CN114561432A - Ring opening method of aromatic compound - Google Patents

Ring opening method of aromatic compound Download PDF

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CN114561432A
CN114561432A CN202011352492.2A CN202011352492A CN114561432A CN 114561432 A CN114561432 A CN 114561432A CN 202011352492 A CN202011352492 A CN 202011352492A CN 114561432 A CN114561432 A CN 114561432A
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carboxylase
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朱蕾蕾
任鹏举
谭子瑊
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Tianjin Institute of Industrial Biotechnology of CAS
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Abstract

The invention discloses a ring opening method of aromatic compounds. The method takes carboxylation-dioxygen coupling reaction as a core, overcomes the thermodynamic reversibility of enzyme catalysis carboxylation reaction, and greatly improves the fixed CO2And build a new model by taking the efficiency as a coreThe naphthalene degradation reaction path and the biological patent utilization realize the quick ring opening of naphthalene and CO2And (4) utilizing.

Description

Ring opening method of aromatic compound
Technical Field
The invention relates to the field of biochemistry, in particular to a ring opening method of an aromatic compound.
Background
Polycyclic Aromatic Hydrocarbons (PAH) are compounds consisting of a plurality of benzene rings in the structure, have carcinogenic and mutagenic activities, and are strictly quarantined in the world. Polycyclic Aromatic Hydrocarbons (PAHs) that pose an ecological hazard are mostly by-products of fossil energy. Since fossil energy is the energy basis for human socioeconomic activity, the PAH hazard is still of widespread concern in the foreseeable future. Due to pi electron conjugation, the energy of the structure is reduced so that the aromatic compound is inert to a certain degree. Moreover, the aromatic compounds are very weak in polarity and mostly sparingly soluble or insoluble in water. This inertness is increasingly pronounced as the number of aromatic rings in the molecule increases. The degradation of the aroma is therefore a slow, complex process.
Naphthalene is the simplest PAH substance in structure, and a molecular structure is formed by two benzene rings. Naphthalene and its derivatives are important chemical raw materials and have wide application in pesticide synthesis, printing and dyeing, rubber and other processes. Naphthalene is produced in large quantities mainly in coal tar and petroleum distillation, which are by-products of coking. However, with the implementation of strict environmental protection policies and regulations, the generation and treatment of naphthalene are strictly regulated. However, aromatic ring substances such as naphthalene and the like generated in the petrochemical industry are mainly cracked in a thermal catalysis mode, and the method has the defects of complex process, high pollution and high energy consumption. In comparison, the biological method for cracking PAH has low energy consumption and is environment-friendly.
Another serious environmental threat from fossil energy use is CO2Emissions and greenhouse effect. The frequency of extreme weather disasters has increased significantly due to global climate deregulation with greenhouse effect. Thus, CO reduction2Emission or increase of CO2The fixation efficiency becomes an urgent need. The insertion of a carboxyl group ortho to a hydroxyl group of a phenolic substance (Kolbe-Schmitt reaction) is a typical carbon-fixing reaction catalyzed by a non-oxidative carboxylase. However, the reverse reaction of this reaction is remarkable, and the conversion rate of the reaction is extremely low. The chemical method needs high temperature and high pressure (90 bar, 120-. The process has high energy consumption, serious pollution and very obvious carbon emission. The enzyme catalysis process can be carried out under mild conditions without 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 fixation of CO is to be improved in the case of an enzyme-catalyzed reaction2Efficiency of (2), the inverse must be solvedAnd should be limited.
Disclosure of Invention
In order to effectively solve the technical problems, the invention aims to provide a novel method for fixing CO through coupled enzyme catalyzed single oxygenation reaction and Kolbe-Schmitt reaction2And dioxygen reaction to open the ring of the naphthalene aromatic ring efficiently, and the path can be used for biodegradation of naphthalene and resource utilization of naphthalene.
In a first aspect, the invention claims a process for ring opening of an aromatic compound.
The ring opening method of the aromatic compound claimed by the invention can comprise the following steps:
(A1) catalyzing aromatic compounds by monooxygenase enzyme, and reacting to generate corresponding phenolic compounds;
(A2) catalyzing the phenolic compound by carboxylase, and reacting to generate a carboxyl-containing aromatic compound;
(A3) the aromatic compound containing carboxyl is catalyzed by dioxygenase to react to generate carboxylic acid compounds, and the carboxylic acid compounds are ring-opening products of the aromatic compound.
In the method, if the aromatic compound contains a phenolic hydroxyl group, the step (a1) may be omitted.
In the method, the catalytic reaction of each enzyme may be carried out by any one of the following means: 1) directly adding corresponding enzyme into a reaction system; 2) cells capable of expressing the corresponding enzyme are added to the reaction system.
In a particular embodiment of the invention, the cell capable of expressing the monooxygenase is an E.coli strain capable of expressing the monooxygenase, such as BL21 Gold (DE 3); the cells capable of expressing the carboxylase are E.coli, such as BL21 Gold (DE3), capable of expressing the carboxylase; the cell capable of expressing the carboxyl-containing aromatic compound is an E.coli strain capable of expressing the carboxylase, such as BL21 Gold (DE 3).
In the method, in step (a1), nad (p) H is required to provide reducing power. NAD (P) H represents NADH or NADPH. Accordingly, NAD (P)+Is indicative of NAD+Or NADP+. The same applies below.
Further, the providing of reducing power by nad (p) H is achieved by any one of the following: 1) adding NAD (P) H directly to the reaction system; 2) a coenzyme NAD (P) H cycle is formed in the reaction system.
Further, the formation of coenzyme NAD (P) H cycle in the reaction system can be realized by any one of the following ways: 1) adding a cell capable of expressing Alcohol Dehydrogenase (ADH) and NAD (P)+(ii) a2) Adding a cell capable of expressing Alcohol Dehydrogenase (ADH) and NAD (P) H to the reaction system.
In a particular embodiment of the invention, the cell capable of expressing an Alcohol Dehydrogenase (ADH) is an escherichia coli capable of expressing an Alcohol Dehydrogenase (ADH), such as BL21 Gold (DE 3). The cell capable of expressing Alcohol Dehydrogenase (ADH) was added to the reaction system in an amount of 0.5g wet weight of cell/mL, NAD (P)+Or NAD (P) H was added in an amount of 30 mM.
In the method, in step (A2), HCO is required3 -Or CO2As another substrate.
Wherein the aromatic compound may be an aromatic compound.
Further, the aromatic hydrocarbon compound may be polycyclic aromatic hydrocarbon.
Still further, the polycyclic aromatic hydrocarbon may be naphthalene.
In a specific embodiment of the invention, the aromatic compound is naphthalene.
In a second aspect, the invention claims a method for degrading naphthalene and/or fixing CO2The method of (1).
Naphthalene degradation and/or CO fixation as claimed in the present invention2The method of (3), may comprise the steps of:
(a1) naphthalene is catalyzed by monooxygenase to react to generate 1-naphthol;
(a2) catalyzing 1-naphthol by carboxylase, and reacting to generate 1-hydroxy-2-benzoic acid;
(a3) the 1-hydroxy-2-benzoic acid is catalyzed by dioxygenase to react to generate 2-carboxyl benzopyruvic acid.
Figure BDA0002801697770000021
In step (a1), NAD (P) H is required to provide reducing power.
Further, the provision of reducing power with nad (p) H may be achieved by any of the following: 1) adding NAD (P) H directly into the reaction system; 2) a coenzyme NAD (P) H cycle is formed in the reaction system.
Further, the formation of coenzyme NAD (P) H cycle in the reaction system can be realized by any one of the following ways: 1) adding a cell capable of expressing Alcohol Dehydrogenase (ADH) and NAD (P)+(ii) a2) Adding a cell capable of expressing Alcohol Dehydrogenase (ADH) and NAD (P) H to the reaction system.
In a particular embodiment of the invention, the cell capable of expressing an Alcohol Dehydrogenase (ADH) is an escherichia coli capable of expressing an Alcohol Dehydrogenase (ADH), such as BL21 Gold (DE 3). The cell capable of expressing Alcohol Dehydrogenase (ADH) was added to the reaction system in an amount of 0.5g wet weight of cell/mL, NAD (P)+Or NAD (P) H was added in an amount of 30 mM.
In step (a2), with HCO3 -Or CO2As another substrate;
in the method, the catalytic reaction of each enzyme may be carried out by any one of the following means: 1) directly adding corresponding enzyme into a reaction system; 2) cells capable of expressing the corresponding enzyme are added to the reaction system.
In a particular embodiment of the invention, the cell capable of expressing the monooxygenase is an E.coli strain capable of expressing the monooxygenase, such as BL21 Gold (DE 3); the cells capable of expressing the carboxylase are E.coli, such as BL21 Gold (DE3), capable of expressing the carboxylase; the cell capable of expressing the carboxyl group-containing aromatic compound is Escherichia coli, such as BL21 Gold (DE3), capable of expressing the carboxyl group-containing aromatic compound.
In a specific embodiment of the present invention, steps (a1) - (a3) are completed in one reaction step in the same reaction system.
The reaction system contains 1) naphthalene, 2) monooxygenase or can be shownA cell expressing the monooxygenase, 3) a carboxylase or a cell capable of expressing the carboxylase, 4) a dioxygenase or a cell capable of expressing the dioxygenase, 5) NAD (P) H, 6) HCO3 -Or CO27) reaction buffer.
Further, in the reaction system, the final concentration of naphthalene was 15mM, the final concentration of NAD (P) H was 60mM, and HCO was3 -Is 50mM or continuously introducing CO into the reaction system during the reaction20.5mg/mL of the monooxygenase enzyme or 0.5g of cell wet weight/mL of the cell capable of expressing the monooxygenase enzyme, 0.03-1U/mL (e.g., 1U/mL, 0.03U/mL, 0.14U/mL) of the carboxylase enzyme or 0.5g of cell wet weight/mL of the cell capable of expressing the carboxylase enzyme, 9U/mL of the dioxygenase enzyme or 0.5g of cell wet weight/mL of the cell capable of expressing the dioxygenase enzyme; the balance being the reaction buffer.
Wherein the pH of the reaction buffer is 6.5-8.0 (e.g., pH 7.0-7.5). Specifically, potassium phosphate buffer (pH7.0 or pH7.5, 100mM) may be used.
The reaction temperature is 25-35 deg.C (such as 30 deg.C), and the reaction time is 3-12h (such as 12 h).
In a third aspect, the invention claims any of the following methods:
the method I comprises the following steps: a method for producing 2-carboxybenzopyrpyruvic acid using naphthalene as a substrate, which comprises the steps (a1) - (a3) of the method as described above.
Method II, a method for producing 2-carboxybenzopyruvate using 1-naphthol as a substrate, may comprise steps (a2) - (a3) of the method described above.
In a specific embodiment of the present invention, the steps (a2) and (a3) of the method II are completed in one reaction system.
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, and 4) HCO3Or CO2 -5) reaction buffer.
Further, in the reaction system, the final concentration of 1-naphthol was 15mM, HCO3 -Final concentration of (2)Is 50mM, 0.03-1U/mL (e.g., 1U/mL, 0.03U/mL, 0.14U/mL) of the carboxylase or 0.5g cell wet weight/mL of a cell capable of expressing the carboxylase, 9U/mL of the dioxygenase or 0.5g cell wet weight/mL of a cell capable of expressing the dioxygenase; the balance being the reaction buffer.
Wherein the pH of the reaction buffer is 6.5-8.0 (e.g., pH 7.0-7.5). Specifically, potassium phosphate buffer (pH7.0 or pH7.5, 100mM) may be used.
The reaction temperature is 25-35 deg.C (such as 30 deg.C), and the reaction time is 3-12h (such as 12 h).
In a fourth aspect, the invention claims a kit of enzymes.
The claimed enzymes of the present invention may be (B1) or (B2) as follows:
(B1) consists of carboxylase and dioxygenase;
(B2) consists of monooxygenase, carboxylase and dioxygenase.
In a fifth aspect, the invention claims a kit of cells.
The claimed cell set may be (C1) or (C2) or (C3) as follows:
(C1) consisting of cells capable of expressing carboxylase and cells capable of expressing dioxygenase;
(C2) consisting of cells capable of expressing a monooxygenase, cells capable of expressing a carboxylase and cells capable of expressing a dioxygenase;
(C3) consisting of cells capable of expressing a monooxygenase, cells capable of expressing a carboxylase, cells capable of expressing a dioxygenase and cells capable of expressing an Alcohol Dehydrogenase (ADH).
In a sixth aspect, the invention claims any of the following applications:
p1 use of a cell according to the method of the first aspect or the enzyme set of the fourth aspect or the cell set of the fifth aspect for degrading aromatic compounds and/or immobilizing CO2The use of (1);
p2 use of a cell of the set of enzymes of the method of the third aspect or the set of enzymes of the fourth aspect or the fifth aspect for degrading naphthalene and/or immobilizing CO2The use of (1).
In the above aspects, the monooxygenase may be a monooxygenase derived from Bacillus megaterium.
Further, the amino acid sequence of the monooxygenase derived from Bacillus megaterium is shown as SEQ ID No. 1.
In the above aspects, the carboxylase may be a carboxylase derived from Aspergillus oryzae (Aspergillus oryzae), a carboxylase derived from rhizobiam sp, or a carboxylase derived from candida albicans moniliforme.
Further, the amino acid sequence of the carboxylase derived from Aspergillus oryzae (Aspergillus oryzae) is shown as SEQ ID No. 2; the amino acid sequence of the carboxylase derived from Rhizobium sp is shown as SEQ ID No. 3; the amino acid sequence of the carboxylase derived from the candida albicans (trichosporine moniliforme) is shown as SEQ ID No. 4.
In the above aspects, the dioxygenase may be a dioxygenase derived from Mycobacterium van basale (Mycobacterium vanbaalenii PYR-1).
Further, the amino acid sequence of the dioxygenase derived from Mycobacterium Vanbalaenii PYR-1 is shown as SEQ ID No. 5.
In the invention, the monooxygenase is obtained by using escherichia coli as a host bacterium to carry out prokaryotic expression and then carrying out Ni column purification. The preparation method specifically comprises the following steps: carrying out induction expression on escherichia coli (such as BL21 Gold (DE3)) expressing the monooxygenase by IPTG with the final concentration of 50 mu M under the induction condition of 20-30 ℃ for 24 h; centrifuging to collect cells, crushing the cells after resuspension, centrifuging to collect supernatant, filtering, purifying by a Ni column, desalting, and freeze-drying to obtain the product.
The invention discloses CO2The method for coupling reaction of fixation and naphthalene cracking takes carboxylation-dioxygen coupling reaction as a core, overcomes the thermodynamic reversibility of enzyme catalysis carboxylation reaction, and greatly improves the fixation of CO2And build up with this as coreA new naphthalene degradation reaction path is provided, naphthalene generates 1-naphthol through oxygenase, and 2-carboxyl benzopyruvic acid is generated through carboxylation-dioxygen coupling reaction, so that the cracking ring opening and CO of naphthalene are realized2And (4) fixing.
Compared with the prior non-oxidation carboxylation reaction, the method has the following advantages:
1. the carboxylase and oxygenase are coupled, so that product inhibition is eliminated, carboxylase catalytic reaction can be carried out under normal environmental conditions, the cost of carboxylase fixing C is reduced, and the efficiency is improved.
2. Takes naphthalene as a substrate to construct a new reaction path for degrading naphthalene and CO2Are coupled together.
3. Takes naphthalene as a substrate, and designs a green and mild method for synthesizing 2-carboxyl benzopyruvic acid.
4. The method disclosed by the invention is independent of the driving of a substrate and temperature, the reaction condition is mild, and the operation is convenient.
Drawings
FIG. 1 shows the effect of enzyme purification by SDS-PAGE electrophoresis. M: standard protein marker; l1: p450 BM-3 (A74G/F87V/L188Q); l2: 2, 3-DHBD; L3.1HNDO, respectively; l4: SAD: l5: 2, 6-DHBD. The arrow indicates the target protein.
FIG. 2 shows the detection of 2-carboxybenzo pyruvic acid pure product and the detection of 1-naphthol standard product by liquid chromatography-mass spectrometry. A is a pure product for detecting 2-carboxyl benzo pyruvic acid by LC-MS; b is a 1-naphthol standard product detected by a liquid phase.
FIG. 3 shows the liquid phase detection of carboxylase 2,3-DHBD and dioxygenase 1HNDO coupled with 1-naphthol cleavage (direct enzymatic reaction).
FIG. 4 shows liquid phase detection of carboxylase 2,6-DHBD and dioxygenase 1HNDO coupled reaction catalyzed 1-naphthol cleavage (direct enzymatic reaction method).
FIG. 5 shows the liquid phase detection of carboxylase SAD and dioxygenase 1HNDO coupled reaction catalyzed 1-naphthol cleavage (direct enzymatic reaction method).
FIG. 6 shows the liquid phase detection of carboxylase 2,3-DHBD and dioxygenase 1HNDO coupled with 1-naphthol cleavage (resting cell reaction method).
FIG. 7 is a liquid phase detection of carboxylase 2,3-DHBD and dioxygenase 1HNDO coupled catalysis 1-naphthol cracking and CO fixation2(direct enzyme reaction method).
FIG. 8 shows liquid phase detection of coupled catalytic naphthalene degradation (direct enzyme reaction method) by monooxygenase P450 BM-3(A74G/F87V/L188Q), carboxylase 2,3-DHBD and dioxygenase 1 HNDO.
FIG. 9 shows liquid phase detection of the coupling catalytic naphthalene degradation (resting cell coenzyme cycling reaction method) of monooxygenase P450 BM-3(A74G/F87V/L188Q), carboxylase 2,3-DHBD and dioxygenase 1 HNDO.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 expression host construction
Through gene optimization, a Bacillus megaterium monooxygenase mutant P450 BM-3(A74G/F87V/L188Q) is entrusted to artificially synthesize a coding gene derived from a Bacillus megaterium monooxygenase mutant (P450 BM-3) shown in SEQ ID No.6 (the protein shown in SEQ ID No.1 is coded by the SEQ ID No. 6), the gene is inserted into a Nde I enzyme digestion site of pET28a, and an obtained recombinant vector is named as pET28a: BM3 after being verified to be correct by sequencing.
A gene encoding the carboxylase (2,3-DHBD) derived from Aspergillus oryzae (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 this gene was inserted into the NcoI cleavage site of pRSFDuet1 vector (New England Biolabs), and the resulting recombinant vector was designated pRSFDuet1::2,3-dhnd after the correctness of sequencing.
A gene encoding carboxylase (2,6-DHBD) derived from Rhizobium sp is shown in SEQ ID No.8 (SEQ ID No.8 encodes a protein shown in SEQ ID No. 3), the gene is inserted into the Nco I cleavage site of pRSFDuet1 vector (New England Biolabs), and the obtained recombinant vector is sequenced and verified to be correct and is named pRSFDuet1::2, 6-dhnd.
A gene encoding a carboxylase (SAD) derived from Trichosporon moniliforme (Trichosporon moniliforme) was synthesized as shown in SEQ ID No.9 (SEQ ID No.9 encodes a protein shown in SEQ ID No. 4), the gene was inserted into the NcoI cleavage site of pRSFDuet1 vector (New England Biolabs), and the resulting recombinant vector was sequenced and verified to be correct and designated pRSFDuet1:: SAD).
A gene encoding Mycobacterium Vanbaalenii PYR-1 oxygenase (1HNDO) derived from Mycobacterium van-Baum is synthesized as shown in SEQ ID No.10 (SEQ ID No.10 encodes a protein shown in SEQ ID No. 5), the gene is inserted into the Nco I cleavage site of pRSFDuet1 vector (New England Biolabs company), and the obtained recombinant vector is named pRSFDuet1::1HNDO after being verified to be correct by sequencing.
And transforming the expression vector vectors into escherichia coli BL21 Gold (DE3), screening positive clones on an LB plate containing kanamycin, culturing, extracting plasmids, and sequencing to determine that the vector construction is successful.
Example 2 expression purification of enzyme
The positive engineering bacteria constructed in example 1 were inoculated into 5mL LB medium, cultured at 37 ℃ and 200r/min for 12h, transferred to 1mL seed bacteria liquid into fresh 100mL LB medium, and cultured at 37 ℃ and 220r/min for about 2h to OD600When the concentration reaches 0.4-0.6, IPTG with the final concentration of 50 mu M is added for induction expression, and the expression condition is low-temperature induction at 20-30 ℃, 220r/min and 24 h.
And (3) centrifugally collecting cells after the expression is finished, re-suspending the cells by using a Binding buffer, concentrating the volume of a cell suspension by 20 times, homogenizing and crushing the cells under high pressure, centrifuging at 12000rpm/min, collecting a supernatant, filtering by using a 0.45-micrometer filter membrane, loading a Ni column, eluting the hybrid protein by using a Washing buffer, eluting and recovering the enzyme by using an Eution buffer, desalting, freeze-drying and storing the enzyme at-80 ℃. Among them, Ni column purified protein buffer is shown in Table 1.
TABLE 1 Ni column purification of protein buffer
Buffer Composition of
Binding buffer 20mM phosphate, 0.5M NaCl, 20mM imidazole, pH 8.0
Washing buffer 20mM phosphate, 0.5M NaCl, 40mM imidazole, pH 8.0
Elution buffer 20mM phosphate, 0.5M NaCl, 500mM imidazole, pH 8.0
The purification effect of the five enzymes involved in this example is shown in FIG. 1. As can be seen, the purity of the other four enzymes except P450 BM-3(A74G/F87V/L188Q) reaches more than 90 percent, which meets the requirement of the experiment. The monooxygenase (P450 BM-3(A74G/F87V/L188Q)) had a purity of 70%.
In addition, the enzyme activity of the purified product is measured, and the detection method uses a fluorescence method to continuously detect the change of a fluorescence signal of a reaction sample, wherein the excitation light is 350nm, and the emission light is 420 nm. Carboxylase: the reaction sample contains KHCO3200mM, 10mM of 1-naphthol, adding an appropriate amount of carboxylase, complementing the volume to 120 μ L with potassium phosphate buffer (ph7.0 or ph7.5, 100mM), transferring to a 96-well fluorescent microplate, and detecting the increase in fluorescence using a microplate reader (the product 1-hydroxy-2-naphthoic acid has a fluorescent property), defined as: enzyme activity required for producing 1. mu.M 1-hydroxy-2-naphthoic acid per minuteIs 1U. Dioxygenase: the reaction sample containing 1-hydroxy-2-naphthoic acid at a final concentration of 0.2mM was added with an appropriate amount of carboxylase, the volume was made up to 120. mu.L with potassium phosphate buffer (pH7.0 or pH7.5, 100mM), transferred to a 96-well fluorescent microplate, and the decrease in fluorescence was detected using a microplate reader. Defining: the enzyme activity required for converting 1 mu M1-hydroxy-2-naphthoic acid per minute is 1U. The enzyme activity results are as follows: 2, 3-DHBD: 0.65U/mgProtein，SAD：0.09U/mgProtein，2,6-DHBD：0.002U/mgProtein，1HNDO：12U/mgProtein。
Example 3, 2,3-DHBD and 1HNDO coupling reaction carbon fixation
1. 1-Naphthol was dissolved in N, N-Dimethylformamide (DMF) to prepare a 500mM solution. Potassium phosphate buffer (pH7.0, 100mM) was prepared. Preparing 3M KHCO3And (3) solution.
2. Enzyme solutions were prepared by dissolving carboxylase 2,3-DHBD and dioxygenase 1HNDO prepared in example 2 in potassium phosphate buffer (pH7.0, 100mM), respectively.
3. The reaction components were premixed to prepare a reaction solution as shown in table 2.
TABLE 2 coupling reaction solution (1mL)
Components Concentration of
1-naphthols 15mM
KHCO3 50mM
Carboxylase (2,3-DHBD) 1U/mL
Dioxygenase (1HNDO) 9U/mL
Potassium phosphate buffer (pH7.0, 100mM) Make up to 1mL
Note: the concentrations of the respective substances in the table are final concentrations in the reaction solution.
Reaction conditions are as follows: reaction at 200rpm and 30 ℃ for 12 h.
And detecting the reaction result by high performance liquid chromatography.
Spectrum column: (ii) a PAH;
eluent: a: 5mM ammonium acetate, B: acetonitrile;
elution procedure: as in table 3.
TABLE 3 HPLC elution procedure 1
Time Rate (ml/min) % B (flow Rate)
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
And (3) detection: UV absorption at 300 nm.
The liquid phase detection results of 2-carboxybenzo pyruvic acid and 1-naphthol are shown in figure 2 (A: the liquid phase detection results of 1-naphthol standard substance and the structural molecular weight of substance are correct by LC-MS identification results after separation and purification of 2-carboxybenzo pyruvic acid prepared by enzyme reaction).
The results are shown in FIG. 3, and in comparison with FIG. 2, it can be seen that the substrate 1-naphthol is almost completely converted into the product 2-carboxybenzopyryruvic acid.
Example 4, 2,6-DHBD and 1HNDO coupling reaction carbon fixation
1. 1-Naphthol was dissolved in N, N-Dimethylformamide (DMF) to prepare a 500mM solution. Potassium phosphate buffer (pH7.0, 100mM) was prepared, and 3M KHCO was prepared3And (3) solution.
2. Enzyme solutions were prepared by dissolving carboxylase 2,6-DHBD and dioxygenase 1HNDO prepared in example 2 in potassium phosphate buffer (pH7.0, 100mM), respectively.
3. The reaction components were premixed to prepare a reaction solution, as shown in table 4.
TABLE 4 coupling reaction solution (1mL)
Figure BDA0002801697770000071
Figure BDA0002801697770000081
Note: the concentrations of the respective substances in the table are final concentrations in the reaction solution.
Reaction conditions are as follows: 200rpm, 30 ℃ for 12 h.
The reaction result was measured by high performance liquid chromatography (the specific measurement method was the same as in example 3).
The results are shown in FIG. 4, and in comparison with FIG. 2, it can be seen that only a portion of the substrate 1-naphthol was converted to the product 2-carboxybenzopyruvate.
Example 5 SAD and 1HNDO coupling reaction carbon sequestration
1. 1-Naphthol was dissolved in N, N-Dimethylformamide (DMF) to prepare a 500mM solution. Potassium phosphate buffer (pH7.0, 100mM) was prepared, and 3M KHCO was prepared3And (3) solution.
2. Enzyme solutions were prepared by dissolving the carboxylase SAD and the dioxygenase 1HNDO prepared in example 2 in potassium phosphate buffer (pH7.0, 100mM), respectively.
3. The reaction components were premixed to prepare a reaction solution as shown in table 5.
TABLE 5 coupling reaction solution (1mL)
Components Concentration of
1-naphthols 15mM
KHCO3 45mM
Carboxylase SAD 0.14U/mL
Dioxygenase (1HNDO) 9U/mL
Potassium phosphate buffer (pH7.0, 100mM) Make up to 1mL
Note: the concentrations of the respective substances in the table are final concentrations in the reaction solution.
Reaction conditions are as follows: 200rpm, 30 ℃ for 12 h.
The reaction result was measured by high performance liquid chromatography (the specific measurement method was the same as in example 3).
The results are shown in FIG. 5, and in comparison with FIG. 2, it can be seen that only a small amount of the substrate 1-naphthol is converted into the product 2-carboxybenzopyryruvic acid.
Example 6, 2,3-DHBD and 1HNDO coupling reaction carbon fixation-resting cells
1. 1-Naphthol was dissolved in N, N-Dimethylformamide (DMF) to prepare a 500mM solution. Potassium phosphate buffer (pH7.0, 100mM) was prepared, and 3M KHCO was prepared3And (3) solution.
2. Resting cells were prepared, and 2,3-DHBD and 1 HNDO-expressing cells (2,3-DHBD and 1 HNDO-expressing E.coli BL21 Gold (DE3) constructed in example 1) were resuspended uniformly in each of potassium phosphate buffer solutions (pH7.0, 100 mM).
3. The reaction components were premixed to prepare a reaction solution as shown in table 6.
TABLE 6 coupling reaction solution (1mL)
Figure BDA0002801697770000082
Figure BDA0002801697770000091
Note: the concentrations of the respective substances in the table are final concentrations in the reaction solution.
Reaction conditions are as follows: reacting at 30 ℃ for 12 h.
The reaction result was measured by high performance liquid chromatography (the specific measurement method was the same as in example 3).
The results are shown in FIG. 6, and in comparison with FIG. 2, it can be seen that all of the 1-naphthol is converted to the product 2-carboxybenzopyruvic acid.
Example 7, 2,3-DHBD and 1HNDO coupling reaction carbon fixation-CO2
1. 1-Naphthol was dissolved in N, N-Dimethylformamide (DMF) to prepare a 500mM solution. Potassium phosphate buffer (pH7.5, 100mM) was prepared.
2. Enzyme solutions were prepared by dissolving carboxylase 2,3-DHBD and dioxygenase 1HNDO prepared in example 2 in potassium phosphate buffer (pH7.5, 100mM), respectively.
3. The reaction components were premixed to prepare a reaction solution as shown in table 7.
TABLE 7 coupling reaction solution (1mL)
Components Concentration of
1-naphthols 15mM
Carboxylase (2,3-DHBD) 1U/mL
Dioxygenase (1HNDO) 9U/mL
Potassium phosphate buffer (pH7.5, 100mM) Make up to 1mL
Note: the concentrations of the respective substances in the table are final concentrations in the reaction solution.
The reaction conditions are as follows: reacting at 30 ℃ for 12h at 200rpm, and continuously introducing CO in the reaction process2。
The reaction result was measured by high performance liquid chromatography (the specific measurement method was the same as in example 3).
The results are shown in FIG. 7, and in comparison with FIG. 2, it can be seen that the substrate 1-naphthol is almost completely converted into the product 2-carboxybenzopyryruvic acid. Visible, CO2Can well replace HCO3 -The reaction is carried out, the invention aims at improving fixed CO2The efficiency of (2) is of great significance.
Example 8 opening of aromatic rings of naphthalene
1. Naphthalene was weighed and dissolved in N, N-Dimethylformamide (DMF) to prepare a solution having a concentration of 500 mM. Potassium phosphate buffer (pH7.0, 100mM) was prepared, and 3M KHCO was prepared3And (3) solution.
2. Enzyme solutions were prepared by dissolving the P450 BM-3(A74G/F87V/L188Q), 2,3-DHBD and 1HNDO prepared in example 2 in potassium phosphate buffer (pH7.0, 100mM), respectively.
3. The reaction components were premixed to prepare a reaction solution, as shown in table 8.
TABLE 8 coupling reaction solution (1mL)
Figure BDA0002801697770000092
Figure BDA0002801697770000101
Note: the concentrations of the respective substances in the table are final concentrations in the reaction solution.
Reaction conditions are as follows: reaction at 200rpm and 30 ℃ for 12 h.
The reaction result was measured by high performance liquid chromatography (the specific detection method is the same as example 3, only the elution procedure is different, and the elution procedure in this example is shown in table 9):
TABLE 9 elution procedure 2 by HPLC
Time Rate (ml/min) % B (flow Rate)
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
The results are shown in FIG. 8, and in comparison with FIG. 2, it can be seen that only naphthalene and HCO are present3 -Under the condition of serving as a substrate, the generation of 2-carboxyl benzopyronic acid is successfully detected through the reaction route designed by the invention, and the success of the route in the aspect of degrading naphthalene and fixed carbon is proved.
Example 9 opening of aromatic rings in naphthalene-coenzyme cycling
1. Weighing naphthaleneThe resulting solution was dissolved in N, N-Dimethylformamide (DMF) to prepare a 500mM solution. Potassium phosphate buffer (pH7.0, 100mM) was prepared, and 3M KHCO was prepared3And (3) solution.
2. Resting cells were prepared, and P450 BM-3(A74G/F87V/L188Q), 2,3-DHBD and 1 HNDO-expressing cells (E.coli BL21 Gold (DE3) expressing P450 BM-3(A74G/F87V/L188Q), 2,3-DHBD and 1 HNDO-expressing cells constructed in example 1) were resuspended uniformly in potassium phosphate buffer (pH7.0, 100mM), respectively. Alcohol Dehydrogenase (ADH) resting cells (recombinant bacteria obtained by introducing the alcohol dehydrogenase ADH-encoding gene into E.coli BL21 Gold (DE3) according to the method of example 1) were prepared for regeneration and recycling of NADH (reduction in cost of reaction).
3. The reaction components were premixed to prepare a reaction solution, as shown in table 10.
TABLE 10 coupling reaction solution (1mL)
Components Concentration of
Naphthalene 15mM
NAD+ 30mM
Isopropanol (I-propanol) 30mM
KHCO3 50mM
P450 BM-3(A74G/F87V/L188Q) cells 0.5gWet weight of cells/mL
ADH cells 0.5gWet weight of cells/mL
2,3-DHBD cells 0.5gWet weight of cells/mL
1HNDO cell 0.5gWet weight of cells/mL
Potassium phosphate buffer (pH7.0, 100mM) Make up to 1mL
Note: the concentrations of the respective substances in the table are final concentrations in the reaction solution.
The reaction conditions are as follows: reacting at 30 ℃ for 12 h.
The reaction results were checked by HPLC (the specific detection method is the same as example 3, only the elution procedure is different, and the elution procedure in this example is shown in Table 9).
The results are shown in FIG. 9, and in comparison with FIG. 2, it can be seen that only naphthalene and HCO are present3 -In the case of using oxidized coenzyme NAD as a substrate+The generation of 2-carboxyl benzopyrone is successfully detected through the reaction route designed by the invention, and the success of the route in the aspect of degrading naphthalene and fixed carbon and the function of a coenzyme circulating system are proved.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences
<120> a ring-opening method of an aromatic compound
<130> GNCLN202053
<160> 10
<170> PatentIn version 3.5
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Met Gly Met Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu
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Leu Lys Asn Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu
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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
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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
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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
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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
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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
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Ile Arg Tyr Gln Ile Ile Thr Phe Leu Ile Ala Gly His Glu Thr Thr
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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
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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
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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
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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
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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
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Glu Glu Lys Gly Arg Tyr Ala Lys Asp Val Trp Ala Gly
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Met Leu Gly Lys Ile Ala Leu Glu Glu Ala Phe Ala Leu Pro Arg Phe
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Glu Glu Lys Thr Arg Trp Trp Ala Ser Leu Phe Ser Thr Asp Ala Glu
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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
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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
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Ser Ala
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Met Gln Gly Lys Val Ala Leu Glu Glu His Phe Ala Ile Pro Glu Thr
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Leu Gln Asp Ser Ala Gly Phe Val Pro Gly Asp Tyr Trp Lys Glu Leu
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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
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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
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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
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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
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Glu Lys Pro Leu Leu His Tyr Phe Arg Asn Asn Ile Trp Leu Thr Thr
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Ser Gly Asn Phe Ser Thr Glu Thr Leu Lys Phe Cys Val Glu His Val
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Gly Ala Glu Arg Ile Leu Phe Ser Val Asp Ser Pro Tyr Glu His Ile
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Asp Val Gly Cys Gly Trp Tyr Asp Asp Asn Ala Lys Ala Ile Met Glu
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Ala Val Gly Gly Glu Lys Ala Tyr Lys Asp Ile Gly Arg Asp Asn Ala
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Lys Lys Leu Phe Lys Leu Gly Lys Phe Tyr Asp Ser Glu Ala
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Met Ser Thr Ala Glu Ser Ser Glu Leu Arg Glu Phe Asp Val Glu Leu
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Glu Ala Ala Asn Leu Arg Gly Gln Trp Ile Tyr Asp Asp Met Leu Glu
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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 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 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 Ala Asn Leu Arg Ala Arg
355 360
<210> 6
<211> 3156
<212> DNA
<213> Artificial sequence
<400> 6
atgggcatga caattaaaga aatgcctcag ccaaaaacgt ttggagagct taaaaattta 60
ccgttattaa acacagataa 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
tatttcttag tgaaaaatcc acatgtatta caaaaagcag cagaagaagc agcacgagtt 900
ctagtagatc ctgttccaag ctacaaacaa gtcaaacagc ttaaatatgt cggcatggtc 960
ttaaacgaag cgctgcgctt atggccaact gctcctgcgt tttccctata tgcaaaagaa 1020
gatacggtgc ttggaggaga atatccttta gaaaaaggcg acgaactaat ggttctgatt 1080
cctcagcttc accgtgataa aacaatttgg ggagacgatg tggaagagtt ccgtccagag 1140
cgttttgaaa atccaagtgc gattccgcag catgcgttta aaccgtttgg aaacggtcag 1200
cgtgcgtgta tcggtcagca gttcgctctt catgaagcaa cgctggtact tggtatgatg 1260
ctaaaacact ttgactttga agatcataca aactacgagc tggatattaa agaaacttta 1320
acgttaaaac ctgaaggctt tgtggtaaaa gcaaaatcga aaaaaattcc gcttggcggt 1380
attccttcac ctagcactga acagtctgct aaaaaagtac gcaaaaaggc agaaaacgct 1440
cataatacgc cgctgcttgt gctatacggt tcaaatatgg gaacagctga aggaacggcg 1500
cgtgatttag cagatattgc aatgagcaaa ggatttgcac cgcaggtcgc aacgcttgat 1560
tcacacgccg gaaatcttcc gcgcgaagga gctgtattaa ttgtaacggc gtcttataac 1620
ggtcatccgc ctgataacgc aaagcaattt gtcgactggt tagaccaagc gtctgctgat 1680
gaagtaaaag gcgttcgcta ctccgtattt ggatgcggcg ataaaaactg ggctactacg 1740
tatcaaaaag tgcctgcttt tatcgatgaa acgcttgccg ctaaaggggc agaaaacatc 1800
gctgaccgcg gtgaagcaga tgcaagcgac gactttgaag gcacatatga agaatggcgt 1860
gaacatatgt ggagtgacgt agcagcctac tttaacctcg acattgaaaa cagtgaagat 1920
aataaatcta ctctttcact tcaatttgtc gacagcgccg cggatatgcc gcttgcgaaa 1980
atgcacggtg cgttttcaac gaacgtcgta gcaagcaaag aacttcaaca gccaggcagt 2040
gcacgaagca cgcgacatct tgaaattgaa cttccaaaag aagcttctta tcaagaagga 2100
gatcatttag gtgttattcc tcgcaactat gaaggaatag taaaccgtgt aacagcaagg 2160
ttcggcctag atgcatcaca gcaaatccgt ctggaagcag aagaagaaaa attagctcat 2220
ttgccactcg ctaaaacagt atccgtagaa gagcttctgc aatacgtgga gcttcaagat 2280
cctgttacgc gcacgcagct tcgcgcaatg gctgctaaaa cggtctgccc gccgcataaa 2340
gtagagcttg aagccttgct tgaaaagcaa gcctacaaag aacaagtgct ggcaaaacgt 2400
ttaacaatgc ttgaactgct tgaaaaatac ccggcgtgtg aaatgaaatt cagcgaattt 2460
atcgcccttc tgccaagcat acgcccgcgc tattactcga tttcttcatc acctcgtgtc 2520
gatgaaaaac aagcaagcat cacggtcagc gttgtctcag gagaagcgtg gagcggatat 2580
ggagaatata aaggaattgc gtcgaactat cttgccgagc tgcaagaagg agatacgatt 2640
acgtgcttta tttccacacc gcagtcagaa tttacgctgc caaaagaccc tgaaacgccg 2700
cttatcatgg tcggaccggg aacaggcgtc gcgccgttta gaggctttgt gcaggcgcgc 2760
aaacagctaa aagaacaagg 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 (10)

1. A method for ring opening an aromatic compound, comprising the steps of:
(A1) catalyzing aromatic compounds by monooxygenase enzyme, and reacting to generate corresponding phenolic compounds;
(A2) catalyzing the phenolic compound by carboxylase, and reacting to generate a carboxyl-containing aromatic compound;
(A3) the aromatic compound containing carboxyl is catalyzed by dioxygenase to react to generate carboxylic acid compounds; the carboxylic acid compounds are ring-opening products of the aromatic compounds.
2. The method of claim 1, wherein: if the aromatic compound contains a phenolic hydroxyl group, the step (A1) is skipped and the step (A2) is directly performed;
and/or
In the method, the catalytic reaction of each enzyme is carried out by any one of the following methods: 1) directly adding corresponding enzyme into a reaction system; 2) cells capable of expressing the corresponding enzyme are added to the reaction system.
3. The method of claim 1, wherein: in the step (A1), NAD (P) H is used for providing reducing power;
further, the provision of reducing power with nad (p) H is achieved by either: 1) adding NAD (P) H directly into the reaction system; 2) forming a coenzyme NAD (P) H cycle in the reaction system;
further, the formation of coenzyme NAD (P) H cycle in the reaction system is realized by any one of the following modes: 1) adding a cell capable of expressing Alcohol Dehydrogenase (ADH) and NAD (P)+(ii) a2) Adding a cell capable of expressing Alcohol Dehydrogenase (ADH) and NAD (P) H to the reaction system;
and/or
In step (A2), HCO is used3 -Or CO2As another substrate.
4. A method according to any one of claims 1-3, characterized in that: the aromatic compound is an aromatic hydrocarbon compound;
further, the aromatic hydrocarbon compound is polycyclic aromatic hydrocarbon;
still further, the polycyclic aromatic hydrocarbon is naphthalene.
5. Naphthalene degradation and/or CO fixation2The method comprises the following steps:
(a1) naphthalene is catalyzed by monooxygenase to react to generate 1-naphthol;
(a2) catalyzing 1-naphthol by carboxylase, and reacting to generate 1-hydroxy-2-benzoic acid;
(a3) the 1-hydroxy-2-benzoic acid is catalyzed by dioxygenase to react to generate 2-carboxyl benzopyruvic acid.
6. The method of claim 5, wherein: in the step (a1), NAD (P) H is used for providing reducing power;
further, the provision of reducing power with nad (p) H is achieved by either: 1) adding NAD (P) H directly into the reaction system; 2) forming a coenzyme NAD (P) H cycle in the reaction system;
further, coenzyme NAD (P) H is formed in the reaction system and circulated through either one of the followingThe formula is realized as follows: 1) adding a cell capable of expressing Alcohol Dehydrogenase (ADH) and NAD (P)+(ii) a2) Adding a cell capable of expressing Alcohol Dehydrogenase (ADH) and NAD (P) H to the reaction system;
and/or
In step (a2), with HCO3 -Or CO2As another substrate;
and/or
In the method, the catalytic reaction of each enzyme is carried out by any one of the following methods: 1) directly adding corresponding enzyme into a reaction system; 2) cells capable of expressing the corresponding enzyme are added to the reaction system.
7. The method according to claim 5 or 6, characterized in that: the steps (a1) - (a3) are completed in one step in the same reaction system;
further, 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 CO27) reaction buffer;
further, in the reaction system, naphthalene was present at a final concentration of 15mM, NAD (P) H was present at a final concentration of 60mM, HCO was present3 -Is 50mM or continuously introducing CO into the reaction system during the reaction2(ii) a And/or
Further, the pH of the reaction buffer is 6.5-8.0;
and/or
The reaction temperature is 25-35 ℃, and the reaction time is 3-12 h.
8. Any one of the following methods:
the method I comprises the following steps: a method for producing 2-carboxybenzopyruvic acid using naphthalene as a substrate, comprising the steps (a1) - (a3) of the method of any one of claims 5 to 7;
method II, a method for producing 2-hydroxybenzophenopyruvic acid using 1-naphthol as a substrate, comprising the steps (a2) - (a3) of the method of claim 4 or 6;
further, in the method II, the steps (a2) and (a3) are completed in the same reaction system through further reaction;
further, the reaction system contains 1) 1-naphthol, 2) carboxylase or a cell capable of expressing the carboxylase, 3) dioxygenase or a cell capable of expressing the dioxygenase, 4) HCO3 -Or CO25) reaction buffer;
more specifically, in the reaction system, the final concentration of 1-naphthol was 15mM, HCO3 -Is 50mM or is continuously fed into the reaction system during the reaction; the balance of the reaction buffer solution;
and/or
Further, the pH of the reaction buffer is 6.5-8.0;
and/or
The reaction temperature is 25-35 ℃, and the reaction time is 3-12 h.
9. The complete enzyme is (B1) or (B2):
(B1) consists of carboxylase and dioxygenase;
(B2) consisting of monooxygenase, carboxylase and dioxygenase;
or
A set of cells, which are (C1) or (C2) or (C3) as follows:
(C1) consisting of cells capable of expressing carboxylase and cells capable of expressing dioxygenase;
(C2) consisting of cells capable of expressing a monooxygenase, cells capable of expressing a carboxylase and cells capable of expressing a dioxygenase;
(C3) consisting of cells capable of expressing a monooxygenase, cells capable of expressing a carboxylase, cells capable of expressing a dioxygenase and cells capable of expressing an alcohol dehydrogenase;
or
Any of the following applications:
p1, the method of any one of claims 1 to 4 or the enzyme set or the cells of the set in degrading aromatic compounds and/orFixation of CO2The use of (1);
p2, the method of claim 8 or the enzyme set or the cell set in degrading naphthalene and/or immobilizing CO2The use of (1).
10. The method or enzyme kit or cell kit or use according to any one of claims 1 to 9, wherein: the monooxygenase is derived from Bacillus megaterium (Bacillus megaterium);
further, the amino acid sequence of the monooxygenase derived from Bacillus megaterium (Bacillus megaterium) is shown as SEQ ID No. 1;
and/or
The carboxylase is carboxylase derived from Aspergillus oryzae (Aspergillus oryzae), carboxylase derived from rhizobiam sp, or carboxylase derived from candida albicans (trichosporium moniliforme);
further, the amino acid sequence of the carboxylase derived from Aspergillus oryzae (Aspergillus oryzae) is shown as SEQ ID No. 2; the amino acid sequence of the carboxylase derived from Rhizobium sp is shown as SEQ ID No. 3; the amino acid sequence of the carboxylase derived from the candida albicans (Trichosporon moniliforme) is shown as SEQ ID No. 4;
and/or
The dioxygenase is a dioxygenase derived from Mycobacterium van-Barn (Mycobacterium vanbaaleni PYR-1);
further, the amino acid sequence of the dioxygenase derived from Mycobacterium Vanbalaenii PYR-1 is shown as SEQ ID No. 5.
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