Algal toxin degrading enzyme, gene, recombinant vector, production strain, preparation method and application thereof

Abstract

The invention provides an algal toxin degrading enzyme, a gene, a recombinant vector, a production strain, a preparation method and application thereof, and relates to the field of gene engineering. The algal toxin degrading enzyme provided by the invention can be applied to the pH environmental condition of pH 6.0-pH 12.0, the optimum pH is 10.0, and even under the strong alkali conditions of pH 11.0 and pH 12.0, the enzyme activity can be kept 80% and 70% of the highest activity. The algal toxin degrading enzyme can be used at the temperature of 15-80 DEG C, the optimum reaction temperature is 50 DEG C, and the enzyme activity can be kept 50% of the highest activity even at the high temperature of 70 DEG C. The algal toxin degrading enzyme disclosed by the invention can degrade algal toxin under high-temperature or / and strong-alkalinity environmental conditions, is widely applied to water bodies, such as lakes, rivers, reservoirs, aquaculture water bodies and the like, which have serious cyanobacterial bloom and are polluted by the algal toxin, and meanwhile, the algal toxin degrading enzyme can be used for degrading the algal toxin. The method can also be used in the fields of non-toxic treatment of algae source products polluted by algal toxins and resource application of algae products, and is ecological, safe, green and low-carbon.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and in particular to an algae toxin degrading enzyme, a gene, a recombinant vector, a production strain, a preparation method and applications thereof. Background Art

[0002] Global warming and eutrophication of water bodies have many adverse effects on the environment, one of which is the increasing frequency and severity of harmful cyanobacteria blooms (HMBs) worldwide. During their growth, cyanobacteria cells release a variety of toxic substances into the water, especially microcystins, which can inhibit the growth and reproduction of other organisms, giving cyanobacteria cells a competitive advantage in growth, resulting in rapid growth and reproduction, and cyanobacteria blooms. At this time, the water quality is alkaline or even strongly alkaline, further damaging the water quality and aquatic ecosystems. During the outbreak of cyanobacteria blooms, the pH value of the water body can usually reach 8 to 10, and even higher in severe cases, devastating the aquatic ecosystem and threatening human health and life safety. In addition, aquatic products and algae-derived products in water bodies contaminated by cyanobacteria blooms and algae toxins will be enriched with algae toxins, which are one of the strongest carcinogens and can cause liver cancer, neurological diseases, etc. How to achieve ecological management of blue algae blooms and effectively remove algal toxins in water bodies, aquatic products, and algae-derived products is an environmental and food safety issue that needs to be urgently addressed in my country and the world.

[0003] Algal toxin degrading enzyme is a new type of protease with dual functions of detoxification and algaecide. It has important application potential in the ecological management of cyanobacterial blooms and the removal of algal toxin pollution. It is expected to achieve the in-situ comprehensive management of cyanobacterial blooms, the non-toxic treatment of aquatic products and algal products, and the goal of happy rivers and lakes and healthy food. However, the outbreak season of cyanobacterial blooms is generally high temperature in summer, and the outbreak of cyanobacterial blooms makes the water body highly alkaline. In addition, the preparation process of algal products may require high temperature and other conditions. Whether the algal toxin degrading enzyme can withstand the harsh environment of high temperature and strong alkalinity poses a huge challenge to its further application. Algal toxin degrading enzymes with high temperature resistance and strong alkali resistance will overcome this defect and have a wider application prospect. They can be used for the removal of algal toxins during cyanobacterial blooms in rivers and lakes, as well as for the detoxification of algal foods and the regulation of water quality in drinking water sources. Therefore, it is becoming increasingly important to find new high-temperature and high-alkali-resistant algae toxin-degrading enzymes to meet the needs of cyanobacterial bloom control. There is an urgent need to develop a new algae toxin-degrading enzyme with excellent properties that is suitable for lakes, rivers, aquaculture waters and drinking water sources where cyanobacterial blooms occur at high temperatures. Summary of the invention

[0004] The purpose of the present invention is to provide an algae toxin degrading enzyme, gene, recombinant vector, production strain, preparation method and application thereof, aiming to solve the problem of how to effectively deal with high temperature and / or strong alkalinity cyanobacterial blooms and algae toxin pollution. It can be applied to application scenarios such as lakes, rivers, reservoirs and aquaculture water bodies that are contaminated by cyanobacterial blooms and algae toxins, and can also be used for the non-toxic treatment of algae-derived products.

[0005] To achieve the above object, the present invention adopts the following technical solutions.

[0006] In a first aspect, the present invention provides a high temperature and strong alkali resistant algae toxin degrading enzyme, wherein the algae toxin degrading enzyme is (a) or (b):

[0007] (a) an algae toxin degrading enzyme consisting of an amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, which has the characteristics of being resistant to high temperature and strong alkali;

[0008] (b) An algal toxin degrading enzyme derived from (a), wherein the amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 is substituted, deleted, or one or more amino acids are added, and the enzyme activity remains unchanged, or an algal toxin degrading enzyme composed of an amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 with a tag connected to the amino terminus and / or carboxyl terminus.

[0009] In some embodiments of the present invention, the unchanged enzyme activity means that under the same assay conditions, the percentage between the enzyme activity of the algae toxin degrading enzyme derived from (a) and the enzyme activity of (a) is not less than 90%.

[0010] In a second aspect, the present invention provides a gene capable of encoding the algae toxin degrading enzyme as described above, wherein the nucleotide sequence of the encoding gene is shown in any one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.

[0011] In a third aspect, the present invention provides a recombinant vector comprising the gene of the algae toxin degrading enzyme as described above.

[0012] In a fourth aspect, the present invention provides a production strain, wherein the production strain contains the recombinant vector as described above.

[0013] In some embodiments of the present invention, the production strain includes any one of lactic acid bacteria, bacilli, yeast or Aspergillus.

[0014] In some embodiments of the present invention, the production strain is any one of Lactobacillus, Streptococcus thermophilus, Escherichia coli, Lactobacillus plantarum, Pichia pastoris, Saccharomyces cerevisiae, Bacillus subtilis, Aspergillus niger, Comamonas aquatic or Exiguobacterium.

[0015] In a fifth aspect, the present invention provides a method for preparing a high temperature and strong alkali resistant algae toxin degrading enzyme, comprising the following steps: S1) culturing a production strain as described above; S2) inducing efficient expression of the gene of the algae toxin degrading enzyme; S3) isolating and purifying the obtained algae toxin degrading enzyme.

[0016] In a sixth aspect, the present invention provides the use of the algae toxin degrading enzyme as described above, the gene capable of encoding the algae toxin degrading enzyme as described above, the recombinant vector as described above, and any of the production strains as described above in inhibiting the growth of cyanobacteria and / or degrading algae toxins, including use in rivers, lakes, reservoirs and aquaculture water bodies threatened by cyanobacterial blooms, use in rivers, lakes, reservoirs and aquaculture water bodies contaminated by algae toxins, and use in the detoxification treatment of algae-derived foods contaminated by algae toxins and the resource utilization of algae products.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] a) In the present invention, the algae toxin degrading enzyme has good high temperature resistance. Compared with the control algae toxin degrading enzyme, the high temperature resistance time of the algae toxin degrading enzyme provided by the present invention is extended by 1.2-1.5 times, and the optimum reaction temperature rises from 35°C to 50°C. Even under the high temperature condition of 70°C, the enzyme activity can maintain 50% of the maximum activity, while the control algae toxin degrading enzyme almost completely loses its activity at 60°C;

[0019] b) In the present invention, the algae toxin degrading enzyme has good alkali resistance. Compared with the control algae toxin degrading enzyme, the algae toxin degrading enzyme provided by the present invention has stronger alkali resistance. Under the strong alkaline condition of pH 12.0, the enzyme activity can also maintain the maximum activity of 70%, while the enzyme activity of the control algae toxin degrading enzyme at pH 10.5 drops to 50% of the maximum activity;

[0020] c) In the present invention, the obtained algae toxin degrading enzyme has higher stability, and the half-life of the algae toxin degrading enzyme at 0°C can be as long as 25 days, and it also has higher thermal stability and pH stability;

[0021] d) In the present invention, the algae toxin degrading enzyme obtained by the present invention not only eliminates the toxicity to human cells, but also can degrade a variety of algae toxins, and at the same time can inhibit the growth of cyanobacteria or kill cyanobacteria, and can be applied to agriculture, fishery production, water environment pollution treatment, non-toxic treatment process of algae products, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0023] Figure 1 It is the SDS-PAGE picture of the algae toxin degrading enzyme in the present invention. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0025] Any value and endpoint value of the range disclosed herein are not limited to the precise range or value, and these ranges or values ​​should be understood to include values ​​close to these ranges. For numerical ranges, between the individual point values ​​of each range, between the endpoint values ​​of each range, and between the endpoint values ​​and the individual point values, one or more new numerical ranges can be combined with each other, and these numerical ranges should be considered as specifically disclosed in this article.

[0026] The algae toxin degrading enzyme provided by the present invention is (a) or (b):

[0027] (a) an algal toxin degrading enzyme consisting of the amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4;

[0028] (b) a cyanotoxin degrading enzyme derived from (a) wherein the amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 is replaced, deleted, or one or more amino acids are added, and the enzyme activity remains unchanged, or a cyanotoxin degrading enzyme consisting of an amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 with a tag connected to the amino terminal and / or carboxyl terminal. Wherein, the enzyme activity remains unchanged means that under the same measurement conditions, the percentage (relative activity) between the enzyme activity of the protein derived from (a) and the enzyme activity of (a) is not less than 90%. It should be noted that, in addition to the relative activity not less than 90% described in this embodiment, the enzyme activity remains unchanged and can also be selected according to actual needs. Standards of not less than 92%, 94%, 96%, 98% or 100%.

[0029] The 20 common amino acid residues that make up proteins can be divided into four categories according to the polarity of their side chains: 1. Positively charged amino acids: histidine (His), arginine (Arg) and lysine (Lys); 2. Negatively charged amino acids: aspartic acid (Asp) and glutamic acid (Glu); 3. Polar uncharged amino acids: glycine (Gly), serine (Ser), threonine (Thr), tyrosine (Tyr), cysteine ​​(Cys), glutamine (Gln) and aspartic acid (Asn); 4. Non-polar amino acids: alanine (Ala), leucine (Leu), isoleucine (Ile), proline (Pro), valine (Val), methionine (Met), phenylalanine (Phe) and tryptophan (Trp). If amino acid residues belonging to the same category are substituted in a protein, such as Lys replacing Arg or Asp replacing Glu, the role of the residue in the protein domain (such as providing positive charge or forming a hydrophobic pocket structure) does not change, and the three-dimensional structure of the protein is not affected, so the function of the protein can still be achieved. The amino acid residue substitution belonging to the same category can occur at any amino acid residue position of the above-mentioned algae toxin degrading enzyme.

[0030] As mentioned above, the derivative protein of the algae toxin degrading enzyme provided by the present invention is derived by modification or mutation. The "derivative protein" described in the present invention refers to a protein having a difference in amino acid sequence from the algae toxin degrading enzyme having the above amino acid sequence, and may also have a difference in the form of modification that does not affect the sequence, or both. These proteins include natural or induced genetic variants obtained by various techniques, such as random mutations produced by high temperature or mutagens, and can also be obtained by techniques such as site-directed mutagenesis or other known molecular biology techniques. The "derivative protein" also includes analogs, such as analogs of natural L-type amino acid residues, such as D-type amino acids, non-naturally occurring or synthetic amino acid analogs, such as β-amino acids or γ-amino acids, etc.

[0031] Usually the primary structure is not changed, that is, the amino acid sequence is not changed in the form of modification, such as acetylation, carboxylation, glycosylation, phosphorylation and dephosphorylation of protein chemical derivatization.

[0032] Preferably, in order to facilitate purification, the algae toxin degrading enzyme (a) can also be modified by adding tags commonly used in the art. For example, the algae toxin degrading enzyme (b) can be modified by adding tags to the amino terminus and / or carboxyl terminus of the algae toxin degrading enzyme (a).

[0033] Preferably, the tag can be selected according to actual needs, such as at least one of Poly-His, Poly-Arg, Strep-tag II and c-myc, so as to obtain the algae toxin degrading enzyme (b). The tag will not affect the activity of the algae toxin degrading enzyme of the present invention. In actual application, whether to add the tag can be selected according to actual needs.

[0034] Preferably, the algae toxin degrading enzyme can be obtained by first synthesizing its encoding gene and then expressing it biologically, or it can be obtained by artificial synthesis.

[0035] The present invention also provides a gene encoding the algae toxin degrading enzyme, which may be (c) or (d) as follows:

[0036] (c) the nucleotide sequence shown by any one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8;

[0037] (d) A nucleotide sequence that hybridizes with the nucleotide sequence shown in any one of (c) under stringent conditions and encodes a cyanotoxin degrading enzyme whose enzymatic activity remains unchanged. The stringent conditions may be: hybridization at 65°C in a solution of 6×SCC, 0.5% SDS, and then washing the membrane once with 2×SCC, 0.1% SDS and once with 1×SCC, 0.1% SDS. The enzyme activity remains unchanged means that under the same assay conditions, the percentage (relative activity) between the enzyme activity of the cyanotoxin degrading enzyme encoded by (d) and the enzyme activity of the cyanotoxin degrading enzyme encoded by (c) is not less than 90%. It should be noted that, in addition to the relative activity not less than 90% described in this embodiment, the enzyme activity remains unchanged and can also be selected according to actual needs. The standard of not less than 92%, 94%, 96%, 98% or 100% can also be selected.

[0038] It is well known in the art that among the 20 different amino acids that make up proteins, except for Met (ATG) or Trp (TGG) which are encoded by a single codon, the other 18 amino acids are encoded by 2-6 codons respectively. Genetic codons are degenerate, and the substitution of the third nucleotide in the triplet codon often does not change the composition of amino acids, so the same protein amino acid sequence is the same, but the nucleotide sequence of the encoding gene can be different. According to the known codon table, the person skilled in the art can completely deduce the nucleotide sequence of the gene that can encode them from the amino acid sequence disclosed in the present invention, and the amino acid sequence with unchanged recombinant algae toxin degrading enzyme activity obtained from the amino acid sequence, and obtain the nucleotide sequence by biological methods (such as PCR method, mutation method) or chemical synthesis method, so the partial nucleotide sequence should be included in the scope of the present invention. On the contrary, using the DNA sequence disclosed herein, the nucleic acid sequence provided by the present invention can also be modified by methods known in the art, such as the method of Sambrook, etc., to obtain an amino acid sequence consistent with the algae toxin degrading enzyme activity of the present invention.

[0039] Preferably, the nucleotide sequence of the gene capable of encoding the algae toxin degrading enzyme is shown in any one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.

[0040] Preferably, the 5' end and / or 3' end of the nucleotide sequence may also be connected to a coding sequence of a tag.

[0041] The nucleotide sequence provided by the present invention can usually be obtained by artificial synthesis, polymerase chain reaction (PCR) amplification, recombination and other methods. For example, those skilled in the art can easily obtain templates and primers based on the nucleotide sequence provided by the present invention, and use PCR to amplify and obtain the relevant sequence. Then, the relevant amino acid sequence is obtained in large quantities by recombination, and the obtained nucleotide sequence is cloned into a vector, and then transferred into genetically engineered bacteria, and then the relevant nucleotide sequence is separated from the propagated host cells by conventional methods.

[0042] In addition, the relevant nucleotide sequences can also be synthesized by known artificial chemical synthesis methods.

[0043] The recombinant vector provided by the invention contains the gene provided by the invention.

[0044] Preferably, the "vector" used in the recombinant vector can be selected from various vectors known in the art, such as various commercially available plasmids, cosmids, phages and retroviruses. The recombinant vector construction can use various nucleases that can have a cleavage site in the vector multiple cloning site, such as SalⅠ, BamHI, EcoRI, etc. for pUC18; NdeⅠ, NheⅠ, EcoRI, BamH, HindⅢ, etc. for pET28a; SalⅠ, BamHI, EcoRI, HindⅢ, etc. for pMAL-p5X; enzyme digestion to obtain a linear plasmid, and ligation with a gene fragment cleaved with the same nuclease to obtain a recombinant plasmid.

[0045] Preferably, pMAL-p5X and the gene fragments connected thereto are double-digested with BamHI and HindIII, and connected with ligase to construct a recombinant vector.

[0046] The production strain provided by the present invention contains the recombinant vector provided by the present invention.

[0047] Preferably, the recombinant vector can be transformed, transduced or transfected into a host cell, ie, a production strain, by conventional methods in the art, such as chemical transformation by the calcium chloride method, high voltage electroporation transformation, preferably electroporation transformation.

[0048] Preferably, the production strain may be a prokaryotic cell or a eukaryotic cell, including any one of lactic acid bacteria, bacilli, yeast or Aspergillus.

[0049] Preferably, the production strain is any one of Lactobacillus, Streptococcus thermophilus, Escherichia coli, Lactobacillus plantarum, Pichia pastoris, Saccharomyces cerevisiae, Bacillus subtilis, Aspergillus niger, Comamonas aquatic or Exiguobacterium.

[0050] The preparation method of the algae toxin degrading enzyme provided by the present invention comprises:

[0051] S1) culturing the production strain provided by the present invention;

[0052] S2) Inducing efficient expression of genes for algal toxin-degrading enzymes;

[0053] S3) separating and purifying the expressed algae toxin degrading enzyme.

[0054] Preferably, the culture conditions are conventional culture conditions, such as using LB medium (peptone 10g / L, yeast extract 5g / L, NaCl 10g / L), culturing at 20-25°C for 8-24h. Preferably, the separation and purification method is to collect cells, disrupt the cells by ultrasound, centrifuge to obtain a crude enzyme solution containing algae toxin degrading enzyme, and purify the crude enzyme solution with a resin containing amylose to obtain an algae toxin degrading enzyme with higher purity.

[0055] The present invention also provides the use of algae toxin degrading enzymes, genes capable of encoding the algae toxin degrading enzymes, recombinant vectors, and production strains in inhibiting the growth of cyanobacteria and / or removing algae toxins.

[0056] Preferably, the cyanobacteria may be various common cyanobacteria that cause water pollution, including Microcystis aeruginosa, Nodularia spumigena, Oscillatoria, Nostoc, and the like.

[0057] Preferably, the algae toxin is one or more of the algae toxins MC-LR, MC-RR, MC-YR, and Nodularin released by cyanobacteria.

[0058] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other. The present invention will be described in detail below with reference to specific embodiments.

[0059] Example 1

[0060] This example is used to illustrate the preparation of the coding gene sequence of the algae toxin degrading enzyme, the construction of the recombinant vector, the preparation of the algae toxin degrading enzyme and the determination of the enzyme activity of the present invention.

[0061] 1. Obtaining genes encoding algal toxin-degrading enzymes and constructing recombinant vectors

[0062] The gene sequence encoding the algae toxin degrading enzyme is synthesized by artificial chemical synthesis. The nucleotide sequence of the gene encoding the algae toxin degrading enzyme is shown in any one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8. The amino acid sequence corresponding to the nucleotide sequence shown in SEQ ID NO: 5 is the amino acid sequence shown in SEQ ID NO: 1; the amino acid sequence corresponding to the nucleotide sequence shown in SEQ ID NO: 6 is the amino acid sequence shown in SEQ ID NO: 2; the amino acid sequence corresponding to the nucleotide sequence shown in SEQ ID NO: 7 is the amino acid sequence shown in SEQ ID NO: 3; the amino acid sequence corresponding to the nucleotide sequence shown in SEQ ID NO: 8 is the amino acid sequence shown in SEQ ID NO: 4.

[0063] Preferably, the nucleotide sequences shown in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8 are loaded into the corresponding vector pMAL-p5X, respectively, to obtain the corresponding recombinant vector of the algae toxin degrading enzyme.

[0064] 2. Obtaining strains producing algae toxin degrading enzymes and preparing algae toxin degrading enzymes

[0065] (1) The recombinant vectors described above were respectively transformed into Escherichia coli TB1 competent cells, which were purchased from New England Biolabs Inc., and cultured and sequenced to obtain the corresponding production strains;

[0066] (2) Inducing the production strains described above with an IPTG inducer at 16 to 37° C. for 16 to 72 hours; the production strains were grown in LB medium, wherein the composition of the medium was: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and 30 to 100 mg / L ampicillin;

[0067] (3) After the induction is completed for 16 to 72 hours, the cells are collected by high-speed refrigerated centrifugation of the induced cell culture medium; the cells are then disrupted by ultrasound and centrifuged to obtain a crude enzyme solution containing algae toxin degrading enzymes; the crude enzyme solution is purified in turn using a resin containing amylose to obtain the corresponding algae toxin degrading enzymes.

[0068] Preferably, the purification step of the crude enzyme solution can be adjusted according to actual needs to obtain algae toxin degrading enzymes of different concentrations.

[0069] Example 2

[0070] This example describes a method for analyzing algae toxin degrading enzymes. The algae toxin degrading enzyme solution obtained in Example 1 is analyzed by polyacrylamide gel electrophoresis (SDS-PAGE).

[0071] Figure 1 The results of polyacrylamide gel electrophoresis (SDS-PAGE) of the algae toxin degrading enzyme obtained in Example 1 are shown. Figure 1 In the figure, M is a protein marker; 1 is a cell rupture solution after induction; 2 is a cell rupture solution without induction; 3 is an algae toxin degrading enzyme solution; 4 is a cell precipitate solution after rupture; and 5 is an algae toxin degrading enzyme solution after purification. The results show that the algae toxin degrading enzyme solution is efficiently expressed in Escherichia coli TB1. After the expressed algae toxin degrading enzyme is purified, the protein content in the algae toxin degrading enzyme solution reaches more than 90% of the total protein. The protein yields obtained by the expression strains encoded by the genes of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8 are 145±15mg / L, 302±12mg / L, 286mg / L±30mg / L, and 290mg / L±11mg / L, respectively.

[0072] Example 3

[0073] This embodiment describes the activity determination and analysis method of the algae toxin degrading enzyme. The activity analysis method of the algae toxin degrading enzyme in the present invention is a method of high performance liquid chromatography (HPLC) analysis. First, the content of algae toxins in the solution before the algae toxin degrading enzyme treatment is determined, then the algae toxin degrading enzyme is added to the solution for treatment, and the amount of algae toxins remaining in the treated solution is determined. Finally, the change in the amount of algae toxins before and after degradation is calculated to evaluate the activity of the algae toxin degrading enzyme.

[0074] The method for determining algal toxins in the present invention adopts HPLC analysis, the instrument is DIONEX UltiMate 3000 (Thermo, USA), the chromatographic column is AcclaimTM120C18 (4.6mm x 250mm, 5μm), the sample volume is 50μL, and the flow rate is 1.0mLmin -1 , the detection wavelength was 238nm. The column temperature was 30℃, and the detection limit of the HPLC system was 0.1μg·L -1 .

[0075] The specific determination method is: take 10μL of standard algal toxin sample and add it to 390μL 50mmol·L -1 Then, set the parameters, load the sample for testing, and calculate the peak area corresponding to the peak time of algae toxin, which is the algae toxin content before treatment. Then, take 10μL of standard algae toxin and 380μL of 50mmol·L -1 Mix the phosphate buffer, add 10 μL of algae toxin degrading enzyme, react at 25°C for 5 to 10 minutes, measure the peak area of ​​algae toxins in the sample after degradation, calculate the amount of remaining algae toxins in the sample based on the peak area, and calculate the enzyme activity of the algae toxin degrading enzyme based on the reduction of algae toxins in the solution before and after treatment.

[0076] The total activity of the algae toxin degrading enzyme obtained in Example 1 produced by the expression strains encoded by the genes described in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8 per liter of culture medium was 810±50U, 4400±150U, 3100±180U, and 960±80U, respectively. The enzyme activity unit U is defined as the amount of enzyme required to degrade 1 μg of algae toxin MC-LR per unit time (1 minute).

[0077] Example 4

[0078] This example describes a method for determining the pH stability of an algae toxin degrading enzyme. The algae toxin degrading enzyme obtained in Example 1 is measured. The specific method is: the algae toxin degrading enzyme obtained in Example 1 and encoded by the gene described in SEQ ID NO: 5 is placed in a buffer solution with a pH of 3.0, 4.0, 5.0, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 10.0, 11.0, and 12.0 and incubated for 2 hours. Then, the corresponding algae toxin degrading enzyme is diluted to a working concentration with 50mM PBS (pH 7.0), and the residual enzyme activity of the algae toxin degrading enzyme is measured. The determination method is as described in Example 4.

[0079] Taking the highest activity of algae toxin degrading enzyme as 100%, the relative activity of algae toxin degrading enzyme after being treated for 2 hours at different pH values ​​was calculated. The experimental results are shown in Table 1. The activity of algae toxin degrading enzyme reaches the maximum at pH 10.0, so the optimum pH is 10.0, which is 100% activity; after being treated for 2 hours at pH 11.0, the activity of algae toxin degrading enzyme is 82.5% of the maximum activity, that is, the residual activity is 82.5% of the maximum activity. After being treated for 2 hours at pH 12.0, the enzyme activity of 73.4% of the maximum activity is still maintained. After being treated for 2 hours at pH 8.0-12.0, the residual enzyme activity is above 70%, indicating that the algae toxin degrading enzyme has strong pH stability.

[0080] Table 1 Relative activities of algal toxin-degrading enzymes at different pH values

[0081]

[0082] Example 5

[0083] The present embodiment describes a method for determining the thermal stability of an algae toxin degrading enzyme. The algae toxin degrading enzyme encoded by the gene described in SEQ ID NO: 5 in Example 1 was determined by placing the algae toxin degrading enzyme in a constant temperature water bath at 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 60°C, 70°C, and 80°C for 1 hour, and then cooling to room temperature to determine the residual enzyme activity of the algae toxin degrading enzyme.

[0084] The relative activity of the algae toxin degrading enzyme at different temperatures was calculated with the highest activity of the algae toxin degrading enzyme as 100%. The results are shown in Table 2: The optimum temperature of the algae toxin degrading enzyme is 50°C, and it has good thermal stability in the range of 30-70°C, and the activity can be maintained at 70% or above, which shows that it has good thermal stability.

[0085] Table 2 Relative activities of algal toxin degrading enzymes at different temperatures

[0086]

[0087] Example 6

[0088] This example describes a method for determining the storage stability of an algae toxin degrading enzyme. The algae toxin degrading enzyme encoded by the gene described in SEQ ID NO: 5 in Example 1 was determined by placing the algae toxin degrading enzyme at 0°C and determining the residual enzyme activity of the algae toxin degrading enzyme on days 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 30, 50, and 70. The determination method is as described in Example 3.

[0089] The relative activity of the algae toxin degrading enzyme at different storage times was calculated with the highest activity of the algae toxin degrading enzyme as 100%. The results are shown in Table 3: The half-life of the algae toxin degrading enzyme at 0°C can be as long as 25 days, which indicates that it has good storage stability.

[0090] Table 3 Relative activity of algae toxin degrading enzymes at different storage times

[0091]

[0092] Example 7

[0093] This embodiment describes an application of an algae toxin degrading enzyme, in which the algae toxin degrading enzyme obtained by the gene encoding described in SEQ ID NO: 5 in Example 1 is applied to the removal of algae toxins, including detoxification of algae toxins in polluted water bodies caused by cyanobacterial blooms, in the preparation of algae-derived foods, and in any one of the detoxification treatments of drinking water sources and water plants. The test algae species used in the embodiment is Microcystis aeruginosa 905.

[0094] The algal toxin degrading enzyme is used to remove algal toxins, and the specific implementation is as follows: (1) the algal toxin degrading enzyme obtained by the gene encoding described in SEQ ID NO: 5 in Example 1 is added to a final concentration of 1 to 100

[0095] mg / L was added to 1L of Microcystis aeruginosa 905 culture solution, and the cyanobacteria density in the culture system was 1-10×10 9 / L, pH 10.5, and placed outdoors at a temperature range of 25-40°C. Algae cultures without algae toxin degrading enzymes were used as the control group, and samples were taken every day during the treatment period of 1 to 8 days. Then, the changes in chlorophyll a and algae toxin content were measured to judge the inhibition of cyanobacteria growth and algae toxin removal by the high temperature and strong alkali algae toxin degrading enzymes. The results are shown in Table 3 below.

[0096] Table 4 Effects of algae toxin degrading enzymes on chlorophyll a, algae toxins and pH

[0097]

[0098] The experimental results of Example 6 show that the chlorophyll a of the control group reached 914 μg / L on the 8th day, the algae toxin content was as high as 394.8 μg / L, and the pH was as high as 11.21. Compared with the control group, the experimental group to which the algae toxin degrading enzyme described above was added had chlorophyll a reduced to 63 μg / L on the 8th day, the algae toxin content was as low as 19.1 μg / L, and the pH was maintained at a weakly alkaline 7.65. This indicates that the algae toxin degrading enzyme described above exhibits a strong inhibitory activity on Microcystis aeruginosa 905, and is in a time-dependent pattern. At the same time, the algae toxin degrading enzyme described above also has a significant algae toxin degradation and removal effect on the algae toxins produced by Microcystis aeruginosa 905, and the effect is significant. In addition, through the treatment of the algae toxin degrading enzyme described above, the pH of the solution gradually decreased to about 7.65, indicating that the enzyme can reduce the pH of the water body from a strong alkaline to a near-neutral range, and has a good water quality regulation effect.

[0099] The embodiments described above are part of the embodiments of the present invention, rather than all of the embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. A high temperature and strong alkali resistant algae toxin degrading enzyme, characterized in that: The algae toxin degrading enzyme is (a) or (b): (a) an algal toxin degrading enzyme consisting of the amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4; (b) An algal toxin degrading enzyme derived from (a), wherein the amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 is substituted, deleted, or one or more amino acids are added, and the enzyme activity remains unchanged, or an algal toxin degrading enzyme composed of an amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 with a tag connected to the amino terminus and / or carboxyl terminus.

2. The high temperature and strong alkali resistant algae toxin degrading enzyme according to claim 1, characterized in that: The enzyme activity remains unchanged means that under the same measurement conditions, the percentage between the enzyme activity of the algae toxin degrading enzyme derived from (a) and the enzyme activity of (a) is not less than 90%.

3. A gene capable of encoding the high temperature and strong alkali resistant algae toxin degrading enzyme as claimed in claim 1 or 2.

4. The gene according to claim 3, characterized in that The nucleotide sequence of the gene is shown in any one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO:

8.

5. A recombinant vector, characterized in that: The recombinant vector contains the gene of the algae toxin degrading enzyme according to claim 3 or 4.

6. A production strain, characterized in that The production strain contains the recombinant vector according to claim 5.

7. The production strain according to claim 6, characterized in that The production strain is any one of lactic acid bacteria, rod-shaped bacteria, yeast or Aspergillus.

8. The production strain according to claim 6 or 7, characterized in that The production strain is any one of Lactobacillus lactis, Streptococcus thermophilus, Escherichia coli, Lactobacillus plantarum, Pichia pastoris, Saccharomyces cerevisiae, Bacillus subtilis, Aspergillus niger, Comamonas aquaticus or Exiguobacterium.

9. A method for preparing a high temperature and strong alkali resistant algae toxin degrading enzyme, characterized in that: The steps include: S1) cultivating the production strain according to any one of claims 5 to 7; S2) Inducing efficient expression of genes for algal toxin-degrading enzymes; S3) Separate and purify the algae toxin degrading enzyme.

10. Use of the algae toxin degrading enzyme according to claim 1, the gene capable of encoding the algae toxin degrading enzyme according to claim 2 or 3, the recombinant vector according to claim 4, and the production strain according to any one of claims 5 to 7 in inhibiting the growth of cyanobacteria and / or degrading algae toxins.

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