Amidase gene adgA, protein encoded thereby and use thereof

Abstract

This invention discloses an atenolol-degrading amidase gene. adgA The amidase gene and the proteins it encodes and their applications. adgA The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein AdgA is shown in SEQ ID NO.2. This invention is the first to clone an amidase gene from strain YD-4. adgA Engineered strains or enzyme preparations constructed using this gene can efficiently degrade atenolol, making them suitable for the removal of atenolol residues in water and soil, and possessing significant theoretical and practical value.

Description

Technical Field

[0001] This invention relates to environmental microorganisms, specifically an amidase gene. adgA And its encoded proteins and applications. Background Technology

[0002] Atenolol is a widely used... β Atenolol, an adrenergic receptor blocker, is used to treat hypertension and angina. Due to its low biodegradability and high solubility in water, it is prevalent in wastewater treatment plant (WWTP) effluents and frequently detected in surface water, groundwater, and other aquatic environments globally, with concentrations ranging from 0.04 to 122 µg / L, far exceeding the maximum acceptable concentration (10 ng / L). Studies have shown that atenolol is toxic to aquatic organisms and may affect human health; therefore, effectively removing atenolol residues from the environment has become a pressing environmental issue. Currently, methods for removing atenolol mainly include physical adsorption and chemical oxidation. Physical adsorption is significantly affected by factors such as ionic strength, pH, and reaction time, resulting in unstable adsorption efficiency. While advanced chemical oxidation can effectively remove atenol, it has high operating costs, requires highly corrosion-resistant equipment, is difficult to control under various operating conditions, and may produce toxic byproducts.

[0003] Biodegradation, as a green and economical remediation method, uses microorganisms and their secreted enzymes to break down complex pollutants into simpler, less toxic products, or even completely mineralize them into carbon dioxide and water. It is considered an effective method for removing harmful compounds from the environment. However, current research on the degradation mechanism of atenolol in microorganisms is relatively weak, mainly focusing on the isolation of some atenolol-degrading bacteria and the inference of metabolic pathways. At present, there are no reports on the molecular-level research of key enzymes in atenolol degradation and their encoding genes. This severely limits a deeper understanding of the biodegradation mechanism of atenolol and hinders the application of technologies such as constructing highly efficient engineered strains and developing enzyme-based bioremediation agents in the remediation of atenolol pollution. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide an amidase gene for degrading atenolol. adgA The study, along with its encoded protein and applications, addresses the problem of the unknown key enzymes and their encoding genes in the degradation of atenolol in existing technologies. It aims to provide applications of these genes and proteins in constructing genetically engineered bacteria and preparing bioremediation agents to achieve efficient and thorough removal of atenolol residues from water and soil.

[0005] Technical solution: The amidase gene described in this invention adgAIts nucleotide sequence is SEQ ID NO.1.

[0006] The amidase gene described in this invention was cloned from a strain capable of efficiently degrading atenolol. Achromobacter sp. YD-4. Based on HPLC and MS / MS data, it was deduced that the first step in the metabolism of atenolol in strain YD-4 is the hydrolysis of the primary amide bond. Therefore, the entire genome of strain YD-4 was sequenced and annotated, and three amidase genes were selected. orf2992 , orf3619 and orf4739 Functional validation was performed using it as a candidate gene. Heterologous expression and in vitro enzyme activity assays showed that... orf4739 The atenolamidinase gene was named adgA, The other two genes do not possess the hydrolytic function of atenolol. Meanwhile, single-protein expression and purification were achieved using a nickel ion exchange column, and based on polyacrylamide gel electrophoresis, the size of the atenolol amidase protein in this strain was preliminarily determined to be approximately 27.4 kDa.

[0007] The amidase gene adgA The encoded protein AdgA, with the amino acid sequence SEQ ID NO.2, was synthesized by Genewiz Biotechnology Co., Ltd. (The gene encoding the amidase was also described.) adgA.

[0008] Contains the aforementioned amidase gene adgA Recombinant expression vectors.

[0009] The recombinant expression vector can be any conventional vector suitable for prokaryotic or eukaryotic expression. Preferably, the recombinant expression vector contains the amidase gene. adgA The result was obtained by linking the NdeI and XhoI sites of pET-29a(+).

[0010] Contains the aforementioned amidase gene adgA Genetically engineered bacteria, wherein the genetically engineered strain is preferably a... Escherichia coli BL21(DE3) is the originating strain.

[0011] Those skilled in the art will understand that, in addition to pET-29a(+) and BL21(DE3), other conventional prokaryotic expression vectors and hosts can also be used to construct the recombinant expression vectors and genetically engineered bacteria of the present invention.

[0012] The amidase gene adgA Application in the degradation of atenolol.

[0013] The aforementioned gene containing amidase adgA Application of recombinant expression vectors in the degradation of atenolol.

[0014] The application of the protein AdgA in the degradation of atenolol.

[0015] The application of the protein AdgA in the preparation of reagents for removing atenolol from water and soil, or in the removal of atenolol from soil and water.

[0016] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention is the first to isolate a strain capable of degrading atenolol from an enrichment solution with atenolol degradation effect, named Achromobacter Based on this, the present invention successfully cloned the amidase gene from strain YD-4 using a combination of bioinformatics and strain genome sequencing. adgA Online amino acid sequence analysis and homology comparison were performed using BLASTP in NCBI (the UniProtKnowledge Base / SwissProt databases). Among enzymes with identified functions, the closest protein to AdgA was the heterobranched acid lyase family protein YecD (Accession number: P0ADI7.2), with only 31% identity and 86% coverage, indicating that the amidase gene... adgA This is a new gene. The amidase gene. adgA The full-length (from start codon to stop codon) protein is 693 bp, encoding 230 amino acids. The amidase protein AdgA provided by this invention exhibits highly efficient catalytic activity against atenolol, degrading 118 μM of atenolol within 192 hours. Furthermore, AdgA can be used to construct genetically engineered strains of atenolol for the removal of atenolol residues from water and soil, demonstrating significant theoretical and practical value. Attached Figure Description

[0017] Figure 1 The HPLC chromatogram shows the degradation of atenolol by strain YD-4.

[0018] Figure 2 The growth and degradation curve of atenolol by strain YD-4.

[0019] Figure 3 This is a phylogenetic diagram of the 16S rDNA of strain YD-4.

[0020] Figure 4 HPLC (A) and MS / MS (B, C) chromatograms of atenolol degradation by strain YD-4.

[0021] Figure 5 For genes orf 4739、 orf3619 , orf2992 The expression.

[0022] Figure 6 The HPLC chromatogram shows the degradation of atenolol by the Orf4739 enzyme.

[0023] Figure 7 The degradation substrate spectrum of Orf4739. Detailed Implementation

[0024] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0025] Example 1: Isolation and screening of atenolol-degrading strain YD-4: (1) Target pollutant gradient acclimatization method Five g of atenolol-rich environmental sludge was inoculated into 100 mL of basal salt medium (MSM), and atenolol, a β-receptor blocker, was supplemented to a final concentration of 300 μM (achieved by adding 1 mL of 30 mM standard stock solution). Selective enrichment culture was performed in a isothermal shaking culture system (30°C, 180 rpm). The culture was iteratively passaged to fresh MSM medium every 72 h at a 5% inoculum rate, and a stable degradation system was constructed after three generations of iterative passages. At the end of the third generation, a blank control group (CK) was simultaneously set up to correct for biodegradation background values. One mL of the final enrichment solution and the control sample were taken, and the substrate residue was determined by full-spectrum scanning with UV spectrophotometer or quantitative analysis by HPLC. The characteristic pollutant metabolism capacity of the microbial community was confirmed by calculating the reduction rate.

[0026] (2) Whole-cell catalysis verification Morphologically different colonies were picked and inoculated into LB liquid medium for amplification. Cells were collected by centrifugation (5000×g, 5 min), resuspended in sterile MSM buffer, and then inoculated into a bioreactor containing 300 μM atenolol (100 mL MSM). A sterile control (CK) was set up for time-series sampling analysis (sampling at 0-14 h intervals). Samples were purified by ultracentrifugation (10,000×g). g After sterilization with a microporous filter membrane (0.22 μm) for 5 min, the dynamic changes in substrate concentration were quantitatively detected by HPLC.

[0027] A highly efficient degrading bacterium, YD-4, was successfully isolated from atenolol-contaminated environmental samples through targeted acclimation using a sequential subculturing enrichment method. This strain exhibited typical behavior on LB medium. β - Colony characteristics of Proteobacteria (milky white, round and raised, with regular edges), whole-cell catalysis experiments showed that it has specific degradation ability for 300 μM atenolol.

[0028] Using total DNA from strain YD-4 as a template, 16S rRNA was amplified using universal primers 27F and 1492R. Primer pair: 27F (5'-AGAGTTTGATCCTGGCTCAG-3') 1492R (5'-GGTTACCTTGTTACGACTT-3') PCR amplification system:

[0029] PCR amplification procedure:

[0030] The process involves 33 cycles of alteration, annealing, and extension.

[0031] The PCR products were detected by electrophoresis on a 1.0% agarose gel. After gel recovery, the products were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. Molecular biological identification confirmed that the 16S rDNA gene sequence (1395 bp, SEQ ID NO.3) was similar to... Achromobacter piechaudii NBRC 102466 T (AY170848) showed a similarity of 99.78%, and phylogenetic tree analysis further confirmed its classification. Achromobacter Genus (Bootstrap=98%). Based on both phenotypic and genotypic characteristics, this strain is tentatively classified as... Achromobacter sp. YD-4 ( Figure 2 ).

[0032] Example 2: Atenolol detection method: (1) Ultraviolet spectral characteristics analysis First, the high-concentration stock solution of atenolol standard (30 mM) was serially diluted with ultrapure water to the working concentration (300 μM). One mL of the test solution was then pipetted into a quartz cuvette, and a UV-Vis spectrophotometer was used to perform a spectral scan in the 200-400 nm band. The maximum absorption wavelength of the compound was determined using characteristic absorption peak identification technology; this parameter will serve as the detection wavelength benchmark for subsequent quantitative chromatographic analysis.

[0033] (2) Chromatographic detection method a. Sample pretreatment: The test solution was purified by high-speed centrifugation (10,000 r / min, 5 min), and then purified by microporous membrane filtration system (0.22 μm pore size). b. Chromatographic conditions and parameters: Separation system: Mobile phase: A buffer system (5 mM ammonium acetate, pH 5.5) and an organic phase (acetonitrile) were mixed at a volume ratio of 9:1. Column temperature: 25℃ constant temperature control Flow rate: 1.0 mL / min isocratic elution Detection system: Ultraviolet detection wavelength: 230 nm (determined based on previous spectral scanning results) Injection volume: 10 μL Degradation characteristics and degradation product analysis of strain YD-4 The growth characteristics and atenolol degradation efficiency of strain YD-4 were determined using a shaker culture method. A 1% inoculum of strain YD-4 was inoculated into 100 mL of LB liquid medium and cultured under constant temperature shaker at 30℃ and 180 rpm until the logarithmic growth phase. The culture was then subjected to a 5000× [temperature missing] [context missing] g Collect bacterial cells by centrifugation for 3 min; resuspend the bacterial cells in MSM liquid medium and wash by centrifugation, repeating the process twice to completely remove residual medium, then resuspend the bacterial cells in MSM liquid medium to OD. 600 The final OD value of the strain was approximately 1.0, and a standardized seed culture was prepared for later use. The seed culture was inoculated into MSM liquid medium containing 600 μM atenolol, and the final OD value of the strain in the system was calculated. 600 The concentration was adjusted to 0.1, and the culture was carried out at 30℃ and 180 rpm in a constant temperature shaker. Samples were taken every 24 h, and the mass concentration changes of each substance in the culture system were determined by LC / MS to analyze the degradation characteristics of atenolol by the strain.

[0034] HPLC chromatograms revealed two compounds (named Compound I and Compound II) during the degradation of atenolol by strain YD-4. The retention times of Compound I and Compound II were 4.71 min and 7.25 min, respectively, consistent with the retention times of the standards atenolol and atenolol acid. Tandem mass spectrometry (MS / MS) analysis provided evidence that the protonated molecular ions of Compound I and Compound II were... m / z 266.16 and m / z 277.15 ( Figure 4 Based on molecular ion analysis, compounds I and II were identified as atenolol (C10-C20). 14 H 22 N2O3, m / z 266.16) and atenolol acid (C 14 H 21 NO4 m / z 277.15).

[0035] The results showed that strain YD-4 could catalyze the conversion of atenolol to atenolol acid (Figure 3 It is predicted that an unidentified amidase will be responsible for this hydrolysis process.

[0036] Example 3: Cloning and functional verification of the atenolamine enzyme gene: The bacterial culture of strain YD-4 was sent to Genewiz Biotechnology for whole-genome draft sequencing using MPS Illumina technology. The results showed that the total genome length of strain YD-4 was 6,338,383 bp, with an average G+C content of 64.02%. 5800 genes were predicted, with an average length of 982 bp and a coding region G+C content of 64.7%. Subsequently, RAST annotation and BLAST analysis were performed to identify potential amidases leading to atenolol degradation. Since atenolol is an aromatic primary amine compound, and its degradation product has been shown to be atenolol acid, three amidase genes from strain YD-4 were selected. orf2992 , orf3619 and orf4739 As a potential atenolamidinase gene.

[0037] Amplified from strain YD-4 orf2992 , orf3619 and orf4739 Candidate genes were selected and constructed into plasmid pET-29a(+), which was then transformed into *E. coli* BL21(DE3) cells for heterologous expression. The corresponding enzymes were purified from the crude extract using nickel-nitrotriacetic acid affinity chromatography. Figure 5 As shown, the purified proteins of Orf2992, Orf3619, and Orf4739 exhibited single bands on SDS-PAGE, with molecular weights consistent with their theoretical molecular weights. Enzymatic assays of the three amidases were performed using 300 μM atenolol as a substrate under aerobic conditions in Tris·HCl buffer. The results showed that only purified Orf4739 hydrolyzed 147.3 μM atenolol after 30 minutes of incubation, accumulating atenolol acid. Orf2992 and Orf3619 did not exhibit atenolol hydrolytic activity. Figure 6 ).therefore, orf4739 Proved to be Achromobacter The amidase gene responsible for atenolol hydrolysis in strain sp. YD-4 is tentatively named... adgA (Atenolol degrading gene A). Amideases are classified into primary, secondary, and tertiary types based on the different types of amide bonds they catalyze, and these types cannot adapt to other substrate types. Furthermore, the amidease described in this invention exhibits substrate specificity for atenolol and can catalyze atenolol and its two chiral isomers, but cannot catalyze acetamide, phenolazine-1-carboxamide, or 4-hydroxy- N -Methylphenylacetamide and N ,N amide bonds in compounds such as -dimethyl-2-phenylacetamide Figure 7 This indicates that the amidase protein AdgA has specific substrate specificity.

Claims

1. An amidase gene adgA Its characteristics are, The nucleotide sequence is SEQ ID NO.

1.

2. The amidase gene according to claim 1 adgA The encoded protein AdgA is characterized by, The amino acid sequence is SEQ ID NO.

2.

3. Contains the amidase gene as described in claim 1 adgA Recombinant expression vectors.

4. The recombinant expression vector according to claim 3, characterized in that, It contains the amidase gene of claim 1. adgA The plasmid obtained by inserting between the NdeI and XhoI sites of pET-29a(+).

5. Containing the amidase gene as described in claim 1 adgA Genetically engineered bacteria.

6. The genetically engineered bacterium according to claim 5, characterized in that, The genetically engineered bacteria are Escherichia coli BL21(DE3) is the starting strain.

7. The amidase gene of claim 1 adgA Application in the degradation of atenolol.

8. The use of the protein AdgA according to claim 2 in the degradation of atenolol.

9. The use of the recombinant expression vector according to claim 3 in the degradation of atenolol.

10. The application of the genetically engineered bacteria according to claim 5 in the degradation of atenolol.

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