Preparation method and application of recombinant catechol-2, 3-dioxygenase

Abstract

The invention discloses a preparation method of recombinant catechol-2, 3-dioxygenase and an application of the recombinant catechol-2, 3-dioxygenase. According to the invention, a novel catechol-2, 3-dioxygenase group C23O927 is identified from a constructed metagenome library, and the catechol-2, 3-dioxygenase group C23O927 is a key enzyme for catalyzing degradation of polycyclic aromatic hydrocarbon pollutants. A C23O927-pET32a (+) recombinant expression vector is constructed and converted into host bacteria, heterologous expression of target protein is achieved, and the prepared recombinase belongs to acid enzymes and has good resistance to high temperature, acid, alkali, high-concentration metal ions, organic solvents and surfactants. The recombinase provided by the invention can be used in an acidic environment, has very good enzymatic activity, can catalytically degrade various catechol compounds, can be better used for treating polycyclic aromatic hydrocarbon polluted environments, and has important significance for developing more products which can tolerate various extreme environments and efficiently catalyze degradation of polycyclic aromatic hydrocarbons.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of recombinant enzyme preparation and pollutant degradation, in particular to a preparation method of a recombinant catechol-2,3-dioxygenase and application thereof. BACKGROUND

[0002] Polycyclic aromatic hydrocarbons refer to aromatic hydrocarbons containing two or more benzene rings (PAHs for short), which are compounds widely and commonly existing in nature. It is generally believed that its main source is generated when fossil fuels (gasoline, coking plants, liquefied petroleum gas, etc.) or plant fuels (wood, forage, animal manure, etc. organic matter) are incompletely combusted. This kind of chemical substance with environmental persistence and biological accumulation has penetrated into the ecological environment system through various ways such as industrial emissions, mining by-products and construction waste.

[0003] In recent years, the research on the microbial degradation pathway of PAHs and its degradation enzyme system has become a hot spot in the field of life science research, and remarkable progress has been made with the addition of molecular biology, genomics and molecular enzymology research techniques. Among them, catechol, also known as 1,2-dihydroxybenzene, is an intermediate produced by most microorganisms in the aerobic degradation of PAHs. Its structural feature is that there are two adjacent hydroxyl groups on the benzene ring, which widely exists in nature and often appears as a drug metabolite or environmental pollutant, posing a potential threat to human health and the natural environment.

[0004] Therefore, how to eliminate the increasing catechol in the environment has become an increasingly serious problem. Catechol and substituted catechol are key intermediates in the catabolic pathway of aromatic compounds. The ortho pathway of catechol degradation is also called the β-ketoadipate pathway. The catabolism of phenolic compounds is usually initiated by hydroxylase enzymes, which incorporate hydroxyl groups into phenolic substrates, resulting in the production of intermediates such as catechol and substituted catechol. Subsequently, these intermediates can be further metabolized as substrates for aromatic ring cleavage by two different groups of enzymes, which are responsible for the ortho and meta cleavage pathways. The intermediate catechol can be catalyzed by catechol-1,2-dioxygenase (C12O) to succinate and acetyl-CoA, thereby initiating the ortho pathway, or by catechol-2,3-dioxygenase (C23O) to pyruvate and acetaldehyde, thereby initiating the meta pathway. It can be seen that catechol-2,3-dioxygenase plays an important role in the complete and efficient degradation of catechol and is widely used in industrial production such as environmental monitoring, biological agents and electrochemical detection.

[0005] However, natural dioxygenases usually require a mild reaction environment, and are easily inactivated under extreme industrial conditions such as high temperature, strong alkalinity, high concentration of organic solvents or surfactants, and the catalytic efficiency is significantly reduced. Most of the catechol-2,3-dioxygenases disclosed in the prior art are alkaline, for example, bpHC-meta from activated sludge metagenomic library has an optimum pH of 9.0; C23O-RW1 from river water metagenome has an optimum pH of 9.0; and BLC23O from lignin Bacillus has an optimum pH of 7.4, and the existing catechol-2,3-dioxygenases have low tolerance to various extreme environments, such as acidic, high-salt, high-concentration organic solvents or surfactants, metal ions and the like, and also have problems such as low enzyme activity and insufficient stability, which are difficult to be applied in acidic environment, such as treating some industrial wastewater and repairing acidic soil, thereby limiting the application range of catechol-2,3-dioxygenase. SUMMARY

[0006] The technical problem to be solved by the present application is to overcome the defects and deficiencies of the existing catechol-2,3-dioxygenase, which has low environmental tolerance (easily inactivated under extreme industrial conditions such as high temperature, strong alkalinity, high concentration of organic solvents or surfactants) and low catalytic efficiency, and to provide a preparation method of recombinant catechol-2,3-dioxygenase and its application.

[0007] The present application aims to provide a recombinant catechol-2,3-dioxygenase.

[0008] Another object of the present application is to provide a preparation method of recombinant catechol-2,3-dioxygenase.

[0009] Still another object of the present application is to provide the application of recombinant catechol-2,3-dioxygenase.

[0010] Still another object of the present application is to provide the application of C23O927 gene or protein in preparing catechol recombinase.

[0011] The above objects of the present application are achieved by the following technical solutions:

[0012] The present application provides a recombinant catechol-2,3-dioxygenase, which is prepared by heterologous expression of the nucleotide sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 2.

[0013] The present application takes the soil of a coking plant as a sample, constructs a high-quality metagenomic library, and selects a novel catechol-2,3-dioxygenase gene C23O927 with a length of 927 bp from the metagenomic library, the gene encodes 308 amino acids, and the theoretical molecular weight is 52.4 kDa, the phylogenetic analysis result shows that C23O927 has a distant genetic relationship with the known catechol dioxygenase and has a very low homology, and may represent a new subtype in the enzyme family. The present application realizes the heterologous expression of the target protein by constructing a C23O927-pET32a(+) recombinant expression vector and transforming the recombinant expression vector into an E. coli. BL21(DE3) host bacterium, the prepared recombinant enzyme has good temperature stability, and has good enzyme activity at a temperature of 30-60 DEG C; the recombinant enzyme belongs to an acidic enzyme, has good pH stability, can resist acid and alkaline, and can also maintain high activity under various high-concentration salinity, metal ions, and organic solvents, surfactants, and has good environmental tolerance; at the same time, the recombinant enzyme has catalytic activity to various catechol compounds, can be used for degrading catechol, 3-chlorocatechol, 3-methylcatechol, 4-methylcatechol, 3,5-dichlorocatechol, 4-chlorocatechol and the like, can be better used for degrading catechol pollutants and polycyclic aromatic hydrocarbon pollutants in a complex pollution environment, and provides a theoretical basis for developing a novel industrial enzyme and an environmental pollution treatment method.

[0014] The present application provides a preparation method of a recombinant catechol-2,3-dioxygenase, comprising the following steps:

[0015] S1. cloning a gene sequence shown in SEQ ID NO: 1 or a protein sequence shown in SEQ ID NO: 2 to prepare a recombinant plasmid;

[0016] S2. transforming the recombinant plasmid into E. coli to obtain a recombinant bacterium;

[0017] S3. performing prokaryotic expression on the recombinant bacterium, collecting the bacterium, washing, and resuspending to obtain the recombinant catechol-2,3-dioxygenase.

[0018] Preferably, the recombinant plasmid in S1 is prepared by double enzyme digestion of the plasmid pET32a(+).

[0019] Preferably, the primer sequence used for cloning the gene in S1 is shown in SEQ ID NO: 3-4.

[0020] Preferably, the E. coli BL21(DE3) competent cell is used in S2.

[0021] Preferably, the prokaryotic expression condition in S3 is: 0.5-1.5 mM IPTG, 20-40 DEG C, and 20-40 h.

[0022] The present application provides the use of a recombinant catechol-2,3-dioxygenase in catalyzing the degradation of catechol compounds or polycyclic aromatic hydrocarbon compounds.

[0023] Preferably, the catechol compound is one or more of catechol, 3-chlorocatechol, 3-methylcatechol, 4-methylcatechol, 3,5-dichlorocatechol, 4-chlorocatechol.

[0024] The present application provides the use of a recombinant catechol-2,3-dioxygenase in preparing a product for degrading catechol compounds or polycyclic aromatic hydrocarbon compounds.

[0025] The present application provides the use of a recombinant catechol-2,3-dioxygenase in degrading catechol compounds or polycyclic aromatic hydrocarbon compounds in a complex contaminated environment.

[0026] Preferably, the complex contaminated environment refers to an environment containing multiple pollutants composed of acidic compounds, salt compounds, metal ions, organic solvents and / or surfactants.

[0027] In particular, the recombinant enzyme prepared by the present application mainly degrades catechol compounds, including but not limited to catechol, 3-chlorocatechol, 3-methylcatechol, 4-methylcatechol, 3,5-dichlorocatechol, 4-chlorocatechol. In addition, the recombinant enzyme prepared by the present application can be used for enzymatic degradation of polycyclic aromatic hydrocarbon pollutants. The degradation of polycyclic aromatic hydrocarbons requires the synergistic action of multiple enzymes, and catechol 2,3-dioxygenase is one of the key enzymes. Therefore, it is expected by those skilled in the art that the recombinant enzyme of catechol 2,3-dioxygenase can also be used for the degradation of polycyclic aromatic hydrocarbon pollutants.

[0028] Preferably, the polycyclic aromatic hydrocarbon compound is selected from Naphthalene, Acenaphthylene, Acenaphthene, Fluorene, Phenanthrene, Anthracene, Fluoranthene, Pyrene, Benzo[a]anthracene, (Chrysene), Benzo[b]fluoranthene, Benzo[k]fluoranthene, Benzo[a]pyrene, Dibenzo[a,h]anthracene, Benzo[g,h,i]perylene, Indeno[1,2,3-cd]pyrene, Benzo[e]pyrene, Benzo[j]fluoranthene, 5-methyl (5-Methylchrysene) one or more of dibenzofuran.

[0029] The present invention also provides the use of the C23O927 gene or protein in preparing catechol 2,3-dioxygenase recombinant enzyme.

[0030] The present invention has the following beneficial effects:

[0031] The present invention identified a novel catechol-2,3-dioxygenase gene, C23O927, from a constructed metagenomic library. By cloning the C23O927 gene and constructing a recombinant plasmid, the gene was heterologously expressed in Escherichia coli. The resulting recombinant enzyme is an acidic enzyme with good resistance to high temperatures, acids, alkalis, high concentrations of metal ions, organic solvents, and surfactants. This is the first time that a recombinant catechol-2,3-dioxygenase has been successfully prepared through heterologous expression. This enzyme can be used in acidic environments, exhibits excellent enzymatic activity, and can catalyze the degradation of various catechol compounds, making it more suitable for the treatment of complex polluted environments. Furthermore, the enzyme is not easily inactivated, which is of great significance for the development of more enzyme preparations that can tolerate a variety of extreme environments and efficiently catalyze the degradation of catechol compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Phylogenetic analysis of catechol-2,3-dioxygenase C23O927 and several dioxygenases from various families with high homology.

[0033] Figure 2SDS-PAGE analysis of C230927 (lanes in the figure: M, protein molecular mass marker (kDa) indicated on the left; 1, recombinant C230927 derived from cell lysate of E. coli BL21(DE3); 2, recombinant C230927 derived from supernatant of cell lysate of E. coli BL21(DE3); 3, recombinant C230927 purified by Ni-NTA affinity chromatography).

[0034] Figure 3 Optimum temperature and temperature stability of C230927 (A, effect of temperature on activity of recombinant C230927, using a temperature gradient of 5°C or 10°C, ranging from 20°C to 70°C; B, effect of temperature on stability of recombinant C230927; error bars represent standard deviation).

[0035] Figure 4 Optimum pH and pH stability of C230927 (A, effect of pH on activity of recombinant C230927; enzyme activity values are expressed as percentage (100%) of maximum activity; B, effect of pH on stability of recombinant C230927; error bars represent standard deviation).

[0036] Figure 5 Fe 2+ Effect of concentration on activity of C230927. DETAILED DESCRIPTION

[0037] The present application is further illustrated by the following description in conjunction with the attached drawing and specific examples, which do not limit the present application in any manner. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0038] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0039] Escherichia coli BL21(DE3) used in the examples is purchased from Sangon Biotech; vector pET32a(+) is purchased from Novagen; pUC118-BamHⅠ / BAP is purchased from TaKaRa.

[0040] LB medium: prepared with sterilized ultrapure water, containing 1% Tryptone, 0.5% Yeast Extract, 20% Agarose, 1% NaCl, adjusted to pH 7.0, 121°C high-pressure sterilization for 20-30 minutes, and formed into solid medium after cooling.

[0041] The soil sample used in the present application is collected from a coking plant in Pingdingshan City, Henan Province (north latitude 33°54'15", east longitude 112°55'15").

[0042] Example 1 Identification of catechol-2,3-dioxygenase

[0043] DNA of the soil sample from a coking plant in Pingdingshan City was extracted using a HiPure Soil DNA Maxi Kits kit (Magen Co., Ltd.), then subjected to enzyme digestion, and the macrogenomic DNA after enzyme digestion of the Pingdingshan coking plant was subjected to 1% agarose gel electrophoresis, followed by gel recovery of the DNA fragments in the range of 2.5-10 kb of the band, which was used for construction of a macrogenomic library. Subsequently, positive clones of catechol-2,3-dioxygenase were picked, plasmids were extracted, and characterized by enzyme digestion and electrophoresis, positive clones of catechol-2,3-dioxygenase were identified, and submitted to Shanghai Sangon Biotech Co., Ltd. (Shanghai, China) for DNA sequencing. Then the ORF-Finder tool provided on the NCBI website was used to identify the open reading frame (ORF) in the positive clone insert (https: / / www.ncbi.nlm.nih.gov / orffinder / ). Then BLAST and ClustalW software were used for homology analysis and multiple sequence alignment, respectively. Then MEGA version 7.0 software and the neighbor-joining method were used to construct a phylogenetic tree.

[0044] Sequencing analysis identified a complete 927 bp open reading frame (ORF) in an approximately 2.0 kb insert in the positive clone, which encoded a catechol-2,3-dioxygenase, designated as C230927 gene. Protein BLAST homology analysis showed that the C230927 protein had the highest similarity to 3,4-dihydroxyphenylacetate-2,3-dioxygenase from Escherichia coli, but the homology of the amino acid sequence was only 30.45%. This result showed that the C230927 enzyme identified in this embodiment had very low homology with known catechol-2,3-dioxygenases, and the constructed phylogenetic tree is shown in Figure 1 The gene encodes 308 amino acids, and the molecular weight of C230927 is predicted to be 52.4 kDa. The nucleotide sequence of the gene is shown as SEQ ID NO: 1, and the amino acid sequence is shown as SEQ ID NO: 2. The GenBank accession number of the C230927 gene is PP952002.

[0045] Example 2 Construction and expression analysis of Escherichia coli expression system

[0046] 1. Construction of recombinant expression vector

[0047] The soil metagenome of Pingdingshan was incompletely enzyme cut, and the enzyme cut reaction system was shown in Table 1 (after screening of restriction enzymes, the finally selected restriction enzyme was BamH I, and the enzyme cutting time was 3 h). After the enzyme reaction was completed, 10x loading buffer was added to terminate the reaction, and the enzyme cut genomic DNA was subjected to 1% agarose gel electrophoresis. Then the DNA fragments in the range of 3-10 kb were cut and recovered (HiPure Gel Pure DNA Mini Kit was used), and the successfully recovered DNA samples were stored at -20℃.

[0048] Table 1 enzyme cutting system of restriction enzyme

[0049]

[0050] The successfully recovered and frozen DNA samples and dephosphorylated pUC118-BamH I / BAP carrier were connected, and the connection system was shown in Table 2. The connection was carried out at 16℃ for 16 hours.

[0051] Table 2 connection system of vector pUC118 and single enzyme cut DNA

[0052]

[0053]

[0054] After the connection was completed, the connected product was purified and recovered using Gel & PCR Clean Up Kit produced by OMGEA company, and the successfully recovered DNA samples were stored at -20℃.

[0055] The gene synthesis service was entrusted to Shanghai Shengong Bioengineering Co., Ltd. The target gene C23O927 was directionally inserted into pET32a(+) expression vector through subcloning technology, and codon optimization design was carried out based on the preference of E. coli expression system. The recombinant plasmid was obtained by sequencing verification.

[0056] 2. Preparation and transformation of E. coli BL21(DE3) chemical competent cells

[0057] The E. coli BL21(DE3) electrotransformation competent cells were prepared by standard electric shock transformation method, and then the purified recombinant plasmid was introduced into the host bacteria by heat shock transformation technology. The experimental process strictly followed the standard operation procedures described in the Guide to Molecular Cloning (third edition), including key steps such as preparation of competent cells, formation of DNA-cell complex and recovery culture, and finally the positive clones were obtained by LB solid plate screening.

[0058] 3. Expression of C23O927 and purification of recombinant catechol-2,3-dioxygenase

[0059] The C23O927 gene was amplified by polymerase chain reaction (PCR) using the following primers: the forward primer for C23O927 (SEQ ID NO: 3) is: 5'-CGC GGATCC ATG GAG GAA CTG ATG GGT GAA G-3', and the reverse primer for C23O927 (SEQ ID NO: 4) is: 5'-CCC AAGCTT GTC CTC GCG GCT GAG CAC GAG-3'. These primers incorporate restriction enzyme sites for BamHI and HindIII enzymes.

[0060] The PCR product was double-digested with BamHI and HindIII and ligated into the prokaryotic expression vector pET32a. The recombinant vector was then transformed into E. coli BL21 (DE3) cells for protein expression. The cells were cultured in LB medium supplemented with 100 μg / mL ampicillin until the optical density at 600 nm (OD 600 ) reached 0.8. IPTG was added to a final concentration of 1.0 mM and gene expression was induced at 30°C and 120 rpm for 30 h. The cells were harvested by centrifugation at 7000 g for 10 min at 4°C, washed twice with sterile water, and then resuspended in 40 mM Britton-Robinson buffer (pH 7.4). Cells were lysed using a 400W ultrasonic disruptor with a pulse time of 3 s, an interval of 10 s, and a total of 30 cycles on ice. The cell lysate was centrifuged at 11000 × g for 10 min at 4°C, and the supernatant was collected as a crude enzyme solution. The crude enzyme solution was then purified using a Ni-NTA affinity chromatography column (Qiagen, Germany) according to the operating procedures in the instructions. The purified recombinant enzyme was stored at 4°C for further analysis.

[0061] 4. SDS-PAGE analysis of C23O927

[0062] To determine the molecular weight of the recombinant enzyme and assess its purity, a 9 μL aliquot of the crude and purified enzyme solutions was mixed with 3 μL of 4× loading buffer. The samples were then heated in a boiling water bath for 10 minutes to denature the proteins. After cooling, the samples were loaded onto the wells of a 12% polyacrylamide gel and electrophoresed at 120 V for 2 hours. Following electrophoresis, the gel was stained with Coomassie Brilliant Blue R-250 for 30 minutes. The gel was then destained to visualize protein bands. The position of the recombinant enzyme bands and the purity of the enzyme were observed and recorded photographically.

[0063] The C230927 gene was heterologously expressed in E. coli BL21(DE3), and the recombinant enzyme samples before and after purification were analyzed by SDS-PAGE electrophoresis, as shown in Figure 2 The electrophoresis results show that in the crude enzyme solution sample of lane 2, a very thick and clear target protein band appears near 50 kDa, indicating that the recombinant C230927 expressed in E. coli has a high solubility, and the expression amount of the recombinant protein is about 580 mg / L. In addition, after purifying the enzyme solution by Ni-NTA affinity chromatography column, most of the impurities are removed. A very obvious pure enzyme band appears at about 50 kDa (lane 3), indicating that the molecular weight of the recombinant enzyme is about 50 kDa, which is consistent with the theoretical molecular weight of the recombinant enzyme. This includes the 34.2 kDa molecular weight of C230927 itself, and an additional fusion protein tag of about 17 kDa from the expression vector.

[0064] Example 3 Enzymatic properties of the recombinant dioxygenase

[0065] 1. Enzyme activity determination method

[0066] The enzyme activity determination system includes 1.7 mL of 100 mM phosphate buffer (pH 4.0), 0.2 mL of 5 mM catechol solution, and 0.1 mL of purified recombinant enzyme solution. The determination procedure is outlined as follows: after mixing the above solutions well, the mixture is incubated at 55°C for 5 min. Then the mixture is immersed in a boiling water bath for 1 min to terminate the reaction. Once the reaction mixture cools to room temperature, the absorbance is measured at 375 nm using a spectrophotometer. Enzyme activity is quantified in units of enzyme activity, where 1 unit of enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 μmol of 2-hydroxy muconic semialdehyde per minute under the specified conditions.

[0067] The calculation formula of enzyme activity is as follows: U = AVt / εtdVs x 10 6 In this formula:

[0068] U represents the enzyme activity, expressed in units of enzyme activity contained per milliliter;

[0069] A represents the optical absorbance of the sample at 375 nm;

[0070] Vt is the total volume of the reaction mixture;

[0071] ε is the molar absorption coefficient of 2-hydroxy muconic semialdehyde, with a value of 36000 M -1 ·cm -1 ;

[0072] t is the duration of the reaction (in minutes);

[0073] D refers to the optical path length of the cuvette, in centimeters;

[0074] Vs is the volume of enzyme solution in liters.

[0075] 2. Effect of temperature on enzyme activity and stability

[0076] The optimum temperature of C230927 was determined by measuring its activity in 0.1 M phosphate buffer (pH 4.0) with catechol as a substrate at different temperatures (20, 30, 40, 50, 55, 60 and 70 °C). To assess its thermal stability, enzyme solution was incubated in 0.1 M phosphate buffer (pH 4.0) at different temperatures (30, 40, 50 and 60 °C) for a specified duration (0, 1, 2, 3 and 4 h) followed by measurement of residual enzyme activity under optimal conditions (55 °C, pH 4.0). The activity of unincubated enzyme solution was set as control (100% activity).

[0077] The results of enzyme activity assay of recombinant C230927 at different temperatures are shown in FIG. 1A, which shows that the enzyme activity of C230927 generally increased with increasing temperature between 20 and 55 °C. However, beyond 55 °C, the activity of the enzyme started to decrease rapidly (FIG. 1A), thus the optimum temperature of C230927 was determined to be 55 °C. Figure 3 Figure 3 A). When C230927 was incubated at temperatures ranging from 30 to 60 °C for 1 h, the enzyme activity decreased significantly, but the residual activity remained above 55% (FIG. 1B). Even when the incubation time was extended to 3 h at the same temperature range, the residual activity of the enzyme remained between 40 and 60%, indicating that C230927 exhibited good thermal stability. Figure 3

[0078] 3. Effect of pH on enzyme activity and stability

[0079] The optimum pH of C230927 was determined by measuring its activity in 0.1 M phosphate buffer at 55 °C with catechol as a substrate by pH gradient (2.0 to 8.0). To determine the pH stability, enzyme solution was pre-incubated in various buffers with pH values ranging from 2.0 to 10.0 at 4 °C for 1 h, followed by measurement of residual activity under optimal conditions of the enzyme (55 °C, pH 4.0). The activity of unincubated enzyme solution was set as reference (100% activity).

[0080] The results of enzyme activity assay of recombinant C230927 at different pH values are shown in FIG. 2A, which shows that the optimum pH of recombinant C230927 was 4.0 (FIG. 2A). Figure 4 Figure 4 ​​​A), indicating that C23O927 is an acidic catechol-2,3-dioxygenase. Notably, the enzyme maintains relatively high activity, exceeding 65% of its maximum activity, even under neutral to slightly alkaline conditions (pH 8.0). In addition, C23O927 exhibits high pH stability over a wide range (pH 2.0 to 10.0). Figure 4 B) After 1 hour of incubation in buffer within this pH range, the residual activity of the enzyme was found to be greater than 70% of its initial activity.

[0081] 4. Effects of metal ions on enzyme activity

[0082] The effects of metal ions on catechol-2,3-dioxygenase activity were examined at the optimal temperature and pH by measuring enzyme activity in phosphate buffer containing 1 mM, 5 mM, and 15 mM metal ions. The metal ions used included CuSO₄·5H₂O, CaCl₂, MgCl₂, FeSO₄·7H₂O, MnCl₂, NaCl, KCl, and CoCl₂. Experiments were performed in triplicate to ensure reproducibility. The enzyme activity of a reaction system without metal ions was used as a control, and its activity was assumed to be 100%.

[0083] The results of enzyme activity assay of recombinant C23O927 to different metal ions are as follows Figure 5 As shown, catechol was used as a substrate to examine the activity of several metal ions (Mn 2+ 、Na + ,K,Co 2+ , Ca 2+ 、Cu 2+ and Mg 2+ ) on the activity of C23O927, and the results are shown in Table 3. All metal ions tested at 1mM, 5mM, and 15mM concentrations significantly enhanced the enzyme activity, and the degree of activation depended on the specific type and concentration of the metal ion. 2+ Effect of concentration on the activity of C23O927, such as Figure 5 As shown, low concentrations of Fe 2+ (1-50 mM) significantly enhanced the activity of C23O927. 2+ When the concentration exceeded 100 mM, the enzyme activity decreased significantly.

[0084] Table 3 Effects of several metal ions on the activity of C23O927

[0085]

[0086]

[0087] 5. Effect of organic solvents and surfactants on the activity of recombinant catechol-2,3-dioxygenase

[0088] The effect of organic solvents and surfactants on the activity of recombinant catechol-2,3-dioxygenase was evaluated by adding appropriate amounts of enzyme and substrate, and different concentrations of organic solvents (v / v) or surfactants (w / v) to 100 mM phosphate buffer at pH 4.0, with final concentrations of 1%, 5%, and 15%, respectively. Residual enzyme activity was measured at 55°C. The organic solvents and surfactants used in this study included methanol (v / v), ethanol (v / v), isopropanol (v / v), acetonitrile, dimethyl sulfoxide (DMSO, v / v), SDS (w / v), Tween-80 (v / v), and Tween-20 (v / v).

[0089] The effect of several organic solvents and surfactants on the activity of C230927 was evaluated using catechol as the substrate, and the results are shown in Table 4, which indicates that recombinant C230927 exhibited significant stability in the presence of various organic solvents. At low (1%, v / v), medium (5%, v / v), and high (15%, v / v) concentrations of several common organic solvents, methanol, ethanol, isopropanol, acetonitrile, and DMSO, except for 15% methanol, which slightly inhibited enzyme activity, the other concentrations of organic solvents significantly enhanced enzyme activity. This suggests that C230927 has good tolerance to organic solvents. In addition, except for three different concentrations of SDS, which significantly inhibited enzyme activity, two non-ionic surfactants, Tween-20 and Tween-80, significantly enhanced enzyme activity at all three tested concentrations.

[0090] Table 4. Effect of several metal ions on the activity of C230927

[0091]

[0092]

[0093] 6. Determination of kinetic parameters and substrate specificity of recombinant catechol-2,3-dioxygenase

[0094] Several different substrates were used: catechol, 3-methylcatechol, 4-methylcatechol, 3-chlorocatechol, 4-chlorocatechol, and 3,5-dichlorocatechol. Determination of various enzymatic parameters, including optimal pH, optimal temperature, kinetic parameters (K m and k cat ), catalytic efficiency (k cat / K m) and specific enzyme activities were determined. The initial linear rates of enzymatic reactions were analyzed at the optimal temperature using different substrate concentrations (0.1-0.5 mM). Each substrate concentration was tested in triplicate. The parameters K m and k cat were calculated using the Holling iterative method. The substrate specificity of C230927 was determined by comparing the catalytic efficiency (k cat / K m ) of the enzyme towards several substrates.

[0095] The molar extinction coefficients of the products were: catechol, 36000 M -1 ·cm -1 at 375 nm, 3-methylcatechol, 13800 M -1 ·cm -1 at 388 nm, 4-methylcatechol, 28100 M -1 ·cm -1 at 382 nm, 3-chlorocatechol, 36800 M -1 ·cm -1 at 290 nm, 4-chlorocatechol, 40000 M -1 ·cm -1 at 379 nm, and 3,5-dichlorocatechol, 10000 M -1 ·cm -1 at 337 nm.

[0096] According to the results of the determination of the catalytic properties of recombinant C230927 towards several common substrates, it was found that the optimal pH values of the enzyme towards different substrates were between 3.0 and 5.0, indicating that C230927 is a typical acidic catechol-2,3-dioxygenase. In addition, the optimal temperatures of the enzyme towards different substrates were 40°C to 55°C, indicating that C230927 is a mesophilic enzyme.

[0097] The specific enzyme activity detection results showed that C230927 exhibited the highest specific enzyme activity towards catechol, as shown in Table 5. By comparing the catalytic efficiency of the enzyme towards various substrates, catechol was determined to be the optimal substrate of C230927, with a corresponding K m value of 0.037 mM and a k cat value of 6100 s -1 . The catalytic efficiency of C230927 towards catechol was 1.65 x 10 5 s -1 ·mM -1 . The catalytic efficiency of the enzyme towards other substrates, from high to low, was as follows: 3-chlorocatechol > 3-methylcatechol > 4-methylcatechol > 3,5-dichlorocatechol > 4-chlorocatechol.

[0098] Table 5 Effect of different substrates on the enzymatic properties of C230927

[0099]

[0100] 7. Effect of salinity on the activity of recombinant catechol-2,3-dioxygenase

[0101] The effect of salinity on the activity of C230927 was investigated in 100 mM phosphate buffer (pH 4.0) supplemented with different concentrations of NaCl: 0 M (control), 1 M, 3 M and 5 M. The relative activity of the enzyme was expressed as a percentage relative to the activity observed at 0% salinity. To assess the stability of C230927 to salt solutions, the enzyme was pre-incubated for 0 h, 6 h and 12 h in 100 mM phosphate buffer (pH 4.0) containing 0 M (control), 1 M, 3 M and 5 M NaCl. The residual activity of the enzyme was then measured using the enzyme activity assay method described earlier.

[0102] The results of the assays are shown in Table 6, which show that NaCl solutions had an activating effect on the activity of C230927. The enzyme activity of C230927 was enhanced substantially regardless of the concentration of NaCl, whether 1 M, 3 M or 5 M. Furthermore, the enzyme activity of C230927 increased gradually as the duration of NaCl treatment was extended. Notably, the activating effect of NaCl solutions on C230927 appeared to be related to both concentration and time. Specifically, the activating effect of NaCl on the enzyme was most pronounced when the enzyme was treated with 1 mM NaCl solution for 12 h, and the activating effect of low concentrations of NaCl solution on the enzyme was more pronounced than that of high concentrations of NaCl solution.

[0103] Table 6 Effect of NaCl concentration and incubation time on the activity of C230927

[0104]

[0105] In summary, the recombinant enzyme C230927 exhibited good thermal stability, with an optimum pH of 4.0, belonging to an acid catechol-2,3-dioxygenase, and also had good tolerance to metal ions, organic solvents, surfactants and salinity, with the highest affinity for the substrate catechol, a fast reaction rate and a catalytic efficiency of 1.65 x 10 5 s -1 ·mM -1 , significantly higher than the existing four similar enzymes of catechol, BLC23O, BpHC-SD3, BpHC-meta and Tcu3516, and comparable to C230Mpc. The recombinant enzyme C230927 provided by the present application has unique substrate specificity and high catalytic efficiency, and has great potential in industrial applications.

[0106] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application.

Claims

1. A recombinant catechol-2,3-dioxygenase, characterized in that, The enzyme is prepared by heterologous expression of the nucleotide sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO:

2.

2. The method of producing the recombinant catechol-2,3-dioxygenase according to claim 1, wherein, The method comprises the following steps: S1. Cloning the gene sequence shown in SEQ ID NO: 1 or the protein sequence shown in SEQ ID NO: 2 to prepare a recombinant plasmid; S2. Transforming the recombinant plasmid into E. coli to obtain a recombinant bacterium; S3. Prokaryotic expression of the recombinant bacterium, collection of bacterial bodies, washing, resuspension, and obtaining.

3. The preparation method according to claim 2, characterized in that: In S1, the recombinant plasmid is prepared by double enzyme digestion of plasmid pET32a (+).

4. The preparation method according to claim 2, characterized in that In S2, E. coli BL21 (DE3) competent cells are used.

5. The preparation method according to claim 2, characterized in that: In S3, the prokaryotic expression conditions are as follows: 0.5-1.5 mM IPTG, 20-40℃, and 20-40 h.

6. Use of the recombinant catechol-2,3-dioxygenase of claim 1 in catalyzing the degradation of catechol compounds or polycyclic aromatic hydrocarbon compounds.

7. Use according to claim 6, characterized in that, the catechol compound is selected from one or more of catechol, 3-chlorocatechol, 3-methylcatechol, 4-methylcatechol, 3,5-dichlorocatechol, 4-chlorocatechol; the polycyclic aromatic compound is selected from one or more of naphthalene, acenylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenzo[a,h]anthracene, benzo[g,h,i]perylene, indeno[1,2,3-cd]pyrene, benzo[e]pyrene, benzo[j]fluoranthene, 5-methyl dibenzofuran.

8. Use of the recombinant catechol-2,3-dioxygenase of claim 1 in preparing products for degrading catechol compounds or polycyclic aromatic hydrocarbon compounds.

9. Use of the recombinant catechol-2,3-dioxygenase according to claim 1 for the degradation of catechol or polycyclic aromatic hydrocarbons in a complex contaminated environment, characterized in that, The complex contaminated environment refers to an environment containing multiple pollutants composed of acidic compounds, salt compounds, metal ions, organic solvents, and / or surfactants.

10. Use of the C230927 gene or protein for the preparation of a reconstituted enzyme of catechol, characterized in that, The nucleotide sequence of the C23O927 gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein is shown in SEQ ID NO: 2.

Images