Novel biological enzyme for effectively degrading algal toxin, gene, expression vector, expression strain, biosynthesis method and application

Abstract

The invention provides a novel biological enzyme capable of effectively degrading algal toxin, a gene, an expression vector, an expression strain, a biosynthesis method and application, and relates to the technical field of biology, the field of environmental governance and the field of food safety. The invention specifically discloses an amino acid sequence and a gene sequence of a novel biological enzyme capable of effectively degrading algal toxin, a preparation method of the biological enzyme and application of the biological enzyme in the aspects of algal toxin degradation and blue-green algae treatment. The invention further discloses application of the biological enzyme to treatment of a mixture containing algal toxin. Therefore, the novel biological enzyme for degrading the algal toxin can be widely applied to agriculture, fishery (including aquaculture and the like) production, river and lake treatment and ecological restoration, detoxification treatment of algal source products polluted by the algal toxin, and detoxification treatment of feeds, fertilizers and other products polluted by the algal toxin; the method is applied to water treatment of rivers, lakes, reservoirs, water sources and the like with algal toxin pollution.

Description

Technical Field

[0001] The present application relates to the technical fields of synthetic biology of bioenzymes, environmental protection, and agricultural and fishery production. Specifically, it relates to a novel bioenzyme, gene, expression vector, expression strain, biosynthesis method, and application for effectively degrading microcystins. Background Art

[0002] In recent decades, with the intensification of global warming and human activities, a large amount of industrial wastewater, domestic sewage, etc. have been discharged, and the eutrophication of lake water bodies has become increasingly serious. The frequency, intensity, and duration of cyanobacterial blooms have been increasing, resulting in serious water quality damage worldwide and frequent outbreaks of cyanobacterial bloom events. The ecological crisis and public health threats caused by harmful cyanobacterial blooms have become environmental problems of global concern. Once a cyanobacterial bloom breaks out, a green blanket will cover the water surface, blocking sunlight and causing almost all organisms in the water to die, seriously damaging the ecosystem and bringing huge economic losses to agricultural, fishery production, and tourism development. Most seriously, the microcystins released after the overgrowth and death of cyanobacteria are the most potent carcinogens, which can easily lead to liver cancer, neurological diseases, etc. Among them, microcystins (MCs) are the most widely detected toxins at present, severely poisoning a variety of animals and plants, and even enriching and transferring through the drinking water supply and / or food chain, ultimately endangering human health (Huisman et al., Nat. Rev. Microbiol. 2018, 16, 471-483; Xiao et al., Biol. Rev. 2018, 93, 1399-1420; Smucker et al., Global Change Biology, 2021, 27, 2507-2519). Microcystin pollution has led to many public health incidents. Reports show that the incidence of primary liver cancer in residents in the eastern region of China is related to MCs in drinking water (Ueno et al., 1996; Chen et al., 2019). The World Health Organization (WHO) has set a limit standard for the content of microcystins in drinking water of 1 μg / L. It is reported that in summer, the MCs concentration in some lakes in China reaches about 15.6 μg / L, and the MCs concentration in some lakes in the United States is also 10-500 μg / L. Therefore, it is urgent to find effective and safe methods to effectively degrade microcystins to protect the safety of water environment and water ecosystem.

[0003] The existing methods for treating cyanobacterial blooms mainly include three categories: physical methods, chemical methods, and biological methods. Physical methods mainly refer to the use of equipment such as dredging and high-pressure algae removal to remove algal cells. This method is simple to operate, but has slow results and high costs. Chemical methods mainly include the use of methods such as copper sulfate and hydrogen peroxide, which have quick results but are prone to causing secondary pollution. Biological methods mainly include the use of plants, fish, etc. to prevent and control cyanobacterial outbreaks, but they have high costs, long cycles, and slow results. At the same time, these methods for treating cyanobacteria can alleviate cyanobacterial blooms to a certain extent, but they cannot remove the harmful effects of toxic secondary metabolites produced by cyanobacteria, such as microcystins. The methods for removing microcystins mainly include physical methods such as activated carbon adsorption and flocculation, but these methods transfer rather than detoxify MCs. In addition, chemical methods such as ozone and permanganate are also used to treat microcystins, but these methods have poor selectivity and are prone to causing secondary pollution. Some natural strains that can effectively degrade microcystins have been identified from eutrophic lakes and reservoirs, but their degradation efficiency is relatively low, and their application potential is limited in waters with high microcystin levels.

[0004] The degradation of microcystins by bioenzymatic methods has attracted much attention due to its characteristics such as targeting, high efficiency, safety, and no secondary pollution (Dexter, et al. Water Research, 2021, 189, 116646). It has been found that the microcystin-degrading enzyme (Microcystinase, MlrA) can degrade cyclic and highly toxic MCs into low-toxic or non-toxic linear MCs (Liu et al., Environ. Sci. Technol., 2020, 54, 8811). As the most important primary enzyme in the microcystin degradation pathway, MlrA can convert cyclic microcystins into linear microcystins, greatly reducing the toxicity of microcystins. It has been found that the highly efficient bioenzyme described in this application can further degrade linear microcystins into almost non-toxic products. However, the yield and enzyme activity of microcystin-degrading enzymes from natural strains are ineffective in the face of high concentrations of linear microcystins. Currently, relatively little research has been done on highly efficient bioenzymes. Among them, Wang et al. attempted heterologous expression of bioenzymes encoding microcystin-degrading enzymes in 2020, but the protein yield in the optimized crude enzyme solution was only 41.175 mg / L. Therefore, obtaining bioenzymes that can effectively degrade microcystins is an effective way to effectively solve the problems of microcystin pollution, cyanobacterial bloom pollution, and the safety of the water ecosystem in the water environment. Summary of the Invention

[0005] The purpose of this application is to provide a novel bioenzyme, gene, expression vector, expression strain, biosynthetic method, and application for effectively degrading microcystins, specifically to solve the problems of low yield, low use efficiency, and secondary pollution of bioenzymes for degrading microcystins in current cyanobacteria treatment.

[0006] To solve the above technical problems, the technical solutions adopted in this application are:

[0007] In a first aspect, the present application provides a novel bioenzyme for effectively degrading microcystin, and the bioenzyme is one of the following a and b;

[0008] The a is a novel bioenzyme (XS25001, XS25002, XS25003) composed of the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3;

[0009] The b is a bioenzyme derived from a, in which the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 is substituted, deleted or added with one or several amino acids and the enzyme activity remains unchanged, or a bioenzyme shown by an amino acid sequence with a tag connected to the amino terminus and / or carboxyl terminus of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

[0010] In a second aspect, the present application provides a gene capable of encoding the above XS25001-XS25003.

[0011] In a third aspect, the present application provides an expression vector containing the above gene.

[0012] In a fourth aspect, the present application provides an expression strain containing the above expression vector. The expression strain is one of Escherichia coli, Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, Lactobacillus plantarum, Lactococcus lactis and Aspergillus niger.

[0013] In a fifth aspect, the present application provides a method for preparing the novel bioenzymes XS25001-XS25003, and the method includes: (1) culturing the above expression strain to induce heterologous expression of the encoding genes of XS25001-XS25003; (2) obtaining the bioenzymes XS25001-XS25003.

[0014] In a sixth aspect, the present application provides the application of the novel bioenzymes XS25001-XS25003 described in the first aspect, the gene described in the second aspect, the expression vector described in the third aspect, and the expression strain described in the fourth aspect in degrading microcystin.

[0015] In a seventh aspect, the present application provides the application of the novel bioenzymes XS25001-XS25003 described in the first aspect, the gene described in the second aspect, the expression vector described in the third aspect, and the expression strain described in the fourth aspect in inhibiting the growth of cyanobacteria and / or killing cyanobacteria.

[0016] Compared with the prior art, the embodiments of the present application at least have the following advantages or beneficial effects:

[0017] 1. The yield of the novel bio-enzyme XS25001-XS25003 obtained in this application is higher than that of the degradation enzymes reported in the existing literature. The protein yield in the crude enzyme solution is above 100 mg / L, which is better than the protein yield of the crude enzyme solution mentioned in the literature and the protein yield of 41.175 mg / L, and it is increased by more than 2 times.

[0018] 2. In the preferred scheme, the enzyme activity of the novel bio-enzyme XS25001-XS25003 is increased by more than 2-5 times. The half-life of the microcystin degradation enzyme XS25001-XS25003 reaches 90 days at 0 °C, and the half-life of the novel bio-enzyme XS25001-XS25003 reaches 30 days at 30 °C, showing good stability. It effectively reduces the production cost and has greater industrial application value.

[0019] 3. This application has developed the application potential of the novel bio-enzyme in inhibiting cyanobacteria and detoxification, and it can be applied to agricultural, fishery (including aquaculture, etc.) production and water environmental pollution treatment. Description of the Drawings

[0020] In order to more clearly illustrate the technical solutions of the embodiments of this application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application, so they should not be regarded as limiting the scope. For those of ordinary skill in the art, other relevant drawings can be obtained based on these drawings without creative efforts.

[0021] Figure 1 It is the SDS-PAGE diagram of the novel bio-enzyme purified in Example 1 of this application;

[0022] Figure 2 It is the inhibition effect diagram of the novel bio-enzyme on Microcystis aeruginosa 905 in Example 2 of this application;

[0023] Figure 3 It is the effect diagram of the novel bio-enzyme on adjusting the pH of water quality in Example 2 of this application;

[0024] Figure 4 It is the diagram showing the effect of the novel bio-enzyme on the cell morphology of Microcystis aeruginosa before and after treatment characterized by the SEM method in Example 2 of this application.

[0025] Figure 5 It is the comparison diagram of the degradation effect of different experimental groups on microcystin in cyanobacteria water in Example 2 of this application;

[0026] Figure 6 It is the schematic diagram of the chlorophyll a content in different cyanobacteria after treatment with the novel bio-enzyme in Example 2 of this application. Detailed Embodiments

[0027] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. For those not specified in the embodiments, the conventional conditions or the conditions recommended by the manufacturer are followed. For reagents or instruments not indicated with the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0028] It should be noted that, without conflict, the embodiments in this application and the features in the embodiments can be combined with each other. The following will refer to specific embodiments to elaborate on this application in detail.

[0029] A novel bioenzyme capable of effectively degrading microcystin, where the novel bioenzyme is one of the following a and b;

[0030] The a is a novel bioenzyme composed of the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, denoted as XS25001, XS25002, XS25003;

[0031] The b is a bioenzyme derived from a by substituting, deleting, or adding one or several amino acids to the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 and having unchanged enzyme activity, or a bioenzyme shown by the amino acid sequences with tags attached to the amino terminus and / or carboxyl terminus of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0032] The 20 amino acid residues that make up the bioenzyme can be divided into four categories according to the polarity of the side chains: 1. Non-polar amino acids: alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), phenylalanine (Phe), tryptophan (Trp), and proline (Pro); 2. Polar uncharged amino acids: glycine (Gly), serine (Ser), threonine (Thr), cysteine (Cys), asparagine (Asn), glutamine (Gln), and tyrosine (Tyr); 3. Positively charged amino acids: arginine (Arg), lysine (Lys), and histidine (His); 4. Negatively charged amino acids: aspartic acid (Asp) and glutamic acid (Glu). If a substitution of amino acid residues belonging to the same category occurs in a protein, for example, Arg substituting Lys or Leu substituting Ile, the role played by the residue in the protein domain (such as providing a positive charge or forming a hydrophobic pocket structure) remains unchanged, and it will not affect the three-dimensional structure of the protein. Therefore, the function of the protein can still be achieved. The substitution of amino acid residues belonging to the same category can occur at any amino acid residue position of the above microcystin-degrading enzyme.

[0033] As described above, the novel bioenzyme provided by the present application can also be modified or mutated to obtain a derived bioenzyme. The "derived bioenzyme" referred to in the present application refers to having differences in amino acid sequence from the highly efficient bioenzyme having the above amino acid sequence, and may also have differences in modification form that do not affect the sequence, or both. These bioenzymes include natural or induced genetic variants. The induced variants can be obtained by various techniques, such as random mutations generated by radiation or mutagenic agents, or by techniques such as site-directed mutagenesis or other known molecular biology techniques. The "derived bioenzyme" also includes analogs having residues of natural L-amino acids (such as D-amino acids), and analogs having non-naturally occurring or synthetic amino acids (such as β-amino acids, γ-amino acids, etc.).

[0034] The above novel bioenzyme can be obtained by artificial synthesis, or its coding gene can be synthesized first and then obtained by biological expression.

[0035] The present application also discloses a gene encoding the novel bioenzyme that can effectively degrade microcystin.

[0036] Correspondingly, the gene can be the following (1) or (2):

[0037] (1) A DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6;

[0038] (2) A DNA molecule that hybridizes with the DNA sequence defined in (1) under stringent conditions and has unchanged enzyme activity of the encoded microcystin-degrading enzyme. Among them, the stringent conditions can be: hybridizing in a solution of 6×SCC and 0.5% SDS at 65°C, and then washing the membrane once with 2×SCC, 0.1% SDS and once with 1×SCC, 0.1% SDS. Unchanged enzyme activity means that under the same measurement conditions, the percentage (relative activity) of the enzyme activity of the protein encoded by (2) to the enzyme activity of the protein encoded by (1) is not less than 95% (or 96%, or 97%, or 98%, or 99%, or 100%).

[0039] As is well known in the art, among the 20 different amino acids that make up proteins, except that Met (ATG) or Trp (TGG) is encoded by a single codon respectively, the other 18 amino acids are each encoded by 2 - 6 codons. That is, due to the degeneracy of the genetic codons, there are usually more than one codon that determines an amino acid, and the substitution of the third nucleotide in the triplet codon often does not change the amino acid composition. Therefore, the nucleotide sequences of genes encoding the same protein can be different. Those skilled in the art can completely deduce the nucleotide sequences of the genes that can encode them from the amino acid sequences disclosed in the present invention and the amino acid sequences with unchanged recombinant high - efficiency bio - enzyme activity obtained from the said amino acid sequences according to the well - known codon table, and obtain the said nucleotide sequences by biological methods (such as PCR method, mutation method) or chemical synthesis method. Therefore, these partial nucleotide sequences should all be included within the scope of the present invention. Conversely, using the DNA sequences disclosed herein, it is also possible to obtain amino acid sequences consistent with the high - efficiency bio - enzyme activity described in the present invention by methods well known in the art, such as the method of Sambrook et al. (Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, Second Edition, 1989), by modifying the nucleic acid sequences provided by the present invention. Further, the nucleotide sequences of the said genes are as shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.

[0040] As described above, correspondingly, the 5' end and / or 3' end of the nucleotide sequence may also be ligated with the coding sequences of the tags shown in Table 1 above.

[0041] Table 1 Common Modifying Tags

[0042] Label Residue number Amino acid sequence Poly-Arg 5 - 6 (usually 5) RRRRR (SEQ ID NO: 7) Poly-His 2 - 10 (usually 6) HHHHHH (SEQ ID NO: 8) FLAG 8 DYKDDDDK (SEQ ID NO: 9) Strep-tagⅡ 8 WSHPQFEK (SEQ ID NO: 10) c-myc 10 EQKLISEEDL (SEQ ID NO: 11)

[0043] The nucleotide sequences provided in the present application can generally be obtained by polymerase chain reaction (PCR) amplification method, recombination method, or artificial synthesis method. For example, those skilled in the art can easily obtain templates and primers according to the nucleotide sequences provided by the present invention and use PCR to amplify the relevant sequences.

[0044] Once the relevant nucleotide sequences are obtained, the relevant amino acid sequences can be obtained in large quantities by the recombination method. Usually, the obtained nucleotide sequences are cloned into vectors, then transferred into genetically engineered bacteria, and then the relevant nucleotide sequences are isolated from the proliferated host cells by conventional methods.

[0045] In addition, the relevant nucleotide sequences can also be synthesized by the well - known artificial chemical synthesis method.

[0046] The present application provides an expression vector containing the above - mentioned gene.

[0047] The "vector" used in the expression vector can be selected from various vectors known in the art, such as various commercially available plasmids, cosmids, phages, retroviruses, etc. The present invention preferably uses the pET21b(+) plasmid. The construction of the expression vector can be carried out using various endonucleases capable of having cleavage sites at the multiple cloning sites of the vector (for example, for pUC18, SalI, BamHI, EcoRI, etc. can be used; for pET28a, NdeI, NheI, EcoRI, BamH, HindIII, etc. can be used) to obtain a linear plasmid by enzymatic digestion, and then ligated with the gene fragment digested with the same endonuclease to obtain a recombinant plasmid. The present invention preferably uses double digestion with NdeI and XhoI on pET21b(+) and the gene fragment ligated thereto, and after ligation with a ligase, an expression vector is constructed.

[0048] This application provides an expression strain containing the above-mentioned expression vector.

[0049] The expression vector can be transformed, transduced or transfected into a host cell (strain) by conventional methods in the art, such as chemical transformation by the calcium chloride method, high-voltage electric shock transformation, preferably electric shock transformation. The host cell can be a prokaryotic cell or a eukaryotic cell, preferably a rod-shaped bacterium (such as Escherichia coli, Lactobacillus plantarum or Bacillus subtilis), a yeast (such as Pichia pastoris or Saccharomyces cerevisiae) or an aspergillus (such as Aspergillus niger). More preferably, the host cell is Escherichia coli (such as Escherichia coli TB1), Bacillus subtilis and Pichia pastoris.

[0050] The present application provides a method for the biosynthesis of a novel bioenzyme that can effectively degrade microcystins. The method comprises the following steps: The culture conditions are conventional culture conditions, such as using LB medium (the solvent is water, and the solutes and their final concentrations are: Tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L), and culturing at 20-25 °C for 8-10 h. Since the expression strain provided by the present invention contains the gene encoding the novel bioenzyme for degrading microcystins, it can efficiently express the novel bioenzyme. After culturing and through separation and purification, the effective novel bioenzyme can be obtained. The separation and purification can be carried out by methods well-known to those skilled in the art (for example, collecting cells from the induced culture solution by high-speed refrigerated centrifugation, then disrupting the cells by ultrasonic treatment, centrifuging to obtain a crude enzyme solution containing the microcystin-degrading enzyme, and purifying the crude enzyme solution with Ni-NTA resin to obtain a novel bioenzyme with a higher purity that can degrade microcystins), which will not be elaborated herein.

[0051] The present invention also provides the use of the above-mentioned recombinant bioenzyme gene, recombinant vector, recombinant strain and composition of the present invention in inhibiting the growth of cyanobacteria and / or degrading microcystins. "Inhibiting the growth of cyanobacteria" means that the growth and reproduction of cyanobacteria are inhibited and they cannot grow normally, including killing cyanobacteria.

[0052] In the present invention, the cyanobacteria can be various common toxic algae that cause water pollution, including Microcystis aeruginosa, Nodulariaspumigena, Oscillatoria, Nostoc, etc. The microcystins are one or more of the microcystins MC-LR, MC-YR, MC-RR, Nodularin released from cyanobacteria.

[0053] The following further describes in detail the features and properties of the present application in conjunction with examples.

[0054] Example 1

[0055] This example is used to illustrate the preparation of the novel bioenzyme gene sequence, the construction of the vector, the preparation of the enzyme and the determination of the enzyme activity of the present invention.

[0056] (1) Obtaining the encoding gene of the effective bioenzyme and constructing the expression vector

[0057] The gene sequences SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 encoding the effective bioenzyme (the corresponding amino acid sequences are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 respectively) are loaded into the pET21b(+) plasmid by artificial chemical synthesis to obtain an expression vector of the novel bioenzyme that can degrade microcystins.

[0058] (2) Obtaining the expression strain and preparing the novel bio-enzyme

[0059] The expression vector of the novel bio-enzyme was transferred into Escherichia coli BL21(DE3) competent cells (purchased from New England Biolabs Inc.). After culturing and sequencing verification, the expression strain was obtained.

[0060] The expression strain containing the novel bio-enzyme was induced by IPTG inducer at 20°C ± 2°C for 12 hours. The expression strain grew in LB medium, and the medium composition was: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, ampicillin 50 - 100 mg / L.

[0061] After 12 - 20 hours of induction, the induced cell culture solution was centrifuged at high speed and frozen to collect the cells. Then the cells were disrupted by ultrasound, and the crude enzyme solution containing the novel bio-enzyme was obtained by centrifugation. The crude enzyme solution was purified with Ni-NTA resin, and a novel bio-enzyme with higher purity could be obtained.

[0062] The novel bio-enzyme was analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The protein content of the bio-enzyme was determined by the Bradford method. More than 90% pure novel bio-enzyme was obtained per liter of medium, and the protein yields were 5.4 mg, 6.4 mg, and 4.8 mg.

[0063] Figure 1 Figure of SDS-PAGE for the purified novel bio-enzyme, where M is Marker; 1 - 3 are high-purity microcystin-degrading enzyme proteins XS25001 - XS25003.

[0064] (3) Determination of the activity of the novel bio-enzyme that can degrade microcystin

[0065] The amount of the product Adda was determined by high performance liquid chromatography (HPLC) analysis method, and the change in the amount of linear microcystin before and after degradation was calculated to evaluate the activity of the highly efficient bio-enzyme.

[0066] In this example, the method for determining microcystin adopted high performance liquid chromatography (HPLC) analysis (DIONEX UltiMate 3000, Thermo, USA). The column was a Thermo Acclaim 120 C18 chromatographic column (4.6 mm x 250 mm, 5 μm), the flow rate was 1.0 mL / min, the sample injection volume was 50 μL, and the detection wavelength was 238 nm. The specific method was as follows: Take 390 μL of PBS (pH 7.5) and mix it with 10 μL of linear microcystin (final concentration 250 μg / L), and measure the peak area and peak time of microcystin. Then take 380 μL of PBS (pH 7.5) and mix it with 10 μL of linear microcystin (final concentration 250 μg / L), add 10 μL of biological enzyme (final concentration 0.03 μg / L), react at 25 °C for 5 minutes, and then measure the peak area and peak time of the degraded product. Calculate the amount of microcystin in the sample according to the peak area, and calculate the enzyme activity according to the amount of microcystin.

[0067] In this example, the total enzyme activity of the biological enzyme obtained per liter of culture medium was 3071.35 ± 98.28 U. The definition of the enzyme activity unit U was the amount of enzyme required to degrade 1 μg of linear microcystin MC-LR within a unit time (1 minute).

[0068] Example 2

[0069] This example was used to illustrate the test of the novel biological enzyme in killing cyanobacteria, degrading microcystin and regulating water quality.

[0070] 1. Add the enzyme preparation of the novel biological enzyme to 50 mL of a culture solution containing a relatively high initial concentration of Microcystis aeruginosa 905 algal cells (about 0.7×10 7 algal cells / mL), and continuously treat for 0, 3, 6, 9 days, and observe and detect the inhibition of Microcystis aeruginosa 905. The inhibition of the enzyme preparation of the novel biological enzyme on Microcystis aeruginosa 905 is shown in Figure 2 , from which it can be seen that the enzyme preparation has a good inhibitory effect on cyanobacteria. In addition, measure the pH value of the algal solution after continuous treatment for 9 days, and the pH change is shown in Figure 3 , from which it can be seen that the enzyme preparation has a good effect on regulating water quality. The effect of the novel biological enzyme on the cell morphology of Microcystis aeruginosa 905 was observed by scanning electron microscopy (SEM). The SEM results showed that before treatment, the number of algal cells was large and the growth state was good, mostly regular spherical, with a smooth surface and many cells in the division stage; after treatment with the novel biological enzyme, the cell surface shrank, deformed, and the cells tended to rupture and die (see Figure 4 ).

[0071] 2. Treat the culture solution of Microcystis aeruginosa 905 according to the method in Step 1. After continuous treatment for 9 days, centrifuge the cell culture solution to obtain supernatant and cell precipitate.

[0072] The supernatant is used for extracting extracellular algal toxins, and the specific operation is as follows: Take 10 mL of methanol and 10 mL of ddH2O to activate the solid-phase extraction column (Waters Oasis HLB 6cc), then pass the supernatant through the activated solid-phase extraction column at a flow rate of 1 mL / min, and then repeat the sample loading 2 - 3 times to ensure that the algal toxins are enriched as completely as possible. Then wash with deionized water and methanol aqueous solutions with volume fractions of 10% and 20% in sequence, and then elute with methanol aqueous solutions with volume fractions of 70% or 80% in sequence. The eluate is dried with nitrogen, redissolved in 70% methanol by volume to a specific volume, and then the content of algal toxins is determined by high-performance liquid chromatography.

[0073] The cell precipitate is used for extracting intracellular algal toxins, and the specific operation is as follows: Freeze-thaw the cells twice, ultrasonically disrupt for 10 minutes, then centrifuge, take the supernatant, and pass it through the activated solid-phase extraction column at a flow rate of 1 mL / min. The remaining steps are the same as above.

[0074] Extract the algal toxins of each experimental group by the above method, and determine the total content of algal toxins (MCs) by high-performance liquid chromatography. Figure 5 shows the change in the total content of algal toxins in the experimental group treated with the MllrC enzyme preparation compared with the two control groups (BG11 and Tris-HCl). It can be seen from Figure 5 that the novel biological enzyme preparation of the present application can well degrade the algal toxins released into the water body.

[0075] 3. Add the enzyme preparation of the novel biological enzyme to the culture solutions of 50 mL of different algal cells. The initial cell concentration is about 0.7×10 7 algal cells / mL. After continuous treatment for 9 days, observe and detect the growth conditions of different algae. Figure 6 shows the influence on the chlorophyll a content in different algae after treatment with the novel biological enzyme. It can be seen from Figure 6It can be seen that the novel bio-enzyme described in the present application has good inhibitory effects on common toxin-producing cyanobacteria such as Microcystis aeruginosa, Oscillatoria, and Nostoc. At the same time, it is found that the novel bio-enzyme has certain selectivity for eukaryotic algae and prokaryotic algae, as well as toxin-producing and non-toxin-producing algae. The novel bio-enzyme has no killing effect on the non-toxic eukaryotic alga Ulothrix acuminate and also has no inhibitory effect on the non-toxin-producing prokaryotic alga Synechocystis. This fully demonstrates that the novel bio-enzyme has excellent effects in the comprehensive treatment of harmful toxin-producing cyanobacteria.

[0076] The embodiments described above are some, but not all, of the embodiments of the present application. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application claimed, but merely represents selected embodiments of the present application. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present application without making creative efforts fall within the scope of protection of the present application.

Claims

1. A novel biological enzyme that effectively degrades algal toxins, characterized in that: The biological enzyme is any one of the following a and b; Said a is a novel biological enzyme consisting of the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; The b is a biological enzyme derived from a, in which the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 is substituted, deleted or added with one or several amino acids and the enzyme activity remains unchanged, or a new biological enzyme shown by the amino acid sequence with a label connected to the amino terminus and / or carboxyl terminus of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO:

3.

2. A gene encoding the novel biological enzyme that effectively degrades algal toxins as claimed in claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:

6.

4. An expression vector, characterized in that: The expression vector contains the gene according to claim 2 or 3.

5. An expression strain, characterized in that: The expression strain contains the expression vector according to claim 4.

6. The expression strain according to claim 5, characterized in that The expression strain is Escherichia coli, Bacillus, lactic acid bacteria, yeast or Aspergillus.

7. The expression strain according to claim 5 or 6, characterized in that: The expression strain is one of Escherichia coli, Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, Lactobacillus plantarum, Lactococcus lactis and Aspergillus niger.

8. A biosynthetic method for the novel bioenzyme for effectively degrading algal toxins as claimed in claim 1, characterized in that: The method comprises the following steps: S1. Cultivating the expression strain according to any one of claims 5 to 7 to induce the expression of the gene encoding the biological enzyme; S2. Separate and purify the enzyme protein of the biosynthesized biological enzyme.

9. Use of the novel biological enzyme according to claim 1, the gene according to claim 2 or 3, the expression vector according to claim 4 or the expression strain according to any one of claims 5 to 7 in degrading algal toxins.

10. Use of the novel biological enzyme according to claim 1, the gene according to claim 2 or 3, the expression vector according to claim 4 or the expression strain according to any one of claims 5 to 7 in inhibiting and / or killing cyanobacteria.

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