Baeyer-Villiger monooxygenase mutant and application thereof in preparation of 9-hydroxynonanoic acid

Abstract

The invention relates to a Baeyer-Villiger monooxygenase mutant and an application of the Baeyer-Villiger monooxygenase mutant in preparation of 9-hydroxynonanoic acid. Specifically, the invention provides a Baeyer-Villiger monooxygenase PpBVMO mutant derived from Pseudomonas putida, a coding nucleic acid of the Baeyer-Villiger monooxygenase PpBVMO mutant, a recombinant expression vector containing the nucleotide sequence, a recombinant expression transformant containing the nucleotide sequence, and an application of the Baeyer-Villiger monooxygenase PpBVMO mutant in the preparation of the Pseudomonas putida. The invention also relates to an application of the recombinant Baeyer-Villiger monooxygenase mutant or the recombinant expression transformant as a catalyst for catalytic synthesis of 9-hydroxynonanoic acid. Compared with the prior art, the Baeyer-Villiger monooxygenase disclosed by the invention has the advantages of high activity, weak substrate inhibition, mild reaction conditions, green and environment-friendly process, simplicity and convenience in operation, easiness in amplification and the like, and has a better application prospect in production of 9-hydroxynonanoic acid.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, 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 preparation method of 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 are an important part of biomass resources and have become an important renewable raw material in the chemical industry. They are widely used in the fields of surfactants, cosmetics, and lubricants. For example, as early as 1864, the industrial production of oilcloth with linseed oil as the main component was realized in London, England. This oilcloth is more durable and environmentally friendly than polyvinyl chloride (PVC).

[0003] With the development of deep processing technology of vegetable oils, its development and utilization has been further expanded to the synthesis of high value-added functional chemicals, such as medium-chain ω-hydroxy fatty acids and α,ω-dibasic acids. As important functional chemicals, the presence of active groups in the molecules of ω-hydroxy fatty acids, α,ω-dibasic acids, and ω-amino fatty acids not only allows them to be used as surfactants, lubricants, adhesives, rust inhibitors, etc., but also can be used to synthesize polyesters, polyamides and other polymer materials with excellent performance. Among them, 1,9-nonanediol is widely used in the production of polyamides and polyesters, pharmaceuticals, plasticizers, lubricants or hydraulic oils, and its synthesis route has received widespread attention.

[0004] At present, there are two main production methods for 1,9-nonanediol intermediate 9-hydroxynonanediol: chemical synthesis and enzymatic hydroxylation of fatty acids. The traditional chemical method for preparing medium-chain ω-hydroxy fatty acids generally requires high temperature, strong acid or toxic oxidants and strong reducing agents, including the following methods: 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); using alkali and castor oil as raw materials, cracking under high temperature and strong base catalysis conditions. The enzymatic terminal hydroxylation of fatty acids is limited by the cytochrome P450 enzyme system's specific requirements for substrates and low catalytic activity (Chem. Commun. (Camb). 2012, 48, 5115). The multi-step enzymatic cascade preparation of 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 a variety of by-products.

[0005] Starting from plant oils and fats, the bio-cleavage pathway of unsaturated fatty acids involving Baeyer-Villiger monooxygenase (BVMO) can be used to prepare 1,9-nonanedioic acid intermediate 9-hydroxynonanoic acid in a green and efficient manner. Compared with traditional chemical synthesis and enzymatic hydroxylation of fatty acids, it has the advantages of mild reaction conditions and a wide and renewable source of raw materials. It is one of the important directions for the development of green chemistry.

[0006] At present, the most important rate-limiting enzyme in the synthesis pathway of 9-hydroxynonanoic acid is Baeyer-Villiger monooxygenase. The Baeyer-Villiger oxidation reaction refers to the reaction in which a ketone compound, under the oxidation of a peroxide (such as peroxycarboxylic acid, hydrogen peroxide, etc.), introduces an oxygen atom between the carbonyl group and its adjacent hydrocarbon group to generate the corresponding ester or lactone. Specifically, NADPH first binds to the enzyme molecule containing FAD, and FAD is reduced. 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 loosely binds to the pocket of the enzyme molecule and triggers the flexible loop to recognize the substrate, so that the enzyme molecule adopts a tighter binding conformation. After the substrate is accepted by the enzyme molecule, the substrate will further react to form a Krieger intermediate, which will further undergo a rearrangement reaction to release the product, and release water molecules and NADP. +(Sci.Rep.2018,8,11). When Baeyer-Villiger monooxygenase catalyzes 10-carbonyl octadecanoic acid, it inserts an oxygen atom on the side of the high-substituent group to convert it into nonanoic acid-9-carboxy nonyl ester, which is then hydrolyzed by alkaline to form 9-hydroxynonanoic acid and n-nonanoic acid. Therefore, 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 them, PpBVMO from Pseudomonas putida KT2400 has been studied more. Researchers found that cysteine ​​at position 302 in PpBVMO is a key amino acid site that affects its stability. The mutant obtained by mutating it to leucine has enhanced oxidative stability and thermal stability (Sci.Rep.2016,6,9), laying the foundation for the efficient synthesis of 9-hydroxynonanoic acid. Seo et al. first reported the multi-level transformation of the Baeyer-Villiger monooxygenase PpBVMO from Pseudomonans putida KT2440 (Metab. Eng. 2019, 54, 137). The researchers obtained the three-dimensional structure of PpBVMO through homology modeling, and based on this, they carried out molecular transformation to obtain mutants with improved oxidative stability and thermal stability. In addition, the stability of the exogenous plasmid in the host Escherichia coli was improved and the expression level of BVMO was optimized through a stable and regulatable plasmid (STAPL) system. On this basis, the whole cell catalyst of fatty acid double bond hydratase, long-chain secondary alcohol dehydrogenase and modified PpBVMO was recombinantly expressed, and the yield of C9 chemicals from oleic acid reached 6mmol / g stem cells, which was about 2.4 times higher than before the transformation (ACS. Synth. Biol. 2019, 8, 1055).

[0007] In summary, in the research on the synthesis of 9-hydroxynonanoic acid from oleic acid, the monooxygenase PpBVMO from Pseudomonas putida is currently widely used in this biotransformation pathway. Researchers have reduced the amount of catalyst and improved the overall conversion efficiency through strategies such as coupling esterases and molecular modification of component enzymes. However, there are still common shortcomings such as severe substrate inhibition and poor catalytic activity of Baeyer-Villiger monooxygenase. The existence of these shortcomings is bound to result in a low concentration of the target product in the reaction system, which brings great difficulties to the separation of the product and increases the cost of separation, thus greatly affecting its industrial application. Therefore, in order to enable the large-scale application of the process of preparing 9-hydroxynonanoic acid by biocatalysis, it is of great significance to develop a Baeyer-Villiger monooxygenase with high activity and weak substrate inhibition. Summary of the invention

[0008] In view of the shortcomings of the Baeyer-Villiger monooxygenase (BVMO) in the prior art, 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 by 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 affinity than PpBVMO. M0 Higher enzyme activity, PpBVMO M0 The amino acid sequence is shown in SEQ ID No.2.

[0012] The present invention is based on E6-PpBVMO reported in the literature C302L / M340L (Metab.Eng.2019,54,137) and the co-expressed GsBVMO constructed in the laboratory 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 Baeyer-Villiger monooxygenase mutant E6-PpBVMO from Pseudomonas putida KT2400 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 PpBVMO M0 , whose nucleotide sequence is shown in SEQ ID No.1.

[0013] On this basis, site-directed saturation mutagenesis was performed on PpBVMO M0 Directed evolution was performed using AlphaFold according to PpBVMO M0The 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. The key sites were selected for transformation and screening to obtain a batch of mutants with improved activity. Specifically, the sequence of the Baeyer-Villiger monooxygenase mutant is shown below:

[0014] (1) replacing the leucine at position 82 of the amino acid sequence shown in SEQ ID No. 2 with isoleucine;

[0015] (2) replacing glutamine at position 223 of the amino acid sequence shown in SEQ ID No. 2 with methionine;

[0016] (3) replacing the glutamine at position 223 of the amino acid sequence shown in SEQ ID No. 2 with isoleucine;

[0017] (4) replacing the glutamine at position 223 of the amino acid sequence shown in SEQ ID No. 2 with threonine;

[0018] (5) replacing glutamine at position 223 of the amino acid sequence shown in SEQ ID No. 2 with cysteine;

[0019] (6) replacing the serine at position 303 of the amino acid sequence shown in SEQ ID No. 2 with arginine;

[0020] (7) replacing the serine at position 303 of the amino acid sequence shown in SEQ ID No. 2 with valine;

[0021] (8) replacing the serine at position 397 of the amino acid sequence shown in SEQ ID No. 2 with cysteine;

[0022] (9) replacing the serine at position 397 of the amino acid sequence shown in SEQ ID No. 2 with threonine;

[0023] (10) The glutamine at position 223 of the amino acid sequence shown in SEQ ID No. 2 is replaced by threonine, and the serine at position 303 is replaced by arginine;

[0024] (11) the leucine at position 82 of the amino acid sequence shown in SEQ ID No. 2 is replaced by isoleucine, and the glutamine at position 223 is replaced by threonine;

[0025] (12) the leucine at position 82 of the amino acid sequence shown in SEQ ID No. 2 is replaced by isoleucine, and the serine at position 303 is replaced by arginine;

[0026] (13) The leucine at position 82 of the amino acid sequence shown in SEQ ID No. 2 is replaced by isoleucine, the glutamine at position 223 is replaced by threonine, and the serine at position 303 is replaced by 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 one.

[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 for obtaining DNA encoding a Baeyer-Villiger monooxygenase mutant by gene cloning technology of the present invention is:

[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 technical solution 1 is amplified using polymerase chain reaction technology.

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

[0036] PCR system (50 μL): 2× Prime Star Mix 20 μL, template plasmid about 100 ng, upstream and downstream primers 1.5 μL each, 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 30 times, followed by 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 of the present invention is provided. The recombinant expression vector can be constructed by connecting the nucleic acid sequence of the Baeyer-Villiger monooxygenase mutant gene of the present invention to various suitable vectors by conventional methods in the art. The vector can be various conventional vectors in the art, preferably a plasmid, more preferably a plasmid pMAL-c5x. The Baeyer-Villiger monooxygenase mutant gene can be operably connected to the downstream of a regulatory sequence suitable for expression in a 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 double-digested with restriction endonucleases EcoR I and Hind III, and the empty plasmid pMAL-c5x is also double-digested with restriction endonucleases EcoR I and Hind III, the above-mentioned enzyme-digested gene DNA fragment and pMAL-c5x plasmid are recovered by gel, and connected with T4DNA 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] A recombinant expression transformant comprising the Baeyer-Villiger mutant gene of the present invention or its recombinant expression vector is provided. 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 various conventional host cells in the art, provided that the recombinant expression vector can be used to stably replicate itself, 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 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 following steps: culturing the recombinant expression transformant of the present invention to obtain the recombinant Baeyer-Villiger monooxygenase mutant. The culture medium used for culturing the recombinant expression transformant can be selected from conventional culture medium in the art, provided that the transformant can grow and produce the Baeyer-Villiger monooxygenase mutant of the present invention. The specific operation of culturing the transformant can be performed according to conventional operations in the art.

[0045] The present invention provides a method for preparing a recombinant Baeyer-Villiger monooxygenase mutant: taking 20 μL of the recombinant strain glycerol bacterial liquid from the recombinant Escherichia coli constructed by the above technical scheme, and inoculating it in 4 mL of LB liquid culture medium (containing 50 μg mL -1 The culture tube was placed in a shaker at 37°C and shaken at 200 rpm for 12 h. 1 mL of the 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 in a 37°C shaker for 3-4 h until the OD 600 Approximately equal to 0.6, use a syringe to add IPTG (isopropyl-β-D-thiogalactoside) with a final concentration of 0.2mM to induce the expression of the target protein. After induction, shake and culture at 200rpm in a 16°C shaker for 24h, and collect the bacteria by centrifugation in a high-speed centrifuge (8000×g, 5min). Resuspend the bacteria in 10mL Gly-NaOH buffer (100mM, pH 9.0), and use an ultrasonic disruptor in an ice water bath at 30% power for 15min to obtain a crude enzyme solution.

[0046] The protein in the present invention contains a histidine tag (His-Tag) at the N-terminus, so a Ni NTABeads6FF nickel column can be used for protein purification. The enzyme protein purification buffers are: A solution: 50mM sodium phosphate buffer (pH8.0), containing 300mM NaCl and 20mM imidazole; B solution: 50mM sodium phosphate buffer (pH8.0), containing 300mM NaCl and 500mM 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 A solution and then use fifteen to twenty times the column volume of B solution for elution, and collect the eluate. Use SDS-PAGE protein electrophoresis to verify the purification of the purified protein, then use ultrafiltration tube centrifugal displacement to remove imidazole, Bradford kit to determine the protein concentration, and store at -80°C for standby 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 is used to catalyze the reaction of 10-carbonyl octadecanoic acid to generate nonanoic acid-9-carboxynonyl ester, which is then hydrolyzed in 1M KOH solution (methanol / water=4 / 1) at 60°C for 2h to form 9-hydroxynonanoic acid and nonanoic acid.

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

[0051] The Baeyer-Villiger monooxygenase mutant of the present invention is used as a catalyst, and the immobilized enzyme obtained by fixing the crude enzyme solution, pure enzyme, etc. on a suitable carrier can also be used as the enzyme catalyst.

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

[0053] During the reaction, samples were taken intermittently, and the conversion rate was analyzed by gas chromatography. The analysis conditions were as follows: In order to detect fatty acids, the reaction samples needed to be 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 taken, and 200 μL of ethyl acetate containing 0.5 mM n-hexadecane (internal standard) was added for extraction, then oscillated and centrifuged (13,000×g, 3 min), and the organic phase was transferred to a new 1.5 mL EP tube, and anhydrous Na2SO4 was added to dry overnight. For the derivatization treatment, the system contained 50 μL of sample, 50 μL of methanol / ether mixture (equal volume), and 20 μL of (trimethylsilyl) diazomethane, and gas phase analysis was performed after standing at room temperature for 30 min. The samples were analyzed using a SHIMA DZUNexis GC-2030 system equipped with a flame ionization detector and an SH-Rtx-1 column (30.0 m×0.25 mm, 0.25 μm). The GC analysis conditions were: first, keep at 150°C for 1 min, increase the temperature to 250°C at 10°C per minute, and finally keep at 250°C for 3 min.

[0054] Compared with the prior art, the positive and progressive effects of the present invention are:

[0055] The Baeyer-Villiger monooxygenase mutant of the present invention has high catalytic activity, strong substrate tolerance, and high comprehensive conversion efficiency, and can directly catalyze the oxygenation of 10-carbonyl octadecanoic acid, and then hydrolyze to form n-nonanoic acid and 9-hydroxynonanoic acid, and 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 to other reported 9-hydroxynonanoic acid synthesis methods, the method described in the present invention is used to prepare medium-chain ω-hydroxy fats such as 9-hydroxynonanoic acid, which has the advantages of mild reaction conditions, no need for pre-treatment of the substrate, and friendly process environment, 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-PpBVMO C302L / M340L Filter

[0059] The E6-PpBVMO reported in the literature was expressed C302L / M340L and laboratory GsBVMO C308L The corresponding recombinant E. coli strain was inoculated into 4 mL LB medium (containing 50 μg mL -1 After shaking for 12 h at 37°C at 200 rpm, 1 mL of the bacterial solution was transferred to 100 mL of TB medium (containing 50 μg mL -1 The cells were induced in a 500 mL shake flask containing ampicillin. After 24 h, the cells were collected by centrifugation. The cells were resuspended in 10 mL of Gly-NaOH buffer (100 mM, pH 9.0), and ultrasonically disrupted in an ice-water bath at 30% power for 15 min to obtain a crude enzyme solution.

[0060] The activity of the two enzyme crude enzyme solutions on the substrate 10-carbonyl octadecanoic acid was verified by biocatalytic reaction. The total reaction system was 1 mL, containing a final concentration of 3 mM 10-carbonyl octadecanoic 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 +, 5g / L BmGDH and crude enzyme of appropriate concentration were added and shaken at 25°C and 1000rpm for 3h. After the reaction was completed, 50μL 20% H2SO4 was added to terminate the reaction, and an equal volume of ethyl acetate was added for shaking extraction. After centrifugation at 12000×g for 3min, the upper layer of ethyl acetate was drawn into an EP tube, and 40μL 1M KOH solution (methanol / water = 4 / 1) was added to dissolve it, and hydrolyzed at 60°C for 2h. After the reaction was completed, it was taken out and cooled on ice for 2min, 160μL 20% H2SO4 was added to terminate the reaction, and an equal volume of ethyl acetate was added for shaking extraction again. The crude enzyme was analyzed by gas chromatography, and the size of the conversion rate of 10-carbonyl octadecanoic acid catalyzed by the crude enzyme and the catalytic activity of the pure enzyme were determined according to the reaction results. The results are shown in Table 1. Among them, E6-PpBVMO reported in the literature C302L / M340L The comprehensive catalytic performance of the enzyme was good, so this enzyme was selected for subsequent research and named PpBVMO. M0 .

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

[0062]

[0063] Example 2 Recombinant Baeyer-Villiger monooxygenase PpBVMO M0 Preparation

[0064] The forward primer 5'- ATGGAATTC GAATTCATGGAAGAAGAAG-3', reverse primer 5'- TTGAAGCTT AAGCTTTTAGTGGTGGTGG-3', the Baeyer-Villiger monooxygenase PpBVMO screened in Example 1 was PCR-treated with M0 The coding gene was amplified, and the obtained amplified coding DNA fragment was double-digested with restriction endonucleases EcoR I and Hind III. At the same time, the empty plasmid pMAL-c5x was also double-digested with restriction endonucleases EcoR I and Hind III. The above-digested gene DNA fragment and pMAL-c5x plasmid were recovered by gel, and connected with T4 DNA ligase to obtain the recombinant expression plasmid pMAL-c5x-PpBVMO M0 .

[0065] The obtained recombinant plasmid was transformed into E. coli BL21, and the constructed recombinant strain was inoculated into 4 mL LB medium (containing 50 μg mL -1 After shaking and culturing at 37°C for 12 h, 1 mL of the bacterial solution was transferred to 100 mL of TB medium (containing 50 μg mL -1Ampicillin) in a 500mL shake flask, culture at 37℃ for 3h, add 10μLIPTG, induce culture at 16℃ for 24h, and collect the bacteria by centrifugation. Resuspend the bacteria in 10mL Gly-NaOH buffer (100mM, pH 9.0), use an ultrasonic disruptor in an ice water bath at 30% power for 15min 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, then rinse with ten column volumes of solution A, and then elute with fifteen to twenty column volumes of solution B, and collect the eluate. Remove imidazole by ultrafiltration tube displacement, determine the protein concentration with a Bradford kit, add glycerol after aliquoting, and store at -80℃ for later use. PpBVMO M0 The catalytic activity is 0.15 U mg -1 .

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

[0067] Apply AlphaFold to build PpBVMO M0 The structural model of the substrate was docked with the substrate 10-carbonyloctadecanoic acid molecule. 19 amino acid residues were selected as targets for site-directed saturation mutagenesis within the range. The target site was subjected to site-directed saturation mutagenesis by designing NNK degenerate codons, and the mutants were cultured in deep-well plates. After shaking culture at 37°C overnight, 50 μL was transferred to a secondary deep-well plate containing 600 μL. After shaking culture at 37°C for 3 hours, IPTG with a final concentration of 0.2 mM was added for induction and cultured at 16°C for 24 hours. The bacteria were then collected by centrifugation, lysozyme was added, and 0.1% TritonX-100 was added as an auxiliary. After breaking for 1 hour, an appropriate amount of enzyme solution was taken and mixed with 3mM 10-carbonyloctadecanoic acid (0.9 g / L, 10% DMSO), 100mM Gly-NaOH buffer (pH9.0), 10g / L Tween 80, 5mM D-glucose, and 0.2mM NADP. + , 5g / L BmGDH, shaken at 25℃, 1000rpm for 3h. After hydrolysis and derivatization, the reaction solution was analyzed by gas chromatography for preliminary screening, and it was found that the activity of thirteen mutants was increased compared with the parent PpBVMO. After the thirteen mutants were purified, the 9-hydroxynonanoic acid synthesis activity of their pure enzymes was measured, and the results are shown in Table 2. The sequence numbers in Table 2 correspond to a series of sequences after Table 2. In the activity column, compared with the parent PpBVMO, 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; three plus signs "+++" indicate that the mutant activity is increased by 5.0-10 times.

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

[0069]

[0070]

[0071] The amino acid sequences of the PpBVMO mutants with corresponding numbers in the table are as follows:

[0072] (1) replacing the leucine at position 82 of the amino acid sequence shown in SEQ ID No. 2 with isoleucine;

[0073] (2) replacing glutamine at position 223 of the amino acid sequence shown in SEQ ID No. 2 with methionine;

[0074] (3) replacing the glutamine at position 223 of the amino acid sequence shown in SEQ ID No. 2 with isoleucine;

[0075] (4) replacing the glutamine at position 223 of the amino acid sequence shown in SEQ ID No. 2 with threonine;

[0076] (5) replacing glutamine at position 223 of the amino acid sequence shown in SEQ ID No. 2 with cysteine;

[0077] (6) replacing the serine at position 303 of the amino acid sequence shown in SEQ ID No. 2 with arginine;

[0078] (7) replacing the serine at position 303 of the amino acid sequence shown in SEQ ID No. 2 with valine;

[0079] (8) replacing the serine at position 397 of the amino acid sequence shown in SEQ ID No. 2 with cysteine;

[0080] (9) replacing the serine at position 397 of the amino acid sequence shown in SEQ ID No. 2 with threonine;

[0081] (10) The glutamine at position 223 of the amino acid sequence shown in SEQ ID No. 2 is replaced by threonine, and the serine at position 303 is replaced by arginine;

[0082] (11) the leucine at position 82 of the amino acid sequence shown in SEQ ID No. 2 is replaced by isoleucine, and the glutamine at position 223 is replaced by threonine;

[0083] (12) the leucine at position 82 of the amino acid sequence shown in SEQ ID No. 2 is replaced by isoleucine, and the serine at position 303 is replaced by arginine;

[0084] (13) the leucine at position 82 of the amino acid sequence shown in SEQ ID No. 2 is replaced by isoleucine, the glutamine at position 223 is replaced by threonine, and the serine at position 303 is replaced by arginine;

[0085] Example 4 Baeyer-Villiger monooxygenase PpBVMO M13 Expression and purification of

[0086] Extract the recombinant plasmid pMAL-c5x-PpBVMO obtained in Example 3 M13 , transformed into E. coli BL21, inoculated the constructed recombinant strain into a test tube containing 4 mL of culture medium, cultured at 37°C with shaking for 12 h, took 1 mL of bacterial solution and transferred it to a shake flask containing 100 mL of TB culture medium, cultured at 37°C for 3 h, added IPTG, induced cultured at 16°C for 24 h, and centrifuged to collect the bacteria. Resuspend the bacteria in 10 mL of Gly-NaOH buffer (100 mM, pH 9.0), and use an ultrasonic disruptor 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, and then eluted with fifteen to twenty column volumes of solution B, wherein the enzyme protein purification buffer A and solution B formulas are: solution A: 50mM sodium phosphate buffer (pH8.0), containing 300mM NaCl and 20mM imidazole; solution B: 50mM sodium phosphate buffer (pH 8.0), containing 300mM NaCl and 500mM imidazole. The eluate was collected. The imidazole was removed by centrifugation through an ultrafiltration tube, and glycerol was added after aliquoting and stored at -80°C for later use.

[0087] Example 5 Baeyer-Villiger monooxygenase PpBVMO M0 and its mutant T m Value determination

[0088] The temperature curve of the protein was verified by circular dichroism spectrometer, and its stability was determined by calculating its Tm value. Prepare protein samples and dilute the samples 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 through light to see if there are any bubbles, set the temperature to 20 ° C ~ 90 ° C, and scan every 2 ° C. Use Global3 analysis software to process relevant data to calculate the T of the protein. m The results are shown in Table 3.

[0089] Table 3 T of PpBVMO and its mutants m Value determination

[0090]

[0091]

[0092] Example 6 Effect of pH on the activity of Baeyer-Villiger monooxygenase PpBVMO M13 The impact of vitality

[0093] 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). M13 Pure enzyme was diluted to 0.05 mg / mL, and appropriate amount of enzyme solution was added to 1 mL of different buffer solutions containing 3.0 mM 10-carbonyl octadecanoic acid, 0.05 mM NADPH, and 0.05 mM FAD. The difference in absorbance of coenzyme NADPH at the characteristic absorption peak of 340 nm was measured by UV-visible spectrophotometer to calculate enzyme activity. Definition of enzyme activity unit (U): The amount of enzyme required to oxidize 1 μmol NADPH per minute is 1 enzyme activity unit.

[0094] The calculation formula of enzyme activity is:

[0095]

[0096] The meaning of each parameter: EW is equal to the change in absorbance of coenzyme NADPH at 340nm in one minute; V is the volume of the activity test reaction solution, unit is mL; 6220 is the molar extinction coefficient, unit is L·mol -1 cm -1 ; l is the optical path, unit is cm.

[0097] The results are shown in Table 4. In KPB buffer at pH 8.0, the relative activity of the enzyme was the highest, which was defined as 100%, and the relative activities at other pH values ​​were calculated.

[0098] Table 4 Baeyer-Villiger monooxygenase PpBVMO M13 Activity at different pH

[0099]

[0100]

[0101] Example 7 Baeyer-Villiger monooxygenase PpBVMO M13 Catalyzes the synthesis of 9-hydroxynonanoic acid from 3 mM 10-carbonyloctadecanoic acid

[0102] The synthesis reaction of 9-hydroxynonanoic acid was carried out in a 2 mL EP tube with a total volume of 1 mL, containing 3 mM 10-carbonyloctadecanoic 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 at a final concentration. + , 5g / L BmGDH freeze-dried enzyme powder and 5g / L crude enzyme PpBVMO M13 , after shaking and reacting at 25°C and 1000rpm for 3h, the sample was taken out and 50μL 20% H2SO4 was added to terminate the reaction, and an equal volume of ethyl acetate was added for shaking extraction. After centrifugation at 12000×g for 3min, the upper layer of ethyl acetate was drawn into an EP tube, and 40μL 1M KOH solution (methanol / water = 4 / 1) was added to dissolve it. It was hydrolyzed at 60°C to generate 9-hydroxynonanoic acid and nonanoic acid, and reacted for 2h. After the reaction was completed, it was taken out and cooled on ice for 2min, 160μL 20% H2SO4 was added to terminate the reaction, an equal volume of ethyl acetate was added for shaking extraction again, and the organic phase was transferred to a new 1.5mL EP tube, and anhydrous Na2SO4 was added to dry overnight. Derivatization treatment was performed, and the system contained 50μL sample, 50μL methanol / ether mixture (equal volume), and 20μL (trimethylsilyl) diazomethane. After being placed at room temperature for 30min, gas phase analysis was performed. PpBVMO was found M13 When catalyzed by 3 mM 10-carbonyloctadecanoic acid, the substrate conversion rate was greater than 99% within 3 h.

[0103] Example 8 Baeyer-Villiger monooxygenase PpBVMO M13 Catalyzes the synthesis of 9-hydroxynonanoic acid from 6 mM 10-carbonyloctadecanoic acid

[0104] The synthesis reaction of 9-hydroxynonanoic acid was carried out in a 2 mL EP tube with a total volume of 1 mL, containing 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 at a final concentration of + , 5g / L BmGDH and 5g / L crude enzyme PpBVMO M13 , after shaking and reacting at 25°C and 1000rpm for 3h, the sample was taken out and 50μL 20% H2SO4 was added to terminate the reaction, and an equal volume of ethyl acetate was added for shaking extraction. After centrifugation at 12000×g for 3min, the upper layer of ethyl acetate was drawn into an EP tube, and 40μL 1M KOH solution (methanol / water = 4 / 1) was added to dissolve it, and it was hydrolyzed at 60°C to generate 9-hydroxynonanoic acid and nonanoic acid, and reacted for 2h. After the reaction was completed, it was taken out and cooled on ice for 2min, 160μL 20% H2SO4 was added to terminate the reaction, an equal volume of ethyl acetate was added for shaking extraction again, and the organic phase was transferred to a new 1.5mL EP tube, and anhydrous Na2SO4 was added to dry overnight. Derivatization treatment was performed, and the system contained 50μL sample, 50μL methanol / ether mixture (equal volume), and 20μL (trimethylsilyl) diazomethane. After being placed at room temperature for 30min, gas phase analysis was performed. PpBVMO was found M13 When catalyzed by 6 mM 10-carbonyloctadecanoic acid, the substrate conversion rate was also greater than 99% within 3 h.

[0105] Comparative Example 1 Baeyer-Villiger monooxygenase PpBVMO M0 Catalytic synthesis of 9-hydroxynonanoic acid

[0106] The synthesis reaction of 9-hydroxynonanoic acid was carried out in a 50 mL conical flask with a total volume of 10 mL, containing 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 + , 5g / L BmGDH and 5g / L crude enzyme PpBVMO M0, shake and react at 25℃, 200rpm for 7h, take samples intermittently during the reaction, add 50μL 20% H2SO4 to the sample to terminate the reaction, add an equal volume of ethyl acetate to shake and extract, centrifuge at 12000×g for 3min, draw the upper ethyl acetate into an EP tube, add 40μL 1M KOH solution (methanol / water=4 / 1) to dissolve, hydrolyze at 60℃ to generate 9-hydroxynonanoic acid and nonanoic acid, and react for 2h. After the reaction is completed, take out and cool on ice for 2min, add 160μL 20% H2SO4 to terminate the reaction, add an equal volume of ethyl acetate to shake and extract again, transfer the organic phase to a new 1.5mL EP tube, add anhydrous Na2SO4 and dry overnight. Derivatization treatment was carried out, the system contained 50μL sample, 50μL methanol / ether mixture (equal volume), 20μL (trimethylsilyl) diazomethane, and gas phase analysis was performed after standing at room temperature for 30min. It was found that the use of PpBVMO M0 When used as a catalyst, only 0.4 mM of 9-hydroxynonanoic acid and n-nonanoic acid were produced, with a conversion rate of 7%.

[0107] The sequences involved in the present invention are as follows:

[0108] SEQ ID No.1:

[0109] Encoding Baeyer-Villiger monooxygenase E6 - PpBVMO C302L / M340L The nucleotide sequence

[0110]

[0111] SEQ ID No.2:

[0112] Amino acid sequence of Baeyer-Villiger monooxygenase E6-PpBVMO C302L / M340L of

[0113] MEEEEEESSHTALPVEPLDVLIMGAGVSGIGAAAYLRRNQPNKTFAILESRERMGGTWDLFRYPGIRSDSDLYTFGFDFKPWTKAKSLADAADILEYLSEAIDEHQLAPFIQYQQKVISANWQSDKGLWSVRVEDGRTAQIRTVECRWLFSAGGYYRYDQGFSPRFEGSEQFKGQIIHPQHWPEDLDYTGKRVVVIGSGATAVTLIPAMADKVASITMLQRTPSYIINQPANDGVAAFLRKVLPAQTAYSLTRYKNAKITLAFWGFCQRF PKLSKKLLLWLTRKELPKDYPVDVHFNPPYKPWDQRLLSVPEGDLFKAISAGNADIVTDHIERFTEHGVLLKSGKLLKADIIVTATGLNVQLFGGITLHKDGKPVVLSETLAYKGMMLSGVPNFAFAVGYTNSSWTLKVCLLCDHFCRLLGLMEREGYNVCEPKAPEGVETRPLLDFGAGYVQRALDSMPRQGPREPWVMSMDYFRDVKLLRRGAVTDKCLKFTAVPNAPLHADVQLQQQGSRR*

[0114] SEQ ID No.3:

[0115] Forward primer

[0116] ATGGAATTCGAATTCATGGAAGAAGAAG

[0117] SEQ ID No.4

[0118] Reverse primer

[0119] TTGAAGCTTAAGCTTTTAGTGGTGGTGG

[0120] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and 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 the leucine at position 82 of the amino acid sequence shown in SEQ ID No. 2 with isoleucine; (2) replacing glutamine at position 223 of the amino acid sequence shown in SEQ ID No. 2 with methionine; (3) replacing the glutamine at position 223 of the amino acid sequence shown in SEQ ID No. 2 with isoleucine; (4) replacing the glutamine at position 223 of the amino acid sequence shown in SEQ ID No. 2 with threonine; (5) replacing glutamine at position 223 of the amino acid sequence shown in SEQ ID No. 2 with cysteine; (6) replacing the serine at position 303 of the amino acid sequence shown in SEQ ID No. 2 with arginine; (7) replacing the serine at position 303 of the amino acid sequence shown in SEQ ID No. 2 with valine; (8) replacing the serine at position 397 of the amino acid sequence shown in SEQ ID No. 2 with cysteine; (9) replacing the serine at position 397 of the amino acid sequence shown in SEQ ID No. 2 with threonine; (10) The glutamine at position 223 of the amino acid sequence shown in SEQ ID No. 2 is replaced by threonine, and the serine at position 303 is replaced by arginine; (11) the leucine at position 82 of the amino acid sequence shown in SEQ ID No. 2 is replaced by isoleucine, and the glutamine at position 223 is replaced by threonine; (12) the leucine at position 82 of the amino acid sequence shown in SEQ ID No. 2 is replaced by isoleucine, and the serine at position 303 is replaced by arginine; (13) The leucine at position 82 of the amino acid sequence shown in SEQ ID No. 2 is replaced by isoleucine, the glutamine at position 223 is replaced by threonine, and the serine at position 303 is replaced by arginine.

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

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

4. A recombinant expression transformant, characterized in that: Comprising the recombinant expression vector as described in 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 to catalyze the reaction of 10-carbonyl octadecanoic acid to generate nonanoic acid-9-carboxynonyl ester, which is then hydrolyzed to form 9-hydroxynonanoic acid and n-nonanoic acid.

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

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

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

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