Novel algae-killing and detoxifying nano composite material as well as preparation method and application thereof

Abstract

The invention relates to the field of material preparation and the field of water environment pollution treatment, in particular to a novel algae-killing and detoxifying nano composite material as well as a preparation method and application thereof. The raw materials comprise one or more of algal toxin degrading enzymes MlrA, MlrB or MlrC, and a nanometer material MSN of an immobilized enzyme. The nanometer material MSN is wide in raw material source, low in price, simple to prepare and high in repeatability, and the MSN has high specific activity and activity recovery rate when being used for immobilization of biological enzyme. Meanwhile, the nano composite material Mlrs (at) MSN disclosed by the invention has good storage stability, thermal stability and acid-base stability, and has excellent performance in the aspects of inhibiting growth of microcystis aeruginosa and degrading algal toxin, and the application range of the Mlrs (at) MSN is integrally widened.

Description

Technical Field

[0001] The present invention relates to the fields of material preparation and water environment pollution control, and in particular to a novel algaecidal and detoxifying nanocomposite material and a preparation method and application thereof. Background Art

[0002] With the increasing globalization of industry and the increasing eutrophication of water bodies, outbreaks of cyanobacteria, such as Microcystis, are becoming frequent. This is particularly true in enclosed or semi-enclosed aquatic environments, such as lakes and reservoirs. Periodic blooms of cyanobacteria can release harmful substances, such as microcystins (MCs), posing a significant threat to ecosystem safety, drinking water safety, and even human health. Existing physical, chemical, and biological methods for the treatment of Microcystis and cyanobacterial toxins each have their own advantages and disadvantages, but they generally suffer from low efficiency, difficulty in recycling, secondary pollution, and long treatment cycles. Among the many treatment strategies, enzymatic degradation, due to its high specificity and environmentally friendly nature, is becoming a hot topic in this field.

[0003] In recent years, studies have shown that microcystin degrading enzymes such as MlrA, MlrB and MlrC have unique spatial structures and high molecular weight characteristics. They can specifically break specific bonds of MCs molecules, thereby achieving efficient and directional degradation of microcystin toxins. They have significant microcystin degradation activity and provide new ideas for the management of algal damage and microcystin toxins. However, the above-mentioned degradation enzymes have also shown a series of bottlenecks in the application process. First, as free enzymes, MlrA, MlrB, and MlrC are naturally fragile in conformation and are easily affected by environmental changes such as temperature, pH, and ionic strength, resulting in deactivation and failure. Their stability is insufficient, resulting in a significant reduction in degradation activity and service life. In addition, it is difficult for free enzymes to achieve efficient spatial anchoring, and they cannot be easily recovered and reused after the reaction is completed, which increases the actual operating cost. In particular, under the actual demand for rapid response to water pollution, the free enzymes are seriously restricted in their industrial promotion due to their lack of immobilization and recycling capabilities.

[0004] Based on this, researchers continue to explore a variety of enzyme immobilization technologies to overcome the above difficulties. Enzyme immobilization can not only improve the stability and environmental adaptability of enzyme catalytic performance, but also facilitate recycling and reuse, thereby improving the economy and controllability of the catalytic system. There are many types of immobilized carriers. For example, CN104569099A discloses a rapid detection phenol electrode, which immobilizes tyrosinase by 1-aminopyrene-modified reduced graphene oxide, and utilizes the high specific surface area and good conductivity of graphene to improve detection sensitivity; CN105154428A discloses a carboxylated three-dimensional ordered mesoporous carbon-lysozyme composite material, which immobilizes lysozyme by covalent cross-linking, and utilizes the unique structure of mesoporous carbon to enhance enzyme activity and mass transfer efficiency; CN100359324C discloses a biosensor enzyme functional membrane containing exfoliated manganese dioxide, which utilizes the large specific surface area and conductivity of manganese dioxide to enhance enzyme fixation and activity.

[0005] However, for macromolecular enzymes such as Mlrs, traditional carriers face a series of key challenges: the pore size does not match the enzyme size. Some carriers have narrow pores and cannot fully encapsulate / anchor macromolecular enzymes, resulting in enzyme activity withdrawal and loss, or conformational changes and decreased activity due to spatial constraints; surface-interface mismatch, such as the limited affinity of carbon materials or inorganic particles for macromolecular enzymes, and even electrical repulsion and active center shielding, resulting in low loading capacity and biological activity; poor environmental adaptability. Some organic or inorganic carriers are easily affected by pH and ion interference, and even have biological toxicity, making it difficult to adapt to the needs of continuous application in complex water environments. In addition, the enzyme immobilization methods involved in existing patents mostly focus on small molecule enzymes or specific reaction systems, and do not solve the problem of efficient and stable immobilization of macromolecular polypeptides such as MlrA, MlrB, and MlrC. At present, there are no published patents or literature that anchor MlrA or MlrC to special carriers and can take into account loading efficiency, activity protection, and environmental stability.

[0006] Therefore, it is urgent to develop a new method for immobilizing MlrA, MlrB or MlrC enzymes and a new algaecidal and detoxifying nanocomposite material to break through the limitations of traditional immobilization carriers and give full play to the anchoring advantages to achieve efficient and green control of microcystins and harmful algal blooms. Summary of the Invention

[0007] To achieve one of the above objectives, the present invention provides a novel algaecidal and detoxifying nanocomposite material, its preparation method, and its application. By adjusting the pore size of the mesoporous silica (MSN) to match the molecular size of the MlrA, MlrB, or MlrC enzyme, the present invention achieves efficient loading and significantly improves enzyme immobilization efficiency and activity retention, thereby achieving efficient degradation and inhibition of cyanobacteria and their toxins in water, achieving the technical effect of efficient, green, and safe algae control and algae killing. The technical solution of the present invention is achieved as follows: In a first aspect, the present invention provides a novel algaecidal and detoxifying nanocomposite material comprising the following raw materials: an algae toxin degrading enzyme and an immobilized enzyme nanomaterial, wherein the algae toxin degrading enzyme comprises one or more of MlrA, MlrB and MlrC, and the immobilized enzyme nanomaterial comprises MSN.

[0008] Preferably, the MlrA, MlrB and MlrC all belong to metalloproteases encoded by the mlr gene cluster, and have highly similar amino acid sequences and catalytic cores, which makes them show similarities in substrate binding patterns and catalytic activities, and they can play similar roles in applications such as bioprocessing and drug development.

[0009] Preferably, the preparation method of the nanomaterial MSN comprises the following steps: S1. Mix the template, alkaline catalyst, and pore-enlarging agent evenly at room temperature, stir and heat, then add the silicon source and stir to obtain the MSN core; S2. Add an etchant to the mixed solution, and perform reflux etching under heating conditions to obtain the nanomaterial MSN.

[0010] Preferably, the mass ratio of the algae toxin degrading enzyme to the nanomaterial of the immobilized enzyme in the raw material is 1:(0.25-4).

[0011] Further preferably, the mass ratio of the template, alkaline catalyst, pore expander and silicon source is 1: (0.08-1): (3-6): (1-4); the template includes one or more of cetyltrimethylammonium bromide, cetyltrimethylammonium chloride, sodium lauryl sulfate, and polyvinylpyrrolidone; the alkaline catalyst includes one or more of triethanolamine, ethylenediamine, tetraethylamine or triethylamine; the pore expander includes one or more of triethanolamine, ethylenediamine, triethylamine and trimethylbenzene; and the silicon source is one or more of tetraethoxysilane, triethoxysilane, sodium silicate, and dichlorosilane.

[0012] Further preferably, the stirring time in step S1 is 10-60 min, and the heating temperature is 50-100° C.; the etching temperature in step S2 is 60-100° C., and the etching time is 1-5 h.

[0013] Further preferably, the etchant in step S2 includes any one or more of the following mixed solutions: ① a mixture of ethanol and hydrochloric acid in a volume ratio of 1: (5-20), ② a mixture of methanol and nitric acid in a volume ratio of 1: (5-20), ③ a mixture of ethanol and ammonia water in a volume ratio of 1: (5-20), and ④ a mixture of ethanol and sodium hydroxide in a volume ratio of 1: (5-20).

[0014] In a second aspect, the present invention provides a method for preparing a novel algaecidal and detoxifying nanocomposite material, based on the above-mentioned method for preparing the nanomaterial MSN, further comprising the following steps: S3. Mix MlrA, MlrB or MlrC with MSN in a mixture of glycerol and water, stir at 30-100 rpm for 10-60 min at room temperature, centrifuge and resuspend the precipitate in buffer to obtain CMlrs@MSN.

[0015] Further preferably, the ratio of glycerol to water in step S3 is 1:(1-3).

[0016] Further preferably, the buffer in step S3 includes one or more of phosphate buffer, Tris buffer, carbonate buffer, and borate buffer, and the pH range of the buffer is 6.5-8.0.

[0017] In a third aspect, the present invention further provides the use of the novel algaecidal and detoxifying nanocomposite material according to the first aspect in the field of degrading algal toxins or inhibiting the growth of cyanobacteria.

[0018] Preferably, the algal toxin is selected from at least one of MC-LR, MC-YR, MC-RR and Nodularin, and the cyanobacteria is selected from at least one of Microcystis aeruginosa, Oscillatoria, Nostoc, Anabaena and Aphanizomenon.

[0019] Compared with the prior art, the advantages of the present invention are: (1) By regulating the ratio of pore-enlarging agents and reaction conditions in the MSN synthesis process, the present invention successfully prepared mesoporous silica with adjustable pore size. This MSN not only achieves precise spatial matching with enzyme molecules, but also has a high specific surface area and good biocompatibility, which is conducive to the efficient loading and activity maintenance of subsequent enzymes.

[0020] (2) The present invention achieves efficient immobilization of MlrA and / or MlrB and / or MlrC on MSN carriers. After immobilization, the enzyme molecules are stably anchored within the carrier, significantly improving the enzyme's catalytic activity, reusability, and environmental stability, while avoiding the problems of free enzymes being easily inactivated and difficult to recycle.

[0021] (3) The Mlrs@MSN nanocomposite material of the present invention has good catalytic activity, is recyclable, and has high environmental stability. It can continuously and efficiently remove algal toxins and inhibit the reproduction of cyanobacteria. It has broad application value in the ecological restoration and water purification of eutrophic water bodies such as drinking water sources, lakes, and ponds.

[0022] (4) The Mlrs@MSN composite material of the present invention combines the excellent mass transfer performance of adjustable-pore MSNs with the efficient biocatalytic properties of immobilized enzymes, achieving "simultaneous degradation and sustained inhibition" of cyanobacteria and their toxins. In particular, the present invention is the first to discover that MlrA and / or MlrC can not only degrade algal toxins but also directly inhibit or even kill cyanobacteria through enzymatic action, providing a new and highly effective technical approach for the treatment of cyanobacterial pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 2 is a scanning electron microscope image of MSN and Mlrs@MSN in Example 1 of the present invention. DETAILED DESCRIPTION

[0025] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0026] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0027] In this document, the terms “contain”, “include” or “include” are open expressions, that is, they include the contents specified in the present invention but do not exclude other contents.

[0028] As used herein, the terms "optionally," "optionally," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0029] Mesoporous silica (MSN) is considered a new generation of highly efficient enzyme immobilization supports due to its unique advantages. These materials offer: pore size tunability—the pore size can be adjusted to 2-50 nm through synthetic processes, precisely matching the size of large enzymes to maximize loading capacity and steric stability; high specific surface area and ordered pore structure—providing ample binding sites and excellent substrate / product exchange efficiency; easy surface functionalization—modification with surface functional groups such as hydroxyl and amino groups enables efficient enzyme anchoring and microenvironmental protection; excellent biocompatibility and environmental safety—eliminating the risk of secondary contamination and adapting to a wide range of applications; and excellent chemical and thermodynamic stability, enabling long-term stable operation in complex aqueous environments.

[0030] By regulating the ratio of pore-enlarging agents and reaction conditions during MSN synthesis, the present invention precisely matches the pore size of MlrA and / or MlrB and / or MlrC molecules at the nanoscale, creating suitable space for enzyme entry and embedding, thereby preventing enzyme inactivation due to inappropriate pore size. Because Mlrs are negatively charged and the MSN surface is positively charged, Mlrs tightly bind to the MSN through electrostatic adsorption, disrupting the photosynthetic metabolic pathway of cyanobacteria and efficiently degrading the algal toxins released by them, thereby achieving a cyanobacterial inhibitory and killing effect.

[0031] The following is a clear and complete description of the technical solutions in the embodiments of the present invention, in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0032] It should be noted that in the embodiments of the present invention, the algal toxin degrading enzyme MlrA used was prepared according to the method of Preparation Example 1 in the specification of patent CN110407329B, and the algal toxin degrading enzymes MlrB and MlrC used were prepared according to the preparation method of MlrA.

[0033] Example 1 This example screened and optimized the conditions for preparing a novel algaecidal and detoxifying nanocomposite material, and prepared a nanomaterial MSN with adjustable pore size and an immobilized algal toxin-degrading enzyme (MlrA@MSN), including the following steps: (1) Take 1g of hexadecyltrimethylammonium chloride (purchased from Shanghai Aladdin Co., Ltd.), 80mg of triethanolamine (purchased from Shanghai Aladdin Co., Ltd.), and 3g of trimethylbenzene (purchased from Shanghai Aladdin Co., Ltd.), mix them evenly at room temperature, stir at 80℃ for 30min, then add 2g of tetraethoxysilane (purchased from Shanghai Aladdin Co., Ltd.), and stir at 80℃ for 20min to obtain MSN core; (2) Etching the MSN core: adding a mixture of 4 ml of anhydrous ethanol (analytical grade) and 40 ml of concentrated hydrochloric acid (analytical grade) to the above mixture, reflow etching at 95 °C for 3 h, then centrifuging at 9000 rpm for 5 min, and washing with pure water to obtain the nanomaterial MSN; (3) Five groups of MlrA samples (0.05 mg, 0.1 mg, 0.2 mg, 0.4 mg, and 0.8 mg) were taken and stirred with 0.2 mg MSN in 1 ml of glycerol and water mixture (500 μL glycerol + 500 μL water). The mixture was incubated at 80 rpm for 30 min and centrifuged at 9000 rpm for 5 min. The precipitate was resuspended in Tris buffer at pH = 7 to obtain the nanocomposite material MlrA@MSN, which was recorded as samples 1, 2, 3, 4, and 5.

[0034] The protein content in the supernatant of MlrA@MSN incubated under the above different conditions was measured by high performance liquid chromatography to determine the immobilization rate of the immobilized enzyme. The MlrA@MSN immobilization capacity was calculated according to the following formula (1), and the activity recovery rate was calculated according to formula (2). The results are shown in Table 1.

[0035] (1)

[0036]

[0037] Where c, v, and U are the concentration, volume, and specific activity of MlrA in the supernatant after centrifugation, respectively. c0, v0, and U0 represent the concentration, volume, and specific activity of the MlrA sample, respectively. MSN and V MSN is the concentration and volume of MSN initially added.

[0038] Table 1 Immobilization amount and activity recovery rate of MlrA@MSN synthesized under different conditions

[0039] As shown in Table 1, for a given MSN mass, the loading capacity increases with increasing MlrA mass. When MlrA reaches 0.4 mg / mL, the mass ratio of MlrA to MSN is 1:0.5, and the MSN-immobilized MlrA loading capacity reaches its maximum, as does the activity recovery rate. When MlrA exceeds 0.4 mg / mL, the specific loading rate of MlrA on MSN decreases, and the activity recovery rate no longer increases. Therefore, the synthesis conditions of Sample 4 are suitable for the synthesis of MlrA@MSN.

[0040] The nanocomposite MSN in this embodiment and the MlrA@MSN of sample 4 were scanned by electron microscope. Figure 1As shown, its microscopic morphology is a spherical shape with uniform mesopores on the surface.

[0041] Example 2 This embodiment provides a method for preparing a novel algaecidal and detoxifying nanocomposite material (MlrB@MSN), comprising the following steps: (1) Take 1 g of hexadecyltrimethylammonium chloride (purchased from Shanghai Aladdin Co., Ltd.), 1 g of triethanolamine (purchased from Shanghai Aladdin Co., Ltd.), and 3 g of trimethylbenzene (purchased from Shanghai Aladdin Co., Ltd.), mix them evenly at room temperature, stir at 100 ° C for 10 minutes, then add 4 g of tetraethoxysilane (purchased from Shanghai Aladdin Co., Ltd.), and stir at 80 ° C for 20 minutes to obtain the MSN core; (2) Etching the MSN core: Add 4 ml of anhydrous ethanol (analytical grade) and 20 ml of concentrated hydrochloric acid (analytical grade) to the above mixture, reflux etch at 100 °C for 1 h, then centrifuge at 9000 rpm for 5 min, and wash with pure water to obtain the nanomaterial MSN; (3) A sample of 0.4 mg of MlrB and 0.2 mg of MSN were stirred in 1 ml of a mixture of glycerol and water (500 μL of glycerol + 500 μL of water), incubated at 80 rpm for 30 min, and centrifuged at 9000 rpm for 5 min. The precipitate was resuspended in Tris buffer at pH = 8 to obtain the nanocomposite material MlrB@MSN.

[0042] Through testing and calculation, it was found that the solid loading capacity of MlrB@MSN in Example 2 was 462.39 mg / g, and the activity recovery rate was 47.62%.

[0043] Example 3 This embodiment provides a method for preparing a novel algaecidal and detoxifying nanocomposite material (MlrC@MSN), comprising the following steps: (1) Take 1g of hexadecyltrimethylammonium chloride (purchased from Shanghai Aladdin Co., Ltd.), 500mg of triethanolamine (purchased from Shanghai Aladdin Co., Ltd.), and 6g of trimethylbenzene (purchased from Shanghai Aladdin Co., Ltd.), mix them evenly at room temperature, stir at 50°C for 60 minutes, then add 1g of tetraethoxysilane, and stir at 80°C for 20 minutes to obtain the MSN core; (2) Etching the MSN core: Add 2 ml of anhydrous ethanol (analytical grade) and 40 ml of concentrated hydrochloric acid (analytical grade) to the above mixture, reflux etching at 60 ° C for 5 h, then centrifuge at 9000 rpm for 5 min, and wash with pure water to obtain the nanomaterial MSN; (3) A 0.4 mg MlrC sample and 0.2 mg MSN were stirred in 1 ml of a glycerol-water mixture (500 μL glycerol + 500 μL water), incubated at 80 rpm for 30 min, and centrifuged at 9000 rpm for 5 min. The precipitate was resuspended in a phosphate buffer solution at pH 6.5 to obtain the nanocomposite material MlrC@MSN.

[0044] Through testing and calculation, it was found that the solid loading capacity of MlrC@MSN in Example 2 was 419.96 mg / g, and the activity recovery rate was 43.25%.

[0045] Example 4 This example verifies the good catalytic ability of MlrA@MSN.

[0046] Take the MlrA@MSN prepared in Sample 4 of Example 1 and add MC-LR to 50 mM phosphate buffer (PBS, pH = 7.0) to a final concentration of 0.25 μg·mL -1 Then, MlrA@MSN was added to a final concentration of 12.5 mg·L -1 The reaction mixture was incubated at 25°C for 5 minutes, followed by the addition of phosphoric acid to a final concentration of 0.2% (volume fraction) to terminate the reaction. After termination, the mixture was centrifuged at 12,000 rpm at 4°C for 30 minutes, and the supernatant was collected for analysis. Residual MC-LR was quantified using high-performance liquid chromatography (HPLC) equipped with a C18 column.

[0047] Chromatographic conditions were as follows: detection wavelength 238 nm, flow rate 1.0 mL min -1 , injection volume 50 μL, column temperature 30°C, detection limit 0.1 μg·L -1 The mobile phase consisted of aqueous phosphoric acid (solvent A, 0.05% volume fraction, pH = 3.84) and acetonitrile (solvent B), with the following gradient program: 10% B for 3 minutes; from 0 to 5 minutes, the B phase was linearly increased from 10% to 40%; from 5 to 12 minutes, the B phase increased from 40% to 70%; from 12 to 12.5 minutes, the B phase was reduced from 70% to 10%; and then maintained at 10% B for 0.5 minutes. The results showed that compared with free MlrA, MlrA@MSN increased the degradation efficiency of intracellular and extracellular MC-LR by 66.19% and 67.92% respectively.

[0048] Example 5 This example verifies the good thermal stability and acid and alkali resistance of MlrA@MSN.

[0049] The novel nanocomposite material MlrA@MSN and free enzyme MlrA were incubated at different temperatures (25-70 °C) and different pH values ​​(4.0-11.0) for 1 h. MC-LR was added to 50 mM phosphate buffer (PBS, pH = 7.0) to a final concentration of 0.25 μg mL -1 Then, the MlrA@MSNs incubated with different additions were added to a final concentration of 12.5 mg·L -1 The reaction mixture was incubated at 25°C for 5 minutes, followed by the addition of phosphoric acid to a final concentration of 0.2% (volume fraction) to terminate the reaction. After termination, the mixture was centrifuged at 12,000 rpm for 30 minutes at 4°C, and the supernatant was collected for analysis. High-performance liquid chromatography (HPLC) equipped with a C18 column was used to quantify the residual MC-LR, determine its residual activity, and assess its thermal stability and acid and alkali resistance.

[0050] Table 2 Thermal stability and acid and alkali resistance of MlrA@MSN and MlrA at different temperatures and pH values

[0051] As shown in Table 2, the activity of MlrA@MSN is higher than that of MlrA within the temperature range of 25-70°C, indicating that MlrA@MSN has high thermal stability. Furthermore, the optimal reaction temperature for MlrA@MSN is 40°C, at which its activity is 1.59 times that of MlrA. The activity of MlrA@MSN is also higher than that of MlrA within the pH range of 3.0-11.0, indicating that MlrA@MSN has strong acid and alkali resistance.

[0052] Example 6 This example verifies the algae-inhibiting effect of MlrA@MSN and free enzyme MlrA, taking Microcystis aeruginosa, a common algae in cyanobacteria blooms, as an example. The specific steps are as follows: Microcystis aeruginosa FACHB-905 was cultured to the logarithmic growth phase, and MlrA@MSN and MlrA were added to the Microcystis aeruginosa solution at the same enzyme concentration (the OD680 value of the algal solution was 0.2 and the number of algal cells was 1×10 8 BG11 was used as a blank control, MSN as a material control, MlrA as a free enzyme control, and MlrA@MSN as an experimental group. Samples were taken on days 0, 2, 4, and 6, and after methanol extraction, the chlorophyll a content was measured by colorimetry to calculate the inhibition rate.

[0053] As shown in Table 3, after 4 days of treatment, the cyanobacteria in the experimental groups with the addition of MlrA@MSN and MlrA were significantly inhibited, with inhibition rates reaching 63.94% and 53.39%, respectively. After 6 days of treatment, the cyanobacteria inhibition rates in the experimental groups with the addition of MlrA@MSN and MlrA were 77.06% and 63.91%, respectively. MlrA@MSN showed a more efficient ability to inhibit the growth of Microcystis aeruginosa.

[0054] Table 3 Algae inhibition efficiency of MlrA@MSN and MlrA at different culture times

[0055] Example 7 This example verifies the detoxification effect of MlrA@MSN and free enzyme MlrA, taking Microcystis aeruginosa, a common algae in cyanobacterial blooms, as an example. The specific steps are as follows: Microcystis aeruginosa FACHB-905 was cultured to the logarithmic growth phase, and MlrA@MSN was added to the Microcystis aeruginosa solution (the OD680 value of the algae solution was 0.2 and the number of algae cells was 1×10 8 BG11 served as a blank control, MSN as a material control, MlrA as a free enzyme control, and MlrA@MSN as an experimental group. Samples were taken on the sixth day and tested for microcystin levels inside and outside the cyanobacteria using an ELISA kit to assess their ability to degrade microcystin during the inhibition process. The results, as shown in Table 4, showed that the microcystin concentration in the BG11 blank control reached 672.19±8.21μg / L, while the microcystin concentration in the MlrA@MSN treatment group was only 227.73±3.2μg / L, a decrease of 66.12%. The microcystin concentration in the MlrA treatment group was 370.61±5.4μg / L, demonstrating that MlrA@MSN not only effectively inhibits algal growth but also more efficiently degrades toxins released by algal cells.

[0056] Table 4 Intracellular, extracellular and total algal toxin contents after 6 days of BG11 treatment

[0057] This comparative example verifies the preferred effect of MSN as an immobilized carrier for algal toxin degrading enzymes. Commercial SBA-15 and KIT-6 were selected as reference materials for multi-parameter comparison. The preparation method of Sample 4 in Example 1 differs from that of Sample 4 in that MSN was replaced with SBA-15 and KIT-6, and the rest is the same.

[0058] Comparative results, as shown in Table 5, show that MSNs achieved 2.4 times the enzyme loading of KIT-6, while their specific activity was 1.6 times that of KIT-6. In terms of activity recovery, MSNs achieved 2.2 times that of SBA-15, and their total activity was further enhanced to 2.6 times that of SBA-15. This phenomenon is attributed to the precise design of multiscale interface engineering in this study. Based on the molecular properties of MlrA, the structural compatibility of MSNs was optimized, thereby achieving a synergistic improvement in the performance of the immobilized enzyme.

[0059] Table 5 Comparison of MlrA immobilization efficiency on different carrier materials

[0060] In summary, the present invention proposes a novel algicidal and detoxifying nanocomposite material and a preparation method, which can achieve the immobilization of MlrA, or / and MlrB, or / and MlrC, and solve the defect of instability of free enzymes. At the same time, MlrA@MSN performs better than free enzymes in inhibiting the growth of Microcystis aeruginosa and degrading algal toxins, thereby expanding the application range of MlrA@MSN.

[0061] The embodiments described above are some embodiments of the present invention, rather than all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but merely represents selected embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative work are within the scope of protection of the present invention.

Claims

1. A novel algaecidal and detoxifying nanocomposite material, characterized in that: The invention comprises the following raw materials: algae toxin degrading enzyme and nanomaterials of immobilized enzyme, wherein the algae toxin degrading enzyme comprises one or more of MlrA, MlrB and MlrC, and the nanomaterials of immobilized enzyme comprises MSN.

2. The novel algaecide and detoxification nanocomposite material according to claim 1, characterized in that: The mass ratio of the algae toxin degrading enzyme to the immobilized enzyme nanomaterial in the raw material is 1:(0.25-4).

3. The novel algaecide and detoxification nanocomposite material according to claim 1, characterized in that: The preparation method of the MSN comprises the following steps: S1, the template agent, alkaline catalyst, and pore-enlarging agent are mixed evenly, stirred and heated, and then the silicon source is added and stirred to obtain the MSN core; S2. Add an etchant to the mixed solution, and perform reflux etching under heating conditions to obtain the nanomaterial MSN.

4. The novel algaecide and detoxification nanocomposite material according to claim 3, characterized in that: The mass ratio of the template, alkaline catalyst, pore expander and silicon source is 1: (0.08-1): (3-6): (1-4); the template includes one or more of cetyltrimethylammonium bromide, cetyltrimethylammonium chloride, sodium lauryl sulfate and polyvinylpyrrolidone; the alkaline catalyst includes one or more of triethanolamine, ethylenediamine, tetraethylamine or triethylamine; the pore expander includes one or more of triethanolamine, ethylenediamine, triethylamine and trimethylbenzene; and the silicon source is one or more of tetraethoxysilane, triethoxysilane, sodium silicate and dichlorosilane.

5. The novel algaecide and detoxification nanocomposite material according to claim 3, characterized in that: The stirring time in step S1 is 10-60 minutes, and the heating temperature is 50-100° C.; the etching temperature in step S2 is 60-100° C., and the etching time is 1-5 hours.

6. The novel algaecide and detoxification nanocomposite material according to claim 3, characterized in that: The etchant in step S2 includes any one or more of the following mixed solutions: ① a mixture of ethanol and hydrochloric acid in a volume ratio of 1: (5-20), ② a mixture of methanol and nitric acid in a volume ratio of 1: (5-20), ③ a mixture of ethanol and ammonia water in a volume ratio of 1: (5-20), and ④ a mixture of ethanol and sodium hydroxide in a volume ratio of 1: (5-20).

7. A method for preparing the novel algaecidal and detoxifying nanocomposite material according to claim 1, characterized in that: The following steps are involved: A1. Place the algal toxin degrading enzyme and MSN in a mixture of glycerol and water, stir and centrifuge; A2. Resuspend the precipitate in buffer to obtain Mlrs@MSN.

8. The novel algaecide and detoxification nanocomposite material according to claim 7, characterized in that: The buffer in step A2 includes one or more of phosphate buffer, Tris buffer, carbonate buffer, and borate buffer, and the pH range of the buffer is 6.5-8.

0.

9. Use of the novel algaecidal and detoxifying nanocomposite material according to any one of claims 1 to 8 in the field of degrading algal toxins or inhibiting the growth of cyanobacteria.

10. The use of the novel algaecide and detoxification nanocomposite material according to claim 9, characterized in that: The algal toxin is selected from at least one of MC-LR, MC-YR, MC-RR and Nodularin, and the cyanobacteria is selected from at least one of Microcystis aeruginosa, Oscillatoria, Nostoc, Anabaena and Aphanizomenon.

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