Tteupo mutants and uses thereof

Abstract

The application discloses a TteUPO mutant and application thereof, and belongs to the technical field of bioengineering and enzyme engineering.The TteUPO mutant disclosed by the application connects the nucleotide sequence of a short-chain non-specific peroxidase with a SUMO and 8HIS tag to a pET28a vector, obtains a recombinant vector, transforms E.coli BL21 (DE3), and obtains a genetically engineered bacterium; and the mutant is obtained through unit point mutation.The application uses an E.coli autoinduction system, can make shake flask culture grow to high density in a short time, contains rich nutrients in the culture medium, and does not need to monitor cell density and add IPTG in the expression process.Meanwhile, a hemin precursor 5ALA is added in the E.coli autoinduction culture medium, and finally, a high-activity short-chain non-specific peroxidase is obtained, and the enzyme after purification is reacted with anisole under the condition of hydrogen peroxide flow addition to obtain a product.

Description

Technical Field

[0001] This invention relates to the fields of bioengineering and enzyme engineering, and more specifically to unspecific peroxidase (UPO) mutants, encoding genes, recombinant engineered bacteria, and their applications in the selective hydroxylation synthesis of p-hydroxyanisole and o-hydroxyanisole using anisole. Background Technology

[0002] Unspecific peroxidases (UPOs) can catalyze a wide range of reactions across a broad substrate spectrum, including epoxidation, hydroxylation, heteroatom oxidation, and halogenation. However, unlike P450 enzymes, UPOs utilize hydrogen peroxide (H₂O₂) instead of NAD(P)H and molecular oxygen as oxygen donors and oxidants, directly catalyzing monooxygenation reactions of various organic substrates without the need for any cofactors. This characteristic makes UPOs highly promising biocatalysts, with their unique catalytic capabilities and simplified coenzyme requirements showing broad application prospects in green chemistry, biomedicine, and environmental remediation. However, the main limitation restricting the application of UPOs to date remains the difficulty of heterologous expression. Since the first UPO (AaeUPO) was discovered in the agaric bacterium *Agrocybe aegerita* in 2004, over 4000 putative UPO enzymes exist in databases, but only about 50 of these can be heterologously expressed. The hydroxylation reaction of anisole mainly yields two products: p-hydroxyanisole (4-methoxyphenol) and o-hydroxyanisole (2-methoxyphenol). p-Hydroxyanisole (4-methoxyphenol) is an important aromatic phenolic fine chemical, used as a highly efficient polymerization inhibitor in the production of olefin monomers such as acrylonitrile, acrylic acid, methacrylic acid and their esters. It also serves as a stabilizer for unsaturated resins and a UV inhibitor, exhibiting a wide range of applications and a stable and continuous market demand in the chemical, materials, and pharmaceutical industries. o-Hydroxyanisole, on the other hand, is a key intermediate in the pharmaceutical industry, used in the synthesis of expectorants such as guaiacol glycerol ether. It also has important applications in the food flavoring industry as a natural smoky flavoring agent.

[0003] Currently, the preparation of p-hydroxyanisole mainly relies on chemical synthesis, typically using anisole as a raw material and hydroxylating it under strong acid, strong base, or strong oxidizing conditions. This method suffers from drawbacks such as harsh reaction conditions, complex byproducts, poor ortho / para selectivity, and severe environmental pollution. Enzymatic methods have become an alternative due to their milder conditions, high selectivity, and environmental friendliness. However, monooxygenases such as cytochrome P450 rely on expensive coenzymes, resulting in complex systems that are difficult to scale up. While UPO only requires hydrogen peroxide as a co-substrate, wild-type TteUPO exhibits low conversion efficiency and insufficient para selectivity when catalyzing anisole, limiting its application in the green synthesis of p-hydroxyanisole. Ortho-hydroxyanisole also suffers from drawbacks such as demethylation and diazonium salt decomposition under strong acid and high temperature conditions, resulting in a yield of only about 30%, significant wastewater pollution (generating large amounts of acidic and phenolic wastewater that is difficult to treat), and severe equipment corrosion due to the corrosive nature of the reaction equipment under strong acid conditions, leading to high equipment maintenance costs. Based on this, developing a green, efficient, simple post-processing method for preparing o-hydroxyanisole and p-hydroxyanisole with high product selectivity is of significant research value.

[0004] Therefore, providing mutants of the nonspecific peroxidase (TteUPO) from Thievaria terrestris and their applications is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a highly active and highly para-selective TteUPO mutant obtained through a single point mutation, which significantly increases the production of p-hydroxyanisole and o-hydroxyanisole in catalytic reactions using anisole as a substrate, thereby improving reaction efficiency and application value; and solves the problems of low catalytic activity, insufficient product yield, and limited industrial application of wild-type Thievaria terrestris nonspecific peroxidase (TteUPO) in the hydroxylation of anisole.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: The TteUPO mutant is based on the amino acid sequence shown in SEQ ID NO.1, with the following mutations: A10R or I37L.

[0007] Furthermore, the coding gene of the TteUPO mutant.

[0008] Furthermore, an expression vector containing the coding gene of the TteUPO mutant described above.

[0009] Furthermore, recombinant cells containing the coding gene of the TteUPO mutant.

[0010] Furthermore, the TteUPO mutant is used in the catalytic synthesis of p-hydroxyanisole and o-hydroxyanisole from anisole.

[0011] The TteUPO mutant was heterologously expressed in Escherichia coli using a two-stage temperature-controlled culture process to achieve heterologous expression of UPO in Escherichia coli.

[0012] Two-stage temperature-controlled culture: The first stage is cultured at 37℃ and 180rpm, which is conducive to rapid bacterial growth. After the bacterial count OD reaches 1.2-1.5, the second stage of low-temperature induction culture is started. The temperature is controlled between 16-25℃ (preferably 16℃). Expression is induced for 24h-72h (preferably 48h) and then stopped. The bacterial cells are collected and purified to obtain pure TteUPO enzyme and its mutant.

[0013] As can be seen from the above technical solution, compared with the prior art, this invention discloses a TteUPO mutant and its application. The nucleotide sequence of a short-chain TteUPO with SUMO and 8His tags is ligated into the pET28a vector. The resulting recombinant vector is transformed into *E. coli* BL21(DE3). Based on this sequence, unit point mutations are performed to obtain genetically engineered bacteria with mutants A10R and I37L. Using an *E. coli* self-induction system, shake-flask cultures are grown at high density in a short time, and protein expression is induced through two-stage temperature-controlled culture. The purified enzyme was reacted with anisole in phosphate buffer at pH 7 and then extracted to obtain p-hydroxyanisole and o-hydroxyanisole. The wild-type pET28a-SUMO-TteUPO-8HIS produced p-hydroxyanisole and o-hydroxyanisole yields of 1.12 mg / L and 0.48 mg / L, respectively. The mutant pET28a-SUMO-TteA10R-8HIS produced p-hydroxyanisole and o-hydroxyanisole yields of 3.52 mg / L and 5.8 mg / L, respectively. The mutant pET28a-SUMO-TteI37L-8HIS produced p-hydroxyanisole and o-hydroxyanisole yields of 1.54 mg / L and 1.65 mg / L, respectively. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention 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 embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0015] Figure 1 The plasmid map of pET28a-SUMO-TteUPO-8HIS.

[0016] Figure 2 The image shows an SDS-PAGE gel image of pET28a-SUMO-TteA10R-8HIS; where M: protein marker; 1: flow-through buffer; 2: supernatant obtained after cell lysis; 3: pure enzyme solution.

[0017] Figure 3 The image shows an SDS-PAGE gel image of pET28a-SUMO-TteI37L-8HIS; where M: protein marker; 1: flow-through buffer; 2: supernatant obtained after cell lysis; 3: pure enzyme solution.

[0018] Figure 4 This is the reaction formula using anisole as a substrate.

[0019] Figure 5 Standard curves fitted for the determination of different concentrations of o-hydroxyanisole and p-hydroxyanisole by gas chromatography-mass spectrometry.

[0020] Figure 6 This is the mass spectrum of o-hydroxyanisole.

[0021] Figure 7 This is the mass spectrum of p-hydroxyanisole.

[0022] Figure 8 This is a peak diagram of the product detected by gas chromatography-mass spectrometry (GC-MS) of the pET28a-SUMO-TteUPO-8HIS reaction sample.

[0023] Figure 9 The peak diagram is obtained by gas chromatography-mass spectrometry detection of the product in the pET28a-SUMO-TteA10R-8HIS reaction sample.

[0024] Figure 10 The peak diagram is obtained by gas chromatography-mass spectrometry detection of the product in the pET28a-SUMO-TteI37L-8HIS reaction sample. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] The amino acid sequence of TteUPO is shown in SEQ ID NO.1.

[0027] AGFDSWHPP A PGDRRGPCPMLNTLANHGFLPHNGRN ITKEITVNALNSALNVNKTLGELLFNFAVTTNPQPNATFFDLDHLSRHNILEHDASLSRADYYFGHDDHTFNQTVFDQTKSYWKTPIIDVQQAANARLARVLTSNATNPTFVLSQIGEAFSFGETAAYILALGDRVSGTVPRQWVEYLFENERLPLELGWRRAKEVISNSDLDQLTNRVINATGALANITRKIKVRDFHAGRFPGEGS; SEQ ID NO.1。

[0028] The gene sequence of pET28a-SUMO-TteUPO-8HIS is shown in SEQ ID NO.2.

[0029]

[0030] In SEQ ID NO.2, 5140-6198 bp is the nucleotide sequence of SUMO-TteUPO-8HIS, and the rest is the pET28a framework. Specifically, 5140-5433 bp is SUMO, 5434-6165 bp is TteUPO, 6166-6174 bp is the linker connecting the target gene to the 8HIS purification tag (the linker's core function is to ensure the tag functions normally and does not interfere with the target protein), and 6175-6198 bp is 8HIS.

[0031] The amino acid sequence of SUMO-TteUPO-8HIS is shown in SEQ ID NO.3.

[0032] MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGAGFDSWHPPAPGDRRGPCPMLNTLANHGFLPHNGRNITKEITVNALNSALNVNKTLGELLFNFAVTTNPQPNATFFDLDH SEQ ID NO.3.

[0033] In SEQ ID NO.3, amino acids 1-98 are SUMO tags, amino acids 99-342 are TteUPO, amino acids 343-345 are linkers, and amino acids 346-353 are 8HIS.

[0034] The nucleotide sequence of Tte-A10R-UPO is shown in SEQ ID NO.4.

[0035] GCTGGTTTCGACTCTTG GCACCCACCACGTCCAGGTGACCGTAGAGGTCCATGCCCAATGTTGAACACTTTGGCTAACCACGGTTTCTTGCCTCACAACGGTAGAAACATTACTAAGGAGATCACTGTTAACGCCTTGAACTCTGCCTTGAACGTTAACAAGACCTTGGGTGAATTGTTGTTCAACTTCGCTGTCACCACCAACCCACAGCCAAACGCCACCTTCTTTGACTTGGACCATTTGTCCAGACACAACATCTTGGAACACGATGCTTCTTTGTCCAGAGCTGACTACTACTTCGGTCACGATGATCACACTTTCAACCAAACCGTTTTCGACCAAACCAAGTCTTACTGGAAAACCCCAATTATTGACGTTCAACAAGCTGCTAACGCTAGATTGGCTAGAGTCTTGACTTCTAACGCTACTAACCCAACTTTCGTTTTGTCTCAGATTGGTGAAGCTTTCTCTTTTGGTGAAACCGCTGCTTACATTTTGGCTTTGGGAGATAGAGTTTCTGGTACTGTTCCAAGACAATGGGTTGAGTACTTGTTCGAAAACGAGAGATTGCCATTGGAACTGGGTTGGAGAAGAGCCAAGGAAGTTATTTCTAACTCTGATTTGGATCAATTGACTAACAGAGTTATTAACGCTACTGGTGCTTTGGCTAACATTACTAGAAAGATTAAGGTTAGAGATTTCCACGCTGGTAGATTCCCAGGTGAAGGTTCT; SEQ ID NO.4。

[0036] The nucleotide sequence of Tte-I37L-UPO is shown in SEQ ID NO.5.

[0037] GCTGGTTTCGACTCTTGGCACCCACCAGCTCCAGGTGACCGTAGAGGTCCATGCCCAATGTTGAACACTTTGGCTAACCACGGTTTCTTGCCTCACAAC GGTAGAAACTTGACTAAGGAGATCACTGTTAACGCCT; SEQ ID NO.5.

[0038] Example 1 Construction based on the protein sequences pET28a-SUMO-TteUPO-8HIS mutants A10R (TteUPO amino acid position 10 is mutated from A to R) and I37L (TteUPO amino acid position 37 is mutated from I to L).

[0039] Using plasmid pET28a-SUMO-TteUPO-8HIS (plasmid map see...) Figure 1 Using the gene sequence of pET28a-SUMO-TteA10R / I37L-8HIS (as shown in SEQ ID NO.2) as a template, the target gene was amplified using primers 28a-TteA10R-F / R and 28a-TteI37L-F / R to obtain a linear fragment of pET28a-SUMO-TteA10R / I37L-8HIS. The primer sequences are as follows: 28a-TteA10R-F: GACCCACCAcgtCCAGGTGACCGTAGAGGTC; SEQ ID NO. 6.

[0040] 28a-TteA10R-R: GGTCACCTGGacgTGGTGGGTGCCAAGAGTCGAAAACCA; SEQ ID NO. 7.

[0041] 28a-TteI37L-F: GGTAGAAACttgACTAAGGAGATCACTGTTAACGCCT; SEQ ID NO. 8.

[0042] 28a-TteI37L-R:TCTCCTTAGTcaaGTTTCTACCGTTGTGAGGCAAGAAAC; SEQ ID NO. 9.

[0043] The total PCR amplification reaction volume was 50 μL, with 25 μL of 2×PrimerStar Mix, 20 μL of ddH2O, 2 μL of upstream primer, 2 μL of downstream primer, and 1 μL of template added. The reaction conditions were: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 60℃ annealing for 15 s, and 72℃ extension for 2 min, for a total of 35 cycles; a final extension at 72℃ for 10 min; and storage at 4℃. After the PCR reaction, 2 μL of DPn1 enzyme and 5 μL of 10×Cutfast Buffer were added, and the mixture was incubated at 37℃ for 1 h to eliminate the template. The template-eliminated fragment was verified by agarose gel electrophoresis and then excised and recovered. The recovered linear fragment was ligated using the Gibson homologous recombination assembly method. The ligation system consisted of 5 µL of pET28a-SUMO-TteA10R / I37L-8HIS linear fragment and 5 µL of 2×Basic Seamless ligase, incubated at 45 °C for 15 min.

[0044] The ligation product was introduced into Trans10 competent cells (Beijing TransGen Biotech Co., Ltd.) and transformed using the E. coli chemical transformation method, as follows: (1) Take Trans 10 competent cells and place them on ice to melt. Add all 10 μL of the above ligation product and incubate on ice for 30 min.

[0045] (2) Heat shock at 42℃ for 30 seconds, followed immediately by ice bath for 2 minutes.

[0046] (3) Add 500 μL of antibiotic-free LB medium that has been pre-chilled and thawed at 37°C and 180 rpm for 1 h.

[0047] (4) After the resuscitation is completed, centrifuge at 6000 rpm for 2 min, remove the supernatant, keep 100 μL, resuspend the bacterial cells, and spread them all on a plate containing Kana resistance (100 µg / ml).

[0048] (5) Incubate overnight at 37℃ for about 12 hours. From the cultured plate, randomly select 10 uniform single colonies to extract the recombinant plasmid pET28a-SUMO-TteA10R / I37L-8HIS. After verification by first-generation sequencing, it was confirmed that the sequencing was correct and could be used for subsequent protein expression.

[0049] Example 2 Protein Expression and Purification 1) Protein expression (1) The TteUPO mutant pET28a-SUMO-TteA10R / I37L-8HIS plasmid constructed in Example 1 was transformed into Escherichia coli BL21(DE3). Positive clones were screened using kana resistance plates (Kana, 100µg / ml), and the bacteria were cultured overnight at 37°C to obtain recombinant bacteria.

[0050] (2) Inoculate the single clones on the plate into 4 mL of LB liquid medium (Kana, 50 µg / mL) and culture in a shaker at 37 °C and 180 r / min for 12 h to obtain seed culture.

[0051] (3) The seed culture obtained in step (2) was inoculated into 100 mL of ZYM5052 liquid medium with a Kana resistance concentration of 50 µg / mL at a ratio of 1:50. At the same time, 600 µmol / L of 5-aminolevulinic acid salt (5ALA) was added to the medium. The first stage was cultured at 37℃ and shaken at 180 rpm until the OD reached 1.2. The second stage was cultured at a low temperature of 16℃ and shaken at 180 rpm for 48 h to induce protein expression.

[0052] The composition of ZYM5052 liquid culture medium is as follows: ZY 50mL, 50×M 1mL, 50×5052 1mL, 1M MgSO4 0.1mL, 1000×Trace metals mixture 0.1mL.

[0053] ZY: 10g / L peptone, 5g / L yeast extract.

[0054] 50×M: 446g / L Na2HPO4·12H2O, 170g / L KH2PO4, 133.76g / L NH4Cl, 35.4g / LNa2SO4.

[0055] 50×5052: 250 g / L glycerol, 25 g / L glucose, 100 g / L α-lactose.

[0056] 1000×Trace metals mixture: 54mg / L FeCl3·6H2O, 6.4mg / L CaCl2·2H2O, 4mg / L MnCl2·4H2O, 6mg / L ZnSO4·7H2O, 1mg / L CoCl2·6H2O, 0.8mg / L CuCl2·2H2O, 1mg / LNiCl2·6H2O, 1mg / L Na2MoO4·2H2O, 0.8mg / L Na2SeO3, 0.4mg / L H3BO3.

[0057] (4) After the induction of expression was completed, the bacterial culture expressed in step (3) was centrifuged at 4500 rpm for 20 min at 4℃. After centrifugation, the supernatant was discarded, the bacterial cells were collected, and the bacterial count was determined. The bacterial cells were resuspended in 100 ml of pre-cooled cell lysis buffer (50 mM phosphate buffer, 300 mM NaCl, 5 mM β-ME, pH=7.5) to obtain a resuspension with an OD of 8.0. Lysozyme (final concentration 0.2 mg / mL) and protease inhibitor (PMSF) (final concentration 0.17 mg / mL) were added respectively. Lysozyme and protease inhibitor were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0058] (5) The cell suspension obtained in step (4) was disrupted (amplifier power 40%) for 40 min, and centrifuged at 4℃ and 12000 rpm. The resulting supernatant was 100 ml of crude enzyme solution (containing a large amount of target protein and other miscellaneous proteins) (supernatant obtained after cell disruption).

[0059] 2) Purification of the mutant SUMO-TteA10R / I37L-8HIS protein (1) Chromatography column treatment: 5 mL of nickel column packing material was rinsed with 20 mL of deionized water, and then rinsed with buffer (50 mM phosphate buffer, 150 mM sodium chloride, pH=7.5) for 20 mL.

[0060] (2) Filter the 100ml crude enzyme solution obtained above through a 0.22μm filter membrane to remove impurities. Add the treated crude enzyme solution into a nickel column and mix it thoroughly. Discard the effluent (flow-through liquid).

[0061] (3) After the sample loading is completed, rinse the nickel column with 15 mL of buffer solution to remove unsuccessfully loaded proteins and impurities, and discard the eluent.

[0062] (4) Add 20 ml of buffer containing 60 mM imidazole to the column to remove impurities and discard the eluent.

[0063] (6) Add 20 mL of buffer containing 200 mM imidazole to the column, elute the target protein, and collect the effluent.

[0064] (7) Add 20 mL of buffer containing 500 mM imidazole to the column to clean the nickel column by washing away the residual proteins.

[0065] (8) Add 20 mL of buffer solution and deionized water to the nickel column in sequence to clean the nickel column; finally add 15 mL of 20% ethanol to preserve the nickel column.

[0066] (9) Place the protein collected in step (6) into a 30kDa ultrafiltration tube and centrifuge at 4500rpm until 500μL of liquid remains in the tube. Add 15mL of cell lysis buffer to dilute the imidazole concentration in the protein. Continue centrifuging until about 500μL of protein remains in the tube (pure enzyme solution).

[0067] (10) Take 10 μL of sample (flow-through solution, supernatant obtained after cell lysis, and pure enzyme solution) for SDS-PAGE gel electrophoresis verification.

[0068] The correct expression of the target protein SUMO-TteA10R / I37L-8HIS was verified by polyacrylamide (SDS) gel electrophoresis.

[0069] According to SDS-PAGE verification and comparison with the marker, a distinct specific band was found at 36.6 kDa. Figure 2 , 3 This demonstrates the successful expression of the fusion protein SUMO-TteA10R / I37L-8HIS. Protein concentrations were determined using a NanoDrop spectrophotometer, with a small sample size (typically 1-2 µL). The results were: SUMO-TteA10R-8HIS: 21 mg / L; SUMO-TteI37L-8HIS: 29 mg / L.

[0070] The SUMO-TteA10R / I37L-8HIS protein was concentrated and then flash-frozen in liquid nitrogen and stored at -80°C.

[0071] Example 3 Anisole Reaction Figure 4 This is the reaction formula using anisole as a substrate.

[0072] Take the enzyme (UPO protein) obtained in the above steps and add 2 mM anisole acetonitrile solution, PBS phosphate buffer (pH 7), 1.2 mM H2O2, and 6 mg of enzyme to a 2.5 ml reaction system in a 5 ml reaction vial. Shake the reaction in a 25 °C metal bath at 600 rpm for 3 h. After the reaction is complete, add 500 μL of ethyl acetate for extraction. Take the upper extract into a 1.5 mL EP tube, centrifuge at 12000 rpm for 2 min, and take 100 μL into the liner tube of a gas chromatography vial for analysis.

[0073] Example 4: Gas chromatography-mass spectrometry determination Agilent GC-MS detection conditions: hold at 35 °C for 2 min, then increase to 150 °C at a rate of 15 °C / min, hold for 3 min, then increase to 29 °C at a rate of 15 °C / min, hold for 1 min. Standard curves for the products were obtained based on the GC-MS results of products at mM values ​​of 0.02, 0.05, 0.1, 0.2, 0.4, 0.5, 1, and 5 mM. Figure 5 The yields in the reaction system were calculated based on the standard curve. The yields of p-hydroxyanisole and o-hydroxyanisole in pET28a-SUMO-TteUPO-8HIS were 1.12 mg / L and 0.48 mg / L, respectively; the yields of p-hydroxyanisole and o-hydroxyanisole in the mutant pET28a-SUMO-TteA10R-8HIS were 3.52 mg / L and 5.8 mg / L, respectively; and the yields of p-hydroxyanisole and o-hydroxyanisole in the mutant pET28a-SUMO-TteI37L-8HIS were 1.54 mg / L and 1.65 mg / L, respectively.

[0074] Figure 6 This is the mass spectrum of o-hydroxyanisole; Figure 7 This is the mass spectrum of p-hydroxyanisole; Figure 8 The peak diagram of the product detected by gas chromatography-mass spectrometry in the pET28a-SUMO-TteUPO-8HIS reaction sample. Figure 9 The peak diagram of the product detected by gas chromatography-mass spectrometry for the pET28a-SUMO-TteA10R-8HIS reaction sample. Figure 10 The peak diagram is obtained by gas chromatography-mass spectrometry detection of the product in the pET28a-SUMO-TteI37L-8HIS reaction sample.

[0075] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A TteUPO mutant, characterized in that, Based on the amino acid sequence shown in SEQ ID NO.1, the following mutations are performed: A10R or I37L.

2. The encoding gene of the TteUPO mutant according to claim 1.

3. An expression vector containing the coding gene of the TteUPO mutant as described in claim 2.

4. Recombinant cells containing the encoding gene of the TteUPO mutant as described in claim 2.

5. The application of the TteUPO mutant according to claim 1 in the catalytic synthesis of p-hydroxyanisole and o-hydroxyanisole from anisole.

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