Phthalate hydrolase gene paeH and application thereof

Abstract

The invention discloses a PAEs hydrolase gene and application thereof. The nucleotide sequence of the dibutyl phthalate hydrolase gene paeH is as shown in SEQ ID NO. 1. PaeH (SEQ ID NO.2) expressed by an engineering strain constructed by using the gene can be used for remarkably degrading PAEs (Polyacrylamide) compounds such as dibutyl phthalate, butyl benzyl phthalate, diamyl phthalate, diisobutyl phthalate and the like. Therefore, the produced PaeH enzyme preparation can be used for removing PAEs compounds such as dibutyl phthalate, butyl benzyl phthalate, diamyl phthalate, diisobutyl phthalate and the like which are remained in soil or water.

Description

Technical Field

[0001] The invention belongs to the field of applied environmental microorganisms and relates to a phthalate hydrolase gene and an application thereof. Background Art

[0002] Phthalates (PAEs), often referred to as phthalates, are synthetic organic compounds. They play a key role as plasticizers in a wide range of fields, including plastics manufacturing, product packaging, cosmetics production, and the processing of lubricants and fragrances. Currently, global annual production of PAEs approaches 300 million tons, and some forecasts indicate that this figure will jump to 500 million tons by 2050. Notably, China has become the world's largest user of plasticizers, accounting for 42% of global consumption in 2017 alone.

[0003] Because there is no stable chemical bond between PAEs and plastic products, they continue to seep and volatilize from the inside of plastic products, and then merge into the natural environment such as soil, water, and atmosphere. Those PAEs left in the soil, water, and atmosphere can enter the human body through channels such as food chain transmission, human respiration, and direct skin contact. Related studies have confirmed that if the human body is exposed to large amounts of PAEs in a short period of time, or if it is continuously exposed to low doses of PAEs for a long time, the reproductive system, liver, kidneys and other organs may be greatly threatened, and even genetic material may be at risk of damage.

[0004] Dibutyl phthalate (DBP), a widely used member of the PAE family, is a significant environmental endocrine disruptor, known for its carcinogenic, teratogenic, and mutagenic properties. Consequently, the U.S. Environmental Protection Agency (EPA), the China National Environmental Monitoring Center, and the European Union have all designated DBP as a priority pollutant.

[0005] Studies have found that under natural conditions, the hydrolysis and photolysis rates of PAEs are very slow, making them difficult to degrade. Microbial degradation is considered the most economical and effective method for removing PAEs. In addition, compared with physical and chemical methods, microbial degradation and remediation technology has the advantages of high efficiency, low cost, and no secondary pollution to the environment. The main mode of microbial decomposition and metabolism is enzymatic reaction, that is, the degradation enzymes extracted from microorganisms are used to remove the residues of toxic and harmful pollutants. The source of the degradation enzyme can be obtained by extracting it from degrading bacteria or by constructing a high-efficiency expression strain through genetic engineering. Therefore, the PAEs hydrolase gene paeH has great application potential in the field of remediation of polluted environments such as degrading PAEs. Summary of the Invention

[0006] The purpose of the present invention is to provide a hydrolase gene paeH cloned from the dibutyl phthalate degrading strain Alicycliphilus sp.LB2.

[0007] Another object of the present invention is to provide the hydrolase PaeH encoded by the hydrolase gene.

[0008] Another object of the present invention is to provide an application of the hydrolase gene.

[0009] The purpose of the present invention can be achieved through the following technical solutions:

[0010] A dibutyl phthalate hydrolase gene paeH has a nucleotide sequence of SEQ ID NO. 1. The gene is 921 bp in length (from the start codon to the stop codon), has a G+C content of 73.0%, encodes 306 amino acids, and has a molecular weight of approximately 32.7 kDa.

[0011] The hydrolase PaeH encoded by the dibutyl phthalate hydrolase gene paeH of the present invention has an amino acid sequence as SEQ ID NO.2.

[0012] A recombinant plasmid containing the dibutyl phthalate hydrolase gene paeH of the present invention.

[0013] As a preferred embodiment of the present invention, the hydrolase gene fragment is ligated with the digested pET-29a(+) to obtain a pET-29a(+) recombinant plasmid (pET-paeH) containing the hydrolase gene.

[0014] A recombinant microorganism Escherichia coli BL21 (DE3) containing the recombinant plasmid of the present invention.

[0015] As a preferred embodiment of the present invention, the enzyme-linked pET-29a(+) recombinant plasmid containing the hydrolase gene is transformed into the expression host bacterium E. coli BL21 (DE3) to obtain the recombinant microorganism E. coli BL21 (pET-paeH).

[0016] The hydrolase gene paeH of the present invention is used for removing PAEs pollution in the environment.

[0017] The invention relates to the application of the hydrolase in removing PAEs pollution in soil and water.

[0018] Wherein, the PAEs are selected from any one or more of dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dipentyl phthalate, dioctyl phthalate, diisobutyl phthalate and butyl benzyl phthalate. More preferably, any one or more of dibutyl phthalate, dipentyl phthalate, diisobutyl phthalate and butyl benzyl phthalate are selected.

[0019] The invention relates to the use of the recombinant microorganism E. coli BL21 (pET-paeH) in removing PAEs residues in soil and water.

[0020] Wherein, the PAEs are selected from any one or more of dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dipentyl phthalate, dioctyl phthalate, diisobutyl phthalate and butyl benzyl phthalate. More preferably, any one or more of dibutyl phthalate, dipentyl phthalate, diisobutyl phthalate and butyl benzyl phthalate are selected.

[0021] Beneficial effects

[0022] 1. The present invention successfully cloned the dibutyl phthalate hydrolase gene paeH from the strain Alicycliphilus sp. LB2 (isolated from the soil of a farmland in Huaibei, Anhui, with the patent strain deposit number CCTCC NO: M 20242749).

[0023] 2. The hydrolase gene paeH was cloned into the high-efficiency expression vector pET29a(+) to obtain the recombinant plasmid pET-paeH, which was then transferred to the high-efficiency expression strain E. coli BL21(DE3) (purchased from General Biotechnology) by transformation to obtain the recombinant expression strain E. coli BL21(pET-paeH).

[0024] 3. After the recombinant expression strain E. coli BL21 (pET-paeH) was induced with 0.5mM IPTG for 18h, a large amount of hydrolase PaeH carrying His6-tag was expressed. 2+ -NTAResin (Shanghai Biotechnology Co., Ltd.) was used for affinity chromatography to obtain pure PaeH-His6.

[0025] 4. The present invention conducted an enzymatic degradation test on the product PaeH expressed by the hydrolase gene paeH, and found that PaeH can significantly degrade dibutyl phthalate, butyl benzyl phthalate, diamyl phthalate and diisobutyl phthalate.

[0026] The engineered strain constructed using the hydrolase gene paeH can efficiently express PaeH-His6, and the enzyme preparation produced can be used to remove residual dibutyl phthalate, butyl benzyl phthalate, dipentyl phthalate and diisobutyl phthalate in soil and water. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a diagram of the cloning strategy of the dibutyl phthalate hydrolase gene paeH of the present invention.

[0028] Figure 2 This is the degradation curve of dibutyl phthalate by the recombinant strain E. coli DH5α (pBBR-paeH) of the present invention.

[0029] Figure 3 The hydrolase gene paeH of the present invention is efficiently expressed and purified in the host bacteria E.coli BL21 (DE3)

[0030] Protein SDS-PAGE electrophoresis.

[0031] Figure 4 This is a liquid chromatography-mass spectrometry detection diagram of the metabolites of dibutyl phthalate degraded by the hydrolase PaeH of the present invention.

[0032] Figure 5 This is the metabolic pathway for the hydrolase PaeH of the present invention to convert dibutyl phthalate.

[0033] Figure 6 It is the optimum catalytic temperature of the hydrolase PaeH of the present invention.

[0034] Figure 7 It is the optimal catalytic pH of the hydrolase PaeH of the present invention.

[0035] Figure 8 The effect of metal ions on the activity of hydrolase PaeH.

[0036] Figure 9 The enzymatic activity of the hydrolase PaeH of the present invention on four preferred PAEs substrates was determined.

[0037] Biomaterial deposit information

[0038] LB2, classified and named Alicycliphilus sp.LB2, is deposited in the China Center for Type Culture Collection, the strain deposit number is CCTCC NO: M 20242749, the deposit date is December 6, 2024, and the deposit address is China Center for Type Culture Collection, Wuhan University, Bayi Road, Hongshan District, Wuhan City, Hubei Province. DETAILED DESCRIPTION

[0039] Example 1 Cloning of the dibutyl phthalate hydrolase gene paeH

[0040] The cloning of dibutyl phthalate hydrolase gene was carried out by shotgun method. The strategy is shown in the figure. Figure 1 , the specific steps are as follows:

[0041] 1.1 Extraction of total genomic DNA from strain LB2

[0042] The strain LB2 was inoculated into liquid LB medium and cultured in a shaking incubator at 30°C and 180 rpm for 24 h. The bacteria were collected by centrifugation, and high-purity, large-fragment total genomic DNA was extracted using a bacterial genome extraction kit (Tiangen Biotechnology), dissolved in TE (pH 8.0), and stored at -20°C.

[0043] 1.2 Shotgun library construction and cloning of target genes

[0044] 1.2.1pUC118 (BamH I) was purchased from Takara Biotechnology (Dalian) Co., Ltd.

[0045] 1.2.2 Enzyme digestion of total DNA

[0046] The total DNA of strain LB2 was partially digested with Sau3AI.

[0047] 1.2.3 DNA recovery

[0048] The total DNA after enzyme digestion was recovered by gel excision (2-6 kb) by electrophoresis (TAE buffer), and then purified and recovered using the Omega gel recovery kit. The recovered DNA was dissolved in TE (pH 8.0) and stored at -20°C.

[0049] 1.2.4 Enzyme ligation

[0050] The following enzyme-linked reaction system was established:

[0051]

[0052]

[0053] The enzyme-linked system was placed in ice water and incubated in a 4°C refrigerator overnight.

[0054] 1.2.5 Conversion

[0055] Take 10 μL of enzyme-linked product to transform 100 μL of E. coli DH5α high-efficiency competent cells (purchased from General Biotechnology (Anhui) Co., Ltd.). For specific methods, refer to the instructions of the competent cells. Spread on a plate containing 50 mg L -1 Dibutyl phthalate (5000 mg L -1mother liquor) and 100 mg L -1 After 24 hours of incubation on an ampicillin-containing LB plate, pick out the colonies that produce a transparent hydrolysis zone around the colonies (dibutyl phthalate has low water solubility, so the LB solid culture medium containing dibutyl phthalate is turbid and opaque. After microbial degradation of dibutyl phthalate, a transparent zone will form around the colonies). This is the positive clone containing the suspected dibutyl phthalate degrading enzyme gene.

[0056] 1.2.6 Nucleotide Sequence Determination of the Dibutyl Phthalate Hydrolase Gene

[0057] Positive transformants were sent to Tongyong Biological (Anhui) Co., Ltd. for sequencing. The insert sequence was obtained through first-generation sequencing. Using online ORF prediction software, the complete ORF contained in the insert sequence was predicted. Finally, based on the NCBI database, a hydrolase gene (designated paeH) suspected to be responsible for dibutyl phthalate degradation was identified. The nucleotide sequence of this hydrolase gene is shown in SEQ ID NO. 1, and the 306-amino acid sequence deduced from this hydrolase gene is shown in SEQ ID NO. 2.

[0058] 1.3 Functional verification of the dibutyl phthalate hydrolase gene paeH

[0059] The paeH hydrolase gene suspected to be responsible for the degradation of dibutyl phthalate was directionally cloned into the broad-host expression vector pBBR1MCS-2 to construct pBBR-paeH, which was then transformed into the expression host E. coli DH5α to obtain the recombinant strain E. coli DH5α (pBBR-paeH). The recombinant strain was then used for whole-cell transformation experiments, and it was finally found that the recombinant strain E. coli DH5α (pBBR-paeH) could completely degrade dibutyl phthalate ( Figure 2 ), indicating that the gene paeH is the key enzyme gene responsible for the degradation of dibutyl phthalate.

[0060] Example 2 High-efficiency expression and purification of dibutyl phthalate hydrolase gene paeH

[0061] 2.1 PCR amplification of the hydrolase gene paeH

[0062] Primer sequences:

[0063] paeH-F:5'-TATCATATGAGGAGAACATCACGCTGC-3'(Nde I)(SEQ ID NO.3); paeH-R:5'-ATA AAGCTTATCGAGCCCGAAG-3'(Hind III) (SEQ ID NO. 4).

[0064] Primers paeH-F and paeH-R were used to amplify the hydrolase gene paeH. The PCR amplification system was as follows:

[0065] Amplification system:

[0066]

[0067] PCR amplification procedure:

[0068]

[0069] 2.2 PCR products and vectors were digested simultaneously with Nde I and Hind III enzymes.

[0070] Enzyme digestion system:

[0071]

[0072] The enzyme digestion products were purified and recovered using a purification and recovery kit.

[0073] 2.3 Conversion

[0074] The DNA fragment digested in 2.2 was ligated with pET-29a(+) by enzyme (refer to 1.2.4). The pET-29a(+) recombinant plasmid containing the paeH hydrolase gene was transformed into the expression host bacteria E. coli BL21(DE3) and spread on a plate containing 50 mg L -1 The recombinant transformant E. coli BL21 (pET-paeH) was obtained by plating on a kanamycin plate.

[0075] 2.4 Expression and purification of hydrolase PaeH

[0076] The recombinant strain E. coli BL21 (pET-paeH) was cultured in LB medium until OD 600 = 0.4, add IPTG to a final concentration of 0.5mM to the culture medium, induce at 30℃ for 18h, and collect the cells by centrifugation. Wash the cells with Tris-HCl (pH 8.0), disrupt with ultrasonic wave, and collect the supernatant by centrifugation. Since the PaeH protein expressed by the constructed recombinant strain E. coli BL21 (pET-paeH) after IPTG induction has a tag consisting of 6 histidine residues at the N-terminus, the recombinant protein can be expressed by Ni 2+-NTAResin (Shanghai Biotechnology) was used for elution and purification. Detailed purification steps and required solutions are provided in the kit instructions. All enzyme solutions collected after purification were poured into dialysis bags and dialyzed in 20mM Tris-HCl buffer (pH 8.0) at 4°C overnight. Finally, SDS-PAGE electrophoresis was performed to detect the purity of the target protein. Finally, pure PaeH-His6 enzyme ( Figure 3 All protein operations were performed in a chromatography cabinet (4°C). Protein concentration was determined by the Bradford method (Bradford, 1976), with bovine serum albumin as the standard protein.

[0077] Degradation of dibutyl phthalate by 2.5PaeH

[0078] An enzymatic reaction system (3 mL) was established in Tris-HCl buffer (pH 8.0, 20 mM) using dibutyl phthalate as the substrate:

[0079] Dibutyl phthalate 50mg L -1

[0080] PaeH 1 μM

[0081] React in a 30°C water bath for 60 min. Adjust the pH to approximately 3.0 with 25% hydrochloric acid, then add an equal volume of ethyl acetate for extraction and termination. Aspirate the upper organic phase and further dehydrate with anhydrous sodium sulfate. After evaporation of the ethyl acetate, reconstitute with methanol. Filter through a 0.22 μm membrane filter and analyze by high-performance liquid chromatography.

[0082] Liquid chromatography detection conditions: Thermo scientific TM Acclaim TM 120C18 reverse phase column (4.6×250mm, 5μm, ), the mobile phase was methanol:water:acetic acid (80:20:0.5, v / v / v), and the flow rate was 1 mL min -1 , column temperature 30 °C, injection volume 20 μL, and UV detection wavelength 235 nm.

[0083] Metabolites were detected and identified by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). MS analysis was performed in ESI mode using an Agilent G6410B Triple Quad Mass Spectrometer.

[0084] The chromatographic detection results showed that the absorption peak of dibutyl phthalate (retention time: 10.073min) decreased significantly, and a product peak with a retention time of 4.807min appeared ( Figure 4a). Mass spectrometry analysis detected two ion peaks ( Figure 4 b and 4c), with m / z 279.1585 and m / z 223.0961, respectively. Based on the molecular weight, m / z = 279.1585 can be determined to be dibutyl phthalate, while m / z = 223.0961 is the product of dibutyl phthalate after the ester bond is broken. This shows that the way PaeH degrades dibutyl phthalate is to hydrolyze the ester bond in the dibutyl phthalate molecule to produce monobutyl phthalate and propanol ( Figure 5 ).

[0085] 2.6 Enzymatic properties of PaeH

[0086] The PaeH-catalyzed reaction of dibutyl phthalate (DBP) was conducted at various temperatures (4-60°C) to investigate its optimal reaction temperature. PaeH was first incubated in a 4-60°C water bath for 30 minutes, and then subjected to the enzymatic reaction at its optimal temperature to investigate its thermal stability. The enzymatic reaction lasted for 90 minutes. After completion, the reaction was terminated by placing the mixture in boiling water for 3 minutes. The pH was adjusted to approximately 3.0, and then an equal volume of ethyl acetate was added for extraction. The upper organic phase was aspirated and further dehydrated with anhydrous sodium sulfate. The ethyl acetate was evaporated and then reconstituted with methanol. The mixture was filtered through a 0.22 μm pore size membrane and analyzed by high-performance liquid chromatography. The degradation rate of DBP by PaeH at different temperatures was determined, with the degradation efficiency at the optimal temperature being considered 100%, and the relative enzyme activity was calculated.

[0087] The experimental results show that PaeH has certain activity towards dibutyl phthalate in the range of 20-37℃, and the optimum reaction temperature is 30℃; when the temperature is 25-35℃, the relative enzyme activity is above 70%; when the temperature is below 16℃ or above 50℃, the relative enzyme activity is below 10% ( Figure 6 Temperature stability experiments showed that PaeH can maintain more than 70% of its enzyme activity at temperatures below 30°C. However, when the temperature rises above 30°C, PaeH activity gradually decreases.

[0088] Four buffers with different pH values were prepared: 50mM citric acid-Na2HPO4 buffer, 50mM citric acid-sodium citrate buffer, 20mM Tris-HCl buffer, and 20mM glycine-NaOH buffer. PaeH was reacted in these buffers at different pH values for 90 minutes at 30°C, followed by terminating the reaction by placing the sample in boiling water for 3 minutes. Samples were processed as above, and the degradation rates of dibutyl phthalate by PaeH at different pH values were determined using high-performance liquid chromatography. The degradation rate at the optimal pH was set as 100%, and relative enzyme activity was calculated.

[0089] The experimental results showed that the optimal reaction pH of PaeH was 7.0; when the pH was between 6.0 and 8.0, the relative activity of PaeH remained above 70%; when the pH was > 9.0, the relative enzyme activity of PaeH was less than 40% ( Figure 7 ).

[0090] Under the optimal reaction temperature and pH conditions, common metal ions (Ni 2+ 、Cu 2+ 、Fe 3+ 、Al 3+ Mg 2+ and Cr 3+ ) and reacted for 90 minutes. The reaction was then terminated by placing the mixture in boiling water for 3 minutes. Samples were processed as above and analyzed by high-performance liquid chromatography (HPLC). The degradation rate of dibutyl phthalate by PaeH under the influence of different metal ions was determined. The relative enzyme activity of each experimental group was calculated, with the enzyme activity in the reaction system without any metal ions defined as 100%.

[0091] The results showed that 0.2 mM of the tested metal ions had a slight inhibitory effect on the enzyme activity of PaeH ( Figure 8 ).

[0092] Add 1% agarose to 30 mL of 20 mM Tris-HCl (pH 8.0) buffer, heat in a microwave oven to dissolve, and add a final concentration of 100 mg L -1 Mix dibutyl phthalate, then pour onto an agarose plate. Follow the same procedure to prepare agarose plates containing other substrates, such as butyl benzyl phthalate, diamyl phthalate, and diisobutyl phthalate. After the plates cool, use a microporator to punch holes in the wells. Add 0.2 mL of PaeH to each well. Add an equal volume of Tris-HCl (20 mM, pH 8.0) buffer without enzyme solution as a negative control.

[0093] The results showed that compared with the control group, the hydrolase PaeH could produce obvious hydrolysis circles on the plates with these four substrates, indicating that the hydrolase PaeH had significant activity on these four substrates ( Figure 9 ).

Claims

1. A dibutyl phthalate hydrolase, characterized in that The amino acid sequence is shown in SEQ ID NO.

2.

2. The gene paeH encoding the dibutyl phthalate hydrolase according to claim 1.

3. The gene paeH according to claim 2, characterized in that The nucleotide sequence is shown in SEQ ID NO.

1.

4. A recombinant plasmid containing the dibutyl phthalate hydrolase gene paeH according to claim 2 or 3.

5. A recombinant microorganism E. coli BL21 (DE3) containing the recombinant plasmid according to claim 4.

6. Use of the dibutyl phthalate hydrolase according to claim 1 in removing PAEs residues in soil and water; the PAEs are selected from any one or more of dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dipentyl phthalate, dioctyl phthalate, diisobutyl phthalate, and butyl benzyl phthalate. More preferably, any one or more of dibutyl phthalate, dipentyl phthalate, diisobutyl phthalate, and butyl benzyl phthalate are selected.

7. Use of the gene paeH according to claim 2 or 3 and the recombinant microorganism E. coli BL21 (DE3) according to claim 4 in removing PAEs residues in soil and / or water.

8. The use according to claim 7, characterized in that The PAEs are selected from any one or more of dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dipentyl phthalate, dioctyl phthalate, diisobutyl phthalate and butyl benzyl phthalate. More preferably, any one or more of dibutyl phthalate, dipentyl phthalate, diisobutyl phthalate and butyl benzyl phthalate are used.

9. Use of the gene paeH according to claim 2 or 3 and the recombinant microorganism E. coli BL21 (DE3) according to claim 4 in preparing a product for removing PAEs residues in soil or water.

10. The use according to claim 9, characterized in that The PAEs are selected from any one or more of dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dipentyl phthalate, dioctyl phthalate, diisobutyl phthalate and butyl benzyl phthalate. More preferably, any one or more of dibutyl phthalate, dipentyl phthalate, diisobutyl phthalate and butyl benzyl phthalate are used.

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