Recombinant enzyme for degrading microcystin in cyanobacteria algal slurry and preparation method thereof

Abstract

The present invention belongs to the technical field of treatment of bloom cyanobacteria, and specifically discloses a recombinant enzyme for degrading microcystin in cyanobacteria slurry and a preparation method thereof. The recombinant enzyme has a solubility-enhancing tag NusA linked to the N-terminus of microcystin-degrading enzyme MlrA and a histidine tag linked to the C-terminus. This recombinant enzyme is characterized by strong stability and high expression level, and can still stably exist and efficiently degrade microcystin in a complex environment with large temperature difference in cyanobacteria accumulation, large pH change, low dissolved oxygen, and high nitrogen and phosphorus. After being treated with the crude extract of the recombinant enzyme for 24 h, the degradation efficiency of microcystin in the accumulated cyanobacteria slurry reaches 89.2%.

Description

Technical Field

[0001] The present invention belongs to the technical field of cyanobacteria bloom treatment, and particularly relates to a recombinant enzyme for degrading microcystin in cyanobacteria slurry and a preparation method thereof. Background Art

[0002] Cyanobacteria bloom is one of the major water environment problems faced globally. Its large-scale outbreak not only restricts the availability of lake water resources but also poses a threat to human health. Currently, the dredging method is one of the most direct, safe, and effective methods for treating cyanobacteria bloom, which can significantly reduce nitrogen and phosphorus pollution in lakes. However, appropriate treatment measures must be found for the algal slurry and dewatered algal sludge after mechanical dredging to achieve harmless treatment, otherwise it will cause environmental pollution. Research shows that a large amount of algal toxins released after cyanobacteria rupture migrate to the sediment interface, threatening the safety of groundwater. When the mixed water after stacking and treating algae is used to irrigate crops, the incompletely removed algal toxins migrate to the surrounding ecosystem, causing ecological safety risks. Therefore, in the treatment of cyanobacteria bloom, how to efficiently treat the salvaged cyanobacteria bloom biomass is one of the key problems faced. Cyanobacteria are rich in nitrogen, phosphorus, potassium, and other substances with potential nutritional value and have application value. For example, phycocyanin in cyanobacteria is one of the rare pigment proteins in nature, contains abundant essential amino acids, and can be widely used in fields such as food and cosmetics; after cyanobacteria are stacked and fermented, they can be used as organic fertilizers to significantly increase the available nitrogen and phosphorus content in the soil.

[0003] Currently, the conventional treatment methods for algal toxins mainly include physical methods (coagulation method, activated carbon adsorption method, reverse osmosis membrane treatment method), chemical methods (photocatalytic oxidation, ozone oxidation method, chlorination method), and biological methods (toxin-degrading bacteria degradation method). The conventional methods for treating microcystin often have many drawbacks, such as high cost, poor effect, and causing secondary environmental pollution. Research shows that the biological enzyme degradation method is an effective and environmentally friendly method, with low cost and beneficial to ecological restoration.

[0004] Microcystin-degrading enzyme (MlrA) is an endopeptidase cloned from microcystin-degrading bacteria. After treatment with this enzyme, microcystin first cleaves the Arg-Adda peptide bond of MC-LR, producing a linear product (H-HN-Adda-Glu-Mdha-Ala-Leu-MeAsp-Arg-OH). The toxicity of the toxin after ring-opening is significantly reduced, and no other harmful by-products are produced. Therefore, MlrA has high application value in the restoration process of aquatic ecosystems. Dziga et al. overexpressed MlrA in bacteria by heterologous expression, immobilized the enzyme in alginate particles, and could stably degrade the algal toxins in flowing lake water within 72 h; Dexter et al. successfully expressed MlrA in cyanobacteria, successfully realizing the application of this enzyme in in-situ water restoration. The above studies show that MlrA has the potential to be applied under field conditions and has specificity and high efficiency.

[0005] However, there is currently no relevant research on using MlrA to degrade the accumulated cyanobacteria after salvage. The reason is that the water content of cyanobacteria after manual or mechanical salvage is relatively high, generally greater than 99%. Ordinary treatment methods, such as burial and incineration, cannot effectively dispose of them. After a large amount of cyanobacteria accumulate, they form aged algae through natural decomposition. Different from the relatively stable environment of field water bodies, the aged algal slurry is rich in various organic substances, has high nitrogen and phosphorus contents, is prone to corruption and odor, and the changes of various components during the accumulation process are complex. General toxin-degrading enzymes cannot play a degrading role in this environment. Research shows that as the accumulation time increases, the temperature of the accumulation environment also rises continuously, and can reach up to 50 °C; during the process of the organic macromolecules released by the rupture of cyanobacteria during the accumulation process being gradually decomposed into small molecules, the pH and dissolved oxygen decrease significantly in the short term, the chlorophyll content decreases, the ammonia nitrogen (NH4 + -N) content increases continuously, the black odor index increases continuously, and the bacteria attached to the surface of cyanobacteria utilize the organic substances released by cyanobacteria, grow continuously and secrete metabolites and various proteases. It is difficult for MlrA to stably exist and degrade toxins in the environment of cyanobacteria accumulation (Zhao Xuanxuan, Zhu Guangcan, Xu Lijuan, et al. Study on the natural decomposition characteristics of cyanobacteria [J]. Jiangsu Journal of Agricultural Sciences, 2013, 29(2): 312-318). At the same time, MlrA is a membrane protein. Because it is prone to misfolding during expression, forming inactive inclusion body precipitates, it cannot play the role of degrading toxins and is difficult to be widely applied in the accumulated algal slurry. Therefore, there is an urgent need for an MlrA recombinant enzyme with high expression level, high solubility, and can stably exist and play a role in cyanobacterial slurry. Summary of the Invention

[0006] The object of the present invention is to provide a recombinant enzyme NusA-MlrA that can stably exist and rapidly degrade microcystin during the accumulation process of cyanobacteria.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The recombinant enzyme NusA-MlrA is obtained by connecting the solubility-enhancing tag NusA to the N-terminus of the microcystin-degrading enzyme MlrA and the histidine tag to the C-terminus. The amino acid sequence of the recombinant enzyme is shown in SEQ ID NO.2, and its preparation method includes: constructing a prokaryotic expression vector containing the gene encoding the recombinant enzyme, and the nucleotide sequence of the gene is shown in SEQ ID NO.3; transforming the prokaryotic expression vector into competent cells of Escherichia coli to obtain a recombinant enzyme expression bacterium, inducing the recombinant enzyme expression bacterium with IPTG, then breaking the cells, and the collected supernatant is the crude extract of the recombinant enzyme.

[0009] Application of the recombinant enzyme NusA-MlrA in degrading microcystin in cyanobacterial pulp: In a specific embodiment of the present invention, after the cyanobacterial pulp is stacked outdoors for 72 h, the crude extract of the recombinant enzyme NusA-MlrA is added according to a volume ratio of 10%. After treatment with the crude extract of the recombinant enzyme NusA-MlrA, the total microcystin content in the stacked pulp is significantly reduced. At 24 h, the total microcystin content is reduced from 429.1 μg / g to 46.3 μg / g, and the degradation efficiency reaches 89.2%.

[0010] Compared with the prior art, the present invention has the following advantages and effects:

[0011] By optimizing the MlrA sequence and replacing the solubility-enhancing tag on the vector, a high-purity microcystin-degrading enzyme NusA-MlrA can be obtained, which can degrade a variety of microcystins without cutting the NusA tag, including the highly toxic MC-LR. After calculation, the enzyme activity of the recombinant enzyme NusA-MlrA is 4933 mU / mL -1 , which is 7.5 times higher than 646 mU / mL reported by Dziga in 2012 -1 (Dziga D, Wladyka B, Zielinska G, Meriluoto J, Wasylewski M. Heterologous expression and characterisation of microcystinase. Toxicon. 2012;59(5):578-86.). At the same time, according to the results of enzyme kinetic parameters, the Kcat / Km value of the optimized recombinant enzyme NusA-MlrA becomes larger, that is, the degradation ability of microcystin is significantly improved.

[0012] Different from relatively stable water bodies, the accumulated cyanobacteria after field salvage and enrichment are characterized by high organic matter, high toxin release, and easy to rot and emit odors during the accumulation process. The temperature, oxygen, pH, etc. change greatly during the accumulation process. Generally, unoptimized MlrA enzyme is easily inactivated in this environment and cannot be used to degrade toxins. However, the stability of the recombinant enzyme NusA-MlrA is increased, and at the same time, the enzyme expression level and degradation efficiency are both improved. The crude extract of NusA-MlrA enzyme can rapidly degrade microcystin released by the rupture of algal slurry within 24 hours. In addition, the nutrients (algal polysaccharides, proteins, carotenoids) in the algal slurry treated with NusA-MlrA do not accelerate their loss. The algal slurry detoxified by NusA-MlrA can be used for the extraction of nutrients, animal feed, etc. for export, providing a certain technical means for cyanobacteria detoxification and resource utilization. Description of the Drawings

[0013] Figure 1 : SDS-PAGE electrophoresis diagram of crude extracts of MlrA recombinant enzymes containing different solubilization tags. Among them, Maker is the protein multi-color pre-stained protein molecular weight standard solution (Yaenzyme, Shanghai), MlrA is the control group, and GST-MlrA, Sumo-MlrA, sfGFP-MlrA, NusA-MlrA, and UB-MlrA are all experimental groups.

[0014] Figure 2 : HPLC chromatogram of the degradation of 10 μg / mL microcystin LR by the crude extract of 1 mg / mL recombinant enzyme NusA-MlrA.

[0015] Figure 3 : Content change of total microcystin in the crude extract of recombinant enzyme NusA-MlrA added to the accumulated cyanobacteria algal slurry within 72 hours.

[0016] Figure 4 : Nutrient content of the algal slurry in the recombinant enzyme NusA-MlrA treatment group and the ddH2O control group. Detailed Embodiments

[0017] Example 1: Preparation method and expression level comparison of MlrA recombinant enzyme

[0018] (1) The gene sequence of the toxin-degrading enzyme MlrA was obtained from the whole genome of the microcystin-degrading bacterium Sphingopyxis sp. HW. The MlrA sequence was sent to a sequencing company (Beijing Tsingke Biotechnology Co., Ltd.) for codon optimization, and then the optimized MlrA gene sequence (SEQ ID NO.1) was amplified. Among them, the upstream primer sequence (MlrA-F) for amplifying the MlrA gene sequence was 5’-CGGGATCGAGGGAAGGATGCGGGAGTTTGTC-3’, and the downstream primer (MlrA-R) sequence was 5’-CACTACCTCCAGATCCACCCTGCGCGTTCGCG-3’. Different solubility-enhancing tags (including GST, Sumo, sfGFP, NusA, UB) were linked to the N-terminus of the microcystin-degrading enzyme MlrA, and a histidine tag was linked to the C-terminus, which was then inserted into the prokaryotic expression vector pMAL-c5X to construct a recombinant vector. The control group was the MlrA sequence with no solubility-enhancing tag linked to the N-terminus and a histidine tag linked to the C-terminus, which was inserted into the expression vector pET28h. The experimental methods refer to Chapters 1 and 15 of the fourth edition of "Molecular Cloning: A Laboratory Manual" (J. Sambrook, D.W. Russell, etc., Science Press, 2016).

[0019] (2) The recombinant vector was transformed into Escherichia coli DH5α competent cells. The specific method was as follows: 5 μL of the recombinant vector was added to 100 μL of Escherichia coli DH5α competent cells, placed on ice for 30 min, then the competent cells were heat-shocked in a 42 °C water bath for 60 s, immediately inserted on ice for cooling for 2 min, and the competent cells were added to an antibiotic-free LB liquid medium under sterile conditions, shaken at 200 rpm for 1 h, the bacterial solution was spread on an LB solid plate containing 100 μg / mL ampicillin, and cultured at 37 °C for 24 h, and single colonies were picked.

[0020] (3) The different single colonies picked were respectively inoculated into 5 mL of LB liquid medium containing ampicillin and cultured at 37 °C for 24 h. 200 μL of the bacterial solution was sent to Wuhan Tsingke Sequencing Company for sequencing, and the remaining bacterial solution was extracted using a plasmid miniprep kit (Jierui, Shanghai). According to the sequencing results, the Vector NTI 10.0 software was used to analyze the sequencing results, and the plasmids extracted from the bacteria with the sequencing result of the MlrA gene were retained. The plasmid concentration was measured using a micro-spectrophotometer NanoDrop 1000 (Thermo, USA), and the gene copy number was calculated based on the concentration of plasmid DNA.

[0021] (4) Transform the constructed plasmid into competent Escherichia coli C43(DE3) cells according to the above transformation method to obtain the MlrA-expressing bacteria. Expand the culture of the MlrA-expressing bacteria and inoculate them into 1 L of LB liquid medium containing 100 μg / mL ampicillin. Culture at 37 °C for 3 h and measure the OD 600 value. When OD 600 ≈ 0.8, induce the MlrA-expressing bacteria with IPTG at a final concentration of 0.5 mM and culture at 18 °C for 12 h. After the induction is completed, centrifuge at a high speed of 6000 rpm, discard the supernatant, collect the bacterial cells, resuspend and wash them with 60 mL of sterile PBS solution (pH = 7.4), and disrupt them by ultrasonic wave. The disruption program is set to run for 3 s and stop for 2 s, disrupt once for 10 min, and disrupt a total of five times, with the power set to 500 W. Collect the supernatant, and the obtained supernatant is the crude extract of MlrA containing different solubility-enhancing tags.

[0022] (5) The SDS-PAGE method is as follows: Install a double vertical plate electrophoresis tank, prepare 5 mL of 12% separating gel and 2 mL of stacking gel. Mix 20 μL of the sample with 5 μL of 5× protein loading buffer (biosharp, Shanghai), boil and heat for 5 minutes, and load 10 μL with a pipette. Among them, the loading order from left to right is: Marker, GST-MlrA, Sumo-MlrA, sfGFP-MlrA, MlrA (control group), NusA-MlrA, UB-MlrA. Then apply a voltage of 60 V / cm on the stacking gel, and increase the voltage to 120 V / cm after the dye enters the separating gel and continue electrophoresis until the dye reaches the bottom; soak it in Coomassie Brilliant Blue R-250 staining solution for 3 hours and then take it out. Boil the stained gel in boiling water for 3 - 5 min, repeat 2 - 3 times until the bands are clear, and then observe and take pictures.

[0023] (6) Take 1 mg / mL of the crude extract of the recombinant enzyme NusA-MlrA and react with the same volume of MC-LR at a concentration of 10 μg / mL, and set the reaction time to 30 min. Use HPLC to determine the change of MC-LR after treatment with the crude extract of NusA-MlrA. Calculate the corresponding enzyme activity and enzyme kinetic constants according to the method in the third chapter of Biochemistry and Molecular Biology, Ninth Edition (Zhou Chunyan et al., People's Medical Publishing House, 2018). The HPLC detection of microcystin LR uses a methanol / trifluoroacetic acid (TFA) aqueous solution system, and the specific implementation method is carried out according to the reference (Mao Jingying. Detection of five microcystins in water by high performance liquid chromatography [J]. Chinese Journal of Environmental Engineering, 2012, (11): 3882 - 3888.).

[0024] By adding different solubility-enhancing tags to the N-terminus of the microcystin-degrading enzyme MlrA, the isoelectric point of the enzyme is adjusted, thereby affecting its expression level. The results are as Figure 1As shown, the expression level of the recombinant enzyme MlrA containing the NusA tag was the highest, with an isoelectric point (PI) value of 5.03 and a protein concentration of 5 mg / mL in the crude enzyme solution. After calculation, the enzyme activity of the crude enzyme solution of the recombinant enzyme NusA-MlrA was 4933 mU / mL -1 , and the Kcat / Km value was 3.672 ± 0.240. The Kcat / Km value of MlrA without the solubility-enhancing tag was 1.406 ± 0.072, indicating that the optimized recombinant enzyme NusA-MlrA had a higher degradation ability for microcystin LR

[0025] Example 2: Degradation ability of MlrA recombinant enzymes containing different solubility-enhancing tags for microcystin LR in cyanobacterial slurry

[0026] The cyanobacteria (Chl-a = 90 mg / L) enriched by fishing were placed in a 250 mL Erlenmeyer flask, and the volume of the algal slurry was controlled at 100 mL. The crude extracts of the MlrA recombinant enzymes containing different solubility-enhancing tags were added to the algal slurry at a ratio of 10% (v / v). After reacting for 72 h, the content of microcystin LR (MC-LR) was determined by HPLC

[0027]

[0028] Among them, C0 is the initial content of MC-LR in the algal cells per unit dry weight, μg / g

[0029] C is the content of MC-LR in the algal cells per unit dry weight in the algal slurry after adding the MlrA recombinant enzyme, μg / g;

[0030] The results showed that the degradation efficiency of the recombinant enzyme NusA-MlrA for MC-LR could reach over 90% after treating the algal slurry for 72 h, while the MC-LR content in the other groups did not change significantly, indicating that only the recombinant enzyme NusA-MlrA had the ability to degrade microcystin LR during the accumulation of cyanobacteria

[0031] Take 1 mg / mL of the crude extract of the recombinant enzyme NusA-MlrA and react with the same volume of MC-LR with a concentration of 10 μg / mL Figure 2 A, Figure 2 B, Figure 2 C respectively represent the HPLC chromatograms of the changes in the reaction of the recombinant enzyme NusA-MlrA with MC-LR at 0, 2, and 30 min. At 0 min of the reaction, MC-LR was not degraded; at 2 min of the reaction, MC-LR was partially degraded into linearized MC-LR, and after 30 min of the reaction, MC-LR was completely degraded

[0032] Example 3: Application of the recombinant enzyme NusA-MlrA in degrading cyanobacterial slurry

[0033] (1) The bloom cyanobacteria were collected from the Guanqiao Pond in the Guanqiao Park of the Institute of Hydrobiology, Chinese Academy of Sciences at the end of October 2022. At this time, the bloom in the pond had reached the middle and late stages. The bloom cyanobacteria on the surface of the pond were fished with a No. 25 phytoplankton net, and after fishing, they were enriched with an 800-mesh sieve. The content of chlorophyll was measured by the acetone method (Nusch, 1980). The specific method was as follows: After taking 1 mL of algal slurry through a GF / C filter membrane, 5 mL of 90% acetone was added, and it was extracted at 4 °C for 24 h, centrifuged at 6000 rpm for 10 min. Finally, the absorbance values at 663, 645, 630, and 750 nm were measured for the supernatant respectively, and the content of chlorophyll a was calculated according to the following formula: Chl-a (μg / L) = [11.64×(OD 663 -OD 750 ) - 2.16×(OD 645 -OD 750 ) + 0.1×(OD 630 -OD 750 )]×v / (V×L)×1000, where the OD value in the formula represents the absorbance value at each wavelength, v represents the volume of constant volume after extraction, V represents the volume of the filtered algal slurry, and L is the optical path of the cuvette. After calculation, the chlorophyll a content of the algal slurry was about 90.1 μg / mL.

[0034] (2) The fresh algal slurry after fishing was evenly divided into six buckets with a volume of 5 L, and each bucket contained 3 L of algal slurry. It was stacked outdoors for 3 days, and the cyanobacteria in the bucket gradually cracked and released various organic substances, metabolites, and microcystins.

[0035] (3) The NusA-MlrA treatment group and the control group were set up, with 3 parallels in each group. NusA-MlrA treatment group: The crude extract of the recombinant enzyme NusA-MlrA was added to the cyanobacteria algal slurry after stacking according to a volume ratio of 10%; Blank control group: The same volume of sterile water was added; MlrA control group: The crude extract of MlrA enzyme without a tag was added. The bucket mouths were sealed with sealing film, and holes were pricked to ensure air circulation. 20 mL of algal slurry was sampled every day and freeze-dried into algal powder with a freeze dryer. One part was used to measure the content of microcystins, and the other part was used to measure the loss of nutrients in the algal powder.

[0036] (4) Weigh 20 mg of algal powder, add 20 mL of 90% methanol solution, add the rotor of a magnetic stirrer, stir at 25 °C for 1 h, centrifuge at 5000 rpm for 20 min, and retain the supernatant; add another 20 mL of 90% methanol to the remaining algal residue, stir magnetically for 1 h, centrifuge at the same speed, retain the supernatant, combine the two supernatants, inject the supernatant solution into the activated C18 chromatographic column (Sep-Tak C18, Waters) using a glass syringe, elute with 20 mL of pure methanol, dry using a rotary evaporator, and then dissolve in 1 mL of 50% chromatographic-grade methanol, transfer to a 2 mL brown CNW sample vial, and determine the total microcystin content by HPLC.

[0037] (5) Extraction and determination of phycocyanin: Weigh 20 mg of freeze-dried algal powder, dissolve it in 1 mL of PBS buffer, add 2 mL of zirconia beads, shake with a shaking crusher for 20 s, with a 10 s interval, shake 5 times; after the algal powder is completely broken, centrifuge at 6000 rpm at room temperature for 3 min, and aspirate the supernatant into a 50 mL brown volumetric flask. Repeat 5 times, make up to 50 mL with PBS, shake well, and measure the absorbance values at 615 nm and 652 nm. The calculation formula is phycocyanin (g / L) = (OD 615 - 0.474 × OD 652 ) / 5.34.

[0038] (6) Extraction and determination of carotenoids in algal cells: Weigh 20 mg of algal powder, add 1 mL of pure methanol, incubate in a 45 °C water bath in the dark for 30 min, centrifuge at 10000 rpm for 3 min, and detect the supernatant at 470 nm, 653 nm, and 666 nm. Chlorophyll a (Chl-a) content (mg / L) = 15.65 × OD 666 - 7.43 × OD 653 , carotenoid content (mg / L) = [1000 × OD 470 - 2.86 × Chl-a - 12.9 × (27.5 × OD 653 - 11.2 × OD 666 )] / 221.

[0039] (7) Extraction and determination of intracellular soluble proteins from algal cells: Weigh 10 mg of algal powder and place it in a 2 mL cryogenic tube. Dissolve it in 400 μL of PBS buffer, then add 4 μL of PMSF protease inhibitor. Add glass beads and zirconium beads, and use a crusher to perform 5 cycles of crushing. Centrifuge at 10,000 rpm for 3 min, and retain the supernatant. Transfer the algal residue to a 15 mL centrifuge glass tube, then add 5 mL of 0.5 M NaOH, and stir magnetically in a water bath at 80 °C for 20 minutes. Centrifuge at 3,000 rpm, transfer the supernatant to a 50 mL volumetric flask again, and add another 5 mL of 0.5 M NaOH. Next, resuspend and wash the algal residue, centrifuge again and add the supernatant to the 50 mL volumetric flask. Finally, add 0.5 M NaOH to make the solution up to 50 mL. Use a BCA protein concentration assay kit (Beyotime, Shanghai) to determine the protein content.

[0040] As Figure 3 shown, after treatment with the crude extract of the recombinant enzyme NusA-MlrA, the total microcystin content in the stacked algal paste decreased significantly. At 24 h, the total microcystin content in the NusA-MlrA treatment group decreased from 429.1 μg / g to 46.3 μg / g, and the degradation efficiency reached 89.2%; the total toxin content at 72 h was 24.3 μg / g, and the toxin degradation rate was 94.3% at this time. There was no significant difference in the total microcystin content between the ddH2O control group and the MlrA control group, indicating that MlrA without a solubility-enhancing tag could not play a role in degrading toxins in the algal paste.

[0041] As Figure 4 shown, there was no significant difference in the phycocyanin content between the ddH2O control group and the recombinant enzyme NusA-MlrA treatment group at 24 h. At 48 h, the phycocyanin content in the recombinant enzyme NusA-MlrA treatment group was slightly lower than that in the ddH2O control group, and the difference between the two groups became insignificant again at 72 h. At 24 h, due to the addition of the crude extract of the recombinant enzyme NusA-MlrA, the soluble protein content in the recombinant enzyme NusA-MlrA treatment group was slightly higher. There was no difference in the soluble protein content between the two groups at 72 h. There was no obvious difference in the carotenoid content between the recombinant enzyme NusA-MlrA treatment group and the ddH2O control group in the algal paste. These results indicate that the addition of the crude extract of the recombinant enzyme NusA-MlrA does not accelerate the loss of nutrients in the bloom-forming cyanobacteria, that is, the risk of harm from the toxins of the bloom-forming cyanobacteria after treatment with the crude extract of the recombinant enzyme NusA-MlrA is significantly reduced, and it can be used for subsequent resource utilization, such as as animal feed or making biological organic fertilizers, etc.

Claims

1. Application of recombinase in degrading microcystin in cyanobacterial pulp, characterized in that, The recombinant enzyme is obtained by linking the solubility-enhancing tag NusA to the N-terminus of the microcystin-degrading enzyme MlrA and linking a histidine tag to the C-terminus. The amino acid sequence of the recombinant enzyme is shown in SEQ ID NO.

2.

2. The application according to claim 1, wherein The method for preparing the recombinant enzyme comprises: constructing a prokaryotic expression vector containing the gene encoding the recombinant enzyme, wherein the nucleotide sequence of the gene is shown in SEQ ID NO.3; transforming the prokaryotic expression vector into competent cells of Escherichia coli to obtain a recombinant enzyme-expressing bacterium, inducing the recombinant enzyme-expressing bacterium with IPTG, disrupting the cells, and collecting the supernatant, which is the crude extract of the recombinant enzyme.

3. A preparation for degrading cyanobacteria algal slurry, characterized in that, It contains the recombinant enzyme as claimed in claim 1.

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