A process for the enzymatic degradation of pet plastics

Abstract

The application relates to the technical field of PET plastic degradation, and discloses a process for degrading PET plastic by using enzymes, which comprises the following steps: etching the surface of PET plastic by using plasma to introduce carboxyl groups and hydroxyl groups on the surface of the PET plastic; covalently combining boric acid-based compounds with the hydroxyl groups on the surface of the PET plastic through boric acid ester bonds to form a grafted layer on the PET surface; and degrading the PET plastic after the grafting treatment by using modified polyester hydrolytic enzymes. The application accurately constructs regular and spaced binding sites on the PET surface by using a mask-guided nanosphere template etching technology, the spacing design is based on the effective catalytic radius of a PETase enzyme molecule, each enzyme molecule has an independent and sufficient catalytic working area, and space steric hindrance and substrate competition between adjacent enzyme molecules are avoided.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of PET plastic degradation, and particularly relates to a process for degrading PET plastic by using enzymes. BACKGROUND

[0002] Polyethylene terephthalate (PET) is one of the largest polyester plastics in the world, which is widely used in packaging, textile and engineering materials, etc. However, its characteristics of being difficult to degrade naturally have caused serious environmental pollution problems. In recent years, the biodegradation of PET plastic by using polyester hydrolase (PETase) has become a research hotspot. This technology has the advantages of mild reaction conditions, environmental friendliness, and recyclable products. The current enzyme degradation technology mainly improves the catalytic activity of PETase by genetic engineering, or improves the enzyme-substrate contact efficiency by using physical and chemical methods to pretreat the surface of PET, and certain progress has been made in laboratory scale.

[0003] However, the existing technology still has significant defects. On the one hand, the surface of PET plastic has high hydrophobicity and chemical inertness, which makes the water-soluble PETase enzyme difficult to effectively adsorb and contact the surface of the substrate, and the enzyme-substrate combination efficiency is low, which seriously limits the degradation rate. On the other hand, the existing surface treatment method often lacks precise control of the enzyme molecule adsorption site, and cannot realize the ordered arrangement and high-density fixation of the enzyme molecules. At the same time, there is a lack of real-time monitoring and condition optimization means in the enzyme hydrolysis reaction process, which leads to low overall degradation efficiency, complex product separation and recovery process, and is difficult to realize industrial application. SUMMARY

[0004] In view of the problems existing in the process for degrading PET plastic by using enzymes and the preparation method thereof, the present application is proposed.

[0005] Therefore, the problem to be solved by the present application is how to improve the combination efficiency of PET plastic and PETase enzyme by surface modification technology, realize the ordered fixation and high-density adsorption of enzyme molecules, and establish a controllable enzyme hydrolysis reaction system.

[0006] To solve the above technical problems, the present application provides the following technical solutions:

[0007] In a first aspect, the present application provides a process for degrading PET plastic by using enzymes, comprising:

[0008] using plasma to etch the surface of the PET plastic, introducing carboxyl groups and hydroxyl groups on the surface of the PET plastic;

[0009] covalently combining a boronic acid compound with the hydroxyl groups on the surface of the PET plastic through a boronic ester bond to form a grafted layer on the surface of the PET;

[0010] The PET plastic after the grafting treatment is subjected to enzymatic degradation by using a modified polyester hydrolase.

[0011] As a preferred solution of the process for degrading PET plastic by using enzymes, in the process, the etching treatment is guided by a mask, and the specific steps include: covering a nanosphere template on the surface of the PET plastic, wherein the nanosphere template has holes with an interval of 50-200 nm; and etching the surface of the PET plastic according to the hole positions of the nanosphere template by using an etching machine.

[0012] The regular arrangement of the etching points provides ordered binding sites for the subsequent grafting of boronic acid-based compounds, improves the surface binding density of enzyme molecules, and reduces excessive etching compared with traditional large-area plasma treatment.

[0013] As a preferred solution of the process for degrading PET plastic by using enzymes, in the process, the ratio of the influence radius of the carboxyl group and the hydroxyl group introduced on the surface of the PET plastic to the regulated etching interval is 1:0.4-1.2.

[0014] The spatial distribution ensures that each region of the PET surface can be effectively degraded, avoiding local over-degradation or insufficient degradation.

[0015] As a preferred solution of the process for degrading PET plastic by using enzymes, in the process, the boronic acid-based compound is selected from one or more of 4-carboxyphenylboronic acid, 3-aminophenylboronic acid, and 4-hydroxyphenylboronic acid, and the boronic acid-based compound is selected by molecular screening during the grafting process, specifically including:

[0016] The boronic acid-based compound is purified by reverse phase chromatography to remove impurities in the synthesis process.

[0017] A gradient elution method is used to obtain a grafting component with a purity of greater than 95%.

[0018] As a preferred solution of the process for degrading PET plastic by using enzymes, in the process, the modified polyester hydrolase is a PETase variant subjected to directed evolution modification, and the PETase variant has an amino acid mutation at one or more combinations of Ile179Phe, Ser214His, and Thr140Asp.

[0019] As a preferred solution of the process for degrading PET plastic by using enzymes, in the process, the adsorption of the modified polyester hydrolase includes the following steps:

[0020] The PET plastic after the grafting treatment is immersed in a diluted enzyme solution with a concentration of 0.1-0.5 U / mL, and is placed at 4-10°C for 30-90 minutes of adsorption to form a primary combination of enzyme molecules and the grafting layer;

[0021] The uncombined enzyme molecules are removed by washing with a phosphate buffer solution with a pH of 7.0-7.5;

[0022] The PET plastic is transferred to a concentrated enzyme solution with a concentration of 2.0-5.0 U / mL, and is subjected to secondary adsorption at 25-35°C for 60-180 minutes;

[0023] The uniform distribution of enzyme molecules at the grafting sites is promoted by gentle stirring for 10-30 minutes.

[0024] The beneficial effect of the preferred technical solution is that the low-temperature diluted solution primary combination reduces the conformational changes of enzyme molecules, the high-temperature concentrated solution secondary adsorption enhances the binding strength, and the two-step adsorption greatly improves the degradation efficiency compared with single-step adsorption.

[0025] As a preferred solution of the process for degrading PET plastic by using enzymes, a synergistic catalyst is added during the enzyme degradation process, which comprises:

[0026] A non-ionic surfactant with a concentration of 0.1-0.5% is added to enhance the enzyme-substrate contact;

[0027] The pH value of the reaction system is maintained in the range of 7.5-8.5 to maintain the active conformation of enzyme molecules.

[0028] As a preferred solution of the process for degrading PET plastic by using enzymes, the enzyme degradation reaction is carried out in a micro-reactor array mode:

[0029] The treated PET plastic is divided into small particles with a size of 0.5-2.0 mm;

[0030] Parallel degradation reactions are carried out in the reaction chambers of the microfluidic chip;

[0031] The reaction conditions of each reaction chamber are adjusted by monitoring the product concentration of each reaction chamber in real time.

[0032] The beneficial effect of the preferred technical solution is that the micro-scale reaction environment greatly shortens the mass transfer distance, accelerates the contact between enzymes and substrates, and accelerates the diffusion of products.

[0033] As a preferred solution of the process for degrading PET plastic by using enzymes, adjusting the reaction conditions of each reaction chamber comprises the following steps:

[0034] The concentrations of terephthalic acid and ethylene glycol in each reaction chamber are detected by an online detection device every 30-60 minutes.

[0035] According to the product concentration data, the degradation rates of each chamber are calculated, and the lag chamber with a degradation rate less than 20% of the average value is screened out;

[0036] One or more of the following adjustment operations are performed on the lag chamber: increasing the reaction temperature by 2-5 DEG C, increasing the enzyme liquid concentration by 0.2-0.8 U / mL, adjusting the pH value by ±0.2 units, and increasing the stirring intensity by 10-30%;

[0037] After each adjustment, 15-30 minutes are monitored, and if the degradation rate still does not reach the target value, the above adjustment steps are repeated;

[0038] When the degradation rate difference of each chamber is controlled within 15%, the adjustment is stopped and the current conditions are maintained until the reaction is completed.

[0039] The preferred technical solution has the beneficial effects that the reaction conditions of the lag chamber are dynamically adjusted, the degradation rate difference of each chamber is controlled within 15%, and the "bucket effect" is avoided.

[0040] As a preferred solution of the process for degrading PET plastic by using enzymes, after the enzyme degradation is completed, product separation and recovery are performed, and the specific steps include:

[0041] The enzyme is inactivated by heating to 85-95 DEG C for 5-10 minutes;

[0042] The reaction system is centrifuged at a separation speed of 8000-12000 rpm for 10-20 minutes to obtain the supernatant;

[0043] The terephthalic acid in the supernatant is separated by an anion exchange resin column, and the eluent is a 0.1-0.5 M NaCl solution;

[0044] The effluent is recovered by distillation or reduced pressure distillation, and the distillation temperature is controlled at 80-120 DEG C;

[0045] The recovered terephthalic acid and ethylene glycol are screened by high performance liquid chromatography detection.

[0046] The present application has the beneficial effects that:

[0047] The nanosphere template etching technology guided by the mask is used to precisely construct regular binding sites with intervals of 50-200 nm on the PET surface. The interval design is based on the effective catalytic radius (25-100 nm) of the PETase enzyme molecule, which ensures that each enzyme molecule has an independent and sufficient catalytic working area, avoiding the steric hindrance and substrate competition between adjacent enzyme molecules. The introduced carboxyl and hydroxyl functional groups improve the hydrophilicity of the PET surface, providing stable binding sites for the covalent grafting of boronic acid compounds. Through the optimized two-step enzyme adsorption process, the ordered arrangement and high-density fixation of the PETase enzyme molecules are achieved. By controlling the ratio of the functional group influence radius to the etching interval (1:0.4-2), the catalytic domain of adjacent enzyme molecules achieves optimal spatial distribution, improving the degradation efficiency. DETAILED DESCRIPTION

[0048] In order to make the above-mentioned objectives, features and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application will be described in detail below. Obviously, the described embodiments are part of the embodiments of the present application, rather than all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor should fall within the scope of protection of the present application.

[0049] In the following description, many specific details are set forth in order to provide a thorough understanding of the present application. However, the present application can be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the present application, so the present application is not limited to the specific embodiments disclosed below.

[0050] Secondly, "one embodiment" or "embodiment" referred to herein means that a specific feature, structure or characteristic can be included in at least one implementation of the present application. "In one embodiment" appearing in different places in the specification does not mean the same embodiment, nor is it an independent or alternative embodiment that excludes other embodiments.

[0051] Embodiment 1 provides a process for degrading PET plastic using enzymes, comprising:

[0052] S1, etching the surface of the PET plastic using plasma to introduce carboxyl groups and hydroxyl groups on the surface of the PET plastic;

[0053] S2, covalently bonding a boronic acid compound to the hydroxyl groups on the surface of the PET plastic through a boronic ester bond to form a grafted layer on the PET surface;

[0054] S3, degrading the PET plastic after grafting treatment using a modified polyester hydrolase.

[0055] It should be noted that PET plastic, as the world's largest polyester plastic, has high chemical stability and hydrophobic surface characteristics, making traditional biodegradation methods extremely inefficient. During enzymatic degradation, the hydrophobicity of the PET surface makes it difficult for water-soluble PETase enzymes to effectively adsorb, the enzyme-substrate contact area is small, the binding strength is weak, and the degradation reaction is severely limited. At the same time, due to the lack of effective enzyme molecule fixation means, the enzyme is prone to dissociation and inactivation during the reaction process, and the degradation efficiency is unstable. Traditional physical and chemical pretreatment methods often damage the molecular structure of PET, affecting the quality of the degradation product, and it is difficult to achieve ordered arrangement and high-density binding of enzyme molecules; therefore, it is very important to establish a PET enzyme degradation process technology that can accurately control the enzyme-substrate interface characteristics, achieve stable enzyme molecule fixation, and improve the degradation efficiency.

[0056] Therefore, in order to solve the problems of low enzyme-substrate contact efficiency and unstable enzyme fixation, through the steps of S1-S3, the surface of PET is modified by plasma etching technology to introduce hydrophilic functional groups to improve the surface wettability and create favorable conditions for subsequent enzyme molecule binding; a stable functional grafting layer is formed by covalent grafting of boronic acid-based compounds to provide specific binding sites for modified polyester hydrolase and achieve ordered arrangement and high-density fixation of enzyme molecules; and through optimized enzyme reaction conditions, the catalytic activity of the modified enzyme is fully utilized to achieve efficient degradation of PET plastic. This process improves the enzyme-substrate binding efficiency, enhances the stability and catalytic activity of the enzyme, and provides an efficient and controllable technical path for the green recycling of PET plastic.

[0057] In Example 2, the surface of the PET plastic is etched by plasma to introduce carboxyl groups and hydroxyl groups on the surface of the PET plastic.

[0058] In the embodiments of the present application, the etching treatment adopts a mask guided mode, and the specific steps include:

[0059] The nanosphere template is covered on the surface of the PET plastic, wherein the nanosphere template has holes with an interval of 50-200 nm on the surface; and the surface of the PET plastic is etched by the etching machine according to the hole position of the nanosphere template. The ratio of the influence radius of the carboxyl groups and the hydroxyl groups introduced on the surface of the PET plastic to the control etching interval is 1:0.4-1.2.

[0060] In a specific implementation, a nanosphere template is first prepared, a dense hexagonal structure is formed on the PET plastic surface by self-assembly technology using polystyrene nanospheres, the nanosphere diameter is 150-500 nm, the distance between the nanospheres is controlled by adjusting the nanosphere concentration and assembly conditions, and regular holes with a spacing of 50-200 nm are opened on the surface of the template. The prepared nanosphere template is covered on the pretreated PET plastic surface to ensure that the template is tightly attached to the substrate and to avoid lateral diffusion during etching.

[0061] Subsequently, the PET plastic surface is precisely etched according to the hole positions of the nanosphere template using a plasma etching machine, the etching gas is a mixture of oxygen and argon (volume ratio 1:3), the plasma power is controlled at 50-150 W, the etching time is 30-120 seconds, and the etching depth is controlled at 5-20 nm. During the etching process, carboxyl (-COOH) and hydroxyl (-OH) functional groups are introduced by RIE (reactive ion etching) technology, and the surface functional group density reaches 2-8 / nm2. After etching, the nanosphere template is removed by sequentially washing with acetone and deionized water, and a PET plastic surface with regular micro-nano structures and abundant functional groups is obtained.

[0062] The ratio of the influence radius of the carboxyl group and the hydroxyl group introduced on the PET plastic surface to the regulated etching interval is 1:0.4-1.2. This ratio is designed based on the effective catalytic radius of the PETase enzyme molecule (about 25-100 nm) and the diffusion influence range of the functional groups. When the ratio is too small (<1:0.4), the spacing between adjacent functionalized regions is too large, which can cause insufficient surface utilization; when the ratio is too large (>1.2), adjacent regions will overlap, causing enzyme molecules to compete for binding and steric hindrance. By optimizing the ratio, the optimal spatial distribution and highest binding density of enzyme molecules can be achieved.

[0063] In an alternative embodiment, the nanosphere template has holes with a spacing of 50 nm; the ratio of the influence radius of the carboxyl group and the hydroxyl group introduced on the PET plastic surface to the regulated etching interval is 1:0.4.

[0064] In a second alternative embodiment, the nanosphere template has holes with a spacing of 200 nm; the ratio of the influence radius of the carboxyl group and the hydroxyl group introduced on the PET plastic surface to the regulated etching interval is 1:1.2.

[0065] In a third alternative embodiment, the nanosphere template has holes with a spacing of 100 nm; the ratio of the influence radius of the carboxyl group and the hydroxyl group introduced on the PET plastic surface to the regulated etching interval is 1:1.

[0066] In Example 3, the boronic acid-based compound is covalently bonded to the hydroxyl groups on the PET plastic surface through boronate ester bonds to form a grafted layer on the PET surface.

[0067] The boronic acid-based compound is selected from one or more of 4-carboxyphenylboronic acid, 3-aminophenylboronic acid, and 4-hydroxyphenylboronic acid, which have molecular weights in the range of 150-200 Da, good water solubility, and reactivity. The boronic acid-based compound is pretreated using a molecular screening method during the grafting process to ensure the purity and uniformity of the grafted component.

[0068] The specific molecular screening step includes first dissolving the crude boronic acid-based compound in a methanol-water mixed solvent (volume ratio 7:3) to prepare a sample solution with a concentration of 10-20 mg / mL.

[0069] Separation and purification are performed by a C18 reverse-phase chromatographic column (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm), using an acetonitrile-0.1% trifluoroacetic acid aqueous solution system for the mobile phase, and a gradient elution method: initial condition of 5% acetonitrile for 5 minutes; then linear gradient to 60% acetonitrile in 20 minutes; and finally 60% acetonitrile for 5 minutes. The flow rate is controlled at 1.0 mL / min, the detection wavelength is 254 nm, and the column temperature is maintained at 35°C. The main peak component is collected, and the solvent is removed by rotary evaporation under reduced pressure to obtain a grafted component with a purity of greater than 95%.

[0070] The grafting reaction is carried out under anhydrous conditions, and the purified boronic acid-based compound is dissolved in anhydrous DMF to prepare a reaction solution with a concentration of 5-15 mM. The catalyst N,N-diisopropylethylamine (DIPEA, 2-5 equiv) is added to the reaction system, and the reaction is carried out at 60-80°C under nitrogen protection for 6-12 hours. The boronic acid group undergoes dehydration condensation with the hydroxyl group on the PET surface to form a stable boronate ester covalent bond (B-O-C).

[0071] After the reaction is completed, the unreacted compounds and catalysts are removed by washing with DMF, ethanol, and deionized water, respectively, and the functionalized PET surface is obtained by drying in a vacuum drying oven at 40°C for 24 hours.

[0072] In an alternative embodiment, 4-carboxyphenylboronic acid is selected as the single grafted component, the reaction concentration is 10 mM, the reaction temperature is 70°C, and the reaction time is 8 hours. The 4-carboxyphenylboronic acid molecule contains a carboxyl functional group, which can provide additional electrostatic attraction for subsequent enzyme molecule binding, and is suitable for binding with a modified PETase enzyme with more positively charged regions.

[0073] In the second alternative embodiment, a mixture of 3-aminobenzoic acid and 4-hydroxybenzoic acid (molar ratio 1:1) is selected as the grafting component, with a total concentration of 12 mM, a reaction temperature of 75 °C, and a reaction time of 10 hours. The mixed grafting can introduce both amino and hydroxyl functional groups, forming a multifunctional surface. The amino groups can enhance enzyme binding through hydrogen bonding and electrostatic interactions, while the hydroxyl groups can provide additional hydrophilicity.

[0074] In the third alternative embodiment, a composite grafting of three boronic acid-based compounds (4-carboxybenzoic acid: 3-aminobenzoic acid: 4-hydroxybenzoic acid = 2:1:1) is used, with a total concentration of 15 mM, a reaction temperature of 80 °C, and a reaction time of 12 hours. The composite grafting layer has surface chemical diversity, which can adapt to the binding requirements of different modified PETase enzymes to improve the binding stability and catalytic efficiency of the enzymes.

[0075] Example 4: Enzymatic degradation of PET plastic after grafting treatment using modified polyester hydrolase.

[0076] The modified polyester hydrolase is a PETase variant that has been directionally evolved to have an amino acid mutation at the following site: Ile179Phe.

[0077] The adsorption of the modified polyester hydrolase includes the following steps:

[0078] The PET plastic after grafting treatment is immersed in a diluted enzyme solution with a concentration of 0.1 U / mL, and is placed at 4 °C for 30 minutes of primary adsorption of enzyme molecules to the grafting layer;

[0079] Unbound enzyme molecules are removed by washing with a pH 7.0 phosphate buffer;

[0080] The PET plastic is transferred to a concentrated enzyme solution with a concentration of 2.0 U / mL, and is subjected to secondary adsorption at 25 °C for 60 minutes;

[0081] Uniform distribution of enzyme molecules at the grafting sites is promoted by gentle stirring for 10 minutes.

[0082] A synergistic catalyst is added during the enzymatic degradation process, including:

[0083] A non-ionic surfactant with a concentration of 0.1% is added to enhance enzyme-substrate contact;

[0084] The pH value of the reaction system is maintained within the range of 7.5 to maintain the active conformation of the enzyme molecules.

[0085] The enzymatic degradation reaction is carried out using a microreactor array:

[0086] The treated PET plastic is divided into small particles of 0.5 mm;

[0087] Parallel degradation reactions are carried out in the reaction chambers of the microfluidic chip respectively;

[0088] The reaction conditions of each reaction chamber are adjusted by monitoring the product concentration of each reaction chamber in real time.

[0089] Adjusting the reaction conditions of each reaction chamber includes the following steps:

[0090] Detect the concentration of terephthalic acid and ethylene glycol in each reaction chamber every 30 minutes through the online detection device;

[0091] According to the product concentration data, calculate the degradation rate of each chamber, and select the lag chamber with a degradation rate less than 20% of the average value;

[0092] One or more of the following adjustment operations are performed on the lag chamber: increase the reaction temperature by 2℃;

[0093] After each adjustment, monitor for 15 minutes. If the degradation rate still does not reach the target value, repeat the above adjustment steps;

[0094] When the difference between the degradation rates of each chamber is controlled within 15%, stop adjusting and maintain the current conditions until the reaction is completed.

[0095] Example 5, using modified polyester hydrolase to enzymatically degrade PET plastic after grafting treatment.

[0096] The modified polyester hydrolase is a PETase variant that has been directionally evolved, with amino acid mutations at the following sites: Ser214His.

[0097] The adsorption of the modified polyester hydrolase includes the following steps:

[0098] After grafting treatment, the PET plastic is immersed in a diluted enzyme solution with a concentration of 0.3 U / mL, and is placed at 8℃ for 60 minutes to form a primary combination of enzyme molecules and the grafting layer;

[0099] Use pH 7.2 phosphate buffer to rinse and remove unbound enzyme molecules;

[0100] Transfer the PET plastic to a concentrated enzyme solution with a concentration of 4.0 U / mL, and perform a secondary adsorption at 30℃ for 100 minutes;

[0101] Promote the uniform distribution of enzyme molecules at the grafting sites by gentle stirring for 20 minutes.

[0102] Add a synergistic catalyst during the enzymatic degradation process, including:

[0103] Add a non-ionic surfactant with a concentration of 0.3% to enhance enzyme-substrate contact;

[0104] The pH value of the reaction system is maintained at 8 to maintain the active conformation of the enzyme molecules.

[0105] The enzyme degradation reaction is carried out in a microreactor array:

[0106] The treated PET plastic is divided into 1 mm micro-particles;

[0107] Parallel degradation reactions are carried out in the reaction chambers of the microfluidic chip;

[0108] The reaction conditions of each reaction chamber are adjusted by monitoring the product concentration in each reaction chamber in real time.

[0109] Adjusting the reaction conditions of each reaction chamber includes the following steps:

[0110] The concentration of terephthalic acid and ethylene glycol in each reaction chamber is detected every 45 minutes by an online detection device;

[0111] According to the product concentration data, the degradation rate of each chamber is calculated, and the lag chamber with a degradation rate more than 20% lower than the average value is identified;

[0112] One or more of the following adjustment operations are performed on the lag chamber: increasing the enzyme solution concentration by 0.2 U / mL, adjusting the pH value by ±0.2 units;

[0113] After each adjustment, monitor for 20 minutes, if the degradation rate still does not reach the target value, repeat the above adjustment steps;

[0114] When the degradation rate difference of each chamber is controlled within 15%, stop adjusting and maintain the current conditions until the reaction is completed.

[0115] Example 6, using modified polyester hydrolase for enzymatic degradation of PET plastic after grafting treatment.

[0116] The modified polyester hydrolase is a PETase variant that has been modified by directed evolution, which has amino acid mutations at the following sites: Thr140Asp.

[0117] The adsorption of the modified polyester hydrolase includes the following steps:

[0118] The PET plastic after grafting treatment is immersed in a diluted enzyme solution with a concentration of 0.5 U / mL, and is placed at 10°C for 90 minutes to form a primary combination of enzyme molecules and the grafting layer;

[0119] Wash with pH 7.5 phosphate buffer to remove unbound enzyme molecules;

[0120] Transfer the PET plastic to a concentrated enzyme solution with a concentration of 5.0 U / mL, and perform a secondary adsorption at 35°C for 180 minutes;

[0121] The uniform distribution of enzyme molecules at the grafting sites is facilitated by gentle stirring for 30 minutes.

[0122] The synergistic catalyst is added during the enzyme degradation process, including:

[0123] The non-ionic surfactant is added at a concentration of 0.5% to enhance enzyme-substrate contact;

[0124] The pH value of the reaction system is maintained at 8.5 to maintain the active conformation of enzyme molecules.

[0125] The enzyme degradation reaction is carried out using a micro-reactor array:

[0126] The treated PET plastic is divided into small particles of 2.0 mm;

[0127] Parallel degradation reactions are carried out in the reaction chambers of the microfluidic chip;

[0128] The reaction conditions of each reaction chamber are adjusted by monitoring the product concentration in real time.

[0129] Adjusting the reaction conditions of each reaction chamber includes the following steps:

[0130] The concentration of terephthalic acid and ethylene glycol in each reaction chamber is detected every 60 minutes by an online detection device;

[0131] The degradation rate of each chamber is calculated based on the product concentration data, and the lagging chamber with a degradation rate more than 20% lower than the average value is identified;

[0132] The lagging chamber is subjected to one or more of the following adjustment operations: increasing the stirring intensity by 30%;

[0133] After each adjustment, monitor for 30 minutes, if the degradation rate still does not reach the target value, repeat the above adjustment steps;

[0134] When the difference in degradation rate of each chamber is controlled within 15%, stop adjusting and maintain the current conditions until the end of the reaction.

[0135] Comparative Example 1: Using wild-type polyester hydrolase for enzyme degradation of PET plastic after grafting treatment.

[0136] The wild-type PETase enzyme without modification does not contain the Ser214His mutation site, and the enzyme activity and thermal stability are at the original level. Enzyme adsorption uses the traditional single-step adsorption method: the PET plastic after grafting treatment is directly immersed in the enzyme solution with a concentration of 4.0 U / mL, and the adsorption is carried out at room temperature (25℃) for 120 minutes, then the unbound enzyme molecules are removed by simple washing with deionized water, without secondary adsorption and uniform distribution treatment steps.

[0137] No co-catalyst was added in the enzymatic degradation process, and the pH value of the reaction system was naturally buffered without special pH control measures. The degradation reaction used the traditional batch reaction method, and the treated PET plastic was placed in a single reactor for degradation. There was no microreactor array design, and the reaction conditions were fixed and unchanged without real-time monitoring and dynamic adjustment functions.

[0138] Comparative Example 2 used a modified polyester hydrolase, but the enzymatic degradation was carried out using a traditional batch reactor.

[0139] The same modified polyester hydrolase (Ser214His mutation) and two-step enzyme adsorption process as in Example 5 were used, and the same co-catalyst (0.3% non-ionic surfactant, pH maintained at 8.0) was added. However, the degradation reaction used a traditional batch reactor, and the treated PET plastic (particle size 1 mm) was placed in a 500 mL glass reactor for degradation. A magnetic stirrer was used for mixing, and the reaction conditions were fixed and unchanged.

[0140] There was no microreactor array design, and parallel processing and independent control could not be achieved. There was no real-time online monitoring system, and only one sample was taken every 12 hours after the reaction started to detect the product concentration, which could not timely discover and correct the reaction abnormalities. There was no dynamic adjustment function during the reaction, and when local areas had poor mass transfer or enzyme deactivation, the conditions could not be adjusted accordingly.

[0141] Table 1: Comparison of experimental results of Example 5 and comparative examples:

[0142]

[0143] From the experimental data in Table 1, it can be clearly seen that in terms of enzyme binding amount, Example 5 reached 1.15 ± 0.08 μg / cm 2 , which was 98.3% higher than that of Comparative Example 1 (0.58 ± 0.12 μg / cm 2 ), which was mainly due to the stronger surface affinity of the modified enzyme (Ser214His mutation) and the optimization effect of the two-step adsorption process. The enzyme binding amount of Example 5 and Comparative Example 2 was similar (only a difference of 2.7%), indicating that the modified enzyme and the optimized adsorption process were fully utilized, but the subsequent microreactor technology further amplified this advantage.

[0144] In terms of degradation efficiency, the 72-hour degradation rate of Example 5 reached 78.2±3.1%, which was 174.4% and 48.1% higher than Comparative Example 1 and Comparative Example 2, respectively. Correspondingly, the product concentration also showed the same trend, with the terephthalic acid concentration reaching 0.42±0.02 mM and the ethylene glycol concentration reaching 0.38±0.02 mM, both being the highest in the three groups of experiments. This result fully demonstrates the important role of microreactor array technology in improving mass transfer effect and achieving precise process control. At the same time, the standard deviation of Example 5 is smaller than that of the two comparative examples, indicating that its reaction process has better reproducibility and stability.

[0145] The data of enzyme activity retention rate and degradation rate further verify the advantages of the technical solution of Example 5. After 72 hours of reaction, the enzyme activity retention rate of Example 5 was 92.5±2.1%, which was much higher than 65.8±6.2% of Comparative Example 1 and 84.3±4.8% of Comparative Example 2, which indicated that the modified enzyme not only had higher intrinsic activity, but also showed better stability. The degradation rate data showed that Example 5 continued at a rate of 1.09±0.04 % / h, which was 2.7 times that of Comparative Example 1 and 49.3% higher than Comparative Example 2. In terms of product recovery purity, the purity of terephthalic acid recovered by Example 5 reached 98.1±0.5%, and the purity of ethylene glycol reached 95.8±0.3%, both meeting the requirements of industrial grade, providing high-quality raw material basis for the complete resource utilization of PET plastics.

[0146] Example 7, after the completion of enzyme degradation, product separation and recovery was carried out, the specific steps including:

[0147] The enzyme was inactivated by heating to 90°C for 5 minutes;

[0148] The reaction system was centrifuged at a speed of 10000 rpm for 15 minutes to obtain the supernatant;

[0149] The supernatant was separated by anion exchange resin column to separate terephthalic acid, and the eluent was 0.3M NaCl solution;

[0150] The effluent was recovered by distillation or reduced pressure distillation, and the distillation temperature was controlled at 100°C;

[0151] The recovered terephthalic acid and ethylene glycol were screened by high performance liquid chromatography.

[0152] In an alternative embodiment, when the processing amount is large (>100 mL of reaction liquid), a continuous centrifugal separator can be used to improve the separation efficiency, the centrifugal speed is adjusted to 12000 rpm, and the treatment time is shortened to 10 minutes, the separation effect is equivalent to batch centrifugation.

[0153] In the second alternative embodiment, for the application requiring high purity (purity of terephthalic acid ≥ 99.5%), a recrystallization step can be added after the anion exchange separation: the crude terephthalic acid is dissolved in hot water, slowly cooled to room temperature to precipitate crystals, washed with cold water and vacuum dried, and the final purity can reach more than 99.8%.

[0154] It should be noted that the above examples are only used to illustrate the technical solutions of the present application but not limit the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or equivalently replaced without departing from the spirit and scope of the present application, and all modifications and equivalent replacements should be covered in the scope of the present application.

Claims

1. A process for degrading PET plastic using enzymes, characterized in that, include: The surface of PET plastic is etched using plasma to introduce carboxyl and hydroxyl groups into the PET plastic surface. A borate compound is covalently bonded to the hydroxyl groups on the surface of the PET plastic via borate ester bonds to form a graft layer on the PET surface; Modified polyester hydrolase was used to enzymatically degrade grafted PET plastic. The etching process employs a mask-guided method, and the specific steps include: A nanosphere template is applied to the surface of a PET plastic, wherein the surface of the nanosphere template has pores spaced 50-200 nm apart. The PET plastic surface is etched using an etching machine according to the hole positions of the nanosphere template; The borate-based compound is selected from one or more of 4-carboxyphenylboronic acid, 3-aminophenylboronic acid, and 4-hydroxyphenylboronic acid, and the borate-based compound is selected using a molecular screening method during the grafting process, specifically including: Boronic acid compounds were separated and purified by reversed-phase chromatography to remove impurities from the synthesis process; Grafted components with a purity greater than 95% were obtained by gradient elution. The modified polyester hydrolase is a PETase variant that has undergone directed evolution, and the PETase variant has an amino acid mutation at one or more of the following sites: Ile179Phe, Ser214His, Thr140Asp. The enzymatic degradation of grafted PET plastic using modified polyester hydrolase includes the following adsorption steps: The grafted PET plastic is immersed in a diluted enzyme solution with a concentration of 0.1-0.5 U / mL and allowed to stand at 4-10℃ for 30-90 minutes to adsorb, forming a primary binding between enzyme molecules and the grafted layer. Wash with phosphate buffer (pH 7.0-7.5) to remove unbound enzyme molecules; PET plastic was transferred to a concentrated enzyme solution with a concentration of 2.0-5.0 U / mL and subjected to secondary adsorption at 25-35℃ for 60-180 minutes. Gentle stirring for 10-30 minutes promotes the uniform distribution of enzyme molecules at the grafting sites; The enzyme degradation process is enhanced by the addition of a co-catalyst, including: Adding 0.1-0.5% nonionic surfactant enhances enzyme-substrate contact; Maintain the pH of the reaction system within the range of 7.5-8.5 to preserve the active conformation of the enzyme molecules; The enzyme degradation is carried out using a microreactor array: The treated PET plastic is cut into tiny particles of 0.5-2.0 mm. Parallel degradation reactions were carried out separately in the reaction chamber of the microfluidic chip. By monitoring the product concentration in each reaction chamber in real time, the reaction conditions in each reaction chamber can be adjusted.

2. The process for enzymatic degradation of PET plastic as described in claim 1, characterized in that, The ratio of the influence radius of the carboxyl and hydroxyl groups introduced on the PET plastic surface to the controlled etching interval is 1:0.4-1.

2.

3. The process for enzymatic degradation of PET plastic as described in claim 1, characterized in that, Adjusting the reaction conditions in each reaction chamber includes the following steps: The concentrations of terephthalic acid and ethylene glycol in each reaction chamber are detected every 30-60 minutes using an online detection device. The degradation rate of each chamber was calculated based on the product concentration data, and the hysteresis chambers with degradation rates less than 20% of the average value were screened out. Perform one or more of the following adjustments to the hysteresis chamber: increase the reaction temperature by 2-5℃, increase the enzyme concentration by 0.2-0.8 U / mL, adjust the pH value by ±0.2 units, or increase the stirring intensity by 10-30%. Monitor for 15-30 minutes after each adjustment. If the degradation rate still does not reach the target value, repeat the above adjustment steps. When the difference in degradation rate between the chambers is controlled within 15%, stop adjusting and maintain the current conditions until the reaction ends.

4. The process for enzymatic degradation of PET plastic as described in claim 3, characterized in that, After the enzyme degradation is completed, the product is separated and recovered. The specific steps include: The enzyme is inactivated by heating to 85-95℃ for 5-10 minutes. The reaction system was centrifuged at a speed of 8000-12000 rpm for 10-20 minutes to obtain the supernatant. The supernatant was passed through an anion exchange resin column to separate terephthalic acid, with the eluent being a 0.1-0.5M NaCl solution; Ethylene glycol is recovered from the effluent by distillation or vacuum distillation, with the distillation temperature controlled at 80-120℃. The recovered terephthalic acid and ethylene glycol were screened by high performance liquid chromatography.

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