Hydroxylase DmpAB, encoding gene and preparation method and application, recombinant expression vector and recombinant strain
The novel hydroxylase DmpAB solves the problem of poor adaptability of existing hydroxylases to substrate substituents, achieving efficient and selective hydroxylation of a variety of aromatic compounds. Its catalytic activity is not affected by substituents, making it suitable for the green synthesis of high-value-added aromatic derivatives.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- WUHAN POLYTECHNIC UNIVERSITY
- Filing Date
- 2026-03-30
- Publication Date
- 2026-06-05
AI Technical Summary
Existing hydroxylases have poor adaptability to substrate substituents and low catalytic activity, which cannot meet the industrial demand for selective hydroxylation of various substituted aromatic hydrocarbons, thus limiting their application in the synthesis of high-value-added aromatic hydrocarbon derivatives.
A novel hydroxylase, DmpAB, is provided, comprising the amino acid sequences of a first component, DmpA, and a second component, DmpB. It can catalyze aromatic ring compounds with hydroxyl substituents, especially selectively catalyzing the ortho-hydroxylation of hydroxyl substituents. It also exhibits good catalytic activity for substrates with para- and meta-hydroxyl substituents replaced by carboxyl or methyl groups, with a broad catalytic range, accurate action sites, and catalytic activity that is not easily affected by substituents.
It achieves efficient and highly selective hydroxylation of benzene rings in a variety of aromatic compounds. The catalytic activity is not affected by substituents, it has a wide range of applications, mild reaction conditions, conforms to the concept of green manufacturing, and is suitable for the synthesis of high-value-added hydroxylated aromatic derivatives.
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Abstract
Description
Technical Field
[0001] This application relates to the field of genetic engineering technology, and in particular to a hydroxylase DmpAB, its encoding gene, preparation method and application, recombinant expression vector, and recombinant strain. Background Technology
[0002] Hydroxylated aromatic compounds are key precursors for high-value-added products such as pharmaceuticals and fine chemicals, and their efficient synthesis has always attracted much attention in the industry. The selective hydroxylation of aromatic compounds is one of the most challenging core reactions in synthetic chemistry. Currently, the hydroxylation of aromatic compounds mainly relies on chemical synthesis methods and traditional bio-enzymatic catalysis.
[0003] Chemical synthesis methods often require harsh reaction conditions such as high temperature and high pressure, which not only consume a lot of energy and have poor process safety, but also easily generate a large number of by-products, resulting in low product yield and difficulty in purification. At the same time, it causes serious secondary environmental pollution, which is not in line with the development trend of green manufacturing.
[0004] While traditional biocatalytic methods offer advantages such as environmental friendliness and mild reaction conditions, existing hydroxylases generally suffer from problems including high substrate specificity, limited catalytic selectivity, and sensitivity to substituent positions. Most reported aromatic hydroxylases can only catalyze a few specific aromatic compounds, and precise control of the hydroxylation site is difficult, often resulting in mixed products from multiple hydroxylation sites. Furthermore, existing hydroxylases exhibit poor adaptability to substrate substituents; when methyl or carboxyl substituents are present on the benzene ring, catalytic activity decreases significantly or even becomes completely inactive. This fails to meet the industrial demand for selective hydroxylation of various substituted aromatics, severely limiting their application in the synthesis of high-value-added aromatic derivatives. Summary of the Invention
[0005] The main purpose of this application is to propose a hydroxylase DmpAB, its encoding gene, preparation method and application, recombinant expression vector, and recombinant strain, aiming to solve the problem that hydroxylases in the prior art have poor adaptability to substrates with few substituents and low catalytic activity.
[0006] To achieve the above objectives, this application proposes a hydroxylase DmpAB, which comprises a first component and a second component. The amino acid sequence of the first component DmpA is shown in SEQ ID NO.1, and the amino acid sequence of the second component DmpB is shown in SEQ ID NO.2.
[0007] This application also provides a hydroxylase DmpAB encoding gene, which is used to encode the aforementioned hydroxylase DmpAB; The hydroxylase DmpAB encoding gene includes a first encoding gene. dmpASecond coding gene dmpB The first coding gene dmpA The second coding gene is used to encode the first component DmpA. dmpB Used to encode the second component DmpB; The first coding gene dmpA The nucleotide sequence is shown in SEQ ID NO.3, the second encoding gene. dmpB The nucleotide sequence is shown in SEQ ID NO.4.
[0008] This application also provides a recombinant expression vector comprising a nucleotide sequence encoding the aforementioned hydroxylase DmpAB or the aforementioned hydroxylase DmpAB encoding gene.
[0009] In one embodiment, the recombinant expression vector further includes an expression vector comprising pET28a(+).
[0010] This application also provides a recombinant strain, which includes a nucleotide sequence encoding the aforementioned hydroxylase DmpAB or the aforementioned hydroxylase DmpAB encoding gene.
[0011] In one embodiment, the host cell of the recombinant strain includes Escherichia coli.
[0012] This application also provides a method for preparing the hydroxylase DmpAB, comprising the following steps: The gene for hydroxylase DmpAB was cloned into an expression vector to obtain a recombinant expression vector; The recombinant expression vector was transferred into host cells to obtain a recombinant bacterial strain; The recombinant strain was cultured to obtain the hydroxylase DmpAB from the culture.
[0013] This application also provides the use of hydroxylase DmpAB in the hydroxylation of aromatic ring compounds with hydroxyl substituents, wherein the hydroxylase DmpAB includes the aforementioned hydroxylase DmpAB or the hydroxylase DmpAB encoded by the aforementioned hydroxylase DmpAB encoding gene.
[0014] In one embodiment, the aromatic ring compound having a hydroxyl substituent further includes a substituent group, the substituent group including a methyl or carboxyl group, the substituent group being located at the para or ortho position of the hydroxyl substituent.
[0015] In one embodiment, the aromatic ring compound having a hydroxyl substituent includes at least one of phenol, 2-methylphenol, 3-methylphenol, 4-methylphenol, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 2,3-dimethylphenol, 2,4-dimethylphenol, 2,5-dimethylphenol, 3,4-dimethylphenol, and 3,5-dimethylphenol.
[0016] This application provides a novel hydroxylase, DmpAB, comprising a first component and a second component. The amino acid sequence of the first component, DmpA, is shown in SEQ ID NO.1, and the amino acid sequence of the second component, DmpB, is shown in SEQ ID NO.2. This hydroxylase DmpAB can effectively catalyze aromatic ring compounds with hydroxyl substituents, particularly selectively catalyzing the ortho-hydroxylation of hydroxyl substituents. Furthermore, DmpAB also exhibits good catalytic activity for substrates where the para- and meta-positions of the hydroxyl substituents are replaced by carboxyl or methyl groups. It has a broad substrate range, precise action sites, and its catalytic activity is not easily affected by substituents. Attached Figure Description
[0017] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.
[0018] Figure 1 This is a product diagram obtained by HPLC analysis of the target aromatic compound catalyzed by DmpAB in Example 6 of this application (wherein, 1 is phenol, 2 is 2-methylphenol, 3 is 3-methylphenol, 4 is 4-methylphenol, 5 is 2,4-dimethylphenol, 6 is 3,5-dimethylphenol, 7 is 2,5-dimethylphenol, and 8-10 are 2-hydroxybenzoic acid, 3-hydroxybenzoic acid, and 4-hydroxybenzoic acid). Figure 2 This is a product diagram obtained by HPLC and LC-MS analysis of the target aromatic compound catalyzed by DmpAB in Example 6 of this application (where 11 is 2,3-dimethylphenol, 12 is 3,4-dimethylphenol, 13 is 2,6-dimethylphenol, 14 is benzene, 15 is toluene, and 16 is aniline).
[0019] The realization of the purpose, functional features and advantages of this application will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation
[0020] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially. Furthermore, the meaning of "and / or" throughout the text includes three parallel solutions; for example, "A and / or B" includes solution A, solution B, or a solution where both A and B are satisfied. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed in this application. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.
[0021] Existing hydroxylases have poor adaptability to substrate substituents. When there are substituents such as methyl or carboxyl groups on the benzene ring of the substrate, the catalytic activity will decrease significantly or even be completely deactivated. They cannot meet the industrial requirements for selective hydroxylation of various substituted aromatic hydrocarbons and severely limit their application in the synthesis of high-value-added aromatic hydrocarbon derivatives.
[0022] In view of this, this application provides a hydroxylase DmpAB, which includes a first component and a second component, wherein the amino acid sequence of the first component DmpA is shown in SEQ ID NO.1 and the amino acid sequence of the second component DmpB is shown in SEQ ID NO.2.
[0023] This application provides a novel hydroxylase, DmpAB, comprising a first component and a second component. The amino acid sequence of the first component, DmpA, is shown in SEQ ID NO.1, and the amino acid sequence of the second component, DmpB, is shown in SEQ ID NO.2. This hydroxylase DmpAB can effectively catalyze aromatic ring compounds with hydroxyl substituents, particularly selectively catalyzing the ortho-hydroxylation of hydroxyl substituents. Furthermore, DmpAB also exhibits good catalytic activity for substrates where the para- and meta-positions of the hydroxyl substituents are replaced by carboxyl or methyl groups. It has a broad substrate range, precise action sites, and its catalytic activity is not easily affected by substituents.
[0024] SEQ ID NO.1 (DmpA): .
[0025] SEQ ID NO.2 (DmpB): MSFVTIDGRLLRNSLGHFATGVTVVTYEIDGEQLGLTLNAFTAVSLDPPLILVSLDRRSNASKNLEGRPFVVNILSGAQLQHAMNFAGKPQQDCIIDWHDCTPGQPPRLAGCAGYFECAPWHSYDGGDHILYLGEVKDFGVAPDVEPLLFYGGKFRTVGGYADDVLQAT.
[0026] This application also provides a hydroxylase DmpAB encoding gene, which encodes the aforementioned hydroxylase DmpAB; the hydroxylase DmpAB encoding gene includes a first encoding gene. dmpA Second coding gene dmpB The first coding gene dmpA The second coding gene is used to encode the first component DmpA. dmpB Used to encode the second component DmpB; the first encoding gene dmpAThe nucleotide sequence is shown in SEQ ID NO.3, the second encoding gene. dmpB The nucleotide sequence is shown in SEQ ID NO.4. It is understood that the hydroxylase DmpAB encoding gene of this application may originate from Rhodococcus (…). Rhodococcus It can also be artificially synthesized from the genome of sp. CH5R1 (accession number: CCTCC NO: M2021012).
[0027] SEQ ID NO.3 ( dmpA ):
[0028] SEQ ID NO.4 ( dmpB ): atgagcttcgtgactattgacggccgccttctgcggaactctctcggacactttgcaaccggtgtcaccgtggtgacttacgaaatcgacggtgaacagttggggctgactttgaacgccttcactg ccgtgtccctcgacccaccgctgatactggtctccctagaccggcgcagcaacgccagcaagaacctggaaggccggcccttcgtggtcaacatcctttccggcgctcagctgcagcacgcgatgaac ttcgcgggaaaaccccagcaggactgcattatcgattggcacgactgcacccccggccaaccgccgagattggctggctgtgccggctatttcgaatgcgcaccgtggcattcgtacgacggcggcga tcacatcttgtatctcggcgaggtcaaggacttcggggtcgccccagacgttgagcccctgctgttttatggcggcaagttccgcacggtggggggatacgccgacgatgtacttcaagccacctga.
[0029] This application also provides a recombinant expression vector, which includes a nucleotide sequence encoding the aforementioned hydroxylase DmpAB or the aforementioned hydroxylase DmpAB encoding gene. Optionally, the recombinant expression vector further includes an expression vector comprising pET28a(+).
[0030] This application also provides a recombinant bacterial strain, which includes a nucleotide sequence encoding the aforementioned hydroxylase DmpAB or the aforementioned hydroxylase DmpAB encoding gene. It is understood that the recombinant bacterial strain can be obtained by chemically transforming the hydroxylase DmpAB encoding gene into a host cell. The host cell can be Escherichia coli, yeast cells, etc.
[0031] Preferably, the host cell of the recombinant strain includes *Escherichia coli*. More preferably, *Escherichia coli* BL21(DE3) competent cells are selected.
[0032] This application also provides a method for preparing hydroxylase DmpAB, comprising the following steps: cloning the gene of hydroxylase DmpAB into an expression vector to obtain a recombinant expression vector; transferring the recombinant expression vector into a host cell to obtain a recombinant strain; culturing the recombinant strain to obtain hydroxylase DmpAB from the culture.
[0033] In this application, the gene of hydroxylase DmpAB was cloned into the expression vector pET28a(+), transformed into host cells Escherichia coli BL21(DE3), and the recombinant strain was cultured to obtain hydroxylase DmpAB from the culture. The obtained hydroxylase DmpAB can effectively catalyze aromatic ring compounds with hydroxyl substituents, especially selectively catalyzing the ortho-hydroxylation of hydroxyl substituents. In addition, hydroxylase DmpAB also has good catalytic activity for substrates with para- and meta-hydroxyl substituents replaced by carboxyl or methyl groups. Its catalytic substrate range is broad, its action site is accurate, and its catalytic activity is not easily affected by substituents.
[0034] The above preparation method is a mature process and is suitable for the rapid production of hydroxylase DmpAB.
[0035] This application also provides an application of hydroxylase DmpAB in the hydroxylation of aromatic ring compounds with hydroxyl substituents, wherein the hydroxylase DmpAB comprises the aforementioned hydroxylase DmpAB or the hydroxylase DmpAB encoded by the aforementioned hydroxylase DmpAB encoding gene. This application has the following advantages and features: (1) Broad substrate spectrum: Hydroxylase DmpAB can catalyze the hydroxylation of benzene rings of a variety of aromatic compounds, adapts to a variety of substituted aromatic structures, and has a much wider range of applications than traditional hydroxylases; (2) High selectivity: It strictly follows the rule of "hydroxyl ortho-position specific catalysis", and introduces new hydroxyl groups only at the ortho position of the existing hydroxyl group on the substrate benzene ring, avoiding the generation of multi-site hydroxylation byproducts, and the product has high purity; (3) Clear substituent adaptability: The influence of substituents such as methyl and carboxyl groups on catalytic activity is clearly defined, and the catalytic effect can be accurately predicted based on the position of the substrate substituents, which facilitates substrate selection and process optimization in industrial applications; (4) Mild reaction conditions: No high temperature or high pressure is required. High efficiency catalysis can be achieved under normal temperature and oxygen conditions. It has low energy consumption, is environmentally friendly, and conforms to the concept of green manufacturing. (5) High application value: It can accurately synthesize a variety of high-value-added hydroxylated aromatic hydrocarbon derivatives, providing key technical support for the green synthesis of pharmaceuticals and fine chemical products.
[0036] Therefore, the hydroxylase DmpAB provided in this application can achieve efficient and selective hydroxylation of benzene rings in a variety of aromatic compounds, clarify the catalytic rules, and meet the demand for green and precise synthesis of high-value-added hydroxylated aromatic derivatives.
[0037] It should be noted that the hydroxylase DmpAB provided in this application catalyzes aromatic compounds only when the hydroxyl group on the benzene ring of the substrate is occupied. Catalysis is not possible when both positions 2 and 6 are occupied. Furthermore, in some embodiments, the aromatic ring compound with the hydroxyl substituent also includes a substituent group, which includes a methyl or carboxyl group, located at the para or ortho position of the hydroxyl substituent. The position of the methyl and carboxyl substituents affects the catalytic activity, with the order of activity being para-substitution > meta-substitution > ortho-substitution. Catalysis is not possible when the carboxyl group is ortho to the hydroxyl substituent.
[0038] Preferably, the aromatic ring compound having a hydroxyl substituent includes at least one selected from phenol, 2-methylphenol, 3-methylphenol, 4-methylphenol, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 2,3-dimethylphenol, 2,4-dimethylphenol, 2,5-dimethylphenol, 3,4-dimethylphenol, and 3,5-dimethylphenol. The hydroxylase DmpAB provided in this application can accurately catalyze the above substrates with high catalytic efficiency.
[0039] The technical solutions of this application will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain this application and are not intended to limit this application.
[0040] The culture medium and buffer formulations are as follows: LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.0.
[0041] LB solid medium containing 50 μg / mL kanamycin: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar powder 1.5%-2.0%, kanamycin 50 μg / mL, pH 7.0.
[0042] LB liquid medium containing 50 μg / mL kanamycin: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, kanamycin 50 μg / mL, pH 7.0.
[0043] Liquid inorganic salt culture medium: Na₂HPO₄·12H₂O 10.2~14.3 g / L, KH₂PO₄ 2~3 g / L, FeSO₄·7H₂O 0.2~0.3 mg / L, MnSO₄ H2O 0.05~0.07mg / L, MgSO4 7H2O 0.015~0.02mg / L, CaCl20.25~0.3mg / L, CuSO40.0125~0.2mg / L, ZnSO40.0125~0.2mg / L, H3BO30.0125~0.2mg / L, (NH4)2SO42~3mM, pH7.0~7.5.
[0044] HEPES-KOH buffer (40mM, pH 8.0): Weigh 1.19g HEPES and dissolve it in 200mL of deionized water. Adjust the pH to 8.0 with KOH and bring the volume to 250mL.
[0045] Example 1: Obtaining the DmpAB encoding gene This application is derived from Rhodococcus (… Rhodococcus The encoding gene for 3,5-dimethylphenol hydroxylase DmpAB was identified and cloned from sp. CH35R1 (accession number: CCTCC NO: M2021012). Recombinant expression strains were constructed using genetic engineering technology to achieve high-efficiency expression of DmpAB, which was then applied to the selective hydroxylation reaction of aromatic compounds.
[0046] With Rhodococcus ( Rhodococcus Using the genomic DNA of sp. CH35R1 (accession number: CCTCC NO: M2021012, depositary: China Center for Type Culture Collection) as a template, specific primers were designed based on the sequence of the DmpAB coding gene (as shown in SEQ ID NO.3 and SEQ ID NO.4). The forward primer sequence is shown in SEQ ID NO.5, and the reverse primer sequence is shown in SEQ ID NO.6. The complete coding gene of DmpAB was obtained by PCR amplification.
[0047] PCR reaction system: 30-50 ng template DNA, 2 μL each of forward and reverse primers (10 μM), 5 μL of 10×Easy Pfu Buffer, 1 μL of dNTPs (10 mM), 1 μL of Easy Pfu DNA Polymerase (2.5 units), and ddH2O to make up to 50 μL.
[0048] PCR reaction conditions: 94℃ pre-denaturation for 5 min; 30 cycles (94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 3 min); 72℃ final extension for 5 min.
[0049] SEQ ID NO.5: ccgcgcggcagccatagcttcgtgactattgacggcc.
[0050] SEQ ID NO.6: tcgagtgcggccgcattagttctgtccgaggcgcttc.
[0051] Example 2 Construction of recombinant expression vector The DmpAB encoding gene obtained by PCR amplification in Example 1 was ligated into the pET28a(+) expression vector, which had been digested with NdeI and HindIII, using the ClonExpress II One Step Cloning Kit to obtain the recombinant expression vector pET28a- dmpAB .
[0052] Example 3 Construction of recombinant strains The recombinant expression vector from Example 2 was transformed into *E. coli* BL21(DE3) competent cells using a chemical transformation method (calcium chloride). Positive transformants were screened on LB agar plates containing 50 μg / mL kanamycin, and after sequencing verification, the recombinant expression strain of DmpAB was obtained. E . coli BL21 (DE3) / pET28a- dmpAB .
[0053] Example 4: Preparation of DmpAB Seed culture: Select a single colony of the recombinant strain and inoculate it into 5 mL of LB liquid medium containing 50 μg / mL kanamycin. Incubate overnight at 37°C and 200 rpm to obtain the seed culture. Expanded culture and induced expression: Seed culture was inoculated at 1% (v / v) into 1L of LB liquid medium containing 50μg / mL kanamycin, and cultured at 37℃ and 200rpm until OD500. 600 =0.4-0.6, add 0.1mM IPTG, and induce culture at 37℃ and 200rpm for 12-16h to obtain the induced culture solution; Preparation of crude enzyme solution: After induction culture, the bacterial culture was centrifuged at 4℃ and 8000rpm for 5min to collect the bacterial cells. The bacterial cells were resuspended in 100mL of 40mM HEPES-KOH buffer (pH 8.0), and the cells were sonicated (400W, 5s sonication / 5s interval, total 30min). The cells were then centrifuged at 4℃ and 12000rpm for 50min. The supernatant was collected, which is the crude enzyme solution of DmpAB.
[0054] Example 5: Purification of DmpAB The crude DmpAB enzyme solution from Example 4 was purified using Ni-agarose gel 6FF (His-tagged purification resin) (Solepro, Beijing), and the target protein was eluted with 250 mM imidazole to obtain DmpAB.
[0055] Example 6: Catalytic application of DmpAB Reaction system preparation: In 40mM HEPES-KOH buffer (pH 8.0), add 0.2mM of the target aromatic substrate, 0.3mM NADPH (reduced nicotinamide adenine dinucleotide phosphate), 0.04mM FAD (flavin adenine dinucleotide), and 100μg DmpAB, and react at room temperature (25℃); Product detection: During the reaction, the consumption of the target aromatic substrate and the formation of the product were monitored by high performance liquid chromatography (HPLC). The HPLC used a Venusil MP C18 column (250 mm × 4.6 mm, 5 μm), the column temperature was 30 ℃, the injection volume was 10 μL, and the appropriate mobile phase (water + 0.3% acetic acid, or water + 0.3% acetonitrile) and flow rate (1.0 mL / min) were selected according to the substrate characteristics. The UV detector wavelength was 280 nm or 230 nm. The product structure was verified by liquid chromatography-mass spectrometry (LC-MS).
[0056] Relative enzyme activity: This is the ratio of the enzyme activity of the test sample to the standardized control group (in this case, the enzyme activity of DmpAB against 3,5-dimethylphenol). It is used to intuitively reflect the degree of influence of the substrate on enzyme activity. The measurement method used in this example is adopted.
[0057] The results are shown in Table 1. Figure 1 , Figure 2 As shown in the figure, red represents the HPLC chromatogram of the product standard, and black represents the HPLC chromatogram of the substrate standard; the experimental group shows the HPLC chromatogram of the product after the catalytic reaction of DmpAB with aromatic compounds, and the control group shows the HPLC chromatogram of the product after the catalytic reaction of aromatic compounds without the addition of DmpAB; blue represents the HPLC detection chromatogram of the product after 0 hours (h) of catalytic reaction of DmpAB with aromatic compounds, green represents the HPLC detection chromatogram of the product after 6 hours of catalytic reaction of DmpAB with aromatic compounds, purple represents the HPLC detection chromatogram of the product after 12 hours of catalytic reaction of DmpAB with aromatic compounds, and orange represents the HPLC detection chromatogram of the product after 24 hours of catalytic reaction of DmpAB with aromatic compounds.
[0058] Table 1. Products obtained from 16 aromatic compounds catalyzed by DmpAB.
[0059] As shown in Table 1, the core catalytic characteristics of DmpAB are as follows: (1) Essential conditions for DmpAB catalysis: The hydroxyl group on the benzene ring of the substrate is an essential structure for DmpAB catalysis. Aromatic compounds without hydroxyl substitution (such as benzene, toluene and aniline) cannot be used as substrates. (2) Site specificity of DmpAB catalysis: The catalytic reaction occurs strictly at the position (position 2 or position 6) adjacent to the hydroxyl group of the substrate. If the positions 2 and 6 of the substrate (such as 2,6-dimethylphenol, where methyl groups are present at both positions 2 and 6) are occupied by other groups, catalysis cannot be initiated. (3) The influence of substituents on DmpAB catalysis: Due to the large steric hindrance of the carboxyl group, the catalytic activity increases when it is far away from the hydroxyl group. Therefore, the activity order is 4-hydroxybenzoic acid > 3-hydroxybenzoic acid > 2-hydroxybenzoic acid. When the carboxyl group is located ortho to the hydroxyl group (such as in 2-hydroxybenzoic acid), the catalytic reaction cannot proceed at all. The presence of the methyl group helps stabilize the binding of the enzyme to the substrate during catalysis, and there is no clear correspondence between its position and catalytic activity.
[0060] The above are merely preferred embodiments of this application and do not limit the patent scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the patent protection scope of this application.
Claims
1. A hydroxylase DmpAB, characterized in that, The hydroxylase DmpAB comprises a first component and a second component, wherein the amino acid sequence of the first component DmpA is shown in SEQ ID NO.1 and the amino acid sequence of the second component DmpB is shown in SEQ ID NO.
2.
2. A gene encoding a hydroxylase DmpAB, characterized in that, The hydroxylase DmpAB encoding gene is used to encode the hydroxylase DmpAB as described in claim 1; The hydroxylase DmpAB encoding gene includes a first encoding gene. dmpA Second coding gene dmpB The first coding gene dmpA The second coding gene is used to encode the first component DmpA. dmpB Used to encode the second component DmpB; The first coding gene dmpA The nucleotide sequence is shown in SEQ ID NO.3, the second encoding gene. dmpB The nucleotide sequence is shown in SEQ ID NO.
4.
3. A recombinant expression vector, characterized in that, The recombinant expression vector includes a nucleotide sequence encoding the hydroxylase DmpAB as described in claim 1 or the hydroxylase DmpAB encoding gene as described in claim 2.
4. The recombinant expression vector as described in claim 3, characterized in that, The recombinant expression vector further includes an expression vector, wherein the expression vector includes pET28a(+).
5. A recombinant bacterial strain, characterized in that, The recombinant strain includes a nucleotide sequence encoding the hydroxylase DmpAB as described in claim 1 or the hydroxylase DmpAB encoding gene as described in claim 2.
6. The recombinant strain according to claim 5, characterized in that, The host cells of the recombinant strain include Escherichia coli.
7. A method for preparing the hydroxylase DmpAB as described in claim 1, characterized in that, Includes the following steps: The gene for hydroxylase DmpAB was cloned into an expression vector to obtain a recombinant expression vector; The recombinant expression vector was transferred into host cells to obtain a recombinant bacterial strain; The recombinant strain was cultured to obtain the hydroxylase DmpAB from the culture.
8. The application of a hydroxylase DmpAB in the hydroxylation of aromatic ring compounds with hydroxyl substituents, characterized in that, The hydroxylase DmpAB includes the hydroxylase DmpAB as described in claim 1 or the hydroxylase DmpAB encoded by the hydroxylase DmpAB encoding gene as described in claim 2.
9. The application of the hydroxylase DmpAB as described in claim 8 in the hydroxylation of aromatic ring compounds with hydroxyl substituents, characterized in that, The aromatic ring compound having a hydroxyl substituent further includes a substituent group, which includes a methyl or carboxyl group, and the substituent group is located at the para or ortho position of the hydroxyl substituent.
10. The use of the hydroxylase DmpAB as described in claim 8 in the hydroxylation of aromatic ring compounds with hydroxyl substituents, characterized in that, The aromatic ring compounds having hydroxyl substituents include at least one of phenol, 2-methylphenol, 3-methylphenol, 4-methylphenol, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 2,3-dimethylphenol, 2,4-dimethylphenol, 2,5-dimethylphenol, 3,4-dimethylphenol, and 3,5-dimethylphenol.