Use of an enzyme in the degradation of microcystins

Abstract

The application discloses application of an enzyme in degrading microcystin. The amino acid sequence of the enzyme comprises: (1) a sequence shown in SEQ ID NO. 1, or (2) an amino acid sequence obtained by substituting, deleting or adding one or at least two amino acid residues from the sequence shown in (1) and being functionally identical or similar to the sequence shown in (1), or (3) an amino acid sequence having at least 90% sequence homology with the sequence shown in (1) or (2) and being functionally identical or similar to the sequence shown in (1). The application firstly finds that an enzyme derived from Sphingosinicellamicrocystinivorans has the function of degrading microcystin, and catalyzes the degradation of microcystin, so that the enzyme is expected to be applied in degrading microcystin, and the degradation rate is more than 90%.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the application of an enzyme in the degradation of microcystin. Background Technology

[0002] Rapid algal blooms in eutrophic waters can lead to algal blooms, releasing various toxins into the water after the rupture of numerous cyanobacterial cells. Among these, microcystin (MC) is the most common and harmful cyanobacterial toxin, causing severe damage to aquatic ecosystems. Of its subtypes, microcystin-LR (MC-LR) is the most toxic and has the highest pollution levels (see: Gu, S. et al. (2022) Microcystin-LR in Primary Liver Cancers: An Overview. Toxins (Basel) 14.10.3390 / toxins14100715). The concentration of MC-LR in drinking water is generally limited to no more than 1 μg / L, and the control and elimination of MC-LR is a global concern.

[0003] Currently used methods for treating microcystin-LR—physicochemical detoxification techniques—have drawbacks such as low specificity, significant resource waste, environmental unfriendliness, and high safety risks, making it difficult to meet the actual needs of water treatment (see: Koshigoe, ASH et al. (2022) Effect of three commercial algaecides on cyanobacteria and microcystin-LR: implications for drinking water treatment using activated carbon. Environ Sci Pollut Res Int. 10.1007 / s11356-022-23281-5). Finding environmentally friendly methods to degrade MC-LR has become a current research hotspot. Microbial biocatalysis has attracted widespread attention in recent years due to its advantages of mild reaction conditions, environmental friendliness, and sustainability. Currently, pure strains of *Sphingomonas*, *Pseudomonas aeruginosa*, and *Burkholderia* have been reported to degrade microcystin. However, the reduction effect of these strains on microcystin is very limited, and detoxification based on microorganisms has problems such as the introduction of toxic microbial metabolites and decreased activity due to mutations. Enzyme-based treatment processes can further improve these problems (see: Manco, G. et al. (2018) Enzymatic detoxification: a sustainable means of degrading toxic organophosphate pesticides and chemical warfare nerve agents. Journal of Chemical Technology & Biotechnology 93, 2064-2082.10.1002 / jctb.5603).

[0004] In conclusion, there is an urgent need to discover enzymes that can effectively degrade microcystin, so as to provide a foundation for the development of enzyme-based microcystin degradation processes. Summary of the Invention

[0005] In response to the shortcomings of existing technologies and practical needs, this invention provides an application of an enzyme in the degradation of microcystin. This invention discovers a novel enzyme for degrading microcystin, which is expected to be applied in water treatment and the prevention and control of harmful algal blooms.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides the application of an enzyme in the degradation of microcystin, wherein the amino acid sequence of the enzyme comprises:

[0008] (1) The sequence shown in SEQ ID NO.1, or,

[0009] (2) An amino acid sequence obtained by substituting, deleting, or adding one or at least two amino acid residues to the sequence described in (1), and having the same or similar function to the sequence described in (1), or,

[0010] (3) An amino acid sequence that has at least 90% sequence homology with the sequence described in (1) or (2) and has the same or similar function as the sequence described in (1).

[0011] This invention is the first to discover an enzyme derived from Sphingosinicella microcystinivorans (amino acid sequence as shown in SEQ ID NO.1) that has the function of degrading microcystin (structural formula as shown in Formula I), catalyzing the degradation of microcystin, and thus has the potential to be applied to the degradation of microcystin, such as water treatment, algal bloom control, etc.

[0012]

[0013] It is understood that this application has discovered that an enzyme derived from *Sphingosinicella microcystinivorans* (amino acid sequence as shown in SEQ ID NO.1) has the function of degrading microcystin. Therefore, enzymes in *Sphingosinicella microcystinivorans* or similar strains with sequences and functions identical or similar to those shown in SEQ ID NO.1 are also expected to have the function of degrading microcystin. In addition, enzymes obtained by modifying the sequence shown in SEQ ID NO.1 by substituting, deleting, or adding one or more amino acid residues using common techniques in the art, while having the same or similar functions as the original protein, are also expected to have the function of degrading microcystin.

[0014] SEQ ID NO.1:

[0015] MREFVRQRPLVSFYVLAILIALAANVLRAMDPTPLGPMFKMLQETHAHLNIVTAIRSTFDYPTAYTFLLFPAAPMLAALIVTGIGYGRAGFRELLSRCAPWRDPVSWRQGVTVIAVCFLVFFALTGMMWVQTYLYAPSGTLDRAFLRYGSDPLSIYAMLAASLLISPG PLLEELGWRGFALPQLLKKFDPLTAAVILGTMWWAWHLPRDLPAMFSGEPGALWGVIVKQFVIAPGMIASTIIAVFVCNKLGGSLWGGLLTHAIHNELGVNVMAEWSPAAAGLGWRPWDFIEFAVAIGLVLICGRSLGAASPDNARLAWGNVPPKLPGGATDKSGANA.

[0016] In a second aspect, the present invention provides a method for degrading microcystin, the method comprising:

[0017] The enzyme described in the first aspect is mixed with a sample containing microcystin degrading agent to achieve microcystin degradation.

[0018] In this invention, after co-incubating the enzyme described in the first aspect with microcystin, a significant reduction in the concentration of microcystin can be detected, and the final degradation rate of microcystin can reach more than 90%.

[0019] Thirdly, the present invention provides the application of the enzyme described in the first aspect in the preparation of microcystin degradation products.

[0020] Fourthly, the present invention provides a microcystin biodegradable agent, wherein the biodegradable agent contains the enzyme described in the first aspect.

[0021] Based on the discovered ability to degrade microcystin, microcystin degradation products can be further prepared.

[0022] Preferably, the biodegradable agent further includes any one or a combination of at least two of a carrier, a preservative, or a protein protectant.

[0023] It is understood that carriers, preservatives, or protein protectants commonly used in the field for biodegradable agents are also applicable to this invention.

[0024] Preferably, the carrier comprises a physiologically acceptable compatible carrier.

[0025] Preferably, the physiologically acceptable carrier includes any one or a combination of at least two of rice husks, rice bran, wheat bran, bentonite, or zeolite powder.

[0026] Preferably, the preservative includes any one or a combination of at least two of potassium sorbate, ethylparaben, methylparaben, or antibiotics.

[0027] Preferably, the antibiotic includes any one or a combination of two of penicillin, gentamicin, or vancomycin.

[0028] Preferably, the protein protectant comprises any one or a combination of at least two of bovine serum albumin, mannitol, glycerol, butylene glycol, sodium chloride, or sodium benzoate.

[0029] Fifthly, the present invention provides the application of the enzyme described in the first aspect in the prevention and control of harmful algal blooms.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] This invention is the first to discover an enzyme derived from Sphingosinicella microcystinivorans (amino acid sequence shown in SEQ ID NO.1) that has the function of degrading microcystin toxins and effectively catalyzes the degradation of microcystin toxins, thus it is expected to be applied to the degradation of microcystin toxins, such as water treatment, algal bloom control, etc. Attached Figure Description

[0032] Figure 1A This is a quantitative detection graph of MC-LR toxin levels before enzyme catalysis in Example 2;

[0033] Figure 1B This is a quantitative detection graph of MC-LR toxin levels after enzyme catalysis in Example 2;

[0034] Figure 2 This is a graph showing the MC-LR toxin content in the enzyme-catalyzed combination control group after the reaction in Example 2;

[0035] Figure 3 The graph shows the degradation rate of MC-LR toxins in wastewater. Detailed Implementation

[0036] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0037] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0038] Example 1

[0039] This embodiment demonstrates the expression of an enzyme derived from Sphingosinicella microcystinivorans (amino acid sequence shown in SEQ ID NO.1).

[0040] In this experiment, the enzyme gene sequence was synthesized by Shanghai Sangon Biotech Co., Ltd., and the D2P expression plasmid and cell-free expression reaction system (refer to Zhang, K. et al. (2021) Systemic Expression, Purification, and Initial Structural Characterization of Bacteriophage T4 Proteins Without Known Structure Homologs. Front Microbiol 12, 674415.10.3389 / fmicb.2021.674415) were obtained from Shanghai Kangma Biotechnology Co., Ltd. After synthesizing the enzyme gene sequence, it was ligated into the protein expression vector plasmid using homologous recombination technology. The recombinant plasmid containing the target gene was used as a reaction template and added to the cell-free reaction system. The reaction was carried out at 37℃ for 3 hours to obtain the target protein. The reaction volume was 1 mL, and the final concentration of the reaction template plasmid was 1 ng / μL. A blank control was set up, i.e., the reaction solution without the template plasmid, to eliminate the influence of enzymes and reaction conditions present in the reaction system.

[0041] SEQ ID NO.2:

[0042]

[0043] Example 2

[0044] This embodiment demonstrates the degradation test of microcystin-releasing enzyme (MC-LR).

[0045] The enzyme-containing reaction system from Example 1 after the reaction was completed was used as the experimental group, and the reaction system from Example 1 without the template plasmid was used as the blank control. After the reaction was completed, the supernatant of the reaction system was collected by low-speed centrifugation into a new centrifuge tube, and MC-LR (final reaction concentration of 2 μg / mL) was added and incubated at 37°C for 3 hours. After incubation, the mixture was centrifuged at high speed. The supernatant was then collected, an equal volume of methanol was added, and the mixture was vigorously shaken and centrifuged again at high speed (12,000 rpm for 5 min). The supernatant was filtered through a 0.22 μm organic filter membrane, and the content of MC-LR before and after the reaction was detected.

[0046] Instrument parameters: The data acquisition instrument system mainly includes ultra-high performance liquid chromatography (Vanquish, UPLC, Thermo, USA) and high-resolution mass spectrometry (Q Exactive, Thermo, USA) (https: / / www.thermofisher.com / ); Column: Waters HSS T3 (50*2.1mm, 1.8μm); Mobile phase: Phase A was ultrapure water (containing 0.1% acetic acid), Phase B was acetonitrile (containing 0.1% acetic acid); Flow rate: 0.3mL / min; Column temperature: 40℃; Injection volume: 2μL; Elution gradient: 0.0min water / acetonitrile (90:10, V / V), 2.0min water / acetonitrile (90:10, V / V), 6.0min water / acetonitrile (40:60, V / V), 8.0min water / acetonitrile (40:60, V / V), 8.1min water / acetonitrile (90:10, V / V), 12.0min water / acetonitrile (90:10, V / V). Throughout the analysis, the sample was placed in an autosampler at 4℃. To avoid the influence of instrument signal fluctuations, continuous analysis of samples was performed in a randomized order. Mass spectrometry data were acquired using a QExactive high-resolution mass spectrometry system from Thermo Fisher Scientific (USA). Electrospray ionization (ESI) conditions were as follows: sheath gas 40 alb; auxiliary gas 10 alb; ion spray voltage -2800 V; temperature 350 °C; ion transmission tube temperature 320 °C. The scanning mode was full scan (Full MS); the scanning method was negative ion. The primary scan range (m / z range) was 100-1500.

[0047] Qualitative and quantitative analysis of metabolites: Mass spectrometry data were processed using TraceFinder software. The concentration of each component in the liquid sample (μg / mL) = C*F / 1000. Where C is the instrument reading concentration in ng / mL; V is the sample extraction volume in mL; M is the total sample weight in mg; and F is the dilution factor.

[0048] The candidate enzyme was derived from *Sphingosinicella microcystinivorans*, an aerobic, mesophilic, Gram-negative bacterium isolated from a eutrophic lake. The enzyme is 336 amino acids in length. Under the conditions described in this example, the quantitative detection results of MC-LR toxin levels before and after enzyme catalysis are as follows: Figure 1A and 1B As shown, the MC-LR toxin content results in the control group and enzyme-catalyzed group after the reaction are as follows: Figure 2 As shown, only the enzyme-catalyzed group MC-LR was effectively degraded by 92.16%, indicating that the enzyme discovered in this invention has good degradation efficiency.

[0049] Example 3

[0050] In this embodiment, the enzyme-containing reaction system after the reaction in Example 1 was completed was used as the experimental group, and the reaction system in Example 1 without the addition of template plasmid was used as the blank control to carry out the degradation experiment of toxins in polluted water samples.

[0051] The specific experimental procedure includes:

[0052] Microcystin standard was added to tap water at a final concentration of 1000 ng / g. 500 μL of the enzyme-containing reaction system was mixed with 5 mL of water sample containing microcystin. The same mixing operation was performed on the reaction system without template plasmid as a control. After incubation at 37°C for 3 hours, 500 μL of methanol was added to stop the reaction.

[0053] Toxin detection and degradation rate calculation are based on Example 2.

[0054] The results are as follows Figure 3 As shown, the toxin degradation rate was 63.83%, indicating that the enzyme discovered in this invention can directly degrade microcystin in polluted water samples when used alone, and has broad application prospects in the development of biodegradable agents.

[0055] In summary, this invention is the first to discover an enzyme derived from Sphingosinicella microcystinivorans (amino acid sequence shown in SEQ ID NO.1) that has the function of degrading microcystin toxins, catalyzing the degradation of microcystin toxins into non-toxic substances, and thus has the potential to be applied to the degradation of microcystin toxins, with a degradation rate of over 90%.

[0056] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. The application of an enzyme in the degradation of microcystin, characterized in that, The amino acid sequence of the enzyme is as follows: The sequence shown in SEQ ID NO.

1.

2. A method for degrading microcystin, characterized in that, The method includes: The enzyme described in claim 1 is mixed with a sample containing microcystin to achieve the degradation of microcystin.

3. The application of an enzyme in the preparation of microcystin biodegradable agents, characterized in that, The biodegrading agent contains the enzyme according to claim 1; the amino acid sequence of the enzyme is shown in SEQ ID NO.

1.

4. The application according to claim 3, characterized in that, The biodegradable agent also includes any one or a combination of at least two of the following: a carrier, a preservative, or a protein protectant.

5. The application according to claim 4, characterized in that, The carriers include physiologically acceptable compatible carriers.

6. The application according to claim 5, characterized in that, Physiologically acceptable carriers include any one or a combination of at least two of rice husks, rice bran, wheat bran, bentonite, or zeolite powder.

7. The application according to claim 4, characterized in that, The preservative includes any one or a combination of at least two of potassium sorbate, ethylparaben, or methylparaben.

8. The application according to claim 4, characterized in that, The protein protectant includes any one or a combination of at least two of bovine serum albumin, mannitol, glycerol, butylene glycol, sodium chloride, or sodium benzoate.

9. The application of the enzyme according to claim 1 in the prevention and control of harmful algal blooms.

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