A composite microbial enzyme preparation and preparation method

Abstract

The present invention discloses a composite microbial enzyme preparation and a preparation method thereof, belonging to the field of bioplastic degradation technology. The enzyme preparation is synergistically constructed by a thermosensitive MOFs / Schiff base network composite carrier and black phosphorus quantum dots, with Fe3O4@ZIF-8 as the core and PNIPAM grafted on the surface to form a temperature-responsive MOFs; a three-dimensional network carrier is constructed by Schiff base crosslinking of CMC and CNF; the composite enzyme is loaded into the MOFs pores and co-encapsulated with the black phosphorus quantum dots in the carrier network. The thermosensitive MOFs open the pores to release the enzyme when the compost temperature rises, and the Schiff base network dissociates under acidic conditions to release the enzyme secondary; the black phosphorus quantum dots promote enzyme activity and enhance the carrier response through the photothermal effect; the composite enzyme contains cutinase, alkaline lipase, neutral lipase, α-amylase, saccharifying enzyme, cellulase, laccase, and protease, and can synergistically degrade PLA, PBAT, and starch-based plastics. The present invention realizes dual environmental responsive controlled release, photothermal synergistic catalysis, and synergistic degradation of the enzyme preparation, shortening the plastic degradation cycle under composting conditions.

Description

Technical Field

[0001] The invention belongs to the technical field of bioplastic degradation, and in particular relates to a composite microbial enzyme preparation and a preparation method thereof. Background Art

[0002] To alleviate the current situation of plastic pollution, biodegradable plastics (such as PLA, PBAT, and starch-based materials) have emerged. These plastics can be decomposed into CO2 and water through the action of microorganisms, shortening the degradation cycle to several months to several years. However, their degradation efficiency under natural conditions is still limited by ambient temperature, humidity, and microbial community activity. For example, PLA takes 60-90 days to degrade under composting conditions (50-60°C), while it may take several years in soil at room temperature. In addition, the microplastics produced during the degradation process may still cause secondary harm to the ecosystem.

[0003] Enzyme-catalyzed degradation is considered a green solution to break through the bottleneck of bioplastic degradation. Enzymes can specifically cut polymer chains and accelerate the degradation process, but free enzymes face multiple challenges in practical application:

[0004] Environmental sensitivity: Enzyme activity is easily inhibited by temperature, pH fluctuations and chemical substances (such as surfactants), and is rapidly inactivated in complex environments;

[0005] Uncontrolled release: Although traditional encapsulation technologies (such as calcium alginate microspheres) can protect enzymes in the short term, the release process relies on passive diffusion and is difficult to match the dynamic requirements of plastic degradation;

[0006] Low substrate contact efficiency: Enzyme molecules have difficulty penetrating the dense plastic surface, resulting in low catalytic efficiency.

[0007] Existing studies have attempted to address the above problems through enzyme immobilization (such as MOFs encapsulation and dynamic hydrogels), but there are still limitations:

[0008] Single response mechanism: Most carriers only respond to a single stimulus (such as pH or temperature) and cannot adapt to multiple environmental changes during the degradation process;

[0009] Poor carrier-enzyme compatibility: hydrophilic enzymes do not bind firmly to hydrophobic carriers, resulting in low loading rates or sudden release;

[0010] Secondary pollution risk: Non-degradable carriers (such as synthetic polymers) remain in the environment, offsetting the environmental benefits of enzymatic degradation. Summary of the Invention

[0011] In view of the above situation, in order to overcome the defects of the existing technology, the present invention constructs a thermosensitive MOFs / Schiff base network composite carrier system and combines it with the photothermal synergistic effect of black phosphorus quantum dots to achieve environmental adaptability protection, dynamic controllable release and improved catalytic efficiency of enzyme preparations, thereby solving the technical problems of poor stability, uncontrollable release and low substrate reaction efficiency of traditional enzyme preparations in complex environments.

[0012] In order to achieve the above object, the following technical solution is adopted: On the one hand, the present invention provides a method for preparing a composite microbial enzyme preparation, comprising the following steps:

[0013] (1) Preparation of thermosensitive MOFs: FeCl3·6H2O and sodium citrate were dissolved in ethylene glycol, reacted at 160-180°C for 8-12 h, and centrifuged at 8000-12000 rpm for 10-30 min to obtain Fe3O4 nanoparticles;

[0014] Fe3O4 nanoparticles were dispersed in methanol, 2-methylimidazole and zinc nitrate were added, stirred at 40-50°C for 12-24 hours, centrifuged at 10000-15000 rpm for 15-20 minutes, and the supernatant was discarded to obtain Fe3O4@ZIF-8;

[0015] Fe3O4@ZIF-8 was dispersed in an ethanol solution containing N-isopropylacrylamide (PNIPAM) and initiator AIBN, and reacted at 60-75°C under nitrogen protection for 6-12 hours. The mixture was then centrifuged at 10,000-15,000 rpm for 15-20 minutes, and the solid was washed three times with ethanol to obtain thermosensitive MOFs with PNIPAM grafted on the surface.

[0016] (2) Preparation of three-dimensional network carrier solution: Carboxymethyl cellulose and glutaraldehyde were dissolved in a first phosphate buffer solution at pH = 7.0, reacted at 40-60°C for 6-8 hours, centrifuged at 5000-8000 rpm for 5-10 minutes to remove insoluble matter, and then the solvent was removed under reduced pressure to obtain aldehyde-modified CMC, which was designated as CHO-CMC;

[0017] Nanocellulose was dispersed in a 3-5% volume fraction ethylenediamine ethanol solution, 1-ethyl-(3-dimethylaminopropyl)carbodiimide was added to activate the hydroxyl groups, and the mixture was reacted at 25-35°C for 12-16 hours. The insoluble matter was removed by centrifugation at 5000-8000 rpm for 5-10 minutes, and the solvent was then removed under reduced pressure to obtain amino-modified CNF, which was designated as NH2-CNF.

[0018] CMC-CHO and CNF-NH2 were mixed in a second phosphate buffer solution with a pH of 7.5, stirred at 25-40°C for 4-6 hours, and a three-dimensional network carrier solution was formed by cross-linking through Schiff base bonds;

[0019] (3) Enzyme complex and co-encapsulation of black phosphorus quantum dots: cutinase, alkaline lipase, neutral lipase, α-amylase, saccharifying enzyme, cellulase, laccase, and protease were mixed to form a complex enzyme, added to a third phosphate buffer solution at pH = 7.0, and the thermosensitive MOFs prepared in step (1) were added. The mixture was shaken and adsorbed at 4°C for 12-16 hours, and the unadsorbed enzyme was removed by centrifugation at 8000-12000 rpm for 10-15 minutes to obtain an enzyme-MOFs complex;

[0020] Black phosphorus quantum dots are added to the three-dimensional network carrier solution prepared in step (2) at a mass ratio of 1-5%, and after ultrasonic dispersion for 20-30 minutes, mixed with the enzyme-MOFs complex. The mixed system is then pre-frozen to -80°C and vacuum freeze-dried for 24-48 hours under a vacuum degree of ≤5Pa to obtain the composite microbial enzyme preparation.

[0021] Furthermore, the mass ratio of the cutinase, alkaline lipase, neutral lipase, α-amylase, saccharifying enzyme, cellulase, laccase and protease is 2-4:1-3:1-3:0.5-1.5:0.5-1.5:0.5-1.5:0.3-0.8:0.3-0.8; the enzymatic activity of the cutinase is 200-400 U / mg; the enzymatic activity of the alkaline lipase is 800-1500 U / mg; the enzymatic activity of the neutral lipase is 500-1000 U / mg; the enzymatic activity of the α-amylase is 3000-4500 U / mg; the enzymatic activity of the saccharifying enzyme is 400-800 U / mg; the enzymatic activity of the cellulase is 30-50 FPU / g; the enzymatic activity of the laccase is 100-300 U / mg; and the enzymatic activity of the protease is 2000-4000 U / mg.

[0022] Wherein, the cutinase is expressed by any one of Fusarium solani (Fusarium solani), Thermobifida fusca (thermophilic actinomycetes) or Aspergillus oryzae (Aspergillus oryzae);

[0023] The alkaline lipase is expressed by any one of Pseudomonas aeruginosa, Bacillus subtilis or Yarrowia lipolytica.

[0024] The neutral lipase is expressed by any one of Candida antarctica (Antarctic Candida), Rhizopus oryzae (Rhizopus oryzae) or Burkholderia cepacia (Burkholderia cepacia).

[0025] The α-amylase is expressed by any one of Bacillus licheniformis (Bacillus licheniformis), Aspergillus niger (Aspergillus niger) or Saccharomyces cerevisiae (Saccharomyces cerevisiae).

[0026] The saccharifying enzyme is expressed by any one of Aspergillus kawachii (Kawachi black aspergillus), Rhizopus delemar (Delemar root mold) or Trichoderma reesei (Reesei Trichoderma).

[0027] The cellulase is expressed by any one of Trichoderma viride (Trichoderma viride), Clostridium thermocellum (Clostridium thermocellum) or Penicillium oxalicum (Penicillium oxalicum).

[0028] The laccase is expressed by any one of Trametes versicolor (cloud fungus), Pleurotus ostreatus (oyster mushroom) or Myceliophthora thermophila (thermophilic myceliophthora).

[0029] The protease is expressed by any one of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Streptomyces griseus (Streptomyces griseus) or Aspergillus sojae (Aspergillus sojae).

[0030] Furthermore, the mass ratio of the FeCl3·6H2O, sodium citrate and ethylene glycol is 1:1.2-1.5:20-30.

[0031] Furthermore, the mass ratio of the Fe3O4 nanoparticles, 2-methylimidazole, zinc nitrate and methanol is 1:5-6:1-1.2:50-100.

[0032] Furthermore, the mass ratio of the Fe3O4@ZIF-8, N-isopropylacrylamide, AIBN and ethanol is 1:0.2-0.5:0.01-0.02:10-20.

[0033] Furthermore, the mass ratio of the carboxymethyl cellulose, glutaraldehyde and the first phosphate buffer is 1:0.3-0.6:50-100.

[0034] Furthermore, the mass ratio of the nanocellulose, ethylenediamine ethanol solution and 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1:20-30:0.1-0.2.

[0035] Furthermore, the mass ratio of the CMC-CHO, CNF-NH2 and the second phosphate buffer is 1:0.8-1.2:20-50.

[0036] Furthermore, the mass ratio of the complex enzyme and the third phosphate buffer is 1:10-20; the mass ratio of the black phosphorus quantum dots, the three-dimensional network carrier solution and the enzyme-MOFs complex is 0.01-0.05:1:1-3.

[0037] On the other hand, the present invention also provides a composite microbial enzyme preparation prepared by the preparation method.

[0038] The beneficial effects of the present invention are:

[0039] (1) The present invention loads enzyme molecules into the pores through the microporous structure of MOFs, and prevents direct erosion of the enzyme molecules by water, surfactants, etc. through the size exclusion effect, thereby improving the enzyme activity retention rate. The PNIPAM grafted on the surface of MOFs has a low critical solution temperature. When the temperature rises, a phase transition and contraction occur, and the PNIPAM chain dehydrates and shrinks, resulting in the opening of the MOFs pores, realizing the controlled release of the enzyme, thereby keeping the enzyme stable at low temperatures and releasing it quickly when the temperature rises in plastic compost, which is highly compatible with the temperature requirements for bioplastic degradation.

[0040] (2) CMC-CNF forms a Schiff base cross-linked network as the outer protective shell of the enzyme-MOFs complex. This highly tough structure can resist the damage caused by mechanical stress during composting, ensuring the integrity of the enzyme preparation during transportation and use. The network pore size allows enzyme molecules to diffuse freely, but can block the invasion of large molecular pollutants such as humic acid. In addition, when organic acids are produced in the early stage of plastic degradation, causing the environmental pH to decrease, the bonds break and the network dissociates to release residual enzymes.

[0041] (3) The present invention embeds black phosphorus quantum dots into the surface of MOFs and the middle layer of the cross-linked network of MOFs and Schiff bases. Black phosphorus quantum dots have good biocompatibility, light absorption and photothermal conversion efficiency, and can produce a local thermal effect under light irradiation. This local thermal effect can promote the dehydration and shrinkage of the PNIPAM chain grafted to MOFs, and at the same time make the temperature of the enzyme active center reach the optimal catalytic temperature, thereby accelerating the reaction rate of the enzyme to decompose plastics from two aspects. The hydroxyl groups on the surface of the black phosphorus quantum dots can form hydrogen bonds with the enzyme molecules, inhibiting the conformational changes of the enzyme at high temperatures. In addition, the black phosphorus quantum dots can naturally and gradually degrade into phosphates, which is harmless to the environment and avoids secondary pollution.

[0042] (4) The present invention uses the synergistic action of multiple enzymes, among which cutinase preferentially cuts the ester bond of PLA, lipase degrades the long-chain fatty acids of PBAT, α-amylase and saccharifying enzyme decompose starch-based materials into glucose, cellulase can assist in the degradation of starch and cellulose-based plastics, protease can reduce protein biofilm or impurities on the plastic surface and reduce the hydrophobicity of the plastic, and laccase can catalyze the oxidative decomposition of aromatic compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The results of the degradation rate of plastics by the enzyme preparations of Examples 1-3, Comparative Examples 1-3 and the control group are shown;

[0044] Figure 2 These are the results of the mechanical damage resistance test of the carriers of Examples 1-3 and Comparative Examples 1-3 of the present invention;

[0045] Figure 3 These are the results of enzyme activity determination of the enzyme preparations of Examples 1-3 and Comparative Examples 1-3 of the present invention after long-term storage.

[0046] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention. DETAILED DESCRIPTION

[0047] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only and are not intended to limit the scope of this application.

[0049] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and the experimental materials used in the following examples, unless otherwise specified, are all purchased from commercial channels.

[0050] Example 1

[0051] A method for preparing a composite microbial enzyme preparation comprises the following steps:

[0052] (1) Preparation of thermosensitive MOFs:

[0053] FeCl3·6H2O and sodium citrate were dissolved in ethylene glycol, wherein the mass ratio of FeCl3·6H2O, sodium citrate and ethylene glycol was 1:1.2:20, the mixture was reacted at 160°C for 8 hours, and the mixture was centrifuged at 8000 rpm for 10 minutes to obtain Fe3O4 nanoparticles;

[0054] The Fe3O4 nanoparticles obtained above were dispersed in methanol, and 2-methylimidazole and zinc nitrate were added according to the mass ratio of Fe3O4 nanoparticles, 2-methylimidazole, zinc nitrate and methanol of 1:5:1:50. The mixture was stirred at 40°C for 12 hours, centrifuged at 10,000 rpm for 15 minutes, and the supernatant was discarded to obtain Fe3O4@ZIF-8.

[0055] Fe3O4@ZIF-8 was dispersed in an ethanol solution containing N-isopropylacrylamide and initiator AIBN. The mass ratio of Fe3O4@ZIF-8, N-isopropylacrylamide, AIBN and ethanol was 1:0.2:0.01:10. The mixture was reacted at 60°C under nitrogen protection for 6 hours, then centrifuged at 10,000 rpm for 15 minutes. The solid was washed three times with ethanol to obtain thermosensitive MOFs with PNIPAM grafted on the surface.

[0056] (2) Preparation of three-dimensional network carrier solution:

[0057] Carboxymethyl cellulose and glutaraldehyde were dissolved in a first phosphate buffer solution at pH 7.0, with a mass ratio of carboxymethyl cellulose, glutaraldehyde, and the first phosphate buffer solution being 1:0.3:50. The reaction was carried out at 40° C. for 6 h, and the insoluble matter was removed by centrifugation at 5000 rpm for 5 min. The solvent was then removed under reduced pressure to obtain aldehyde-modified CMC, designated as CHO-CMC.

[0058] Nanocellulose was dispersed in a 3% volume fraction ethylenediamine ethanol solution. The mass ratio of nanocellulose, ethylenediamine ethanol solution, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide was 1:20:0.1. 1-ethyl-(3-dimethylaminopropyl)carbodiimide was added to activate the hydroxyl groups. The reaction was incubated at 25°C for 12 hours, and the insoluble matter was removed by centrifugation at 5000 rpm for 5 minutes. The solvent was then removed under reduced pressure to obtain amino-modified CNF, which was designated as NH2-CNF.

[0059] CMC-CHO and CNF-NH2 were mixed in a second phosphate buffer solution with a pH of 7.5, with a mass ratio of CMC-CHO, CNF-NH2, and the second phosphate buffer solution being 1:0.8:20. The mixture was stirred at 25°C for 4 h to form a three-dimensional network carrier solution through Schiff base cross-linking.

[0060] (3) Co-encapsulation of enzyme complex and black phosphorus quantum dots:

[0061] Cutinase, alkaline lipase, neutral lipase, α-amylase, saccharifying enzyme, cellulase, laccase and protease were mixed in a mass ratio of 2:1:1:0.5:0.5:0.5:0.3:0.3 to form a composite enzyme, wherein the enzyme activity of cutinase was 200 U / mg and the enzyme activity of Fusarium solani (Fusarium solani); alkaline lipase with an activity of 800 U / mg, expressed by Pseudomonas aeruginosa; neutral lipase with an activity of 500 U / mg, expressed by Candida antarctica; α-amylase with an activity of 3000 U / mg, expressed by Bacillus licheniformis; saccharification enzyme with an activity of 400 U / mg, expressed by Aspergillus skawachii (Kawachi Aspergillus niger); cellulase with an activity of 30 FPU / g, expressed by Trichoderma viride (Trichoderma viride); laccase with an activity of 100 U / mg, expressed by Trametes versicolor (Trametes versicolor); and protease with an activity of 2000 U / mg, expressed by Bacillus amyloliquefaciens (Bacillus amyloliquefaciens).

[0062] The complex enzyme was added to a third phosphate buffer solution with a pH of 7.0, with a mass ratio of the complex enzyme to the third phosphate buffer solution of 1:10, and the thermosensitive MOFs prepared in step (1) were added, and the mixture was adsorbed at 4° C. for 12 h, and then centrifuged at 8000 rpm for 10 min to remove the unadsorbed enzyme, thereby obtaining an enzyme-MOFs complex;

[0063] Black phosphorus quantum dots are added to the three-dimensional network carrier solution prepared in step (2) at a mass ratio of 1%, and after ultrasonic dispersion for 20 minutes, they are mixed with the enzyme-MOFs complex. The mass ratio of black phosphorus quantum dots, three-dimensional network carrier solution and enzyme-MOFs complex is 0.01:1:1. Then, the mixed system is pre-frozen to -80°C and vacuum freeze-dried for 24 hours under a vacuum degree of ≤5Pa to obtain the composite microbial enzyme preparation.

[0064] Example 2

[0065] A method for preparing a composite microbial enzyme preparation comprises the following steps:

[0066] (1) Preparation of thermosensitive MOFs:

[0067] FeCl3·6H2O and sodium citrate were dissolved in ethylene glycol, wherein the mass ratio of FeCl3·6H2O, sodium citrate and ethylene glycol was 1:1.5:30, the mixture was reacted at 180°C for 12 hours, and the mixture was centrifuged at 12000 rpm for 30 minutes to obtain Fe3O4 nanoparticles;

[0068] The Fe3O4 nanoparticles obtained above were dispersed in methanol, and 2-methylimidazole and zinc nitrate were added according to the mass ratio of Fe3O4 nanoparticles, 2-methylimidazole, zinc nitrate and methanol of 1:6:1.2:100. The mixture was stirred at 50°C for 24 hours, centrifuged at 15000 rpm for 20 minutes, and the supernatant was discarded to obtain Fe3O4@ZIF-8;

[0069] Fe3O4@ZIF-8 was dispersed in an ethanol solution containing N-isopropylacrylamide and initiator AIBN. The mass ratio of Fe3O4@ZIF-8, N-isopropylacrylamide, AIBN, and ethanol was 1:0.5:0.02:20. The mixture was reacted at 75°C under nitrogen protection for 12 hours. The mixture was then centrifuged at 15,000 rpm for 20 minutes and the solid was washed three times with ethanol to obtain thermosensitive MOFs with PNIPAM grafted on the surface.

[0070] (2) Preparation of three-dimensional network carrier solution:

[0071] Carboxymethyl cellulose and glutaraldehyde were dissolved in a first phosphate buffer solution at pH 7.0, with a mass ratio of carboxymethyl cellulose, glutaraldehyde, and the first phosphate buffer solution being 1:0.6:100. The mixture was reacted at 60°C for 8 h, and the insoluble matter was removed by centrifugation at 8000 rpm for 10 min. The solvent was then removed under reduced pressure to obtain aldehyde-modified CMC, designated as CHO–CMC.

[0072] Nanocellulose was dispersed in a 5% volume fraction ethylenediamine ethanol solution. The mass ratio of nanocellulose, ethylenediamine ethanol solution, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide was 1:30:0.2. 1-ethyl-(3-dimethylaminopropyl)carbodiimide was added to activate the hydroxyl groups. The reaction was incubated at 35°C for 16 hours, and the insoluble matter was removed by centrifugation at 8000 rpm for 10 minutes. The solvent was then removed under reduced pressure to obtain amino-modified CNF, which was designated as NH2-CNF.

[0073] CMC-CHO and CNF-NH2 were mixed in a second phosphate buffer solution with a pH of 7.5, with a mass ratio of CMC-CHO, CNF-NH2, and the second phosphate buffer solution being 1:1.2:50, and stirred at 40°C for 6 h to form a three-dimensional network carrier solution through Schiff base cross-linking.

[0074] (3) Co-encapsulation of enzyme complex and black phosphorus quantum dots:

[0075] Cutinase, alkaline lipase, neutral lipase, α-amylase, saccharifying enzyme, cellulase, laccase and protease are mixed in a mass ratio of 4:3:3:1.5:1.5:1.5:0.8:0.8 to form a composite enzyme, wherein the cutinase has an enzyme activity of 400 U / mg and is expressed by Thermobifida fusca (thermophilic actinomycetes); the alkaline lipase has an enzyme activity of 1500 U / mg and is expressed by Bacillus subtilis (Bacillus subtilis); the neutral lipase has an enzyme activity of 1000 U / mg and is expressed by Rhizopus oryzae (Rhizopus oryzae); the α-amylase has an enzyme activity of 4500 U / mg and is expressed by Aspergillus niger (Aspergillus niger); the saccharifying enzyme has an enzyme activity of 800 U / mg and is expressed by Rhizopus delemar (Rhizopus delemar); cellulase with an activity of 50 FPU / g was expressed by Clostridium thermocellum (Clostridium thermocellum); laccase with an activity of 300 U / mg was expressed by Pleurotus ostreatus (Pleurotus ostreatus); and protease with an activity of 4000 U / mg was expressed by Streptomyces griseus (Streptomyces griseus).

[0076] The complex enzyme was added to a third phosphate buffer solution with a pH of 7.0, with a mass ratio of the complex enzyme to the third phosphate buffer solution of 1:20, and the thermosensitive MOFs prepared in step (1) were added, and the mixture was adsorbed at 4° C. for 16 h, and then centrifuged at 12,000 rpm for 15 min to remove the unadsorbed enzyme, thereby obtaining an enzyme-MOFs complex;

[0077] Black phosphorus quantum dots are added to the three-dimensional network carrier solution prepared in step (2) at a mass ratio of 5%, and after ultrasonic dispersion for 30 minutes, they are mixed with the enzyme-MOFs complex. The mass ratio of black phosphorus quantum dots, three-dimensional network carrier solution and enzyme-MOFs complex is 0.05:1:3. Then, the mixed system is pre-frozen to -80°C and vacuum freeze-dried for 48 hours under a vacuum degree of ≤5Pa to obtain the composite microbial enzyme preparation.

[0078] Example 3

[0079] A method for preparing a composite microbial enzyme preparation comprises the following steps:

[0080] (1) Preparation of thermosensitive MOFs:

[0081] FeCl3·6H2O and sodium citrate were dissolved in ethylene glycol, wherein the mass ratio of FeCl3·6H2O, sodium citrate and ethylene glycol was 1:1.3:25, the mixture was reacted at 170°C for 10 hours, and the mixture was centrifuged at 10,000 rpm for 20 minutes to obtain Fe3O4 nanoparticles;

[0082] The Fe3O4 nanoparticles obtained above were dispersed in methanol, and 2-methylimidazole and zinc nitrate were added according to the mass ratio of Fe3O4 nanoparticles, 2-methylimidazole, zinc nitrate and methanol of 1:5.5:1.1:75. The mixture was stirred at 45°C for 20 h and centrifuged at 12000 rpm for 18 min. The supernatant was discarded to obtain Fe3O4@ZIF-8.

[0083] Fe3O4@ZIF-8 was dispersed in an ethanol solution containing N-isopropylacrylamide and initiator AIBN. The mass ratio of Fe3O4@ZIF-8, N-isopropylacrylamide, AIBN, and ethanol was 1:0.35:0.015:15. The mixture was reacted at 70°C under nitrogen protection for 10 hours, then centrifuged at 12,000 rpm for 18 minutes. The solid was washed three times with ethanol to obtain thermosensitive MOFs with PNIPAM grafted on the surface.

[0084] (2) Preparation of three-dimensional network carrier solution:

[0085] Carboxymethyl cellulose and glutaraldehyde were dissolved in a first phosphate buffer solution at pH 7.0, with a mass ratio of carboxymethyl cellulose, glutaraldehyde, and the first phosphate buffer solution being 1:0.45:75. The mixture was reacted at 50° C. for 7 h, and the insoluble matter was removed by centrifugation at 6500 rpm for 7 min. The solvent was then removed under reduced pressure to obtain aldehyde-modified CMC, designated as CHO-CMC.

[0086] Nanocellulose was dispersed in a 4% volume fraction of ethylenediamine ethanol solution. The mass ratio of nanocellulose, ethylenediamine ethanol solution and 1-ethyl-(3-dimethylaminopropyl)carbodiimide was 1:25:0.15. 1-ethyl-(3-dimethylaminopropyl)carbodiimide was added to activate the hydroxyl groups. The reaction was incubated at 30°C for 15h. The insoluble matter was removed by centrifugation at 6500rpm for 7min. The solvent was then removed under reduced pressure to obtain amino-modified CNF, which was designated as NH2-CNF.

[0087] CMC-CHO and CNF-NH2 were mixed in a second phosphate buffer solution with a pH of 7.5, with a mass ratio of CMC-CHO, CNF-NH2 and the second phosphate buffer of 1:1:35, and stirred at 30°C for 5 h to form a three-dimensional network carrier solution through Schiff base cross-linking.

[0088] (3) Co-encapsulation of enzyme complex and black phosphorus quantum dots:

[0089] Cutinase, alkaline lipase, neutral lipase, α-amylase, saccharification enzyme, cellulase, laccase and protease are mixed in a mass ratio of 3:2:2:1:1:1:0.5:0.5 to form a composite enzyme, wherein the cutinase has an enzyme activity of 300 U / mg and is expressed by Aspergillus oryzae; the alkaline lipase has an enzyme activity of 1150 U / mg and is expressed by Yarrowia lipolytica; the neutral lipase has an enzyme activity of 750 U / mg and is expressed by Burkholderia cepacia; the α-amylase has an enzyme activity of 3750 U / mg and is expressed by Saccharomyces cerevisiae; the saccharification enzyme has an enzyme activity of 600 U / mg and is expressed by Trichoderma reesei; the cellulase has an enzyme activity of 40 FPU / g and is expressed by Penicillium oxalicum (Penicillium oxalicum); laccase with an activity of 200 U / mg was expressed from Myceliophthora thermophila (Myceliophthora thermophila); protease with an activity of 3000 U / mg was expressed from Aspergillus sojae (Aspergillus sojae);

[0090] The complex enzyme was added to a third phosphate buffer solution with a pH of 7.0, with a mass ratio of the complex enzyme to the third phosphate buffer solution of 1:15, and the thermosensitive MOFs prepared in step (1) were added, and the mixture was adsorbed at 4° C. for 16 h, and then centrifuged at 10,000 rpm for 12 min to remove the unadsorbed enzyme, thereby obtaining an enzyme-MOFs complex;

[0091] Black phosphorus quantum dots are added to the three-dimensional network carrier solution prepared in step (2) at a mass ratio of 3%, and after ultrasonic dispersion for 25 minutes, they are mixed with the enzyme-MOFs complex. The mass ratio of black phosphorus quantum dots, three-dimensional network carrier solution and enzyme-MOFs complex is 0.03:1:2. Then, the mixed system is pre-frozen to -80°C and vacuum freeze-dried for 36 hours under a vacuum degree of ≤5Pa to obtain the composite microbial enzyme preparation.

[0092] Comparative Example 1

[0093] The difference between this comparative example and Example 3 is that PNIPAM is not grafted onto Fe3O4@ZIF-8, but Fe3O4@ZIF-8 is used instead of the thermosensitive MOFs with PNIPAM grafted on the surface for subsequent preparation. The rest is the same as Example 3.

[0094] Comparative Example 2

[0095] The difference between this comparative example and Example 3 is that the aldehyde / amino group modification and Schiff base crosslinking steps are omitted, and unmodified carboxymethyl cellulose and nanocellulose are directly used instead of CMC-CHO and CNF-NH2. The rest is the same as Example 3.

[0096] Comparative Example 3

[0097] The difference between this comparative example and Example 3 is that black phosphorus quantum dots are not added in step (3), and the rest are the same as Example 3.

[0098] Result Analysis

[0099] Test Example 1

[0100] Determination of the degradation rate of plastics by enzyme preparations

[0101] PLA, PBAT, and starch-based plastic were made into 10mm×10mm×0.1mm film sheets, respectively, and mixed in equal proportions. The enzyme preparations of Examples 1-3 and Comparative Examples 1-3 were mixed with the substrate at a ratio of 1% (w / w) to simulate a composting environment. The mixture was placed in a constant temperature and humidity chamber at 55°C and 60% humidity, and a sterile compost matrix (composed of the following components in the following volume ratios: 50% wheat straw powder, 30% decomposed chicken manure, 10% peat soil, and 10% perlite) was laid on the bottom. Simultaneously, sunlight was simulated. A control group was set up without adding any enzyme preparation. Ten parallel samples were set up for each group to conduct the test. The degradation rates of different groups were compared after 60 days and 180 days. The results are shown in Table 1. Figure 1 .

[0102] The degradation rates of Examples 1-3 reached over 50% at 60 days and exceeded 89% at 180 days, significantly higher than those of Comparative Examples 1-3. The natural degradation rate in the control group was extremely low. This is because the PNIPAM phase transition is triggered when the compost temperature rises, and the MOFs pores open to release the enzyme, matching the high temperature period of the compost and ensuring that the enzyme acts at high concentrations during the critical stage of plastic degradation. Under simulated light, the black phosphorus quantum dots convert light energy into localized high temperatures, raising the temperature of the enzyme active center to the optimal range and promoting the opening of the MOFs pores, resulting in accelerated enzyme release through both light-heat and temperature-responsiveness.

[0103] Test Example 2

[0104] Enzyme preparation stability verification

[0105] The mixed solution prepared in step (3) of Example 1-3 and Comparative Example 1-3 without vacuum freeze drying was subjected to ultrasonic treatment (200W, 30min). The free enzyme concentration in the solution was measured to evaluate the ability of the carrier to resist mechanical damage. The results are shown in Table 1. Figure 2 .

[0106] The enzyme preparations of Examples 1-3 and Comparative Examples 1-3 were sealed and stored in a dark environment at 20°C and 30% relative humidity. Samples were taken at 6 months and 12 months to test the enzyme activity. Figure 3 .

[0107] from Figure 2 It can be seen that the free enzyme concentrations of Examples 1-3 are all lower than 15 μg / mL, indicating that the three-dimensional network carrier effectively resists ultrasonic damage. Comparative Example 2 has the highest leakage rate, demonstrating the key role of the Schiff base cross-linked network in mechanical stability.

[0108] from Figure 3 As can be seen, Examples 1-3 retained >85% activity after 12 months, thanks to the dual protection of the three-dimensional support and MOFs, which reduced enzyme denaturation. Comparative Example 1, which lacked PNIPAM grafting, experienced enzyme leakage and inactivation due to temperature fluctuations, resulting in an activity retention of only 52.6%. Comparative Example 2, which lacked a modified support, exposed the enzyme to the environment due to the lack of a cross-linked network, resulting in a 12-month activity of only 34.8%. Comparative Example 3, which lacked black phosphorus quantum dots, exhibited a lower activity retention than the Examples due to insufficient local temperature control.

[0109] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

[0110] The present invention and its embodiments are described above. Such description is not restrictive. The drawings show only one embodiment of the present invention, and actual applications are not limited thereto. In short, if a person skilled in the art is inspired by the above, and does not deviate from the purpose of the present invention, any method and embodiment similar to the technical solution without creative design shall fall within the scope of protection of the present invention.

Claims

1. A method for preparing a composite microbial enzyme preparation, characterized in that: The steps include: (1) Preparation of thermosensitive MOFs: FeCl3·6H2O and sodium citrate were dissolved in ethylene glycol, reacted at 160-180°C for 8-12 h, and centrifuged at 8000-12000 rpm for 10-30 min to obtain Fe3O4 nanoparticles; Fe3O4 nanoparticles were dispersed in methanol, 2-methylimidazole and zinc nitrate were added, stirred at 40-50°C for 12-24 hours, centrifuged at 10000-15000 rpm for 15-20 minutes, and the supernatant was discarded to obtain Fe3O4@ZIF-8; Fe3O4@ZIF-8 was dispersed in an ethanol solution containing N-isopropylacrylamide and initiator AIBN, and reacted at 60-75°C under nitrogen protection for 6-12 hours. The mixture was then centrifuged at 10,000-15,000 rpm for 15-20 minutes, and the solid was washed three times with ethanol to obtain thermosensitive MOFs with PNIPAM grafted on the surface. (2) Preparation of three-dimensional network carrier solution: Carboxymethyl cellulose and glutaraldehyde were dissolved in a first phosphate buffer solution at pH = 7.0, reacted at 40-60°C for 6-8 hours, centrifuged at 5000-8000 rpm for 5-10 minutes to remove insoluble matter, and then the solvent was removed under reduced pressure to obtain aldehyde-modified CMC, which was designated as CHO-CMC; Nanocellulose was dispersed in a 3-5% volume fraction ethylenediamine ethanol solution, 1-ethyl-(3-dimethylaminopropyl)carbodiimide was added to activate the hydroxyl groups, and the mixture was reacted at 25-35°C for 12-16 hours. The insoluble matter was removed by centrifugation at 5000-8000 rpm for 5-10 minutes, and the solvent was then removed under reduced pressure to obtain amino-modified CNF, which was designated as NH2-CNF. CHO-CMC and NH2-CNF are mixed in a second phosphate buffer solution with a pH of 7.5, stirred at 25-40°C for 4-6 hours, and cross-linked by Schiff base bonds to form a three-dimensional network carrier solution; (3) Enzyme complex and co-encapsulation of black phosphorus quantum dots: cutinase, alkaline lipase, neutral lipase, α-amylase, saccharifying enzyme, cellulase, laccase and protease were mixed to form a complex enzyme, added to a third phosphate buffer solution of pH = 7.0, and the thermosensitive MOFs prepared in step (1) were added. The mixture was shaken and adsorbed at 4°C for 12-16 hours, and the unadsorbed enzyme was removed by centrifugation at 8000-12000 rpm for 10-15 minutes to obtain an enzyme-MOFs complex; Black phosphorus quantum dots are added to the three-dimensional network carrier solution prepared in step (2) at a mass ratio of 1-5%, and after ultrasonic dispersion for 20-30 minutes, mixed with the enzyme-MOFs complex. The mixed system is then pre-frozen to -80°C and vacuum freeze-dried for 24-48 hours under a vacuum degree of ≤5Pa to obtain the composite microbial enzyme preparation.

2. The method for preparing the composite microbial enzyme preparation according to claim 1, wherein: The mass ratio of the cutinase, alkaline lipase, neutral lipase, α-amylase, saccharifying enzyme, cellulase, laccase and protease is 2-4:1-3:1-3:0.5-1.5:0.5-1.5:0.5-1.5:0.3-0.8:0.3-0.8; the enzymatic activity of the cutinase is 200-400 U / mg; the enzymatic activity of the alkaline lipase is 800-1500 U / mg; the enzymatic activity of the neutral lipase is 500-1000 U / mg; the enzymatic activity of the α-amylase is 3000-4500 U / mg; the enzymatic activity of the saccharifying enzyme is 400-800 U / mg; the enzymatic activity of the cellulase is 30-50 FPU / g; the enzymatic activity of the laccase is 100-300 U / mg; and the enzymatic activity of the protease is 2000-4000 U / mg.

3. The method for preparing the composite microbial enzyme preparation according to claim 2, wherein: The mass ratio of the FeCl3·6H2O, sodium citrate and ethylene glycol is 1:1.2-1.5:20-30.

4. The method for preparing the composite microbial enzyme preparation according to claim 3, wherein: The mass ratio of the Fe3O4 nanoparticles, 2-methylimidazole, zinc nitrate and methanol is 1:5-6:1-1.2:50-100.

5. The method for preparing the composite microbial enzyme preparation according to claim 4, wherein: The mass ratio of the Fe3O4@ZIF-8, N-isopropylacrylamide, AIBN and ethanol is 1:0.2-0.5:0.01-0.02:10-20.

6. The method for preparing the composite microbial enzyme preparation according to claim 5, characterized in that: The mass ratio of the carboxymethyl cellulose, glutaraldehyde and the first phosphate buffer is 1:0.3-0.6:50-100.

7. The method for preparing the composite microbial enzyme preparation according to claim 6, wherein: The mass ratio of the nanocellulose, ethylenediamine ethanol solution and 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1:20-30:0.1-0.

2.

8. The method for preparing the composite microbial enzyme preparation according to claim 7, characterized in that: The mass ratio of the CHO-CMC, NH2-CNF and the second phosphate buffer is 1:0.8-1.2:20-50.

9. The method for preparing the composite microbial enzyme preparation according to claim 8, characterized in that: The mass ratio of the complex enzyme and the third phosphate buffer is 1:10-20; the mass ratio of the black phosphorus quantum dots, the three-dimensional network carrier solution and the enzyme-MOFs complex is 0.01-0.05:1:1-3.

10. A composite microbial enzyme preparation, characterized in that: Prepared according to the preparation method according to any one of claims 1 to 9.

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