Microcystic toxin degrading enzyme MlrA mutant and application thereof

Abstract

The invention discloses a microcystic toxin degrading enzyme MlrA mutant and application thereof, and belongs to the technical field of gene engineering and enzyme engineering. The microcystic toxin degrading enzyme MlrA mutant is obtained by carrying out single-point mutation on the 179th or 284th amino acid residue of the amino acid sequence of the wild microcystic toxin degrading enzyme MlrA as shown in SEQ ID No.1; the single point mutation is that phenylalanine at the 179th site is mutated into tyrosine, and arginine at the 284th site is mutated into glutamic acid. Two genetically engineered bacteria for expressing the mutant microcystic toxin degrading enzyme MlrA are obtained through a genetic engineering technology, MC-LR is degraded by utilizing the genetically engineered bacteria, the service life of cells is greatly prolonged, and the requirements of current industrial application are met.

Description

Technical Field

[0001] The invention belongs to the technical fields of genetic engineering and enzyme engineering, and particularly relates to a microcystin degrading enzyme MlrA mutant and application thereof. Background Art

[0002] The eutrophication of water bodies is now intensifying, leading to frequent occurrence of cyanobacterial blooms, which can release toxic microcystins (MCs), posing a serious threat to the ecological environment and human health.

[0003] MCs are mainly produced by harmful cyanobacteria such as Microcystis, Anabaena, Nostoc, and Phytosporum. They are hepatotoxic, nephrotoxic, neurotoxic, and potentially carcinogenic. They can inhibit the intracellular phosphatases PP1 and PP2A, causing hyperphosphorylation of intracellular proteins, disrupting cytoskeleton stability, damaging DNA, and causing cell death (Christoffersen K, et al. Microbial activity and bacterial community structure during degradation of microcystins. Aquatic Microbial Ecology, 2002, 27(2):125-136.). MCs are cyclic polypeptides composed of seven different amino acids. Due to their unique cyclic structure, MCs have stable physical and chemical properties and can withstand various natural factors such as high temperature, extreme pH, and light (Rastogi RP, et al. The cyanotoxin-microcystins: current overview. Reviews in Environmental Science and Bio / Technology, 2014, 13(2):215-249.). Nowadays, there are more than 250 substances with unique chemical structures called MCs, the most common of which are MC-LR, MC-RR and MC-YR. Among them, MC-LR has been widely studied due to its wide distribution and high toxicity (Merel S, et al. State of knowledge and concerns on cyanobacterial blooms and cyanotoxins. Environment International, 2013, 59: 303-327.). MCs exposed to the environment enter the body through skin penetration, respiratory inhalation and other pathways, posing a potential threat to the life safety of the organism. It has been reported that MCs have an adverse effect on fish (Li Y, et al. Integration of Multi-Omics, Histological, and Biochemical Analysis Reveals the Toxic Responses of Nile Tilapia Liver to Chronic Microcystin-LR Exposure. Toxins, 2024, 16 (3): 149.), shrimp (Zhang D, et al.Integrated analysis of mRNA and microRNA expression profiles in hepatopancreas of Litopenaeus vannameiunder acute exposure to MC-LR. Frontiers in Genetics, 2023, 14: 1088191.) and other aquatic animals, frogs (Li J, et al. Subchronic Toxicity of Microcystin-LR on Young Frogs (Xenopus laevis) and Their Gut Microbiota. Frontiers in Microbiology, 2022, 13: 895383.) and other amphibians, earthworms (Liu X, et al. Revealing microcystin-LR ecotoxicity to earthworm (Eisenia fetida) at the intestinal cell level. Chemosphere, 2023, 311 (Pt1): 137046.) and other terrestrial invertebrates, and celery (Xiang L, et al. High ecological and human health risks from microcystins in vegetable fields in southern China. Environment International, 2019, 133: 105142.), rice (Jiang J, et al. Bioaccumulation of Microcystin-LR and Induced Physio-Biochemical Changes in Rice (Oryza sativa L.) at Vegetative Stage under Hydroponic Culture Conditions. Toxins, 2024, 16(2): 82.) and other crops. MCs accumulate in edible aquatic organisms and crops, ultimately affecting human health through the food chain. The World Health Organization and my country's National Health Commission have stipulated that the safe limit of MC-LR in drinking water is 1μg / L (World Health O. Cyanobacterial toxins: microcystins. Geneva: World Health Organization, 2020.).

[0004] Given the impact of MCs on human production and life, the study of MCs degradation is of great significance to environmental governance. Existing conventional water treatment methods for removing MCs include flocculation sedimentation, activated carbon adsorption, ultrasonic treatment, flotation, ozone oxidation, photodegradation, etc. (Chang SC, et al. Effective removal of Microcystis aeruginosa and microcystin-LR using nanosilicate platelets. Chemosphere, 2014, 99: 49-55.). Although these methods have a certain effect on the degradation of MCs, their practical application is limited due to the high cost, difficulty in recycling and reuse, complex process, and easy secondary pollution to the environment. In contrast, biodegradation technology has attracted much attention due to its advantages such as efficient degradation ability, environmental friendliness and economy.

[0005] Bacterial degradation of MCs primarily relies on the mlr+ gene cluster first discovered by Bourne et al., which mediates the cascade degradation of MC-LR by encoding the MlrA / B / C / D enzymes (Bourne DG, et al. Characterization of a gene cluster involved in bacterial degradation of the cyanobacterial toxin microcystin LR. Environmental Toxicology, 2001, 16(6): 523-534.). Among them, the microcystin-degrading enzyme MlrA is the most critical enzyme, capable of degrading cyclic MC-LR into linear MC-LR, reducing its toxicity by 2100-fold and generating a substantially non-toxic degradation product (Dziga D, et al. Heterologous expression and characterization of microcystinase. Toxicon, 2012, 59(5): 578-586.).

[0006] It has been reported that about 35 bacterial species carry mlr gene clusters or gene sequences homologous to mlrA, among which Sphingopyxis sp. is the strain with the highest MC-LR degradation efficiency reported so far (Yang F, et al. A complete route for biodegradation of potentially carcinogenic cyanotoxin microcystin-LR in a novel indigenous bacterium. Water Research, 2020, 174: 115638.). However, the reported MlrA has poor stability and poor sustained degradation ability in water bodies, which greatly limits its working efficiency. Therefore, how to improve the stability of MlrA is an urgent problem to be solved in the biodegradation of algal toxins. It has been reported that a mutant of the algal toxin-degrading enzyme MlrA with improved thermal stability was obtained through error-prone PCR and site-directed mutagenesis technology. However, the activity of the constructed genetically engineered bacteria was relatively low and the stability was not significantly improved (Bao Wenna et al. A mutant of an algal toxin-degrading enzyme and its application. Application No. 202211589150.1, 2023.05.16).

[0007] With the development of protein engineering technology and molecular biology, the artificial evolution and modification of enzyme molecules using directed evolution and rational / semi-rational design has become a hot topic in the field of enzyme engineering. Among them, semi-rational design has become a favorable method for enzyme modification, greatly accelerating the evolution of proteins. Summary of the Invention

[0008] In view of the problems existing in the prior art, the object of the present invention is to design and provide a technical solution for a microcystin degrading enzyme MlrA mutant and its application.

[0009] The present invention is specifically implemented by the following technical solutions:

[0010] In a first aspect, the present invention provides a microcystin degrading enzyme MlrA mutant, which is obtained by performing a single point mutation on the 179th or 284th amino acid residue of the amino acid sequence of the wild-type microcystin degrading enzyme MlrA shown in SEQ ID No. 1; the single point mutation is to mutate the phenylalanine at position 179 to tyrosine and the arginine at position 284 to glutamate.

[0011] Furthermore, the microcystin degrading enzyme MlrA mutant is one of the following:

[0012] 1) mutating the phenylalanine at position 179 of the amino acid sequence of the wild-type microcystin-degrading enzyme MlrA shown in SEQ ID No. 1 to tyrosine. The amino acid sequence of the microcystin-degrading enzyme MlrA mutant is shown in SEQ ID No. 2;

[0013] 2) mutating the arginine at position 284 of the amino acid sequence of the wild-type microcystin-degrading enzyme MlrA shown in SEQ ID No. 1 to glutamic acid; the amino acid sequence of the microcystin-degrading enzyme MlrA mutant is shown in SEQ ID No. 3;

[0014] The second aspect of the present invention provides a gene encoding the above-mentioned microcystin degrading enzyme MlrA mutant.

[0015] The third aspect of the present invention provides a recombinant vector containing the above encoding gene.

[0016] The fourth aspect of the present invention provides a host cell containing the above-mentioned recombinant vector.

[0017] A fifth aspect of the present invention provides a use of the above-mentioned microcystin degrading enzyme MlrA mutant in degrading MC-LR.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. Two microcystin degrading enzyme MlrA mutants were obtained through bioinformatics analysis and site-directed mutagenesis. Their amino acid sequences are shown in SEQ ID No. 2 or SEQ ID No. 3, and they are encoded by the nucleotide sequences shown in SEQ ID No. 5 or SEQ ID No. 6.

[0020] 2. Through genetic engineering technology, a new recombinant vector was obtained, which contains a DNA molecule with a nucleotide sequence as shown in SEQ ID No. 5 or SEQ ID No. 6.

[0021] 3. Through genetic engineering technology, a new host cell was obtained, which contains a recombinant vector containing a DNA molecule with a nucleotide sequence as shown in SEQ ID No. 5 or SEQ ID No. 6. The host cell can express the mutant microcystin degrading enzyme MlrA.

[0022] 4. The present invention mutates the phenylalanine at position 179 of the wild-type microcystin degrading enzyme MlrA to tyrosine or the arginine at position 284 to glutamate, thereby greatly improving the thermal stability of the enzyme.

[0023] 5. Through genetic engineering technology, two genetically engineered bacteria expressing the mutant microcystin degrading enzyme MlrA of the present invention were obtained. The use of these genetically engineered bacteria to degrade MC-LR greatly increased the service life of the cells, meeting the requirements of current industrial applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the MC-LR degradation experiment of the microcystin degrading enzyme MlrA with GST tag (GST-M) and MBP tag (MBP-M).

[0025] Figure 2 This is an SDS-PAGE image of the purification of wild-type and mutant microcystin-degrading enzyme MlrA with an MBP tag. In the image, M represents protein molecular weight markers, which, from top to bottom, are 180 kDa, 130 kDa, 100 kDa, 70 kDa, 55 kDa, 40 kDa, 35 kDa, 25 kDa, 15 kDa, and 10 kDa.

[0026] Figure 3 The results show the thermal stability of wild-type and mutant microcystin-degrading enzyme MlrA. DETAILED DESCRIPTION

[0027] In the following embodiments of the present invention, the relevant detection methods are as follows:

[0028] (1) Enzyme activity assay: A certain amount of recombinant enzyme MlrA and a certain amount of MC-LR substrate were mixed evenly with an appropriate amount of 20 mmol / L Tris-HCl (pH 7.4) buffer, and the solution containing the recombinant enzyme was added and mixed evenly to form a degradation system. Each 150 μL degradation system contained 10 μL of recombinant enzyme solution, with a final concentration of 3 μg / mL MC-LR. The blank control did not add recombinant enzyme solution, and was supplemented with an equal volume of 20 mM Tris-HCl (pH 7.4) buffer. The reaction was carried out at 37°C for 2 minutes, and the reaction was terminated by boiling in a water bath for 10 minutes. The supernatant was centrifuged at 12,000 × g for 5 minutes, and the supernatant was passed through a 0.22 μm organic membrane and analyzed by HPLC. Under the above reaction conditions, the MC-LR degradation enzyme activity was defined as 100% relative activity.

[0029] (2) Detection method of MC-LR content: Shimadzu LC-20A liquid chromatography system was used for analysis, and the detection method was as follows: the chromatographic column used was a Titank-C18 column (4.6*250 mm, 5 μm), the mobile phase ratio was methanol: 0.1% (v / v) trifluoroacetic acid aqueous solution = 60:40 (v / v), the flow rate was 0.8 mL / min, the column temperature was 35°C, the ultraviolet detection wavelength was 238 nm, and the injection volume was 20 μL.

[0030] MC-LR, initially at a concentration of 20 μg / mL, was diluted with ultrapure water to create a series of standard solutions with concentrations of 0, 0.4, 1, 2, 4, 10, and 20 μg / mL. The peak areas of the standard MC-LR solutions were measured using the aforementioned chromatographic analysis method, and a calibration curve was constructed comparing MC-LR concentration and peak area. Subsequently, the peak values ​​were substituted into the calibration curve equation to calculate the MC-LR concentration.

[0031] (3) Protein concentration determination method: The protein concentration was determined using a modified Bradford protein concentration determination kit.

[0032] The present invention will be further described below with reference to the following examples. It should be noted that these examples are only used to explain the present invention, rather than to limit the scope of the present invention.

[0033] Example 1: Synthesis of microcystin degrading enzyme genes and construction of different genetically engineered bacteria

[0034] Based on the mlrA gene from Sphingobacterium sphingomonas YF1 (GenBank accession number KY491637.1), Nanjing Qingke Biotechnology Co., Ltd. was commissioned to synthesize the gene sequence shown in SEQ ID No. 4. The gene was then inserted into the pGEX-4T-1 and pMAL-c5X plasmids, respectively, and finally constructed in Escherichia coli BL21(DE3). For the pGEX-4T-1 vector, a BamHI restriction enzyme site was designed at the N-terminus of the mlrA gene, and an XhoI restriction enzyme site was designed at its C-terminus. The resulting recombinant strain was named E. coli-pGEX-4T-1-mlrA. The expressed mlrA protein was fused to a GST tag at its N-terminus, denoted by "GST-M." For the vector pMAL-c5X, a BamH I restriction enzyme site was designed at the N-terminus of the mlrA gene, and a Hind III restriction enzyme site was designed at the C-terminus. The obtained recombinant bacteria were named E. coli-pMAL-c5X-mlrA. The final expressed protein had an MBP fusion tag at the N-terminus, and the protein was represented by "MBP-M".

[0035] Example 2: Comparison of enzyme activities of different genetically engineered bacteria

[0036] The recombinant E. coli-pGEX-4T-1-mlrA and the recombinant E. coli-pMAL-c5X-mlrA in Example 1 were inoculated into a culture medium containing 100 μg / mL ampicillin. When the bacterial concentration (OD 600) reached 0.6, 0.05mM isopropylthio-β-D-galactoside was added and cultured at 16℃ with shaking for 20h. The cells were collected by centrifugation at 4000rpm for 10min at 4℃ and suspended with an appropriate amount of 20mmol / L Tris-HCl (pH7.4) to make the concentrations of the two engineered bacteria consistent. Under the same conditions, the cells were broken by ultrasonication in an ice bath and centrifuged at 12000rpm for 20min at 4℃. Equal amounts of the supernatant of the two engineered bacteria were taken for MC-LR degradation experiments. The results are shown in Figure 2. Figure 1 As shown, when the initial MC-LR concentration was 3 μg / mL, the MBP-tagged recombinant enzyme MBP-M degraded MC-LR by 88.13% within 1 minute. In contrast, the GST-tagged recombinant enzyme GST-M only degraded MC-LR by 25.41% within 1 minute. Therefore, MBP-M exhibits significantly greater activity than GST-M, approximately 3.47 times that of GST-M. Subsequent modifications will target the more active MBP-M.

[0037] Example 3: Bioinformatics analysis of potential stabilizing sites

[0038] Using Discovery Studio software, all amino acids of the MlrA protein (except the MBP tag part) were scanned for alanine at 20°C, 37°C, 50°C, 70°C, and 90°C, and amino acid residues that were unstable under different temperature conditions were selected. Then, saturation mutations were performed on the unstable sites at 37°C, 50°C, and 70°C, and potential stable mutants were screened out through mutation energy analysis. In addition, the microcystin degrading enzyme MlrA was molecularly docked with the substrate MC-LR to screen amino acid residues that were unstable in the interaction between the protein and the substrate, and mutated them into amino acids with stable interaction. The mutants screened above and the wild-type MlrA were subjected to molecular dynamics simulation analysis, and the specific conditions were: the Amber force field was set to a size of TIP3P water box model and add Na + and Cl - To neutralize the system's charge, a protein system was formed. Energy minimization of the protein system was performed, and molecular dynamics simulations were performed on the Amber software platform at a constant temperature of 310 K and a constant pressure of 1 bar for 100 ns. Based on the root mean square deviations of the wild-type and mutant forms, two stable mutants, F179Y and R284E, were obtained.

[0039] Example 4: Construction of mutants of the microcystin degrading enzyme MlrA

[0040] Nanjing Qingke Biotechnology Co., Ltd. was commissioned to synthesize the two stable mutants screened in Example 3, insert them into the pMAL-c5X plasmid, and finally construct them on Escherichia coli BL21 (DE3). Two mutant strains were thus obtained, wherein the codon (TTT) encoding phenylalanine at position 179 in the mutant F179Y mutated to a codon (TAT) encoding tyrosine, and its nucleotide sequence is shown in SEQ ID No.5, and its amino acid sequence is shown in SEQ ID No.2. The codon (CGC) encoding arginine at position 284 in the mutant R284E mutated to a codon (GAA) encoding glutamic acid, and its nucleotide sequence is shown in SEQ ID No.6, and its amino acid sequence is shown in SEQ ID No.3. The amino acid sequence of the wild-type microcystin degrading enzyme MlrA is shown in SEQ ID No.1.

[0041] Example 5: Expression and purification of wild-type and mutant microcystin degrading enzyme MlrA

[0042] The recombinant E. coli-pMAL-c5X-mlrA in Example 1 and the two single mutant strains F179Y and R284E in Example 4 were inoculated into LB medium containing 100 μg / mL ampicillin. When the bacterial concentration (OD 600 ) reaches 0.6, add 0.05mM isopropylthio-β-D-galactoside and culture with shaking at 16℃ for 20h. Collect the bacteria by centrifugation at 4000rpm for 10min at 4℃, rinse three times with physiological saline, add lysis buffer (20mmol / L Tris-HCl, pH7.4) at a ratio of 5mL per gram of bacteria, pre-ultrasonicate on ice to break the bacteria, centrifuge at 12000rpm at 4℃ for 20min, and take the supernatant. Add ammonium sulfate to the supernatant in small amounts and multiple times to mix and dissolve, so that the final concentration of ammonium sulfate is 0.25M, and let it stand on ice for 30min, then centrifuge at 12000rpm at 4℃ for 20min. Take the supernatant and continue to add ammonium sulfate to the supernatant in small amounts and multiple times to mix and dissolve, so that the final concentration of ammonium sulfate is 1.5M. Then continue to stand on ice for 30 minutes, centrifuge at 4°C, 12000rpm, for 20 minutes, and redissolve the precipitate with an appropriate amount of 20mmol / LTris-HCl (pH 7.4) buffer until it is completely dissolved. Use a 10kDa dialysis bag to dialyze and desalt, load the desalted protein onto a DEAE anion exchange chromatography column, and elute the protein with Tris-HCl buffer with 0-400mM NaCl, pour the target protein into an ultrafiltration tube, centrifuge and concentrate, add 20mmol / L Tris-HCl (pH 7.4) buffer to the concentrate, and concentrate multiple times until the salt ions are removed. Add glycerol to a final concentration of 20% to the protein after desalting for storage. The results of 10% SDS-PAGE are as follows Figure 2 As shown, lane 1 is the wild-type microcystin degrading enzyme MlrA, lane 2 is the single mutant protein F179Y, and lane 3 is the single mutant protein R284E. After purification, the bands of the three proteins are single.

[0043] Example 6: Activity and thermal stability analysis of wild-type and mutant microcystin degrading enzyme MlrA

[0044] The enzyme activities of the wild-type MlrA, single mutant F179Y, and single mutant R284E purified in Example 5 were determined, and the wild-type and mutant MlrA enzymes were placed under different temperature conditions (20-70°C, with a sample taken every 10°C) for 10 minutes, and then immediately placed on ice to cool for 10 minutes. The control group was not incubated and was directly cooled on ice for 10 minutes. The activity of the wild-type and mutant MlrA enzymes was determined according to the above-mentioned detection method, and the residual enzyme activity was calculated based on the control group. The enzyme activity of the control group in degrading MC-LR was set to 100%. Under the temperature condition of 55°C, after incubation for different times (sampling every 20 minutes, a total of 2 hours), it was immediately placed on ice to cool for 10 minutes (the control group was not incubated and was directly cooled on ice for 10 minutes), and the residual enzyme activity of the recombinant enzyme after incubation at 55°C for different times was determined according to the above-mentioned detection method. The measured residual enzyme activity data were fitted to the origin2021Boltzmann sigmoidal equation to obtain the 10-min half-inactivation temperature of the recombinant enzyme ( Each experiment was repeated for three parallel samples.

[0045] Wild type and different mutants Value Figure 3 Shown in A, wild type The value was 65.507℃. Compared with the wild type, the F179Y and R284E mutants The values ​​increased by 0.199℃ and 0.397℃ respectively. Figure 3B shows the half-life of the wild type and mutants at 55°C. The half-life of the wild-type enzyme is 37.117 min, and the half-lives of the F179Y and R284E mutants are 74.474 min and 87.207 min, which are 2.006 times and 2.35 times that of the wild type, respectively. The results reported (application number 202211589150.1) show that the obtained mutant has a residual activity of about 83.2% after incubation at 50°C for 30 min, while the R284E mutant constructed by the present invention still has a residual activity of 99.1% after incubation at 50°C for 30 min, and a residual activity of 84.29% after incubation at 55°C for 30 min, showing higher temperature stability than the previously reported microcystin degrading enzyme MlrA mutant. In addition, the enzyme activity of the F179Y mutant is comparable to that of the wild type, and the enzyme activity of the R284E mutant is slightly higher than that of the wild type.

[0046] The above results show that the temperature stability of the mutant microcystin-degrading enzyme MlrA is significantly improved compared with the wild-type microcystin-degrading enzyme MlrA, and its high enzyme activity is maintained.

[0047] The sequence involved in the present invention is as follows:

[0048] SEQ ID NO.1:

[0049]

[0050] SEQ ID NO.2:

[0051]

[0052]

[0053] SEQ ID NO.3:

[0054]

[0055] SEQ ID NO.4:

[0056]

[0057]

[0058] SEQ ID NO.5:

[0059]

[0060]

[0061] SEQ ID NO.6:

[0062]

[0063]

Claims

1. A microcystin degrading enzyme MlrA mutant, characterized in that: The microcystin degrading enzyme MlrA mutant is obtained by performing a single-point mutation on the 179th or 284th amino acid residue of the amino acid sequence of the wild-type microcystin degrading enzyme MlrA shown in SEQ ID No. 1; the single-point mutation is to mutate the phenylalanine at position 179 to tyrosine and the arginine at position 284 to glutamic acid.

2. The microcystin degrading enzyme MlrA mutant according to claim 1, characterized in that: The microcystin degrading enzyme MlrA mutant is one of the following: 1) mutating the phenylalanine at position 179 of the amino acid sequence of the wild-type microcystin-degrading enzyme MlrA shown in SEQ ID No. 1 to tyrosine. The amino acid sequence of the microcystin-degrading enzyme MlrA mutant is shown in SEQ ID No. 2; 2) The arginine at position 284 of the amino acid sequence of the wild-type microcystin degrading enzyme MlrA shown in SEQ ID No. 1 is mutated to glutamic acid. The amino acid sequence of the microcystin degrading enzyme MlrA mutant is shown in SEQ ID No.

3.

3. A gene encoding the microcystin degrading enzyme MlrA mutant according to claim 1.

4. A recombinant vector containing the coding gene according to claim 3. A host cell containing the recombinant vector according to claim 4.

6. Use of the microcystin degrading enzyme MlrA mutant according to claim 1 in degrading MC-LR.

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