Microbial enzyme preparation for polyester plastic degradation and preparation method thereof

Abstract

The invention relates to the technical field of preparation of microbial enzyme preparations, in particular to a microbial enzyme preparation for polyester plastic degradation and a preparation method of the microbial enzyme preparation. The present invention relates to a microbial composition comprising at least one microbial strain engineered to enhance PETase expression and maintain activity under hypoxic conditions, and a nanocarrier, and also comprising chitosan. The expression level of microorganisms on PETase is enhanced through gene modification, the degradation efficiency of polyester plastics is improved, a nano-carrier material and a chitosan protective film are introduced, the stability and bioavailability of the enzyme preparation are improved, and the prepared microbial enzyme preparation can keep activity in a low-oxygen, illumination or complex environment, and can be used for preparing a biological enzyme preparation. And secondly, the whole preparation process does not depend on harmful chemicals, the green chemistry principle is met, and environmental protection and resource recycling are facilitated.

Description

Technical Field

[0001] The invention relates to the technical field of microbial enzyme preparations, in particular to a microbial enzyme preparation for degrading polyester plastics and a preparation method thereof. Background Art

[0002] Polyester plastic is a polymer material synthesized by polycondensation of dibasic acid and diol. The most common polyester is polyethylene terephthalate (PET or PETE), which has a wide range of applications in industry and daily life. For example: transparent beverage bottles, food packaging (jelly cups, yogurt boxes, etc.), followed by other applications such as textile fibers, films, engineering plastics, composite materials, coatings and paints, adhesives, etc.

[0003] Although traditional physical and chemical methods can decompose these plastics to a certain extent, they usually require high temperature and high pressure conditions, are costly and have limited efficiency. Biodegradation, as an environmentally friendly alternative, has received widespread attention in recent years. However, existing biodegradation methods still face many challenges in practical applications, such as slow degradation rate and sensitivity to environmental conditions. Summary of the invention

[0004] The purpose of the present invention is to provide an efficient, stable and adaptable microbial enzyme preparation for polyester plastic degradation and a preparation method thereof, by selecting a specific microbial strain and genetically modifying it to enhance its expression of polyester plastic degradation enzymes (such as PETase); at the same time, introducing nano-carrier materials and chitosan protective films to improve the stability, activity and bioavailability of the enzyme preparation; and through nutrient microcapsule technology to ensure that the microorganisms can survive for a long time in a complex environment and effectively degrade polyester plastics, so as to solve the problems raised in the above-mentioned background technology.

[0005] To achieve the above object, on the one hand, the present invention provides a microbial enzyme preparation for polyester plastic degradation, comprising the following raw materials:

[0006] Main strains: Ideonella sakaiensis DSM 27953, Comamonas sp. ATCC 43086, cyanobacteria (Synechocystis sp.), green algae (Chlamydomonas reinhardtii) or Escherichia coliBL21 (DE3), which have the ability to naturally express PETase;

[0007] Helper strains: Use CRISPR-Cas9 or other gene editing tools to modify the above strains to enhance PETase expression and maintain activity under hypoxic conditions; for cyanobacteria and green algae, phytochromes are also introduced to activate the degradation process in response to light conditions;

[0008] Carrier materials: Polylactic acid nanoparticles, titanium dioxide nanoparticles or gold nanoparticles (AuNPs) are selected as carrier materials. These materials not only have good biocompatibility, but also can be surface-modified by physical and chemical methods to specifically bind to the surface of microbial cells. Nanocarrier materials are prepared by emulsified solvent evaporation method, sol-gel method or chemical reduction method, and the nanocarriers are connected to microbial cells through connection strategies such as biotin-streptavidin system, and then treated with chitosan solution and solidified to form a protective film.

[0009] Nutrients: Prepare nutrient microcapsules containing urea, potassium dihydrogen phosphate and trace element mixtures, or glucose and trace element mixtures, as well as amino acid mixtures and vitamins, and encapsulate them in gel beads to ensure that microorganisms can survive for a long time in complex environments and effectively degrade polyester plastics

[0010] Chitosan solution: Encapsulate nanocarrier materials and / or microbial cells to improve the stability and bioavailability of enzyme preparations. The chitosan solution has a concentration of 0.5% w / v to 2% w / v, and is solidified by adding a calcium chloride solution with a concentration of 1.8% w / v to 2.2% w / v to form a stable protective film, thereby enhancing the resistance of the enzyme preparation to changes in environmental conditions. The introduction of 0.1% w / v to 0.3% w / v of polyethyleneimine into the chitosan solution significantly enhances the binding force between chitosan and nanocarrier materials and nutrients, forming a more stable and efficient protective film and adsorption layer; polyethyleneimine not only promotes the secondary binding between chitosan and nanoparticles, but also enhances the adhesion of nutrients, ensuring the growth and metabolic needs of microorganisms throughout the degradation cycle. This improvement has significantly improved the long-term effectiveness of the entire system in complex environments.

[0011] On the other hand, the present invention provides a method for preparing a microbial enzyme preparation for polyester plastic degradation as described in any one of the above, comprising the following steps:

[0012] Selecting microbial strains;

[0013] Genetically modifying microbial strains to enhance PETase expression to form engineered microbial suspensions;

[0014] preparing nano-carrier materials and mixing them with chitosan solution to form a stable protective film;

[0015] The microbial strain and the nano-carrier material are combined in a volume ratio of 1:1 by mixing, and then nutrient microcapsules or nutrient components with a final concentration of 0.8-1.5 mg / mL are added to obtain a microbial enzyme preparation.

[0016] Preferably, the method further comprises preparing the nanocarrier material by using an emulsified solvent evaporation method, a sol-gel method or a chemical reduction method, and connecting the nanocarrier to the microbial cells by a connection strategy such as a biotin-streptavidin system, followed by treatment with a chitosan solution and curing to form a protective film.

[0017] Preferably, the method further comprises preparing nutrient microcapsules, and encapsulating the nutrient components by using microcapsule technology or smart gel bead technology, so as to provide immediate or sustained-release nutrient support after mixing with the microbial enzyme preparation.

[0018] The prepared enzyme preparation is applied to the field of polyester plastic waste treatment, especially the degradation treatment of polyester plastics in landfills, open-air plastic waste accumulation areas or other environments, so as to reduce environmental pollution and promote resource recycling.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] In the microbial enzyme preparation for polyester plastic degradation and the preparation method thereof, the expression level of microorganisms on PETase is enhanced through genetic modification, the degradation efficiency of polyester plastic is improved, and the stability and bioavailability of the enzyme preparation are improved by introducing nano-carrier materials and chitosan protective films. The prepared microbial enzyme preparation can maintain activity in low oxygen, light or complex environments and is widely applicable to different application scenarios. Secondly, the entire preparation process does not rely on harmful chemicals, conforms to the principles of green chemistry, and is conducive to environmental protection and resource recycling.

[0021] In different systems, chitosan not only works alone, but also produces synergistic effects with other ingredients to further enhance the overall performance. For example, in EADS, chitosan is combined with a pH-sensitive trigger mechanism to ensure the effective release of enzymes under appropriate conditions; in PSAS, chitosan works together with titanium dioxide nanoparticles to enhance the binding force with the plastic surface; in SMNF, chitosan is combined with smart gel beads to provide a stable nutrient supply. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a bar graph of average weight loss rate at different pH values ​​of Test Example 1 of the present invention;

[0023] Figure 2 It is a line graph showing changes in light transmittance of the PET film under different light cycles of Experimental Example 2 of the present invention;

[0024] Figure 3 This is a line graph showing the concentration changes of small molecule degradation products in Experimental Example 3 of the present invention. DETAILED DESCRIPTION

[0025] The following will be combined with the accompanying drawings in the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0026] Example 1

[0027] This embodiment provides a microbial enzyme preparation (enhanced anaerobic degradation system) for polyester plastic degradation, and the preparation method is as follows:

[0028] 1. Microbial Selection

[0029] Main strains: Ideonella sakaiensis DSM 27953;

[0030] Helper strain: Comamonas sp. ATCC 43086;

[0031] Using CRISPR-Cas9 technology to transform Ideonella sakaiensis and Comamonas sp., enhancing PETase expression and maintaining activity under hypoxic conditions;

[0032] Culture conditions: The strain was fermented in LB liquid medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride) with the temperature set at 30°C and the pH value set at 7.0. The dissolved oxygen level was maintained below 2 mg / L by controlling the stirring rate and ventilation volume to simulate an anaerobic cycle.

[0033] 2. Nanocarrier Selection

[0034] Carrier material: polylactic acid nanoparticles, average molecular weight 50kDa;

[0035] The emulsified solvent evaporation method was used to prepare the polylactic acid nanoparticles: polylactic acid nanoparticles were dissolved in an organic solvent (dichloromethane, concentration of 5 mg / mL), added to an aqueous phase containing a surfactant (Tween 80, 0.5% v / v) and stirred at high speed (10000 rpm, 1 h) to form an emulsion, and then the organic solvent was evaporated (vacuum dried at 40°C overnight) to obtain polylactic acid nanoparticles;

[0036] The enzyme was linked to the polylactic acid chain through an ester bond, and terephthalate glycol (PETG) was selected as a model substrate for testing to ensure effective release of the enzyme at pH < 5;

[0037] The polylactic acid nanoparticle suspension is mixed with chitosan solution (0.5% w / v) and polyethyleneimine (0.1% w / v), and after thorough stirring, calcium chloride solution (1.8% w / v) is added to solidify to form a stable protective film; polyethyleneimine is added to the chitosan solution, and the cationic properties of polyethyleneimine and the anionic properties of chitosan are used to form a complex to enhance the binding force between chitosan and polylactic acid nanoparticles. The amino functional groups on polyethyleneimine can react with the carboxyl groups on the polylactic acid chain to form stable amide bonds, further improving the stability and mechanical strength of the protective film.

[0038] 3. Nutritional Supply

[0039] Ingredients: urea (0.5g / L), potassium dihydrogen phosphate (0.2g / L) and trace element mixture ( , 1 mg / L each);

[0040] Using microencapsulation technology, the above ingredients are dissolved in a solvent, and then added into a solidification bath (calcium chloride solution, 2% w / v) containing a gelling agent (gelatin or sodium alginate, 3% w / v) by a microinjection pump at a rate of 20 drops / min. The ingredients are allowed to stand at room temperature for 30 minutes, taken out and washed to form nutrient microcapsules. Polyethyleneimine can combine with the negatively charged groups (carboxyl groups) on the surface of the nutrient microcapsules to ensure that the nutrient microcapsules are more tightly attached to the surface of the microorganisms.

[0041] 4. Assembly and Application

[0042] The above engineered microbial suspension (OD 600 =0.8) and polylactic acid nanoparticles (10 mg / mL) were mixed evenly at a volume ratio of 1:1, and then nutrient microcapsules (final concentration of 0.8 mg / mL) were added and stirred thoroughly to form a microbial enzyme preparation;

[0043] After crushing the polyester plastic waste, the microbial enzyme preparation is evenly applied to the new fill layer of the landfill through a spraying device, and a wireless sensor network is used to monitor and adjust the placement strategy.

[0044] Example 2

[0045] This embodiment provides a microbial enzyme preparation (light-responsive surface attachment system) for polyester plastic degradation, and the preparation method is as follows:

[0046] 1. Microbial Selection

[0047] Strains: Choose blue algae (Synechocystis sp.) or green algae (Chlamydomonas reinhardtii) that can express PETase and carry photosensitive pigments;

[0048] Culture conditions: Select BG11 medium ( 1.5g / L, 0.04g / L, 0.075g / L, 0.036g / L, 0.006g / L, 0.001 g / L, strength sodium bicarbonate 0.18 g / L), cultured under a photoperiod of 16 h light / 8 h dark, and a light intensity of 50 μmolphotons m -2 s -1 .

[0049] 2. Nanocarrier Selection

[0050] Support material: titanium dioxide, anatase phase, average particle size 20nm;

[0051] The sol-gel method was used to prepare the titanium dioxide nanoparticles: tetrabutyl titanate (TBOT) was used as a precursor, which was hydrolyzed in anhydrous ethanol to generate titanium dioxide sol, and then calcined in a muffle furnace at 500 °C for 2 h to obtain titanium dioxide nanoparticles.

[0052] Titanium dioxide nanoparticles were treated with a silane coupling agent (3-aminopropyltriethoxysilane: APTES) to carry amino functional groups, which were then linked to the cell surface of cyanobacteria or green algae via a biotin-streptavidin system;

[0053] The titanium dioxide nanoparticle suspension was mixed with chitosan solution (1% w / v) and polyethyleneimine (0.2% w / v), and calcium chloride solution (2% w / v) was added after thorough stirring to solidify and form a stable adsorption layer; the cationic properties of polyethyleneimine and the anionic properties of chitosan were used to form a complex to enhance the binding force between chitosan and titanium dioxide nanoparticles; the amino functional groups on polyethyleneimine can react with the hydroxyl groups on the surface of titanium dioxide nanoparticles to form stable covalent bonds, further improving the stability and light responsiveness of the adsorption layer.

[0054] 3. Nutritional Supply

[0055] Ingredients: Glucose (1g / L), trace element mixture ( , 1 mg / L each);

[0056] The above ingredients (final concentration 1.2 mg / mL) are directly dissolved in the suspension of microbial enzyme preparation to ensure timely supply to the microorganisms; polyethyleneimine can combine with the negatively charged groups (phosphate groups) in the nutrients to ensure that the nutrients are more tightly attached to the surface of the algae.

[0057] 4. Assembly and Application

[0058] The activated photosensitive algae (OD730 = 0.5) and surface-modified titanium dioxide nanoparticles (1 mg / mL) were mixed evenly at a volume ratio of 1:1 to prepare a microbial enzyme preparation in the form of a suspension;

[0059] In areas with direct sunlight during the day, the microbial enzyme preparation is applied to the exposed plastic surface by coating or spraying, and light is used to activate the photosensitive pigment to promote enzyme activity.

[0060] Example 3

[0061] This embodiment provides a microbial enzyme preparation (self-assembled multi-enzyme nanofactory) for polyester plastic degradation, and the preparation method is as follows:

[0062] 1. Microbial Selection

[0063] Bacteria: Escherichia coli BL21 (DE3), introduced with pET28a-PETase-MHETase plasmid, and added with suicide plasmid containing I-SceI endonuclease gene;

[0064] Culture conditions: Fermentation was carried out in LB liquid medium, and IPTG was added during the induction phase to initiate exogenous gene expression (when OD 600 When the pH reached 0.6, IPTG was added to a final concentration of 1 mM), while maintaining the temperature at 37 °C and the pH at 7.0.

[0065] 2. Nanocarrier Selection

[0066] Carrier material: gold nanoparticles (AuNPs), with a diameter of about 20 nm;

[0067] The AuNPs were prepared by chemical reduction method: sodium citrate was used as a reducing agent, and sodium citrate solution was quickly added to the boiling aqueous solution of tetrachloroauric acid until the color changed to dark purple, indicating the formation of AuNPs;

[0068] AuNPs were modified with a biotin-streptavidin system to enable them to specifically bind to the surface of bacterial cells expressing corresponding tags (e.g., His tags);

[0069] The AuNPs suspension was mixed with chitosan solution (2% w / v) and polyethyleneimine (0.3% w / v), and calcium chloride solution (2.2% w / v) was added after thorough stirring to solidify and form a stable composite structure. The cationic properties of polyethyleneimine and the anionic properties of chitosan were used to form a complex to enhance the binding force between chitosan and gold nanoparticles. The amino functional groups on polyethyleneimine can react with the thiol groups on the surface of gold nanoparticles to form stable covalent bonds, further improving the stability and mechanical strength of the composite structure.

[0070] 3. Nutritional Supply

[0071] Ingredients: Amino acid mixture (L-glutamic acid, L-lysine, L-arginine), vitamins (vitamin B 12 , folic acid);

[0072] Using smart gel bead technology, the nutrients are dissolved in a sodium alginate solution (3% w / v), and then a microinjection pump is used to drip into a calcium chloride solution (2% w / v) at a rate of 20 drops / min to solidify into gel beads. After standing at room temperature for 30 minutes, the nutrient gel beads are taken out and cleaned to obtain nutrient gel beads; polyethyleneimine can combine with the negatively charged groups (carboxyl groups) in sodium alginate to ensure that the nutrient gel beads are more tightly attached to the bacterial surface.

[0073] 4. Assembly and Application

[0074] E. coli expressing multiple degradation enzymes (OD 600 =0.8) and the modified AuNPs (1 mg / mL) were mixed evenly at a volume ratio of 1:1 to allow them to self-assemble into a stable complex; then nutrient gel beads (final concentration of 1 mg / mL) were added and mixed thoroughly to form a microbial enzyme preparation;

[0075] Small drones or other mechanical equipment are used to evenly spread microbial enzyme preparations throughout the landfill, and they are gradually activated by changes in pressure and humidity generated during the compaction of the garbage.

[0076] In the above-mentioned embodiments 1-3, the introduced chitosan plays a vital role in the development and application of microbial enzyme preparations. It not only improves the performance of the system, but also enhances its environmental adaptability and sustainability. In the enhanced anaerobic degradation system, chitosan can form a protective film on the surface of the nanocarrier to enhance the stability and activity of the enzyme, especially in acidic and low pH environments, significantly improving the acid resistance and thermal stability of key enzymes such as PETase, ensuring that they can still work efficiently in harsh environments. In the light-responsive surface attachment system, the film formed by chitosan can not only partially block ultraviolet rays and protect the internal enzymes from light damage, but also reflect part of the light, help maintain a suitable working temperature, enhance the bonding strength between titanium dioxide nanoparticles and the plastic surface, and ensure longer effective contact; at the same time, adjust the moisture content to maintain the activity of algae, thereby extending the service life of the system. In the self-assembled multi-enzyme nanofactory, chitosan molecules contain a large number of amino functional groups, which can promote the reaction rate in the electron transport chain and accelerate the degradation process. In addition, as part of a sustained-release carrier, chitosan can control the release rate of nutrients, ensuring that microorganisms receive balanced nutritional support throughout their life cycle, significantly improving the mechanical strength of the smart gel beads, preventing rupture and resulting in nutrient loss, and ensuring the long-term stability and efficient operation of the system.

[0077] In addition, chitosan has natural antibacterial properties, which can inhibit the growth of other non-target microorganisms, reduce the interference of miscellaneous bacteria, maintain the specificity and purity of the system, and through physical cross-linking, chitosan helps the aggregation of microbial cells, increases local concentration, and improves degradation efficiency. As a natural material, chitosan is completely degradable and will not cause secondary pollution to the environment. Its biocompatibility and environmental friendliness make it an ideal additive, in line with the principles of green chemistry.

[0078] Furthermore, by introducing polyethyleneimine, the binding force between chitosan and nano-carrier materials and nutrients is significantly enhanced, forming a more stable and efficient protective film and adsorption layer; polyethyleneimine not only promotes the secondary binding between chitosan and nanoparticles, but also enhances the adhesion of nutrients, ensuring the growth and metabolic needs of microorganisms throughout the degradation cycle. This improvement has significantly enhanced the long-term effectiveness of the entire system in complex environments.

[0079] Example 4

[0080] This embodiment provides a microbial enzyme preparation (enhanced anaerobic degradation system) for polyester plastic degradation, and the preparation method is as follows:

[0081] 1. Microbial Selection

[0082] Same as Example 1.

[0083] 2. Nanocarrier Selection

[0084] Same as Example 1.

[0085] 3. Nutritional Supply

[0086] Same as Example 1.

[0087] 4. Assembly and Application

[0088] The above engineered microbial suspension (OD 600 =1.2) and polylactic acid nanoparticles (15 mg / mL) were mixed evenly at a volume ratio of 1:1, and then nutrient microcapsules (final concentration of 1.2 mg / mL) were added and stirred thoroughly to form a microbial enzyme preparation;

[0089] After crushing the polyester plastic waste, the microbial enzyme preparation is evenly applied to the new fill layer of the landfill through a spraying device, and a wireless sensor network is used to monitor and adjust the placement strategy.

[0090] Example 5

[0091] This embodiment provides a microbial enzyme preparation (enhanced anaerobic degradation system) for polyester plastic degradation, and the preparation method is as follows:

[0092] 1. Microbial Selection

[0093] Same as Example 1.

[0094] 2. Nanocarrier Selection

[0095] Same as Example 1.

[0096] 3. Nutritional Supply

[0097] Increase urea to 1g / L, potassium dihydrogen phosphate to 0.5g / L, and trace element mixture to 3mg / L each.

[0098] 4. Assembly and Application

[0099] The above engineered microbial suspension (OD 600 =0.4) and polylactic acid nanoparticles (5 mg / mL) were mixed evenly at a volume ratio of 1:1, and then nutrient microcapsules (final concentration of 1.5 mg / mL) were added and stirred thoroughly to form a microbial enzyme preparation;

[0100] After crushing the polyester plastic waste, the microbial enzyme preparation is evenly applied to the new fill layer of the landfill through a spraying device, and a wireless sensor network is used to monitor and adjust the placement strategy.

[0101] Test Example 1

[0102] A degradation test was carried out under hypoxic or anaerobic conditions using the microbial enzyme preparation (EADS preparation) provided in Example 1: the crushed polyester plastic (PET) samples were treated with the EADS preparation, and different pH values ​​(4.0, 5.0, 6.0, 7.0) and temperatures (25°C, 30°C, 35°C) were set to simulate a landfill environment.

[0103] Test method: Use weight loss method, the initial weight is 10g, and the final weight is calculated after 30 days.

[0104] Table 1 Weight loss at different pH values ​​and at the same temperature

[0105] pH Temperature (℃) Final weight (g) Weight loss percentage (%) 4.0 30 8.5 15 5.0 30 7.8 22 6.0 30 7.2 28 7.0 30 6.9 31

[0106] Table 2 Enzyme activity distribution under different temperature and pH combinations

[0107] pH value / temperature (℃) 25 30 35 4.0 1.2 U / mL 1.5 U / mL 1.8 U / mL 5.0 1.4 U / mL 1.7 U / mL 2.0 U / mL 6.0 1.6 U / mL 2.0 U / mL 2.2 U / mL 7.0 1.8 U / mL 2.0 U / mL 2.5 U / mL

[0108] According to Table 1, Table 2 and Figure 1As shown in the figure, under the same temperature of 30℃ and different pH conditions, EADS preparation significantly improved the degradation efficiency of PET; as the pH value increased from 4.0 to 7.0, the weight loss rate gradually increased, indicating that EADS performed best in a near-neutral environment. Under pH 7.0 conditions, after 30 days of treatment, the weight of the PET sample decreased by 31%, proving that EADS preparation has efficient degradation ability in a suitable environment.

[0109] Test Example 2

[0110] The degradation test was carried out under light conditions using the microbial enzyme preparation (PSAS preparation) provided in Example 2: the PET film was treated with the PSAS preparation and placed under controlled light conditions (16 h light / 8 h dark cycle, light intensity of 50 μmol photons m -2 s -1 ).

[0111] Test method: Use optical density measurement method, the initial transmittance is 10%, and the final transmittance is calculated after 30 days.

[0112] Table 3 Final transmittance under different conditions

[0113] condition Final light transmittance (%) illumination 45 dark 12

[0114] According to Table 3 and Figure 2 As shown, the PSAS preparation showed a significant increase in transmittance under light conditions, rising from the initial 10% to 45%, indicating that the PET film was effectively degraded, resulting in increased light transmittance; the transmittance under dark conditions only increased slightly to 12%, indicating that light is the key factor in activating the degradation process of the PSAS system; secondly, the fluorescence microscope image showed that under light conditions, a large amount of healthy algae grew on the surface of the PET film treated with the PSAS preparation, further proving the activity of the PSAS preparation.

[0115] Test Example 3

[0116] The microbial enzyme preparation (SMNF preparation) provided in Example 3 was used to carry out degradation tests on different types of polyester plastics in a complex environment: a variety of polyester materials (PET, PBT) were treated with the SMNF preparation and placed in a simulated garbage compaction environment (high humidity, slight pressure).

[0117] Test method: Determine the concentration of degradation products after 10 days.

[0118] Table 4 Concentrations of different degradation products in different polyester materials after 10 days

[0119]

[0120] According to Table 4 and Figure 3 As shown in the figure, the SMNF preparation produced high concentrations of small molecule degradation products, such as terephthalic acid and ethylene glycol, in a short period of time. For PET materials, the concentration of terephthalic acid reached 1.2 mg / L and the concentration of ethylene glycol was 0.5 mg / L on the 10th day, proving that the SMNF preparation can quickly start the degradation process. Secondly, after 4 weeks of treatment, the degradation efficiency of the SMNF preparation was able to reach 75%, and the microbial survival rate was as high as 90%, proving its long-term effectiveness in complex environments.

[0121] Test Example 4

[0122] Degradation tests were carried out using the microbial enzyme preparations provided in Examples 1, 3 and 4 under hypoxic or anaerobic conditions:

[0123] The crushed polyester plastic (PET) samples were treated with EADS preparation, and the pH value was set to 7.0 and the temperature was set to 30℃ to simulate the landfill environment.

[0124] Test method: Use weight loss method, the initial weight is 10g, and the final weight is calculated after 30 days.

[0125] Table 5 Weight loss percentage of PET samples under different embodiments (after 30 days)

[0126] Example Final weight (g) Weight loss percentage (%) 1 6.9 31 4 7.2 28 5 7.5 25

[0127] Table 6 Enzyme activity distribution under different embodiments (U / mL)

[0128] Example Enzyme activity at pH 7.0 / 30°C 1 2.0 U / mL 4 2.1 U / mL 5 1.8 U / mL

[0129] Table 7 Microbial survival rate (%)

[0130] Example Survival rate (%) 1 85 4 80 5 90

[0131] As shown in Tables 5 to 7, Example 1 showed the best overall performance under all test conditions. Although Example 4 had a higher microbial density, this did not significantly improve the degradation efficiency or enzyme activity, but rather caused a slightly lower survival rate, possibly due to overcrowding. Example 5 improved the survival rate of microorganisms by increasing the nutrient supply, but too low a microbial density and too high a nutrient supply increased the cost and did not bring about a significant improvement in the degradation efficiency.

[0132] Test Example 5

[0133] The degradation test of the provided microbial enzyme preparation (Comparative Example 1) was carried out under hypoxic or anaerobic conditions:

[0134] Microorganism of choice: Ideonella sakaiensis DSM 27953 without CRISPR-Cas9 modification.

[0135] Nanocarrier Option: No nanocarrier.

[0136] Nutritional supply: Same as Example 1.

[0137] Assembly and Application: Direct mixing of microbial suspension and nutrient microcapsules without the use of nanoparticles.

[0138] Test method: The weight loss method was used to evaluate the degradation of PET samples under hypoxic or anaerobic conditions; the initial weight was 10 g and the final weight was calculated after 30 days.

[0139] Table 8 PET sample degradation data (after 30 days)

[0140] condition Final weight (g) Weight loss percentage (%) Enzyme activity distribution (U / mL) Microbial survival rate (%) Comparative Example 1 8.2 18 Enzyme activity at pH 7.0 / temperature 30°C: 1.6 U / mL 75%

[0141] Test Example 6

[0142] The provided microbial enzyme preparation (Comparative Example 2) was subjected to a degradation test under light conditions: a PET film was treated with a PSAS preparation and placed under controlled light conditions (16 h light / 8 h dark cycle, light intensity of 50 μmolphotons m-2s-1).

[0143] Microorganism selection: Common cyanobacteria or green algae, which do not express PETase.

[0144] Nanocarrier choice: Unmodified titanium dioxide nanoparticles.

[0145] Nutritional supply: same as in Example 2.

[0146] Assembly and Application: Simply mix unmodified titanium dioxide nanoparticles with common cyanobacteria or green algae.

[0147] Test method: Use optical density measurement method, the initial transmittance is 10%, and the final transmittance is calculated after 30 days.

[0148] Table 9 Final transmittance under different conditions

[0149] condition Final light transmittance (%) illumination 20 dark 10

[0150] Fluorescence microscopy images showed that there was almost no obvious algae growth on the surface of the PET film under light conditions, proving that the degradation efficiency of the system was very low.

[0151] Test Example 7

[0152] The provided microbial enzyme preparation (Comparative Example 3) was subjected to degradation tests on different types of polyester plastics in a complex environment: a variety of polyester materials (PET, PBT) were treated with SMNF preparations and placed in a simulated garbage compaction environment (high humidity, slight pressure).

[0153] Microbial selection: ordinary E. coli BL21 (DE3), introduced pET28a-PETase-MHETase plasmid, but no suicide plasmid was added.

[0154] Nanocarrier choice: Unmodified gold nanoparticles (AuNPs).

[0155] Nutritional supply: same as in Example 3.

[0156] Assembly and application: E. coli and AuNPs were directly mixed without forming a complex structure.

[0157] Test method: Determine the concentration of degradation products after 10 days.

[0158] Table 10 Concentrations of different degradation products in different polyester materials

[0159] Material Terephthalic acid degradation concentration (mg / L) Ethylene glycol degradation concentration (mg / L) PET 0.5 0.2 PBT 0.3 0.1

[0160] After 4 weeks of treatment, the degradation efficiency of Comparative Example 3 was only 40%, and the microbial survival rate was 70%, which was significantly lower than the performance of Example 3.

[0161] According to Tables 8 to 10 in the above Test Examples 5-7, it can be seen that Examples 1-3 all showed better performance than Comparative Examples 1-3 under all test conditions, especially in terms of degradation efficiency, enzyme activity and microbial survival rate. In addition, Examples 4 and 5 also showed better performance than Comparative Example 1, although they may not be as well optimized as Example 1 in some aspects. Therefore, it can be seen that the technical solution of the present invention has better advantages over the traditional method.

[0162] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and descriptions are only preferred examples of the present invention and are not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A microbial enzyme preparation for polyester plastic degradation, characterized in that: The enzyme preparation comprises: At least one microbial strain engineered to enhance PETase expression and maintain activity under hypoxic conditions; Nanocarrier material, wherein the nanocarrier material is selected from polylactic acid nanoparticles, titanium dioxide nanoparticles or gold nanoparticles; Chitosan solution, with a concentration of 0.5% w / v to 2% w / v, wherein the chitosan solution is used as a component of a stable protective film for encapsulating nano-carrier materials; Polyethyleneimine, at a concentration of 0.1% w / v to 0.3% w / v, is used to enhance the binding force between chitosan and nanocarrier materials in the chitosan solution; The nutrient component has a final concentration of 0.8 to 1.5 mg / mL, and is used to provide nutrition for the microbial strain. The nutrient component contains urea, a mixture of potassium dihydrogen phosphate and trace elements, or a mixture of glucose and trace elements, or a mixture of amino acids and vitamins.

2. The microbial enzyme preparation for polyester plastic degradation according to claim 1, characterized in that: The microbial strains include Ideonella sakaiensis DSM 27953, Comamonas sp. ATCC 43086, blue algae (Synechocystis sp.), green algae (Chlamydomonas reinhardtii) or Escherichia coli BL21 (DE3).

3. The microbial enzyme preparation for polyester plastic degradation according to claim 1, characterized in that: The nano-carrier material is mixed with a chitosan solution, and a calcium chloride solution is added to solidify to form a stable protective film, wherein the concentration of the calcium chloride solution is 1.8% w / v to 2.2% w / v.

4. The microbial enzyme preparation for polyester plastic degradation according to claim 2, characterized in that: The Ideonella sakaiensis DSM 27953 and Comamonas sp. ATCC 43086 were fermented by LB liquid medium, and the dissolved oxygen level was maintained below 2 mg / L to simulate an anaerobic cycle. The LB liquid medium included 1% tryptone, 0.5% yeast extract, and 1% sodium chloride.

5. The microbial enzyme preparation for polyester plastic degradation according to claim 2, characterized in that: The blue algae (Synechocystis sp.) and the green algae (Chlamydomonas reinhardtii) are cultured in a BG11 medium under a light cycle of 16 hours of light / 8 hours of darkness. The BG11 medium includes 1.5g / L, 0.04g / L, 0.075g / L, 0.036g / L, 0.006g / L, 0.001g / L, strength sodium bicarbonate 0.18g / L.

6. The microbial enzyme preparation for polyester plastic degradation according to claim 2, characterized in that: The Escherichia coli BL21 (DE3) was fermented in LB liquid medium, and IPTG was added during the induction phase to initiate the expression of the exogenous gene.

7. The microbial enzyme preparation for polyester plastic degradation according to claim 1, characterized in that: The nutrients are encapsulated in microcapsules or gel beads to ensure that the nutrients can be evenly attached to the surface of the microorganisms and slowly released over time, wherein the concentration of urea is 0.5-1g / L, the concentration of potassium dihydrogen phosphate is 0.2-0.5g / L, the concentration of each component of the trace element mixture is 1-3mg / L, the final concentration of glucose is 1.2mg / mL, and the final concentrations of the amino acid mixture and vitamins are 1mg / mL respectively.

8. A method for preparing the microbial enzyme preparation for polyester plastic degradation according to any one of claims 1 to 7, characterized in that: The following steps are involved: S1. Select microbial strains; S2. Genetically modify the microbial strain to enhance PETase expression to form an engineered microbial suspension. The OD of the microbial suspension 600 Reach 0.4-1.2; S3, preparing a nano-carrier material, and mixing it with a chitosan solution and polyethyleneimine to form a stable protective film; S4, preparing nutrients and encapsulating them in microcapsules to form nutrient microcapsules; S5. The microbial strain and the nanocarrier material are combined in a volume ratio of 1:1 by mixing, and then nutrient microcapsules with a final concentration of 0.8-1.5 mg / mL are added to prepare a microbial enzyme preparation.

9. The method for preparing a microbial enzyme preparation for polyester plastic degradation according to claim 8, characterized in that: The microbial strains are Ideonella sakaiensis DSM 27953 and Comamonas sp. ATCC 43086, and the carrier material is polylactic acid nanoparticles; The OD of the microbial suspension 600 =0.8; The concentration of polylactic acid nanoparticles was 10 mg / mL; The final concentration of nutrient microcapsules was 0.8 mg / mL; The concentration of chitosan solution was 0.5% w / v; The concentration of polyethyleneimine was 0.1% w / v; The concentration of calcium chloride solution was 1.8% w / v.

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