A kind of ring opening method of aromatic compound

Abstract

The present invention discloses a ring-opening method for aromatic compounds. The method is based on the carboxylation-dioxygenation coupling reaction, overcomes the thermodynamic reversibility of the enzyme-catalyzed carboxylation reaction, greatly improves the efficiency of CO2 fixation, and builds a new naphthalene degradation reaction pathway and biological patent utilization based on this, realizing the rapid ring-opening of naphthalene and CO2 utilization.

Description

Technical Field

[0001] The invention relates to the field of biochemistry, and in particular to a ring-opening method for aromatic compounds. Background Art

[0002] Polycyclic aromatic hydrocarbons (PAH) are a class of compounds composed of multiple benzene rings in their structure. They are carcinogenic and mutagenic and are subject to strict quarantine worldwide. Most of the polycyclic aromatic hydrocarbons (PAH) that cause ecological hazards are byproducts of fossil energy. Since fossil energy is the energy basis of human social and economic activities, the hazards of PAH will continue to receive widespread attention in the foreseeable future. Due to the conjugation of π electrons, the energy of the structure is reduced, so aromatic compounds show a certain degree of inertness. Moreover, the polarity of aromatic compounds is very weak, and most of them are slightly soluble or insoluble in water. This inertness becomes more significant as the number of aromatic rings in the molecule increases. Therefore, the degradation of aromatic substances is a slow and complex process.

[0003] Naphthalene is the simplest PAH substance, with a molecular structure composed of two benzene rings. Naphthalene and its derivatives are important chemical raw materials, and are widely used in pesticide synthesis, printing and dyeing, rubber and other processes. Naphthalene is mainly produced in large quantities in coal tar, a byproduct of coking, and petroleum distillation. However, with the implementation of strict environmental protection policies and regulations, the production and management of naphthalene are subject to strict supervision. However, aromatic ring substances such as naphthalene produced in the petrochemical industry are mainly cracked by thermal catalysis, which is a complex process and has the disadvantages of high pollution and high energy consumption. In comparison, biological cracking of PAH has low energy consumption and is environmentally friendly.

[0004] Another serious environmental threat caused by the use of fossil energy is CO2 emissions and the greenhouse effect. Due to the greenhouse effect, the global climate is abnormal and the frequency of extreme meteorological disasters has increased significantly. Therefore, reducing CO2 emissions or improving the efficiency of CO2 fixation has become an urgent need. The insertion of carboxyl groups at the ortho-hydroxyl position of phenolic substances (Kolbe–Schmitt reaction) catalyzed by non-oxidative carboxylase is a typical carbon fixation reaction. However, the reverse reaction of this reaction is significant, and the conversion rate of the reaction is very low. The chemical method requires high temperature and high pressure conditions (~90 bar, 120–300 ° C) to improve the conversion rate of the reaction. This process has high energy consumption, serious pollution, and very obvious carbon emissions. Enzyme catalysis can be carried out under mild conditions and does not require a large amount of energy input, but the reverse reaction is obvious and the problem of low conversion rate is difficult to avoid. If the efficiency of enzyme-catalyzed CO2 fixation is to be improved, the limitation of the reverse reaction must be solved. Summary of the invention

[0005] In order to effectively solve the above technical problems, the present invention aims to provide a new pathway for efficiently opening the aromatic ring of naphthalene by coupling enzyme-catalyzed monooxygenation reaction, Kolbe–Schmitt reaction to fix CO2 and dioxygenation reaction, which can be used for the biodegradation of naphthalene and its resource utilization.

[0006] In a first aspect, the present invention claims a method for ring-opening an aromatic compound.

[0007] The ring-opening method of the aromatic compound claimed in the present invention may include the following steps:

[0008] (A1) Aromatic compounds are catalyzed by monooxygenase to generate corresponding phenolic compounds;

[0009] (A2) the phenolic compound is catalyzed by carboxylase to generate a carboxyl-containing aromatic compound;

[0010] (A3) The carboxyl group-containing aromatic compound is catalyzed by dioxygenase to generate a carboxylic acid compound, which is the ring-opening product of the aromatic compound.

[0011] In the method, if the aromatic compound contains a phenolic hydroxyl group, step (A1) can be omitted.

[0012] In the method, the catalytic reaction of various enzymes can be carried out by any of the following methods: 1) directly adding the corresponding enzymes into the reaction system; 2) adding cells capable of expressing the corresponding enzymes into the reaction system.

[0013] In a specific embodiment of the present invention, the cell capable of expressing the monooxygenase is Escherichia coli capable of expressing the monooxygenase, such as BL21 Gold (DE3); the cell capable of expressing the carboxylase is Escherichia coli capable of expressing the carboxylase, such as BL21 Gold (DE3); the cell capable of expressing the carboxyl-containing aromatic compound is Escherichia coli capable of expressing the carboxylase, such as BL21 Gold (DE3).

[0014] In the method, in step (A1), NAD(P)H is required to provide reducing power. NAD(P)H represents NADH or NADPH. + NAD + or NADP + The same applies below.

[0015] Furthermore, the providing of reducing power by NAD(P)H is achieved by any of the following methods: 1) directly adding NAD(P)H to the reaction system; 2) forming a coenzyme NAD(P)H cycle in the reaction system.

[0016] Furthermore, the formation of the coenzyme NAD(P)H cycle in the reaction system can be achieved by any of the following methods: 1) adding cells capable of expressing alcohol dehydrogenase (ADH) and NAD(P) + ; 2) adding cells capable of expressing alcohol dehydrogenase (ADH) and NAD(P)H to the reaction system.

[0017] In a specific embodiment of the present invention, the cell capable of expressing alcohol dehydrogenase (ADH) is an Escherichia coli capable of expressing alcohol dehydrogenase (ADH), such as BL21 Gold (DE3). The amount of the cell capable of expressing alcohol dehydrogenase (ADH) added to the reaction system is 0.5 g cell wet weight / mL, NAD(P) + Or the amount of NAD(P)H added was 30 mM.

[0018] In the method, in step (A2), HCO3 is required - or CO2 as another substrate.

[0019] Wherein, the aromatic compound may be an aromatic hydrocarbon compound.

[0020] Furthermore, the aromatic hydrocarbon compound may be a polycyclic aromatic hydrocarbon.

[0021] Furthermore, the polycyclic aromatic hydrocarbon may be naphthalene.

[0022] In a specific embodiment of the present invention, the aromatic compound is naphthalene.

[0023] In a second aspect, the present invention claims a method for degrading naphthalene and / or fixing CO2.

[0024] The method for degrading naphthalene and / or fixing CO2 claimed in the present invention may include the following steps:

[0025] (a1) Naphthalene is catalyzed by monooxygenase to produce 1-naphthol;

[0026] (a2) 1-naphthol is catalyzed by carboxylase to generate 1-hydroxy-2-benzoic acid;

[0027] (a3) 1-Hydroxy-2-benzoic acid is catalyzed by dioxygenase to generate 2-carboxybenzopyruvic acid.

[0028]

[0029] In step (a1), NAD(P)H is required to provide reducing power.

[0030] Furthermore, providing reducing power with NAD(P)H can be achieved by any of the following methods: 1) directly adding NAD(P)H to the reaction system; 2) forming a coenzyme NAD(P)H cycle in the reaction system.

[0031] Furthermore, the formation of the coenzyme NAD(P)H cycle in the reaction system can be achieved by any of the following methods: 1) adding cells capable of expressing alcohol dehydrogenase (ADH) and NAD(P) + ; 2) adding cells capable of expressing alcohol dehydrogenase (ADH) and NAD(P)H to the reaction system.

[0032] In a specific embodiment of the present invention, the cell capable of expressing alcohol dehydrogenase (ADH) is an Escherichia coli capable of expressing alcohol dehydrogenase (ADH), such as BL21 Gold (DE3). The amount of the cell capable of expressing alcohol dehydrogenase (ADH) added to the reaction system is 0.5 g cell wet weight / mL, NAD(P) + Or the amount of NAD(P)H added was 30 mM.

[0033] In step (a2), HCO3 - or CO2 as another substrate;

[0034] In the method, the catalytic reaction of various enzymes can be carried out by any of the following methods: 1) directly adding the corresponding enzymes into the reaction system; 2) adding cells capable of expressing the corresponding enzymes into the reaction system.

[0035] In a specific embodiment of the present invention, the cell capable of expressing the monooxygenase is Escherichia coli capable of expressing the monooxygenase, such as BL21 Gold (DE3); the cell capable of expressing the carboxylase is Escherichia coli capable of expressing the carboxylase, such as BL21 Gold (DE3); the cell capable of expressing the carboxyl-containing aromatic compound is Escherichia coli capable of expressing the carboxyl-containing aromatic compound, such as BL21 Gold (DE3).

[0036] In a specific embodiment of the present invention, steps (a1) to (a3) ​​are completed in the same reaction system through a one-step reaction.

[0037] The reaction system contains 1) naphthalene, 2) monooxygenase or cells capable of expressing the monooxygenase, 3) carboxylase or cells capable of expressing the carboxylase, 4) dioxygenase or cells capable of expressing the dioxygenase, 5) NAD(P)H, 6) HCO3 - or CO2, 7) reaction buffer.

[0038] Furthermore, in the reaction system, the final concentration of naphthalene is 15 mM, the final concentration of NAD(P)H is 60 mM, and the final concentration of HCO3 is 15 mM. - The final concentration is 50 mM or CO2 is continuously introduced into the reaction system during the reaction, the monooxygenase is 0.5 mg / mL or 0.5 g cell wet weight / mL of cells capable of expressing the monooxygenase, the carboxylase is 0.03-1 U / mL (such as 1 U / mL, 0.03 U / mL, 0.14 U / mL) or 0.5 g cell wet weight / mL of cells capable of expressing the carboxylase, the dioxygenase is 9 U / mL or 0.5 g cell wet weight / mL of cells capable of expressing the dioxygenase; and the remainder is the reaction buffer.

[0039] The pH of the reaction buffer is 6.5-8.0 (such as pH 7.0-7.5), and can be specifically a potassium phosphate buffer (pH 7.0 or pH 7.5, 100 mM).

[0040] The reaction temperature is 25-35°C (eg 30°C), and the reaction time is 3-12h (eg 12h).

[0041] In a third aspect, the present invention claims any of the following methods:

[0042] Method I: A method for producing 2-carboxybenzopyruvic acid using naphthalene as a substrate, which may include steps (a1) to (a3) ​​of the method described above.

[0043] Method II, a method for producing 2-carboxybenzopyruvic acid using 1-naphthol as a substrate, may include steps (a2) to (a3) ​​of the method described above.

[0044] In a specific embodiment of the present invention, steps (a2) and (a3) ​​of method II are completed in the same reaction system through a one-step reaction.

[0045] The reaction system contains 1) 1-naphthol, 2) carboxylase or cells capable of expressing the carboxylase, 3) dioxygenase or cells capable of expressing the dioxygenase, 4) HCO3 or CO2 - , 5) reaction buffer.

[0046] Furthermore, in the reaction system, the final concentration of 1-naphthol is 15 mM, HCO3 - The final concentration is 50mM, the carboxylase is 0.03-1U / mL (such as 1U / mL, 0.03U / mL, 0.14U / mL) or 0.5g cell wet weight / mL of cells capable of expressing the carboxylase, the dioxygenase is 9U / mL or 0.5g cell wet weight / mL of cells capable of expressing the dioxygenase; and the remainder is the reaction buffer.

[0047] The pH of the reaction buffer is 6.5-8.0 (such as pH 7.0-7.5), and can be specifically a potassium phosphate buffer (pH 7.0 or pH 7.5, 100 mM).

[0048] The reaction temperature is 25-35°C (eg 30°C), and the reaction time is 3-12h (eg 12h).

[0049] In a fourth aspect, the present invention claims a set of enzymes.

[0050] The enzyme set claimed in the present invention may be as follows (B1) or (B2):

[0051] (B1) consists of carboxylase and dioxygenase;

[0052] (B2) consists of monooxygenase, carboxylase and dioxygenase.

[0053] In a fifth aspect, the present invention claims a set of cells.

[0054] The set of cells claimed in the present invention may be as follows (C1) or (C2) or (C3):

[0055] (C1) consists of cells capable of expressing carboxylase and cells capable of expressing dioxygenase;

[0056] (C2) consists of cells capable of expressing monooxygenase, cells capable of expressing carboxylase, and cells capable of expressing dioxygenase;

[0057] (C3) consists of cells capable of expressing monooxygenase, cells capable of expressing carboxylase, cells capable of expressing dioxygenase, and cells capable of expressing alcohol dehydrogenase (ADH).

[0058] In a sixth aspect, the present invention claims protection for any of the following applications:

[0059] P1. Use of the method described in the first aspect above, the set of enzymes described in the fourth aspect above, or the set of cells described in the fifth aspect above in the degradation of aromatic compounds and / or the fixation of CO2;

[0060] P2. Use of the method described in the third aspect above, the set of enzymes described in the fourth aspect above, or the set of cells described in the fifth aspect above in degrading naphthalene and / or fixing CO2.

[0061] In the above aspects, the monooxygenase may be a monooxygenase derived from Bacillus megaterium.

[0062] Furthermore, the amino acid sequence of the monooxygenase derived from Bacillus megaterium is shown in SEQ ID No.1.

[0063] In the above aspects, the carboxylase may be a carboxylase derived from Aspergillus oryzae, a carboxylase derived from Rhizobium sp., or a carboxylase derived from Trichosporon moniliforme.

[0064] Furthermore, the amino acid sequence of the carboxylase derived from Aspergillus oryzae is shown in SEQ ID No.2; the amino acid sequence of the carboxylase derived from Rhizobium sp. is shown in SEQ ID No.3; and the amino acid sequence of the carboxylase derived from Trichosporon moniliiforme is shown in SEQ ID No.4.

[0065] In the above aspects, the dioxygenase may be a dioxygenase derived from Mycobacterium vanbaalenii PYR-1.

[0066] Furthermore, the amino acid sequence of the dioxygenase derived from Mycobacterium vanbaalenii PYR-1 is shown in SEQ ID No.5.

[0067] In the present invention, the monooxygenase is obtained by prokaryotic expression using Escherichia coli as a host bacterium and then purified by Ni column. Specifically, it can be prepared according to a method comprising the following steps: inducing the expression of Escherichia coli (such as BL21 Gold (DE3)) expressing the monooxygenase with IPTG at a final concentration of 50 μM, and the expression condition is 20-30°C for 24 hours; collecting cells by centrifugation, breaking the cells after resuspending, collecting the supernatant by centrifugation, filtering, purifying by Ni column, desalting, and freeze-drying to obtain.

[0068] The invention discloses a method for a coupling reaction of CO2 fixation and naphthalene cracking. The method takes a carboxylation-dioxygenation coupling reaction as a core, overcomes the thermodynamic reversibility of an enzyme-catalyzed carboxylation reaction, greatly improves the efficiency of CO2 fixation, and constructs a new naphthalene degradation reaction path based on this core. Naphthalene is converted into 1-naphthol through oxygenase, and then into 2-carboxybenzopyruvic acid through a carboxylation-dioxygenation coupling reaction, thereby realizing the cracking and ring-opening of naphthalene and CO2 fixation.

[0069] Compared with the existing non-oxidative carboxylation reaction, the present invention has the following advantages:

[0070] 1. By coupling carboxylase and oxygenase, product inhibition is eliminated, and the carboxylase-catalyzed reaction can be carried out under normal environmental conditions, which greatly reduces the cost of carboxylase fixation of C and improves efficiency.

[0071] 2. Using naphthalene as substrate, a new reaction pathway was constructed to couple the degradation of naphthalene and the fixation of CO2.

[0072] 3. A green and mild method for synthesizing 2-carboxybenzopyruvic acid was designed using naphthalene as substrate.

[0073] 4. The method of the present invention does not rely on substrate and temperature drive, the reaction conditions are mild and the operation is convenient. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 The results of enzyme purification were detected by SDS-PAGE electrophoresis. M: standard protein marker; L1: P450 BM-3 (A74G / F87V / L188Q); L2: 2,3-DHBD; L3.1HNDO; L4: SAD: L5: 2,6-DHBD. The arrows indicate the target protein.

[0075] Figure 2 A is the detection of pure 2-carboxybenzopyruvic acid by LC-MS and the detection of standard 1-naphthol by liquid chromatography. A is the detection of pure 2-carboxybenzopyruvic acid by LC-MS; B is the detection of standard 1-naphthol by liquid chromatography.

[0076] Figure 3 Liquid phase detection of carboxylase 2,3-DHBD and dioxygenase 1HNDO coupled catalytic 1-naphthol cleavage (direct enzyme reaction method).

[0077] Figure 4 Liquid phase detection of carboxylase 2,6-DHBD and dioxygenase 1HNDO coupled reaction catalyzed 1-naphthol cleavage (direct enzyme reaction method).

[0078] Figure 5 Liquid phase detection of the coupled reaction of carboxylase SAD and dioxygenase 1HNDO catalyzing the cleavage of 1-naphthol (direct enzyme reaction method).

[0079] Figure 6 To detect the coupled catalytic cleavage of 1-naphthol by carboxylase 2,3-DHBD and dioxygenase 1HNDO in liquid phase (resting cell reaction method).

[0080] Figure 7 The liquid phase detection was carried out to couple the catalytic cleavage of 1-naphthol and fixation of CO2 by carboxylase 2,3-DHBD and dioxygenase 1HNDO (direct enzyme reaction method).

[0081] Figure 8 Liquid phase detection of monooxygenase P450 BM-3 (A74G / F87V / L188Q), carboxylase 2,3-DHBD and dioxygenase 1HNDO catalyzed naphthalene degradation (direct enzyme reaction method).

[0082] Fig. 9 The liquid phase detection was carried out to detect the coupled catalytic degradation of naphthalene by monooxygenase P450 BM-3 (A74G / F87V / L188Q), carboxylase 2,3-DHBD and dioxygenase 1HNDO (resting cell coenzyme cycle reaction method). DETAILED DESCRIPTION

[0083] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0084] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0085] Example 1. Expression host construction

[0086] After gene optimization, Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to artificially synthesize the coding gene of the monooxygenase mutant [P450 BM-3 (A74G / F87V / L188Q)] from Bacillus megaterium as shown in SEQ ID No.6 (SEQ ID No.6 encodes the protein shown in SEQ ID No.1), and the gene was inserted into the Nde Ⅰ restriction site of pET28a. The obtained recombinant vector was named pET28a::bm3 after sequencing verification.

[0087] The coding gene of carboxylase (2,3-DHBD) derived from Aspergillus oryzae was synthesized as shown in SEQ ID No.7 (SEQ ID No.7 encodes the protein shown in SEQ ID No.2), and the gene was inserted into the Nco Ⅰ restriction site of pRSFDuet1 vector (New England Biolabs). The obtained recombinant vector was named pRSFDuet1::2,3-dhnd after being verified as correct by sequencing.

[0088] The coding gene of carboxylase (2,6-DHBD) synthesized from Rhizobium sp. is shown in SEQ ID No. 8 (SEQ ID No. 8 encodes the protein shown in SEQ ID No. 3), and the gene is inserted into the Nco Ⅰ restriction site of pRSFDuet1 vector (New England Biolabs). The obtained recombinant vector is named pRSFDuet1::2,6-dhnd after being verified by sequencing.

[0089] The coding gene of the carboxylase (SAD) synthesized from Trichosporon moniliiforme is shown in SEQ ID No.9 (SEQ ID No.9 encodes the protein shown in SEQ ID No.4), and the gene is inserted into the NcoⅠ restriction site of the pRSFDuet1 vector (New England Biolabs). The obtained recombinant vector is named pRSFDuet1::sad after being verified as correct by sequencing.

[0090] The coding gene of the oxygenase (1HNDO) derived from Mycobacterium vanbaalenii PYR-1 was synthesized as shown in SEQ ID No.10 (SEQ ID No.10 encodes the protein shown in SEQ ID No.5), and the gene was inserted into the NcoⅠ restriction site of the pRSFDuet1 vector (New England Biolabs). The obtained recombinant vector was named pRSFDuet1::1hndo after being verified as correct by sequencing.

[0091] The above expression vectors were transformed into Escherichia coli BL21 Gold (DE3), positive clones were screened and cultured on LB plates containing kanamycin, plasmids were extracted, and sequencing was performed to confirm that the vectors were successfully constructed.

[0092] Example 2: Enzyme expression and purification

[0093] Each positive engineered bacteria constructed in Example 1 was inoculated into 5 mL LB medium, cultured at 37°C and 200 r / min for 12 h, and 1 mL of seed bacteria solution was transferred to 100 mL fresh LB medium, and cultured at 37°C and 220 r / min for about 2 h until OD 600 When it reached 0.4-0.6, IPTG was added at a final concentration of 50 μM to induce expression. The expression conditions were low-temperature induction at 20-30°C, 220 r / min, and 24 h.

[0094] After the expression, the cells were collected by centrifugation, resuspended in Binding buffer, concentrated 20 times, and broken by high-pressure homogenization. The supernatant was collected and filtered through a 0.45 μm filter membrane. The sample was then loaded onto the Ni column, the impurities were eluted with Washing buffer, the enzyme was eluted with Eution buffer, and then desalted, freeze-dried, and stored at -80°C. The buffer for protein purification by Ni column is shown in Table 1.

[0095] Table 1. Ni column protein purification buffer

[0096] Buffer composition Binding buffer 20 mM phosphate, 0.5 M NaCl, 20 mM imidazole, pH 8.0 Washing buffer 20 mM phosphate, 0.5 M NaCl, 40 mM imidazole, pH 8.0 Elution buffer 20 mM phosphate, 0.5 M NaCl, 500 mM imidazole, pH 8.0

[0097] The purification effects of the five enzymes involved in this example are shown in Figure 1 As can be seen from the figure, except for P450 BM-3 (A74G / F87V / L188Q), the purity of the other four enzymes reached more than 90%, meeting the experimental requirements. The purity of monooxygenase (P450 BM-3 (A74G / F87V / L188Q)) was 70%.

[0098] In addition, the enzyme activity of the purified product was determined by fluorescence detection, and the change of the fluorescence signal of the reaction sample was continuously detected, with an excitation light of 350nm and an emission light of 420nm. Carboxylase: The reaction sample contained a final concentration of 200mM KHCO3 and a final concentration of 1-naphthol of 10mM. An appropriate amount of carboxylase was added, and the volume was supplemented to 120μL with potassium phosphate buffer (pH7.0 or pH7.5, 100mM), and transferred to a 96-well fluorescence ELISA plate. The increase of fluorescence was detected using an ELISA reader (the product 1-hydroxy-2-naphthoic acid has fluorescence characteristics). Definition: The enzyme activity required to generate 1μM 1-hydroxy-2-naphthoic acid per minute is 1U. Dioxygenase: The final concentration of 1-hydroxy-2-naphthoic acid in the reaction sample is 0.2mM. Add an appropriate amount of carboxylase and use potassium phosphate buffer (pH7.0 or pH7.5, 100mM) to make up the volume to 120μL. Transfer to a 96-well fluorescent microplate and use a microplate reader to detect the decrease in fluorescence. Definition: The enzyme activity required to convert 1μM 1-hydroxy-2-naphthoic acid per minute is 1U. Enzyme activity results: 2,3-DHBD: 0.65U / mg 蛋白 , SAD: 0.09U / mg 蛋白 , 2,6-DHBD: 0.002U / mg 蛋白 , 1HNDO: 12U / mg 蛋白 .

[0099] Example 3: Carbon fixation by coupling reaction of 2,3-DHBD and 1HNDO

[0100] 1. Weigh 1-naphthol and dissolve it in N,N-dimethylformamide (DMF) to prepare a 500 mM solution. Prepare potassium phosphate buffer (pH 7.0, 100 mM). Prepare 3 M KHCO3 solution.

[0101] 2. Prepare enzyme solution: dissolve the carboxylase 2,3-DHBD and dioxygenase 1HNDO prepared in Example 2 in potassium phosphate buffer (pH 7.0, 100 mM), respectively.

[0102] 3. Premix the reaction components to prepare a reaction solution as shown in Table 2.

[0103] Table 2. Coupling reaction solution (1 mL)

[0104] Components concentration 1-Naphthol 15mM <![CDATA[KHCO3]]> 50mM Carboxylase (2,3-DHBD) 1U / mL Dioxygenase (1HNDO) 9U / mL Potassium phosphate buffer (pH 7.0, 100 mM) Make up 1mL

[0105] Note: The concentration of each substance in the table is the final concentration in the reaction solution.

[0106] Reaction conditions: 200 rpm, 30°C for 12 h.

[0107] The reaction results were detected by high performance liquid chromatography.

[0108] Column: PAH;

[0109] Eluent: A: 5 mM ammonium acetate, B: acetonitrile;

[0110] Elution program: as shown in Table 3.

[0111] Table 3. HPLC elution program 1

[0112] time Rate (ml / min) %B(flow rate ratio) 0.0 0.6 5.0 7.0 0.6 5.0 12.0 0.8 45.0 24.0 0.8 50.0 30.0 0.6 5.0

[0113] Detection: UV absorption, 300nm.

[0114] The liquid phase detection results of 2-carboxybenzopyruvic acid and 1-naphthol are shown in Figure 2 (A: After the 2-carboxybenzopyruvic acid prepared by the enzyme reaction was separated and purified, the LC-MS identification result showed that the structure and molecular weight of the substance were correct; B: The liquid phase detection result of the 1-naphthol standard product).

[0115] The results are as follows Figure 3 As shown, the control Figure 2 It can be seen that the substrate 1-naphthol is almost completely converted into the product 2-carboxybenzopyruvic acid.

[0116] Example 4: Carbon fixation by coupling reaction of 2,6-DHBD and 1HNDO

[0117] 1. Weigh 1-naphthol and dissolve it in N,N-dimethylformamide (DMF) to prepare a solution with a concentration of 500mM. Prepare potassium phosphate buffer (pH 7.0, 100mM) and 3M KHCO3 solution.

[0118] 2. Prepare enzyme solution: dissolve the carboxylase 2,6-DHBD and dioxygenase 1HNDO prepared in Example 2 in potassium phosphate buffer (pH 7.0, 100 mM), respectively.

[0119] 3. Premix the reaction components to prepare a reaction solution as shown in Table 4.

[0120] Table 4. Coupling reaction solution (1 mL)

[0121]

[0122]

[0123] Note: The concentration of each substance in the table is the final concentration in the reaction solution.

[0124] Reaction conditions: 200 rpm, 30°C for 12 h.

[0125] The reaction results were detected by high performance liquid chromatography (the specific detection method is the same as in Example 3).

[0126] The results are as follows Figure 4 As shown, the control Figure 2 , it can be seen that only part of the substrate 1-naphthol is converted into the product 2-carboxybenzopyruvate.

[0127] Example 5: Carbon fixation by coupling reaction of SAD and 1HNDO

[0128] 1. Weigh 1-naphthol and dissolve it in N,N-dimethylformamide (DMF) to prepare a solution with a concentration of 500mM. Prepare potassium phosphate buffer (pH 7.0, 100mM) and 3M KHCO3 solution.

[0129] 2. Prepare enzyme solution: dissolve the carboxylase SAD and dioxygenase 1HNDO prepared in Example 2 in potassium phosphate buffer (pH 7.0, 100 mM), respectively.

[0130] 3. Premix the reaction components to prepare a reaction solution as shown in Table 5.

[0131] Table 5. Coupling reaction solution (1 mL)

[0132] Components concentration 1-Naphthol 15mM <![CDATA[KHCO3]]> 45mM SAD 0.14U / mL Dioxygenase (1HNDO) 9U / mL Potassium phosphate buffer (pH 7.0, 100 mM) Make up 1mL

[0133] Note: The concentration of each substance in the table is the final concentration in the reaction solution.

[0134] Reaction conditions: 200 rpm, 30°C for 12 h.

[0135] The reaction results were detected by high performance liquid chromatography (the specific detection method is the same as in Example 3).

[0136] The results are as follows Figure 5 As shown, the control Figure 2 , it can be seen that only a small amount of substrate 1-naphthol is converted into the product 2-carboxybenzopyruvate.

[0137] Example 6: Carbon fixation by coupling reaction of 2,3-DHBD and 1HNDO - resting cells

[0138] 1. Weigh 1-naphthol and dissolve it in N,N-dimethylformamide (DMF) to prepare a solution with a concentration of 500mM. Prepare potassium phosphate buffer (pH 7.0, 100mM) and 3M KHCO3 solution.

[0139] 2. Prepare resting cells, and resuspend the cells expressing 2,3-DHBD and 1HNDO (Escherichia coli BL21 Gold (DE3) expressing 2,3-DHBD and 1HNDO constructed in Example 1) in potassium phosphate buffer (pH 7.0, 100 mM).

[0140] 3. Premix the reaction components to prepare a reaction solution as shown in Table 6.

[0141] Table 6. Coupling reaction solution (1 mL)

[0142]

[0143]

[0144] Note: The concentration of each substance in the table is the final concentration in the reaction solution.

[0145] Reaction conditions: 30°C for 12h.

[0146] The reaction results were detected by high performance liquid chromatography (the specific detection method is the same as in Example 3).

[0147] The results are as follows Figure 6 As shown, the control Figure 2 , it can be seen that all 1-naphthol is converted into the product 2-carboxybenzopyruvic acid.

[0148] Example 7: Carbon fixation by coupling reaction of 2,3-DHBD and 1HNDO - CO2

[0149] 1. Weigh 1-naphthol and dissolve it in N,N-dimethylformamide (DMF) to prepare a solution with a concentration of 500 mM. Prepare potassium phosphate buffer (pH 7.5, 100 mM).

[0150] 2. Prepare enzyme solution: dissolve the carboxylase 2,3-DHBD and dioxygenase 1HNDO prepared in Example 2 in potassium phosphate buffer (pH 7.5, 100 mM), respectively.

[0151] 3. Premix the reaction components to prepare a reaction solution as shown in Table 7.

[0152] Table 7. Coupling reaction solution (1 mL)

[0153] Components concentration 1-Naphthol 15mM Carboxylase (2,3-DHBD) 1U / mL Dioxygenase (1HNDO) 9U / mL Potassium phosphate buffer (pH 7.5, 100 mM) Make up 1mL

[0154] Note: The concentration of each substance in the table is the final concentration in the reaction solution.

[0155] Reaction conditions: 200 rpm, 30°C for 12 h, with CO2 continuously introduced during the reaction.

[0156] The reaction results were detected by high performance liquid chromatography (the specific detection method is the same as in Example 3).

[0157] The results are as follows Figure 7 As shown, the control Figure 2 , it can be seen that the substrate 1-naphthol is almost completely converted into the product 2-carboxybenzopyruvic acid. It can be seen that CO2 can replace HCO3 very well. - The present invention is of great significance for improving the efficiency of fixing CO2.

[0158] Example 8: Opening of the Aromatic Ring of Naphthalene

[0159] 1. Weigh naphthalene and dissolve it in N,N-dimethylformamide (DMF) to prepare a 500 mM solution. Prepare potassium phosphate buffer (pH 7.0, 100 mM) and 3 M KHCO3 solution.

[0160] 2. Prepare enzyme solution: dissolve P450 BM-3 (A74G / F87V / L188Q), 2,3-DHBD and 1HNDO prepared in Example 2 in potassium phosphate buffer (pH 7.0, 100 mM), respectively.

[0161] 3. Premix the components of the reaction to prepare a reaction solution, as shown in Table 8.

[0162] Table 8. Coupling reaction solution (1 mL)

[0163]

[0164]

[0165] Note: The concentration of each substance in the table is the final concentration in the reaction solution.

[0166] Reaction conditions: 200 rpm, 30°C for 12 h.

[0167] The reaction results were detected by high performance liquid chromatography (the specific detection method is the same as in Example 3, only the elution procedure is different, and the elution procedure of this embodiment is shown in Table 9):

[0168] Table 9. HPLC elution procedure 2

[0169] time Rate (ml / min) %B(flow rate ratio) 0.0 0.5 1.0 10.0 0.5 20.0 15.0 0.8 80.0 24.0 0.8 80.0 30.0 0.5 10.0

[0170] The results are as follows Figure 8 As shown, the control Figure 2 , it can be seen that only naphthalene and HCO3 - In the case of 2-carboxybenzopyruvic acid as a substrate, the generation of 2-carboxybenzopyruvic acid was successfully detected through the reaction route designed by the present invention, which proves the effectiveness of this route in degrading naphthalene and fixing carbon.

[0171] Example 9: Opening of the Aromatic Ring of Naphthalene - Coenzyme Cycle

[0172] 1. Weigh naphthalene and dissolve it in N,N-dimethylformamide (DMF) to prepare a 500 mM solution. Prepare potassium phosphate buffer (pH 7.0, 100 mM) and 3 M KHCO3 solution.

[0173] 2. Prepare resting cells. Resuspend the expression cells of P450 BM-3 (A74G / F87V / L188Q), 2,3-DHBD and 1HNDO (the E. coli BL21 Gold (DE3) expressing P450 BM-3 (A74G / F87V / L188Q), 2,3-DHBD and 1HNDO constructed in Example 1) in potassium phosphate buffer (pH 7.0, 100 mM). Prepare alcohol dehydrogenase (ADH) resting cells (the recombinant bacteria obtained by introducing the alcohol dehydrogenase ADH encoding gene into E. coli BL21 Gold (DE3) according to the method of Example 1) for the regeneration and circulation of NADH (to reduce the cost of the reaction).

[0174] 3. Premix the components of the reaction to prepare a reaction solution, as shown in Table 10.

[0175] Table 10, coupling reaction solution (1mL)

[0176] Components concentration Naphthalene 15mM <![CDATA[NAD + ]]> 30mM Isopropyl alcohol 30mM <![CDATA[KHCO3]]> 50mM P450 BM-3 (A74G / F87V / L188Q) cells <![CDATA[0.5g 细胞湿重 / mL]]> ADH Cells <![CDATA[0.5g 细胞湿重 / mL]]> 2,3-DHBD cells <![CDATA[0.5g 细胞湿重 / mL]]> 1HNDO cells <![CDATA[0.5g 细胞湿重 / mL]]> Potassium phosphate buffer (pH 7.0, 100 mM) Make up 1mL

[0177] Note: The concentration of each substance in the table is the final concentration in the reaction solution.

[0178] Reaction conditions: 30°C for 12h.

[0179] The reaction results were detected by high performance liquid chromatography (the specific detection method was the same as in Example 3, only the elution procedure was different, and the elution procedure of this example is shown in Table 9).

[0180] The results are as follows Fig. 9 As shown, the control Figure 2 , it can be seen that only naphthalene and HCO3 - When using oxidized coenzyme NAD as a substrate + Through the reaction route designed by the present invention, the generation of 2-carboxybenzopyruvic acid was successfully detected, proving the effectiveness of this route in degrading naphthalene and fixing carbon and the role of the coenzyme circulation system.

[0181] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims. <110> Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences <120> A kind of ring opening method of aromatic compound <130> GNCLN202053 <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 1051 <212> PRT <213> Artificial sequence <400> 1 Met Gly Met Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu 1 5 10 15 Leu Lys Asn Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu 20 25 30 Met Lys Ile Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro 35 40 45 Gly Arg Val Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala 50 55 60 Cys Asp Glu Ser Arg Phe Asp Lys Asn Leu Ser Gln Gly Leu Lys Phe 65 70 75 80 Val Arg Asp Phe Ala Gly Asp Gly Leu Val Thr Ser Trp Thr His Glu 85 90 95 Lys Asn Trp Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln 100 105 110 Gln Ala Met Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln 115 120 125 Leu Val Gln Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val 130 135 140 Pro Glu Asp Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly 145 150 155 160 Phe Asn Tyr Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe 165 170 175 Ile Thr Ser Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Gln Gln 180 185 190 Arg Ala Asn Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe 195 200 205 Gln Glu Asp Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala 210 215 220 Asp Arg Lys Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met 225 230 235 240 Leu Asn Gly Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn 245 250 255 Ile Arg Tyr Gln Ile Ile Thr Phe Leu Ile Ala Gly His Glu Thr Thr 260 265 270 Ser Gly Leu Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His 275 280 285 Val Leu Gln Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro 290 295 300 Val Pro Ser Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val 305 310 315 320 Leu Asn Glu Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu 325 330 335 Tyr Ala Lys Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys 340 345 350 Gly Asp Glu Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr 355 360 365 Ile Trp Gly Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn 370 375 380 Pro Ser Ala Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln 385 390 395 400 Arg Ala Cys Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val 405 410 415 Leu Gly Met Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr 420 425 430 Glu Leu Asp Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val 435 440 445 Val Lys Ala Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro 450 455 460 Ser Thr Glu Gln Ser Ala Lys Lys Val Arg Lys Lys Ala Glu Asn Ala 465 470 475 480 His Asn Thr Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala 485 490 495 Glu Gly Thr Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe 500 505 510 Ala Pro Gln Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg 515 520 525 Glu Gly Ala Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro 530 535 540 Asp Asn Ala Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp 545 550 555 560 Glu Val Lys Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn 565 570 575 Trp Ala Thr Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu 580 585 590 Ala Ala Lys Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala 595 600 605 Ser Asp Asp Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp 610 615 620 Ser Asp Val Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp 625 630 635 640 Asn Lys Ser Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met 645 650 655 Pro Leu Ala Lys Met His Gly Ala Phe Ser Thr Asn Val Val Ala Ser 660 665 670 Lys Glu Leu Gln Gln Pro Gly Ser Ala Arg Ser Thr Arg His Leu Glu 675 680 685 Ile Glu Leu Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly 690 695 700 Val Ile Pro Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg 705 710 715 720 Phe Gly Leu Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu 725 730 735 Lys Leu Ala His Leu Pro Leu Ala Lys Thr Val Ser Val Glu Glu Leu 740 745 750 Leu Gln Tyr Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg 755 760 765 Ala Met Ala Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu 770 775 780 Ala Leu Leu Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg 785 790 795 800 Leu Thr Met Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Lys 805 810 815 Phe Ser Glu Phe Ile Ala Leu Leu Pro Ser Ile Arg Pro Arg Tyr Tyr 820 825 830 Ser Ile Ser Ser Ser Pro Arg Val Asp Glu Lys Gln Ala Ser Ile Thr 835 840 845 Val Ser Val Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys 850 855 860 Gly Ile Ala Ser Asn Tyr Leu Ala Glu Leu Gln Glu Gly Asp Thr Ile 865 870 875 880 Thr Cys Phe Ile Ser Thr Pro Gln Ser Glu Phe Thr Leu Pro Lys Asp 885 890 895 Pro Glu Thr Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro 900 905 910 Phe Arg Gly Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln 915 920 925 Ser Leu Gly Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu 930 935 940 Asp Tyr Leu Tyr Gln Glu Glu Leu Glu Asn Ala Gln Ser Glu Gly Ile 945 950 955 960 Ile Thr Leu His Thr Ala Phe Ser Arg Met Pro Asn Gln Pro Lys Thr 965 970 975 Tyr Val Gln His Val Met Glu Gln Asp Gly Lys Lys Leu Ile Glu Leu 980 985 990 Leu Asp Gln Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met 995 1000 1005 Ala Pro Ala Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val 1010 1015 1020 His Gln Val Ser Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu 1025 1030 1035 Glu Glu Lys Gly Arg Tyr Ala Lys Asp Val Trp Ala Gly 1040 1045 1050 <210> 2 <211> 338 <212> PRT <213> Artificial sequence <400> 2 Met Leu Gly Lys Ile Ala Leu Glu Glu Ala Phe Ala Leu Pro Arg Phe 1 5 10 15 Glu Glu Lys Thr Arg Trp Trp Ala Ser Leu Phe Ser Thr Asp Ala Glu 20 25 30 Thr His Val Lys Glu Ile Thr Asp Ile Asn Lys Ile Arg Ile Glu His 35 40 45 Ala Asp Lys His Gly Val Gly Tyr Gln Ile Leu Ser Tyr Thr Ala Pro 50 55 60 Gly Val Gln Asp Ile Trp Asp Pro Val Glu Ala Gln Ala Leu Ala Val 65 70 75 80 Glu Ile Asn Asp Tyr Ile Ala Glu Gln Val Arg Val Asn Pro Asp Arg 85 90 95 Phe Gly Ala Phe Ala Thr Leu Ser Met His Asn Pro Lys Glu Ala Ala 100 105 110 Asp Glu Leu Arg Arg Cys Val Glu Lys Tyr Gly Phe Lys Gly Ala Leu 115 120 125 Val Asn Asp Thr Gln Arg Ala Gly Pro Asp Gly Asp Asp Met Ile Phe 130 135 140 Tyr Asp Asn Ala Asp Trp Asp Ile Phe Trp Gln Thr Cys Thr Glu Leu 145 150 155 160 Asp Val Pro Phe Tyr Met His Pro Arg Asn Pro Thr Gly Thr Ile Tyr 165 170 175 Glu Lys Leu Trp Ala Asp Arg Lys Trp Leu Val Gly Pro Pro Leu Ser 180 185 190 Phe Ala His Gly Val Ser Leu His Val Leu Gly Met Val Thr Asn Gly 195 200 205 Val Phe Asp Arg His Pro Lys Leu Gln Ile Ile Met Gly His Leu Gly 210 215 220 Glu His Val Pro Phe Asp Met Trp Arg Ile Asn His Trp Phe Glu Asp 225 230 235 240 Arg Lys Lys Leu Leu Gly Leu Ala Glu Thr Cys Lys Lys Thr Ile Arg 245 250 255 Asp Tyr Phe Ala Glu Asn Ile Trp Ile Thr Thr Ser Gly His Phe Ser 260 265 270 Thr Thr Thr Leu Asn Phe Cys Met Ala Glu Val Gly Ser Asp Arg Ile 275 280 285 Leu Phe Ser Ile Asp Tyr Pro Phe Glu Thr Phe Ser Asp Ala Cys Glu 290 295 300 Trp Phe Asp Asn Ala Glu Leu Asn Gly Thr Asp Arg Leu Lys Ile Gly 305 310 315 320 Arg Glu Asn Ala Lys Lys Leu Phe Lys Leu Asp Ser Tyr Lys Asp Ser 325 330 335 Ser Ala <210> 3 <211> 327 <212> PRT <213> Artificial sequence <400> 3 Met Gln Gly Lys Val Ala Leu Glu Glu His Phe Ala Ile Pro Glu Thr 1 5 10 15 Leu Gln Asp Ser Ala Gly Phe Val Pro Gly Asp Tyr Trp Lys Glu Leu 20 25 30 Gln His Arg Leu Leu Asp Ile Gln Asp Thr Arg Leu Lys Leu Met Asp 35 40 45 Ala His Gly Ile Glu Thr Met Ile Leu Ser Leu Asn Ala Pro Ala Val 50 55 60 Gln Ala Ile Pro Asp Arg Arg Lys Ala Ile Glu Ile Ala Arg Arg Ala 65 70 75 80 Asn Asp Val Leu Ala Glu Glu Cys Ala Lys Arg Pro Asp Arg Phe Leu 85 90 95 Ala Phe Ala Ala Leu Pro Leu Gln Asp Pro Asp Ala Ala Thr Glu Glu 100 105 110 Leu Gln Arg Cys Val Asn Asp Leu Gly Phe Val Gly Ala Leu Val Asn 115 120 125 Gly Phe Ser Gln Glu Gly Asp Gly Gln Thr Pro Leu Tyr Tyr Asp Leu 130 135 140 Pro Gln Tyr Arg Pro Phe Trp Gly Glu Val Glu Lys Leu Asp Val Pro 145 150 155 160 Phe Tyr Leu His Pro Arg Asn Pro Leu Pro Gln Asp Ser Arg Ile Tyr 165 170 175 Asp Gly His Pro Trp Leu Leu Gly Pro Thr Trp Ala Phe Ala Gln Glu 180 185 190 Thr Ala Val His Ala Leu Arg Leu Met Ala Ser Gly Leu Phe Asp Glu 195 200 205 His Pro Arg Leu Asn Ile Ile Leu Gly His Met Gly Glu Gly Leu Pro 210 215 220 Tyr Met Met Trp Arg Ile Asp His Arg Asn Ala Trp Val Lys Leu Pro 225 230 235 240 Pro Arg Tyr Pro Ala Lys Arg Arg Phe Met Asp Tyr Phe Asn Glu Asn 245 250 255 Phe His Ile Thr Thr Ser Gly Asn Phe Arg Thr Gln Thr Leu Ile Asp 260 265 270 Ala Ile Leu Glu Ile Gly Ala Asp Arg Ile Leu Phe Ser Thr Asp Trp 275 280 285 Pro Phe Glu Asn Ile Asp His Ala Ser Asp Trp Phe Asn Ala Thr Ser 290 295 300 Ile Ala Glu Ala Asp Arg Val Lys Ile Gly Arg Thr Asn Ala Arg Arg 305 310 315 320 Leu Phe Lys Leu Asp Gly Ala 325 <210> 4 <211> 350 <212> PRT <213> Artificial sequence <400> 4 Met Arg Gly Lys Val Ser Leu Glu Glu Ala Phe Glu Leu Pro Lys Phe 1 5 10 15 Ala Ala Gln Thr Lys Glu Lys Ala Glu Leu Tyr Ile Ala Pro Asn Asn 20 25 30 Arg Asp Arg Tyr Phe Glu Glu Ile Leu Asn Pro Cys Gly Asn Arg Leu 35 40 45 Glu Leu Ser Asn Lys His Gly Ile Gly Tyr Thr Ile Tyr Ser Ile Tyr 50 55 60 Ser Pro Gly Pro Gln Gly Trp Thr Glu Arg Ala Glu Cys Glu Glu Tyr 65 70 75 80 Ala Arg Glu Cys Asn Asp Tyr Ile Ser Gly Glu Ile Ala Asn His Lys 85 90 95 Asp Arg Met Gly Ala Phe Ala Ala Leu Ser Met His Asp Pro Lys Gln 100 105 110 Ala Ser Glu Glu Leu Thr Arg Cys Val Lys Glu Leu Gly Phe Leu Gly 115 120 125 Ala Leu Val Asn Asp Val Gln His Ala Gly Pro Glu Gly Glu Thr His 130 135 140 Ile Phe Tyr Asp Gln Pro Glu Trp Asp Ile Phe Trp Gln Thr Cys Val 145 150 155 160 Asp Leu Asp Val Pro Phe Tyr Leu His Pro Glu Pro Pro Phe Gly Ser 165 170 175 Tyr Leu Arg Asn Gln Tyr Glu Gly Arg Lys Tyr Leu Ile Gly Pro Pro 180 185 190 Val Ser Phe Ala Asn Gly Val Ser Leu His Val Leu Gly Met Ile Val 195 200 205 Asn Gly Val Phe Asp Arg Phe Pro Lys Leu Lys Val Ile Leu Gly His 210 215 220 Leu Gly Glu His Ile Pro Gly Asp Phe Trp Arg Ile Glu His Trp Phe 225 230 235 240 Glu His Cys Ser Arg Pro Leu Ala Lys Ser Arg Gly Asp Val Phe Ala 245 250 255 Glu Lys Pro Leu Leu His Tyr Phe Arg Asn Asn Ile Trp Leu Thr Thr 260 265 270 Ser Gly Asn Phe Ser Thr Glu Thr Leu Lys Phe Cys Val Glu His Val 275 280 285 Gly Ala Glu Arg Ile Leu Phe Ser Val Asp Ser Pro Tyr Glu His Ile 290 295 300 Asp Val Gly Cys Gly Trp Tyr Asp Asp Asn Ala Lys Ala Ile Met Glu 305 310 315 320 Ala Val Gly Gly Glu Lys Ala Tyr Lys Asp Ile Gly Arg Asp Asn Ala 325 330 335 Lys Lys Leu Phe Lys Leu Gly Lys Phe Tyr Asp Ser Glu Ala 340 345 350 <210> 5 <211> 361 <212> PRT <213> Artificial sequence <400> 5 Met Ser Thr Ala Glu Ser Ser Glu Leu Arg Glu Phe Asp Val Glu Leu 1 5 10 15 Glu Ala Ala Asn Leu Arg Gly Gln Trp Ile Tyr Asp Asp Met Leu Glu 20 25 30 Ser Val Val Gly Gly Pro Lys Pro Ala Gly Val Pro Phe Leu Trp Arg 35 40 45 Trp His Asp Val Tyr Ala Lys Leu Leu Lys Ser Cys Asp Val Met Pro 50 55 60 Glu Ser Leu Thr Ala Arg Arg Asn Leu Ser Phe Ile Asn Pro Asp Ala 65 70 75 80 Arg Gly Thr Thr His Thr Ile Asn Met Gly Met Gln Met Leu Lys Pro 85 90 95 Gly Glu Ile Ala Tyr Ala His Arg His Thr Met Ala Ala Leu Arg Phe 100 105 110 Ala Ile Gln Gly Gly Pro Gly Leu Val Thr Val Val Asp Gly Glu Pro 115 120 125 Cys Gln Met Asp Thr Tyr Asp Leu Val Leu Thr Pro Arg Trp Thr Trp 130 135 140 His Asp His Glu Asn Ala Thr Ser Glu Asn Val Val Trp Leu Asp Val 145 150 155 160 Leu Asp Ile Gly Leu Val Leu Gly Leu Asn Val Pro Phe Tyr Glu Pro 165 170 175 Tyr Gly Glu Met Arg Gln Pro Gln Arg Glu Asp Pro Gly Glu His Leu 180 185 190 Ala Asp Arg Gly Gly Met Leu Arg Pro Ala Trp Glu Gln Val Lys Ala 195 200 205 Ala Asn Phe Pro Tyr Arg Tyr Pro Trp Arg Asp Val Glu Arg Gln Leu 210 215 220 Gln Arg Met Ala Gly Leu Ala Gly Ser Pro Tyr Asp Gly Val Val Leu 225 230 235 240 Arg Tyr Ala Asn Pro Val Thr Gly Gly Ser Thr Met Pro Thr Leu Asp 245 250 255 Cys Trp Val Gln Leu Leu Arg Pro Gly Gln Gln Thr Glu Ala His Arg 260 265 270 His Thr Ser Ser Ala Val Tyr Phe Val Arg Gly Glu Gly Thr Thr 275 280 285 Val Val Asp Gly Val Glu Leu Asp Trp Gly Pro His Asp Ser Phe Val 290,295,300 Val Pro Asn Trp Ser Thr His Phe Val Asn Arg Ser Ala Glu Asn 305 310 315 320 Ala Leu Leu Phe Ser Val Asn Asp Ile Pro Thr Leu Lys Ala Leu Asp 325 330 335 Leu Tyr Tyr Glu Glu Pro Glu Leu Ser Leu Gly Thr Gln Pro Phe Pro 340 345 350 Pro Val Pro Wing Leu Arg Wing Arg 355,360 <210> 6 <211> 3156 <212> DNA <213> Artificial Sequence <400> 6 atgggcatga cattaaaga aatgcctcag ccaaaacgt ttggagct taaaaattta 60 ccgttattaa accagataa accggttcaa gctttgatga aaattgcgga tgaattagga 120 gaaatcttta aattcgaggc gcctggtcgt gtaacgcgct acttatcaag tcagcgtcta 180 attaaagaag catgcgatga atcacgcttt gataaaaact taagtcaagg tcttaaattt 240 gtacgtgatt ttgcaggaga cgggttagtg acaagctgga cgcatgaaaa aaattggaaa 300 aaagcgcata atatcttact tccaagcttc agtcagcagg caatgaaagg ctatcatgcg 360 atgatggtcg atatcgccgt gcagcttgtt caaaagtggg agcgtctaaa tgcagatgag 420 catattgaag taccggaaga catgacacgt ttaacgcttg atacaattgg tctttgcggc 480 tttaactatc gctttaacag cttttaccga gatcagcctc atccatttat tacaagtatg 540 gtccgtgcac tggatgaagc aatgaacaag cagcagcgag caaatccaga cgacccagct 600 tatgatgaaa acaagcgcca gtttcaagaa gatatcaagg tgatgaacga cctagtagat 660 aaaattattg cagatcgcaa agcaagcggt gaacaaagcg atgatttatt aacgcatatg 720 ctaaacggaa aagatccaga aacgggtgag ccgcttgatg acgagaacat tcgctatcaa 780 attattacat tcttaattgc gggacacgaa acaacaagtg gtcttttatc atttgcgctg 840 tattcttag tgaaaaatcc acatgtatta caaaagcag cagaagaagc agcacgagtt 900 ctagtagatc ctgttccaag ctacaaaaaa gtcaacagc ttaatatgt cggcatggtc 960 ttaaacgaag cgctgcgctt atggccaact gctcctgcgt ttccctata tgcaaagaa 1020 gatacggtgc ttggagga atatccttta gaaaaaggcg acgaactaat ggttctgatt 1080 cctcagcttc accgtgataa aacaatttgg ggagacgatg tggagagtt ccgtccagag 1140 cgttttgaaa atccaagtgc gattccgcag catgcgttta aaccgttttgg aaacggtcag 1200 cgtgcgtgta tcggtcagca gttcgctctt catgaagcaa cgctggtact tggtatgatg 1260 ctaaaacact tgacttga agatcataca acacacgagc tggattaa agaacttta 1320 acgttaaaac ctgaaggctt tgtgtaaaa gcaaatcga aaaaaattcc gcttggcggt 1380 attccttcac ctagcactga acagtctgct aaaaagtac gcaaaaggc agaaaacgct 1440 cataatacgc cgctgcttgt gctatacggt tcaatatgg gaacagctga aggaacggcg 1500 cgtgatttag cagatattgc atgagcaaa ggatttgcac cgcaggtcgc aacgcttgat 1560 tcacacgccg gaaatctcc gcgcgaagga gctgtatta ttgtaacggc gtcttataac 1620 ggtcatccgc ctgataacgc aaagcaattt gtcgactggt tegccaagc gtctgctgat 1680 gaagtaaag gcgttcgcta ctccgtattt ggatgcggcg ataaaactg gctactacg 1740 tatcaaaag tgcctgcttt tatcgatgaa acgcttgccg ctaaaggggc agaaaaacatc 1800 gctgaccgcg gtgaagcaga tgcaagcgac gactttgaag gcacatatga agaatggcgt 1860 gaacatatgt ggagtgacgt agcagcctac tttaacctcg acattgaaaa cagtgaagat 1920 aaaatcta ctctttcact tcaatttgtc gagcgccg cggatatgcc gcttgcgaaa 1980 atgcacggtg cgttttcaac gaacgtcgta gcaagcaag aacttcaa gccaggcagt 2040 gcacgaagca cgcgacatct tgaaattgaa cttccaaag aagctctta tcagaagga 2100 gatcatttag gtgttattcc tcgcactat gagaatag taaaccgtgt aacagcaagg 2160 ttcggcctag atgcatcaca gcaatccgt ctggaagcag aagagaaaa atgctcat 2220 ttgccactcg ctaaaacagt atccgtagaa gagcttctgc atacgtgga gcttcagat 2280 cctgttacgc gcacgcagct tcgcgcaatg gctgctaaaa cggtctgccc gccgcataaa 2340 gtagagcttg aagccttgct tgaaaagcaa gcctacaaag aacaagtgct ggcaaaacgt 2400 ttaaaatgc ttgaactgct tgaaaaatac ccggcgtgtg aaatgaaatt cagcgaattt 2460 atcgcccttc tgccaagcat acgcccgcgc tattactcga tttcttcatc acctcgtgtc 2520 gatgaaaaac aagcaagcat cacggtcagc gttgtctcag gagaagcgtg gagcggatat 2580 ggagaatata aaagaattgc gtcgaactat cttgccgagc tgcaagaagg agatacgatt 2640 acgtgcttta tttccacacc gcagtcagaa tttacgctgc caaaagaccc tgaaacgccg 2700 cttatcatgg tcggaccggg aacaggcgtc gcgccgttta gaggctttgt gcaggcgcgc 2760 aaaagctaa aagaaagg acagtcactt ggagaagcac atttatactt cggctgccgt 2820 tcacctcatg aagactatct gtatcaagaa gagcttgaaa acgcccaaag cgaaggcatc 2880 attacgcttc ataccgcttt ttctcgcatg ccaaatcagc cgaaaacata cgttcagcac 2940 gtaatggaac aagacggcaa gaaattgatt gaacttcttg atcaaggagc gcacttctat 3000 atttgcggag acggaagcca aatggcacct gccgttgaag caacgcttat gaaaagctat 3060 gctgacgttc accaagtgag tgaagcagac gctcgcttat ggctgcagca gctagaagaa 3120 aaaggccgat acgcaaaaga cgtgtgggct gggtaa 3156 <210> 7 <211> 1017 <212> DNA <213> Artificial sequence <400> 7 atgctgggta aaatcgctct ggaagaagct ttcgctctgc cgcgtttcga agaaaaaacc 60 cgttggtggg cttctctgtt ctctaccgac gctgaaaccc acgttaaaga aatcaccgac 120 atcaacaaaa tccgtatcga acacgctgac aaacacggtg ttggttacca gatcctgtct 180 tacaccgctc cgggtgttca ggacatctgg gacccggttg aagctcaggc tctggctgtt 240 gaaatcaacg actacatcgc tgaacaggtt cgtgttaacc cggaccgttt cggtgctttc 300 gctaccctgt ctatgcacaa cccgaaagaa gctgctgacg aactgcgtcg ttgcgttgaa 360 aaatacggtt tcaaaggtgc tctggttaac gacacccagc gtgctggtcc ggacggtgac 420 gacatgatct tctacgacaa cgctgactgg gacatcttct ggcagacctg caccgaactg 480 gacgttccgt tctacatgca cccgcgtaac ccgaccggta ccatctacga aaaactgtgg 540 gctgaccgta aatggctggt tggtccgccg ctgtctttcg ctcacggtgt ttctctgcac 600 gttctgggta tggttaccaa cggtgttttc gaccgtcacc cgaaactgca gatcatcatg 660 ggtcacctgg gtgaacacgt tccgttcgac atgtggcgta tcaaccactg gttcgaagac 720 cgtaaaaaac tgctgggtct ggctgaaacc tgcaaaaaaa ccatccgtga ctacttcgct 780 gaaaacatct ggatcaccac ctctggtcac ttctctacca ccaccctgaa cttctgcatg 840 gctgaagttg gttctgaccg tatcctgttc tctatcgact acccgttcga aaccttctct 900 gacgcttgcg aatggttcga caacgctgaa ctgaacggta ccgaccgtct gaaaatcggt 960 cgtgaaaacg ctaaaaaact gttcaaactg gactcttaca aagactcttc tgcttaa 1017 <210> 8 <211> 984 <212> DNA <213> Artificial sequence <400> 8 atgcagggta aagttgctct ggaagaacac ttcgctatcc cggaaaccct gcaggactct 60 gctggtttcg ttccgggtga ctactggaaa gaactgcagc accgtctgct ggacatccag 120 gacacccgtc tgaaactgat ggacgctcac ggtatcgaaa ccatgatcct gtctctgaac 180 gctccggctg ttcaggctat cccggaccgt cgtaaagcta tcgaaatcgc tcgtcgtgct 240 aacgacgttc tggctgaaga atgcgctaaa cgtccggacc gtttcctggc tttcgctgct 300 ctgccgctgc aggacccgga cgctgctacc gaagaactgc agcgttgcgt taacgacctg 360 ggtttcgttg gtgctctggt taacggtttc tctcaggaag gtgacggtca gaccccgctg 420 tactacgacc tgccgcagta ccgtccgttc tggggtgaag ttgaaaaact ggacgttccg 480 ttctacctgc acccgcgtaa cccgctgccg caggactctc gtatctacga cggtcacccg 540 tggctgctgg gtccgacctg ggctttcgct caggaaaccg ctgttcacgc tctgcgtctg 600 atggcttctg gtctgttcga cgaacacccg cgtctgaaca tcatcctggg tcacatgggt 660 gaaggtctgc cgtacatgat gtggcgtatc gaccaccgta acgcttgggt taaactgccg 720 ccgcgttacc cggctaaacg tcgtttcatg gactacttca acgaaaactt ccacatcacc 780 acctctggta acttccgtac ccagaccctg atcgacgcta tcctggaaat cggtgctgac 840 cgtatcctgt tctctaccga ctggccgttc gaaaacatcg accacgcttc tgactggttc 900 aacgctacct ctatcgctga agctgaccgt gttaaaatcg gtcgtaccaa cgctcgtcgt 960 ctgttcaaac tggacggtgc ttaa 984 <210> 9 <211> 1053 <212> DNA <213> Artificial sequence <400> 9 atgcgtggta aagtttctct ggaagaagct ttcgaactgc cgaaattcgc tgctcagacc 60 aaagaaaaag ctgaactgta catcgctccg aacaaccgtg accgttactt cgaagaaatc 120 ctgaacccgt gcggtaaccg tctggaactg tctaacaaac acggtatcgg ttacaccatc 180 tactctatct actctccggg tccgcagggt tggaccgaac gtgctgaatg cgaagaatac 240 gctcgtgaat gcaacgacta catctctggt gaaatcgcta accacaaaga ccgtatgggt 300 gctttcgctg ctctgtctat gcacgacccg aaacaggctt ctgaagaact gacccgttgc 360 gttaaagaac tgggtttcct gggtgctctg gttaacgacg ttcagcacgc tggtccggaa 420 ggtgaaaccc acatcttcta cgaccagccg gaatgggaca tcttctggca gacctgcgtt 480 gacctggacg ttccgttcta cctgcacccg gaaccgccgt tcggttctta cctgcgtaac 540 cagtacgaag gtcgtaaata cctgatcggt ccgccggttt ctttcgctaa cggtgtttct 600 ctgcacgttc tgggtatgat cgttaacggt gttttcgacc gtttcccgaa actgaaagtt 660 atcctgggtc acctgggtga acacatcccg ggtgacttct ggcgtatcga acactggttc 720 gaacactgct ctcgtccgct ggctaaatct cgtggtgacg ttttcgctga aaaaccgctg 780 ctgcactact tccgtaacaa catctggctg accacctctg gtaacttctc taccgaaacc 840 ctgaaattct gcgttgaaca cgttggtgct gaacgtatcc tgttctctgt tgactctccg 900 tacgaacaca tcgacgttgg ttgcggttgg tacgacgaca acgctaaagc tatcatggaa 960 gctgttggtg gtgaaaaagc ttacaaagac atcggtcgtg acaacgctaa aaaactgttc 1020 aaactgggta aattctacga ctctgaagct taa 1053 <210> 10 <211> 1086 <212> DNA <213> Artificial sequence <400> 10 atgtctaccg ctgaatcttc tgaactgcgt gaattcgacg ttgaactgga agctgctaac 60 ctgcgtggtc agtggatcta cgacgacatg ctggaatctg ttgttggtgg tccgaaaccg 120 gctggtgttc cgttcctgtg gcgttggcac gacgtttacg ctaaactgct gaaatcttgc 180 gacgttatgc cggaatctct gaccgctcgt cgtaacctgt ctttcatcaa cccggacgct 240 cgtggtacca cccacaccat caacatgggt atgcagatgc tgaaaccggg tgaaatcgct 300 tacgctcacc gtcacaccat ggctgctctg cgtttcgcta tccagggtgg tccgggtctg 360 gttaccgttg ttgacggtga accgtgccag atggacacct acgacctggt tctgaccccg 420 cgttggacct ggcacgacca cgaaaacgct acctctgaaa acgttgtttg gctggacgtt 480 ctggacatcg gtctggttct gggtctgaac gttccgttct acgaaccgta cggtgaaatg 540 cgtcagccgc agcgtgaaga cccgggtgaa cacctggctg accgtggtgg tatgctgcgt 600 ccggcttggg aacaggttaa agctgctaac ttcccgtacc gttacccgtg gcgtgacgtt 660 gaacgtcagc tgcagcgtat ggctggtctg gctggttctc cgtacgacgg tgttgttctg 720 cgttacgcta acccggttac cggtggttct accatgccga ccctggactg ctgggttcag 780 ctgctgcgtc cgggtcagca gaccgaagct caccgtcaca cctcttctgc tgtttacttc 840 gttgttcgtg gtgaaggtac caccgttgtt gacggtgttg aactggactg gggtccgcac 900 gactctttcg ttgttccgaa ctggtctacc caccacttcg ttaaccgttc tgctgaaaac 960 gctctgctgt tctctgttaa cgacatcccg accctgaaag ctctggacct gtactacgaa 1020 gaaccggaac tgtctctggg tacccagccg ttcccgccgg ttccggctaa cctgcgtgct 1080 cgttaa 1086

Claims

1. A method for degrading naphthalene and / or fixing CO2, comprising the steps of: (a1) Naphthalene is catalyzed by monooxygenase to produce 1-naphthol; (a2) 1-naphthol is catalyzed by carboxylase to generate 1-hydroxy-2-benzoic acid; (a3) 1-Hydroxy-2-benzoic acid is catalyzed by dioxygenase to generate 2-carboxybenzopyruvic acid.

2. The method according to claim 1, characterized in that: In step (a1), NAD(P)H is used to provide reducing power.

3. The method according to claim 2, characterized in that: The providing reducing power with NAD(P)H is achieved by any of the following methods: 1) directly adding NAD(P)H to the reaction system; 2) forming a coenzyme NAD(P)H cycle in the reaction system.

4. The method according to claim 3, characterized in that: The formation of the coenzyme NAD(P)H cycle in the reaction system is achieved by any of the following methods: 1) adding cells capable of expressing alcohol dehydrogenase and NAD(P) + ; 2) adding cells capable of expressing alcohol dehydrogenase and NAD(P)H to the reaction system.

5. The method according to claim 1, characterized in that: In step (a2), HCO3 - or CO2 as another substrate.

6. The method according to claim 1, characterized in that: In the method, the catalytic reaction of various enzymes is carried out by any of the following methods: 1) directly adding the corresponding enzymes into the reaction system; 2) adding cells capable of expressing the corresponding enzymes into the reaction system.

7. The method according to claim 1, characterized in that: Steps (a1) to (a3) ​​are completed in the same reaction system through a one-step reaction.

8. The method according to claim 7, characterized in that: The reaction system contains 1) naphthalene, 2) the monooxygenase or a cell capable of expressing the monooxygenase, 3) the carboxylase or a cell capable of expressing the carboxylase, 4) the dioxygenase or a cell capable of expressing the dioxygenase, 5) NAD(P)H, 6) HCO3 - or CO2, 7) reaction buffer.

9. The method according to claim 8, characterized in that: In the reaction system, the final concentration of naphthalene is 15mM, the final concentration of NAD(P)H is 60mM, and the final concentration of HCO3 is 15mM. - The final concentration is 50 mM or CO2 is continuously introduced into the reaction system during the reaction.

10. The method according to claim 8, characterized in that: The pH of the reaction buffer is 6.5-8.

0.

11. The method according to claim 7, characterized in that: The reaction temperature is 25-35°C and the reaction time is 3-12h.

12. The method according to any one of claims 1 to 11, characterized in that: The monooxygenase is derived from Bacillus megaterium.

13. The method according to claim 12, characterized in that: The amino acid sequence of the monooxygenase derived from Bacillus megaterium is shown in SEQ ID No.

1.

14. The method according to any one of claims 1 to 11, characterized in that: The carboxylase is a carboxylase derived from Aspergillus oryzae, a carboxylase derived from Rhizobium sp., or a carboxylase derived from Trichosporon moniliiforme.

15. The method according to claim 14, characterized in that: The amino acid sequence of the carboxylase derived from Aspergillus oryzae is shown in SEQ ID No.2; the amino acid sequence of the carboxylase derived from Rhizobium sp. is shown in SEQ ID No.3; the amino acid sequence of the carboxylase derived from Trichosporon moniliforme is shown in SEQ ID No.

4.

16. The method according to any one of claims 1 to 11, characterized in that: The dioxygenase is a dioxygenase derived from Mycobacterium vanbaalenii PYR-1.

17. The method according to claim 16, characterized in that: The amino acid sequence of the dioxygenase derived from Mycobacterium vanbaalenii PYR-1 is shown in SEQ ID No.

5.

18. A method for producing 2-carboxybenzopyruvic acid using naphthalene as a substrate, comprising steps (a1) to (a3) ​​of any one of claims 1 to 17.

19. A method for producing 2-hydroxybenzopyruvic acid using 1-naphthol as a substrate, comprising steps (a2) to (a3) ​​of any one of claims 1 to 17.

20. The method according to claim 19, characterized in that: In the method, steps (a2) and (a3) ​​are completed in the same reaction system through a one-step reaction.

21. The method according to claim 20, characterized in that: The reaction system contains 1) 1-naphthol, 2) the carboxylase or cells capable of expressing the carboxylase, 3) the dioxygenase or cells capable of expressing the dioxygenase, 4) HCO3 - or CO2, 5) reaction buffer.

22. The method according to claim 21, characterized in that: In the reaction system, the final concentration of 1-naphthol is 15 mM, HCO3 - The final concentration is 50 mM or is continuously introduced into the reaction system during the reaction; the remainder is the reaction buffer.

23. The method according to claim 21, characterized in that: The pH of the reaction buffer is 6.5-8.

0.

24. The method according to claim 20, characterized in that: The reaction temperature is 25-35°C and the reaction time is 3-12h.

25. Use of the method or the set of enzymes or the set of cells according to any one of claims 18 to 24 in the degradation of naphthalene and / or the fixation of CO2; The enzyme set is as follows (B1) or (B2): (B1) consists of carboxylase and dioxygenase; (B2) consists of monooxygenases, carboxylases, and dioxygenases; The set of cells is as follows (C1) or (C2) or (C3): (C1) consists of cells capable of expressing carboxylase and cells capable of expressing dioxygenase; (C2) consists of cells capable of expressing monooxygenase, cells capable of expressing carboxylase and cells capable of expressing dioxygenase; (C3) consists of cells capable of expressing monooxygenase, cells capable of expressing carboxylase, cells capable of expressing dioxygenase, and cells capable of expressing alcohol dehydrogenase.

26. The use according to claim 25, characterized in that: The monooxygenase is derived from Bacillus megaterium.

27. The use according to claim 26, characterized in that: The amino acid sequence of the monooxygenase derived from Bacillus megaterium is shown in SEQ ID No.

1.

28. The use according to claim 25, characterized in that: The carboxylase is a carboxylase derived from Aspergillus oryzae, a carboxylase derived from Rhizobium sp., or a carboxylase derived from Trichosporon moniliiforme.

29. The use according to claim 28, characterized in that: Furthermore, the amino acid sequence of the carboxylase derived from Aspergillus oryzae is shown in SEQ ID No.2; the amino acid sequence of the carboxylase derived from Rhizobium sp. is shown in SEQ ID No.3; and the amino acid sequence of the carboxylase derived from Trichosporon moniliiforme is shown in SEQ ID No.

4.

30. The use according to claim 25, characterized in that: The dioxygenase is a dioxygenase derived from Mycobacterium vanbaalenii PYR-1.

31. The use according to claim 25, characterized in that: The amino acid sequence of the dioxygenase derived from Mycobacterium vanbaalenii PYR-1 is shown in SEQ ID No.5.

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