with restriction enzymes Eco RI and Hind The enzyme digestion was performed by double enzyme digestion with 1:133, and the enzyme-digested gene DNA fragment and the pMAL-c5x plasmid were recovered by gel electrophoresis. The fragments were ligated with T4 DNA ligase to obtain a recombinant expression plasmid containing the Baeyer-Villiger monooxygenase mutant.

[0041] The fourth technical solution adopted by the present invention is:

[0042] Provided is a recombinant expression transformant comprising the Baeyer-Villiger mutant gene of the present invention or its recombinant expression vector. The recombinant expression transformant can be obtained by transforming the recombinant expression vector of the present invention into a host cell. The host cell can be any conventional host cell in the art, provided that the recombinant expression vector can stably replicate on its own and the Baeyer-Villiger monooxygenase gene carried by the host cell can be effectively expressed. The preferred host cell of the present invention is Escherichia coli, more preferably Escherichia coli. E. coli BL21 (DE3).

[0043] The fifth technical solution adopted by the present invention is:

[0044] A method for preparing a recombinant Baeyer-Villiger monooxygenase mutant is provided, comprising the steps of culturing the recombinant expression transformant described herein to obtain the recombinant Baeyer-Villiger monooxygenase mutant. The culture medium used to culture the recombinant expression transformant can be selected from conventional culture media in the art, provided that the transformant can grow and produce the Baeyer-Villiger monooxygenase mutant of the present invention. The specific operations for culturing the transformant can be performed according to conventional procedures in the art.

[0045] The present invention provides a method for preparing a recombinant Baeyer-Villiger monooxygenase mutant: 20 μL of the recombinant strain glycerol culture of the recombinant Escherichia coli constructed by the above technical solution is inoculated into 4 mL of LB liquid medium (containing 50 μg mL -1The culture was shaken at 200 rpm at 37°C for 12 h, and 1 mL of bacterial solution was transferred to 100 mL of LB liquid medium (containing 50 μg mL -1 The cells were cultured in a 500 mL shake flask containing ampicillin and shaken at 200 rpm at 37°C for 3-4 h until the OD 600 The expression of the target protein was induced by adding IPTG (isopropyl-β-D-thiogalactopyranoside) to a final concentration of 0.2 mM using a syringe. After induction, the cells were cultured in a shaker at 16°C and 200 rpm for 24 h. The cells were centrifuged at 8000 × g The cells were collected by centrifugation (100 mM, pH 9.0) and resuspended in 10 mL of Gly-NaOH buffer (100 mM, pH 9.0). The cells were ultrasonically disrupted in an ice-water bath at 30% power for 15 min to obtain a crude enzyme solution.

[0046] The protein of the present invention contains a histidine tag (His-Tag) at the N-terminus, so Ni NTA Beads6FF nickel column can be used for protein purification. The enzyme protein purification buffers are: Solution A: 50 mM sodium phosphate buffer (pH 8.0), containing 300 mM NaCl and 20 mM imidazole; Solution B: 50 mM sodium phosphate buffer (pH 8.0), containing 300 mM NaCl and 500 mM imidazole. Use five times the column volume of deionized water to rinse the nickel column, load the crude enzyme solution onto the nickel column, then rinse with ten times the column volume of Solution A and then elute with fifteen to twenty times the column volume of Solution B, and collect the eluate. Use SDS-PAGE protein electrophoresis to verify the purification of the purified protein, then use ultrafiltration tube centrifugation to remove imidazole, determine the protein concentration with a Bradford kit, and store at -80°C for future use after aliquoting.

[0047] The sixth technical solution adopted by the present invention is:

[0048] Application of the Baeyer-Villiger monooxygenase mutant in the synthesis of 9-hydroxynonanoic acid.

[0049] Specifically, the Baeyer-Villiger monooxygenase mutant was used to catalyze the reaction of 10-carbonyloctadecanoic acid to produce 9-carboxynonyl nonyl ester, which was then hydrolyzed in a 1 M KOH solution (methanol / water = 4 / 1) at 60° C. for 2 h to form 9-hydroxynonanoic acid and n-nonanoic acid.

[0050] The buffer salt system of the buffer solution is not limited, as long as its pH range is 6.0 9.0; the preferred buffer salt system is Gly-NaOH, pH 9.0. The reaction temperature is 20 65 ° C, preferably 25 ° C. An appropriate amount of water-soluble solvent or water-insoluble solvent can be added to the buffer solution. Other reaction conditions such as substrate concentration, enzyme dosage, etc. can be selected according to the conventional conditions of this type of reaction in the art.

[0051] The Baeyer-Villiger monooxygenase mutant of the present invention is used as a catalyst. Alternatively, an immobilized enzyme obtained by immobilizing a crude enzyme solution or pure enzyme onto a suitable carrier can be used as the enzyme catalyst.

[0052] In some embodiments of the present invention, the conditions for the catalytic reaction are: enzyme concentration of 1 to 100 g / L, substrate 10-carbonyloctadecanoic acid concentration of 3-20 mM, reaction temperature of 20 to 45° C., pH 3 to 9.5, and reaction time of 0.5 to 24 h.

[0053] Samples were taken intermittently during the reaction, and the conversion was analyzed by gas chromatography under the following conditions: To detect fatty acids, the reaction samples were derivatized with methyl esters before gas chromatography analysis. Methylation method: The reaction solution was acidified to pH 2.0 with 20% H2SO4. 100 μL of the acidified reaction solution was extracted with 200 μL of ethyl acetate containing 0.5 mM n-hexadecane (internal standard). The mixture was then shaken and centrifuged (13,000 × 4°C). g , 3 min). The organic phase was transferred to a new 1.5 mL EP tube and dried overnight over anhydrous Na₂SO₄. For derivatization, the system consisted of 50 μL of sample, 50 μL of a methanol / diethyl ether mixture (equal volumes), and 20 μL of (trimethylsilyl)diazomethane. The mixture was allowed to stand at room temperature for 30 min before gas chromatography analysis. Samples were analyzed using a SHIMA DZU Nexis GC-2030 system equipped with a flame ionization detector and an SH-Rtx-1 column (30.0 m × 0.25 mm, 0.25 µm). GC analysis conditions were: first, hold at 150°C for 1 min, then increase the temperature to 250°C at 10°C / min, and finally hold at 250°C for 3 min.

[0054] Compared with the prior art, the positive progress of the present invention is:

[0055] The Baeyer-Villiger monooxygenase mutant described in the present invention has high catalytic activity, strong substrate tolerance, and high comprehensive conversion efficiency. It can directly catalyze the oxygenation of 10-carbonyl octadecanoic acid, which is then hydrolyzed to form n-nonanoic acid and 9-hydroxynonanoic acid. The product n-nonanoic acid can be hydroxylated and normalized to 9-hydroxynonanoic acid. This effectively avoids the steps of protecting and deprotecting the substrate in the chemical synthesis method and is more environmentally friendly. Compared with other reported 9-hydroxynonanoic acid synthesis methods, the preparation of medium-chain ω-hydroxy fats such as 9-hydroxynonanoic acid using the method described in the present invention has the advantages of mild reaction conditions, no need for substrate pretreatment, and an environmentally friendly process, and has good application development prospects. DETAILED DESCRIPTION

[0056] The present invention is described in detail below with reference to specific embodiments.

[0057] The following examples will help to further understand the present invention, but are not intended to limit the present invention.

[0058] Example 1 Bacillus putida E6- Pp BVMO C302L / M340L Filter

[0059] The E6- Pp BVMO C302L / M340L and laboratory Gs BVMO C308L The corresponding recombinant E. coli strain was inoculated into 4 mL LB medium (containing 50 μg mL -1 After shaking at 200 rpm for 12 h at 37°C, 1 mL of the bacterial suspension was transferred to 100 mL of TB medium (containing 50 μg mL -1 Induce the culture in a 500 mL shake flask containing ampicillin. After 24 hours, harvest the cells by centrifugation. Resuspend the cells in 10 mL of 100 mM Gly-NaOH buffer (pH 9.0) and sonicate at 30% power for 15 minutes in an ice-water bath to obtain a crude enzyme solution.

[0060] The activity of the crude enzymes of the two enzymes on the substrate 10-oxooctadecanoic acid was verified by biocatalytic reaction. The total reaction system was 1 mL, containing a final concentration of 3 mM 10-oxooctadecanoic acid (0.9 g / L, 10% DMSO), 100 mM Gly-NaOH buffer (pH 9.0), 10 g / L Tween 80, 5 mM D-glucose, 0.2 mM NADP +, 5 g / L BmGDH and an appropriate concentration of crude enzyme were added and shaken at 25°C and 1000 rpm for 3 h. After the reaction was completed, 50 μL of 20% H2SO4 was added to terminate the reaction, and an equal volume of ethyl acetate was added for extraction and shaken. 12000 × g After centrifugation for 3 minutes, the upper ethyl acetate layer was aspirated into an EP tube and dissolved in 40 μL of 1M KOH solution (methanol / water = 4 / 1). Hydrolysis was performed at 60°C for 2 hours. After the reaction was complete, the tube was removed and cooled on ice for 2 minutes. 160 μL of 20% H2SO4 was added to terminate the reaction. An equal volume of ethyl acetate was added and extracted again by shaking. Gas chromatography was used to analyze the reaction results. The crude enzyme catalyzed 10-carbonyl octadecanoic acid conversion rate and the catalytic activity of the pure enzyme were determined based on the reaction results. The results are shown in Table 1. Pp BVMO C302L / M340L The comprehensive catalytic performance is good, and this enzyme was selected for subsequent research and named Pp BVMO M0 .

[0061] Table 1 Comparison of catalytic performance of candidate strains

[0062]

[0063] Example 2 Recombinant Baeyer-Villiger Monooxygenase Pp BVMO M0 Preparation

[0064] With forward primer 5'- ATGGAATTC GAATTCATGGAAGAAGAAG-3', reverse primer 5'- TTGAAGCTT AAGCTTTTAGTGGTGGTGG-3', the Baeyer-Villiger monooxygenase screened in Example 1 was quenched by polymerase chain reaction Pp BVMO M0 The coding gene was amplified and the obtained amplified coding DNA fragment was cleaved with restriction endonucleases. Eco RI and Hind III double enzyme digestion, and the empty plasmid pMAL-c5x was also digested with restriction enzymes Eco RI and Hind The enzyme digestion was performed by double enzyme digestion with III, and the above enzyme digested gene DNA fragment and pMAL-c5x plasmid were recovered by gel electrophoresis, and connected with T4 DNA ligase to obtain the recombinant expression plasmid pMAL-c5x- Pp BVMO M0 .

[0065] The obtained recombinant plasmid was transformed into Escherichia coli E. coli The recombinant strain was inoculated into 4 mL of LB medium (containing 50 μg mL -1 After shaking and culturing at 37°C for 12 h, 1 mL of bacterial suspension was transferred to 100 mL of TB medium (containing 50 μg mL -1 The cells were cultured in a 500 mL shake flask containing ampicillin (100 mM HCl, 1% HCl, and 1% HCl). After incubation at 37°C for 3 h, 10 μL IPTG was added and the cells were induced at 16°C for 24 h. The cells were then collected by centrifugation. The cells were resuspended in 10 mL of Gly-NaOH buffer (100 mM, pH 9.0) and sonicated in an ice-water bath at 30% power for 15 min to obtain a crude enzyme solution. The nickel column was rinsed with five column volumes of deionized water, the crude enzyme solution was loaded onto the nickel column, and then rinsed with ten column volumes of Solution A, followed by elution with fifteen to twenty column volumes of Solution B. The eluate was collected. Imidazole was removed by ultrafiltration, and the protein concentration was determined using a Bradford kit. The cells were aliquoted and stored at -80°C with glycerol. Pp BVMO M0 The catalytic activity was 0.15 U mg -1 .

[0066] Example 3 Baeyer-Villiger monooxygenase Pp BVMO M0 Molecular modification

[0067] Apply AlphaFold to build Pp BVMO M0 A structural model of the 10-oxooctadecanoic acid substrate was constructed and docked to the substrate 10-oxooctadecanoic acid molecule. Nineteen amino acid residues within 5 Å of the substrate binding site were selected as targets for site-directed saturation mutagenesis. Degenerate NNK codons were designed for site-directed saturation mutagenesis of the target sites. Mutants were cultured in deep-well plates and shaken overnight at 37°C. After transfer, 50 μL was transferred to a secondary deep-well plate containing 600 μL. After shaking at 37°C for 3 h, IPTG was added for induction at a final concentration of 0.2 mM and incubated at 16°C for 24 h. The cells were then harvested by centrifugation, lysozyme was added, and 0.1% Triton X-100 was added to disrupt the cells for 1 h. An appropriate amount of enzyme solution was then mixed with 3 mM 10-oxooctadecanoic acid (0.9 g / L, 10% DMSO), 100 mM Gly-NaOH buffer (pH 9.0), 10 g / L Tween 80, 5 mM D-glucose, and 0.2 mM NADP. + , 5 g / L Bm GDH was shaken at 25°C and 1000 rpm for 3 h. The reaction solution was hydrolyzed and derivatized and then analyzed by gas chromatography. Preliminary screening was performed and thirteen mutants were found to be different from the parent Pp Compared with BVMO, its activity was increased. After the thirteen mutants were purified, the 9-hydroxynonanoic acid synthesis activity of their pure enzymes was determined. The results are shown in Table 2. The sequence numbers in Table 2 correspond to the series of sequences after Table 2. In the activity column, the activity of the mutants was significantly different from that of the parent. Pp Compared with BVMO, one plus sign “+” indicates that the mutant activity is increased by 1.0-2.0 times; two plus signs “++” indicate that the mutant activity is increased by 2.0-5.0 times; and three plus signs “+++” indicate that the mutant activity is increased by 5.0-10 times.

[0068] Table 2 List of Baeyer-Villiger monooxygenase mutants with improved activity

[0069]

[0070] The corresponding numbers in the table Pp The amino acid sequences of the BVMO mutants are as follows:

[0071] (1) replacing leucine at position 88 of the amino acid sequence shown in SEQ ID No. 2 with isoleucine;

[0072] (2) replacing glutamine at position 229 of the amino acid sequence shown in SEQ ID No. 2 with methionine;

[0073] (3) replacing glutamine at position 229 of the amino acid sequence shown in SEQ ID No. 2 with isoleucine;

[0074] (4) replacing glutamine at position 229 of the amino acid sequence shown in SEQ ID No. 2 with threonine;

[0075] (5) replacing glutamine at position 229 of the amino acid sequence shown in SEQ ID No. 2 with cysteine;

[0076] (6) replacing serine at position 309 of the amino acid sequence shown in SEQ ID No. 2 with arginine;

[0077] (7) replacing serine at position 309 of the amino acid sequence shown in SEQ ID No. 2 with valine;

[0078] (8) replacing serine at position 403 of the amino acid sequence shown in SEQ ID No. 2 with cysteine;

[0079] (9) replacing the serine at position 403 of the amino acid sequence shown in SEQ ID No. 2 with threonine;

[0080] (10) The glutamine at position 229 of the amino acid sequence shown in SEQ ID No. 2 is replaced by threonine, and the serine at position 309 is replaced by arginine;

[0081] (11) The leucine at position 88 of the amino acid sequence shown in SEQ ID No. 2 is replaced by isoleucine, and the glutamine at position 229 is replaced by threonine;

[0082] (12) replacing the leucine at position 88 of the amino acid sequence shown in SEQ ID No. 2 with isoleucine, and replacing the serine at position 309 with arginine;

[0083] (13) The amino acid sequence of SEQ ID No. 2 is substituted with isoleucine at position 88, glutamine at position 229 with threonine, and serine at position 309 with arginine;

[0084] Example 4 Baeyer-Villiger monooxygenase Pp BVMO M13 Expression and purification

[0085] Extract the recombinant plasmid pMAL-c5x- Pp BVMO M13 , which was transformed into Escherichia coli E. coli For BL21, the constructed recombinant strain was inoculated into a test tube containing 4 mL of culture medium. After shaking and culturing at 37°C for 12 hours, 1 mL of the culture was transferred to a shake flask containing 100 mL of TB medium. After culturing at 37°C for 3 hours, IPTG was added. After induction at 16°C for 24 hours, the cells were harvested by centrifugation. The cells were resuspended in 10 mL of Gly-NaOH buffer (100 mM, pH 9.0) and sonicated at 30% power in an ice-water bath for 15 minutes to obtain a crude enzyme solution. Rinse the nickel column with five column volumes of deionized water. Load the crude enzyme solution onto the nickel column, rinse with ten column volumes of Solution A, and then elute with fifteen to twenty column volumes of Solution B. The enzyme protein purification buffers A and B are as follows: Solution A: 50 mM sodium phosphate buffer (pH 8.0), containing 300 mM NaCl and 20 mM imidazole; Solution B: 50 mM sodium phosphate buffer (pH 8.0), containing 300 mM NaCl and 500 mM imidazole. Collect the eluate, remove the imidazole by centrifugation through an ultrafiltration tube, aliquot, add glycerol, and store at -80°C until needed.

[0086] Example 5 Baeyer-Villiger monooxygenase Pp BVMO M0 and its mutants T m Value determination

[0087] The temperature curve of protein was verified by circular dichroism spectrometry. T The m value determines its stability. Prepare protein samples and dilute the sample to 0.3–0.5 mg / mL with purified C solution, where the C solution formula is 50 mM sodium phosphate buffer (pH 8.0), containing 150 mM NaCl and 1 mM DTT. Set the scanning wavelength to 190 nm–260 nm, draw 200 μL of protein sample into a 1 mm thick cuvette, observe it through light to see if there are any bubbles, set the temperature to 20°C ~ 90°C, and scan once every 2°C. Use Global3 analysis software to process the relevant data to calculate the protein's m value. T m The results are shown in Table 3.

[0088] Table 3 PpBVMO and its mutants T m Value determination

[0089]

[0090] Example 6 Effect of pH on Baeyer-Villiger Monooxygenase Pp BVMO M13 The impact of vitality

[0091] Prepare buffer solutions of different pH values: pH 6.0-8.0 (KPB), pH 8.0-9.0 (Tris-HCl), pH 9.0-11.0 (Gly-NaOH). Pp BVMO M13 Pure enzyme was diluted to 0.05 mg / mL. Appropriate amounts of enzyme solution were added to 1 mL of different buffer solutions containing 3.0 mM 10-oxooctadecanoic acid, 0.05 mM NADPH, and 0.05 mM FAD. Enzyme activity was calculated by measuring the difference in absorbance at the characteristic absorption peak of the coenzyme NADPH at 340 nm using a UV-visible spectrophotometer. One unit of enzyme activity (U) is defined as the amount of enzyme required to oxidize 1 μmol of NADPH per minute.

[0092] The calculation formula of enzyme activity:

[0093] Enzyme activity (U) = (EW × V ×1000) / (6220 ×l)

[0094] The meaning of each parameter: EW is equal to the change in absorbance of the coenzyme NADPH at 340 nm in one minute; V is the volume of the activity test reaction solution, in mL; 6220 is the molar extinction coefficient, in L·mol ‒1 cm ‒1 ; l is the optical path, unit is cm.

[0095] The results are shown in Table 4. The relative activity of the enzyme was the highest in KPB buffer at pH 8.0, which was defined as 100%, and the relative activities at other pH values ​​were calculated.

[0096] Table 4 Baeyer-Villiger monooxygenases Pp BVMO M13 Activity at different pH

[0097]

[0098] Example 7 Baeyer-Villiger monooxygenase Pp BVMO M13 Catalyzes the synthesis of 9-hydroxynonanoic acid from 3 mM 10-carbonyloctadecanoic acid

[0099] The synthesis reaction of hydroxynonanoic acid was carried out in a 2 mL EP tube with a total volume of 1 mL, containing a final concentration of 3 mM 10-oxooctadecanoic acid (0.9 g / L, 10% DMSO), 100 mM KPB buffer (pH 8.0), 10 g / L Tween 80, 5 mM D-glucose, 0.2 mM NADP + , 5 g / L BmGDH freeze-dried enzyme powder and 5 g / L crude enzyme Pp BVMO M13 After shaking at 25°C and 1000 rpm for 3 h, the sample was removed and 50 μL of 20% H2SO4 was added to terminate the reaction. An equal volume of ethyl acetate was added for extraction and 12000 × gAfter centrifugation for 3 minutes, the upper ethyl acetate was aspirated into an EP tube, and 40 μL of 1 M KOH solution (methanol / water = 4 / 1) was added to dissolve it. It was hydrolyzed at 60°C to generate 9-hydroxynonanoic acid and n-nonanoic acid, and the reaction was continued for 2 hours. After the reaction was completed, the tube was taken out and cooled on ice for 2 minutes. 160 μL of 20% H2SO4 was added to terminate the reaction. An equal volume of ethyl acetate was added and the mixture was shaken and extracted again. The organic phase was transferred to a new 1.5 mL EP tube and dried overnight with the addition of anhydrous Na2SO4. Derivatization treatment was performed, and the system contained 50 μL of sample, 50 μL of methanol / ether mixture (equal volume), and 20 μL of (trimethylsilyl)diazomethane. After standing at room temperature for 30 minutes, gas phase analysis was performed. It was found that Pp BVMO M13 When catalyzed by 3 mM 10-carbonyloctadecanoic acid, the substrate conversion rate was greater than 99% within 3 h.

[0100] Example 8 Baeyer-Villiger Monooxygenase Pp BVMO M13 Catalyzes the synthesis of 9-hydroxynonanoic acid from 6 mM 10-carbonyloctadecanoic acid

[0101] The synthesis reaction of 9-hydroxynonanoic acid was carried out in a 2 mL EP tube with a total volume of 1 mL, containing a final concentration of 6 mM 10-oxooctadecanoic acid (0.18 g / L, 10% DMSO), 100 mM KPB buffer (pH 8.0), 10 g / L Tween 80, 10 mM D-glucose, 0.2 mM NADP + , 5 g / L Bm GDH and 5 g / L crude enzyme Pp BVMO M13 After shaking at 25°C and 1000 rpm for 3 h, the sample was removed and 50 μL of 20% H2SO4 was added to terminate the reaction. An equal volume of ethyl acetate was added for extraction and 12000 × g After centrifugation for 3 minutes, the upper ethyl acetate was aspirated into an EP tube, and 40 μL of 1 M KOH solution (methanol / water = 4 / 1) was added to dissolve it. It was hydrolyzed at 60°C to generate 9-hydroxynonanoic acid and n-nonanoic acid, and the reaction was continued for 2 hours. After the reaction was completed, the tube was taken out and cooled on ice for 2 minutes. 160 μL of 20% H2SO4 was added to terminate the reaction. An equal volume of ethyl acetate was added and the mixture was shaken and extracted again. The organic phase was transferred to a new 1.5 mL EP tube and dried overnight with the addition of anhydrous Na2SO4. Derivatization treatment was performed, and the system contained 50 μL of sample, 50 μL of methanol / ether mixture (equal volume), and 20 μL of (trimethylsilyl)diazomethane. After standing at room temperature for 30 minutes, gas phase analysis was performed. It was found that Pp BVMO M13 When catalyzing 6 mM 10-carbonyloctadecanoic acid, the substrate conversion rate was also greater than 99% within 3 h.

[0102] Comparative Example 1 Baeyer-Villiger monooxygenase Pp BVMO M0 Catalytic synthesis of 9-hydroxynonanoic acid

[0103] The synthesis reaction of 9-hydroxynonanoic acid was carried out in a 50 mL conical flask with a total volume of 10 mL, containing a final concentration of 6 mM 10-carbonyloctadecanoic acid (0.18 g / L, 10% DMSO), 100 mM KPB buffer (pH 8.0), 10 g / L Tween 80, 10 mM D-glucose, 0.2 mM NADP + , 5 g / L Bm GDH and 5 g / L crude enzyme Pp BVMO M0 The reaction was carried out at 25°C and 200 rpm for 7 h. Samples were taken intermittently during the reaction. 50 μL of 20% H2SO4 was added to the sample to terminate the reaction. An equal volume of ethyl acetate was added for extraction. 12000 × g After centrifugation for 3 minutes, the upper ethyl acetate was drawn into an EP tube, and 40 μL of 1 M KOH solution (methanol / water = 4 / 1) was added to dissolve it. It was hydrolyzed at 60°C to generate 9-hydroxynonanoic acid and n-nonanoic acid, and the reaction was continued for 2 hours. After the reaction was completed, the tube was taken out and cooled on ice for 2 minutes. 160 μL of 20% H2SO4 was added to terminate the reaction. An equal volume of ethyl acetate was added and the mixture was shaken and extracted again. The organic phase was transferred to a new 1.5 mL EP tube, and anhydrous Na2SO4 was added to dry overnight. Derivatization treatment was performed, and the system contained 50 μL of sample, 50 μL of methanol / ether mixture (equal volume), and 20 μL of (trimethylsilyl)diazomethane. After standing at room temperature for 30 minutes, gas phase analysis was performed. It was found that the use of Pp BVMO M0 When used as a catalyst, only 0.4 mM of 9-hydroxynonanoic acid and n-nonanoic acid was produced, with a conversion rate of 7%.

[0104] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. A Baeyer-Villiger monooxygenase mutant, characterized in that The sequence of the Baeyer-Villiger monooxygenase mutant is shown below: (1) replacing leucine at position 88 of the amino acid sequence shown in SEQ ID No. 2 with isoleucine; (2) replacing the leucine at position 88 of the amino acid sequence shown in SEQ ID No. 2 with isoleucine, and replacing the glutamine at position 229 with threonine; (3) replacing the leucine at position 88 of the amino acid sequence shown in SEQ ID No. 2 with isoleucine, and replacing the serine at position 309 with arginine; (4) The leucine at position 88 of the amino acid sequence shown in SEQ ID No. 2 is replaced by isoleucine, the glutamine at position 229 is replaced by threonine, and the serine at position 309 is replaced by arginine.

2. An isolated nucleic acid, characterized in that The nucleic acid encodes the Baeyer-Villiger monooxygenase mutant according to claim 1.

3. A recombinant expression vector, characterized in that: Comprising the nucleic acid according to claim 2.

4. A recombinant expression transformant, characterized in that: Comprising the recombinant expression vector according to claim 3.

5. A method for preparing the Baeyer-Villiger monooxygenase mutant according to claim 1, characterized in that: The method comprises the following steps: culturing the recombinant expression transformant according to claim 4 to obtain a Baeyer-Villiger monooxygenase mutant.

6. Use of the Baeyer-Villiger monooxygenase mutant according to claim 1 in the synthesis of 9-hydroxynonanoic acid.

7. The use according to claim 6, characterized in that The Baeyer-Villiger monooxygenase mutant according to claim 1 is used as a catalyst to catalyze the reaction of 10-carbonyl octadecanoic acid to generate 9-carboxynonyl nonyl ester of nonanoic acid, which is then hydrolyzed to form 9-hydroxynonanoic acid and n-nonanoic acid.

8. The use according to claim 7, characterized in that The crude enzyme solution of the Baeyer-Villiger monooxygenase mutant and the pure enzyme of the Baeyer-Villiger monooxygenase mutant were fixed on a carrier to obtain an immobilized enzyme used as an enzyme catalyst.

9. The use according to claim 7, characterized in that The conditions for the catalytic reaction are: enzyme concentration of 1 ~ 100 g / L, substrate 10-carbonyloctadecanoic acid concentration of 3-20 mM.

10. The use according to claim 7, characterized in that The reaction temperature is 20 ~ 45°C, pH 3 ~ 9.5, and the reaction time is 0.5 ~ 24 h.

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