CN115896193A - Method for complete depolymerization of thermoplastic polyester type polyurethane plastic by double enzymes - Google Patents
Method for complete depolymerization of thermoplastic polyester type polyurethane plastic by double enzymes Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/50—Reuse, recycling or recovery technologies
- Y02W30/62—Plastics recycling; Rubber recycling
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Abstract
The invention belongs to the field of environmental science, and relates to a method for completely depolymerizing thermoplastic polyester polyurethane plastic by double enzymes, in particular to a method for depolymerizing polyester polyurethane plastic by taking polyester polyurethane plastic as a substrate and adding esterase PETase and esterase Aes72. Wherein PETase hydrolyzes ester bond part, aes72 mainly hydrolyzes urethane bond, and double enzymes synergistically promote complete depolymerization of thermoplastic polyester type polyurethane plastic. According to the invention, the PBA-PU is degraded by using double enzymes, the enzymolysis efficiency is improved, meanwhile, the PBA-PU can be completely depolymerized into monomer MDA, and the monomer generated after depolymerization is utilized for repolymerization or other high-value utilization, so that the product utilization rate is improved, and the competitive advantage of the enzymatic method for degrading the thermoplastic polyester type polyurethane plastic is improved.
Description
Technical Field
The invention belongs to the field of environmental science, and relates to a method for completely depolymerizing thermoplastic polyester polyurethane plastic by double enzymes.
Background
Polyurethane (PU) plastic is one of five major plastics with the largest global production and consumption, and the product application relates to many fields of textile, building materials, automobiles, national defense and the like. According to statistics, the global plastic yield in 2019 reaches 3.68 hundred million tons, wherein the PU plastic yield accounts for 6-7% of the total yield, the PU plastic becomes the polyester type plastic with the second largest global yield, the yield reaches 1470 ten thousand tons in 2020 of China, and the consumption is about 1175 ten thousand tons. Most of plastic products are used once or temporarily, huge consumption wastes are inevitably brought by huge use, 63 hundred million tons of plastic wastes are produced globally until 2015, 91% of the plastic wastes are incinerated, buried or discarded in soil and sea, not only is the problem of global carbon resource waste caused, but also ecological environment problems represented by micro plastic pollution are paid attention to all countries. In 9% of the recycling proportion of waste plastics, most of the waste plastics are waste plastics with single structure, clean products and clear components, such as leftover materials of plastic factories, food-grade polyethylene terephthalate (PET) packages and the like, but PU plastics have complex structures and various types and are difficult to effectively recycle by the existing physical and chemical recycling modes.
With the establishment of an enzymatic recovery technology system of PET plastics, the enzymatic recovery of waste plastics has become the focus of domestic and foreign research. The French Carbios company utilizes cutinase LCC from plant compost to decompose 97% of PET plastics within 16h, and the decomposed product is reacted again to generate new plastics. The enzymatic recovery of PU plastics is still in the excavation stage of high-efficiency depolymerase elements at present, but the success of the enzymatic recovery technology of PET plastics provides important feasibility reference and technical reference for the enzymatic recovery technology of PET plastics.
The advantage of enzymatic degradation over microorganisms is that the enzymatic depolymerization leads to valuable degradation products and is more controllable and reproducible than microbial degradation. Because of the complex structure of PU plastics, enzymes with single function can not completely depolymerize the PU plastics into monomer substances, so valuable degradation products can not be obtained. Therefore, the biological recovery of PU by using the two-enzyme system has great practical significance.
Disclosure of Invention
The invention aims to solve the technical problem of the prior art and provides a method for completely depolymerizing thermoplastic polyester polyurethane plastic by double enzymes.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
the invention discloses a method for completely depolymerizing thermoplastic polyester polyurethane plastic by double enzymes, which comprises the following steps:
(1) Dissolving polyurethane plastic in an organic solvent, and dissolving by ultrasonic waves to obtain a first mixed solution; pouring the first mixed solution into a glass flat plate, and naturally drying to obtain a polyurethane plastic film;
(2) And (2) dissolving esterase and the polyurethane plastic film obtained in the step (1) in a phosphate buffer solution to carry out depolymerization reaction.
Specifically, in the step (1), the polyurethane plastic is polyester polyurethane.
Preferably, in the step (1), the polyurethane plastic is PBA-PU.
Specifically, in the step (1), the organic solvent is dichloromethane; the mass volume ratio of the polyurethane plastic to the organic solvent is 1.5-2 g:20mL, preferably 1.5g:20mL.
Specifically, in the step (1), the ultrasonic dissolving is performed at an ultrasonic frequency of 40Hz and an ultrasonic temperature of 25-30 ℃, preferably 25 ℃.
In the step (1), the ultrasonic dissolution is carried out for a period of time until the solid in the first mixed solution is completely dissolved.
Wherein, in the step (1), the specification of the glass flat plate is 9cm in diameter.
Wherein, in the step (1), the polyurethane plastic film is cut into a square block of 1cm multiplied by 1cm after being naturally dried and is used as a degradation substrate.
Specifically, in the step (2), the esterase is PETase and Aes72; in the esterase, the mass ratio of PETase to Aes72 is 1: 37-1: 40, preferably 1:37.
specifically, in the step (2), the enzyme activity of the PETase and the Aes72 is measured by using p-NPB, and the enzyme activity is defined as: hydrolysis of p-NPB at 37 ℃ per minute produced 1. Mu. Mol p-nitrophenol defined as one protease activity unit.
Wherein, the protease activity formula is as follows: protease activity = a/(B × C), where a is the amount of p-nitrophenol produced (μmol)), B is the reaction time (min), and C is the amount of enzyme added to the reaction (mL).
Wherein, specific enzyme activity = protease activity (U/mL)/protein concentration (mg/mL).
Specifically, in the step (2), the specific enzyme activity of the PETase is 33-40U/mg; the specific enzyme activity of the Aes72 is 67-75U/mg.
Wherein, the preparation method of the PETase refers to A bacterium that is used as a classifier and is used as a template for preparing polymers, SCIENCE,351 (6278) and 1196-1199.
Wherein, the Aes72 is prepared by a method referred to patent EP 3587570A1.
Specifically, in the step (2), the mass-to-volume ratio of the esterase to the phosphate buffer is 3.8-4.1 mg:5mL, preferably 3.8mg:5mL.
Specifically, in the step (2), the mass-to-volume ratio of the polyurethane plastic film to the phosphate buffer solution is 3-6 mg:1mL, preferably 4 to 5mg:1mL.
Specifically, in the step (2), the depolymerization reaction is carried out at a temperature of 37-40 ℃ for 24-48 h.
Wherein, in the step (2), the phosphate buffer (PB buffer), 50mM phosphate buffer, pH =7.74.
Wherein, in the step (2), the depolymerization reaction is preferably carried out in a shaker at a rotation speed of 200rpm.
In the step (2), after the depolymerization reaction is finished, taking out the reacted polyurethane plastic film, washing the film with pure water, putting the film into an incubator at 30 ℃ for drying, weighing the film to calculate the mass loss before and after the reaction, and performing Fourier infrared spectroscopy (FTIR). After the supernatant of the reaction mixture was centrifuged at 12000rpm for 2min, the supernatant was subjected to High Performance Liquid Chromatography (HPLC).
Wherein the chromatographic conditions of the High Performance Liquid Chromatography (HPLC) are as follows:
liquid chromatography column: agilent 5HC-C18 (2) 150X 4.6mm;
detection wavelength: 240nm;
column temperature: 30 ℃;
flow rate: 1mL/min;
sample injection amount: 10 mu L of the solution;
the mobile phase is water and acetonitrile respectively;
gradient elution: 0-5 min, the volume fraction of water is 90%, and the volume fraction of acetonitrile is 10%; 5-14 min, the volume fraction of water is changed from 90% to 35%, and the volume fraction of acetonitrile is changed from 10% to 65%.
Has the advantages that:
(1) The double-enzyme system used in the invention can improve the enzymolysis effect and simultaneously can completely depolymerize the thermoplastic polyester type polyurethane plastic, thereby improving the utilization rate of the product. Wherein esterase PETase in the double-enzyme system can hydrolyze ester bonds in PBA-PU but cannot hydrolyze urethane bonds. The esterase Aes72 has the activity of carbamate enzyme, can completely depolymerize the oligomer with carbamate bonds generated by hydrolysis of PETase into monomer substances, removes the product inhibition of the oligomer on PETase, improves the degradation efficiency, improves the yield of the monomer substances and improves the product utilization rate.
(2) The double-enzyme system used in the invention can improve the biodegradation efficiency of the thermoplastic polyurethane plastic and simultaneously lead the thermoplastic polyurethane plastic to be completely depolymerized so as to improve the product utilization rate, and has important significance for the biological recovery and reconstruction of PU plastic.
(3) The plastic biological enzyme depolymerization and high-valued technology of the invention has mild conditions, few byproducts and environmental protection, is an ideal means for upgrading and utilizing the waste plastics at the same level, and is a research hotspot at home and abroad.
(4) The plastic is a high molecular material formed by polyaddition or polycondensation of monomer raw materials, for example, the waste can be depolymerized to obtain monomers for reproduction, so that the whole industrial chain becomes a closed loop, and the method is the most ideal waste recycling treatment mode. The double-enzyme system used in the invention can completely depolymerize thermoplastic polyester polyurethane plastic into monomer 4, 4-Methylenedianiline (MDA), and can utilize the monomer generated after depolymerization to perform repolymerization or other high-value utilization, thereby realizing harmless recovery of polyurethane waste plastic.
Drawings
The foregoing and/or other advantages of the invention will become further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.
FIG. 1 is a schematic diagram of PETase and Aes72 double-enzyme degradation PBA-PU.
FIG. 2 shows the mass loss of the PBA-PU film before and after degradation.
FIG. 3 is FTIR analysis of PBA-PU films after degradation.
FIG. 4 is an HPLC analysis of the supernatant after degradation.
FIG. 5 is a MDA concentration calibration curve.
FIG. 6 shows the MDA concentration in the supernatant after degradation.
Detailed Description
The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.
The esterase used by the invention is PETase and Aes72; wherein, the enzyme activity of p-NPB used by PETase and Aes72 is determined, and the enzyme activity is defined as: hydrolysis of p-NPB at 37 ℃ per minute produced 1. Mu. Mol p-nitrophenol defined as one protease activity unit.
Wherein, the protease activity formula is as follows: protease activity = a/(B × C), where a is the amount of p-nitrophenol produced (μmol)), B is the reaction time (min), and C is the amount of enzyme added to the reaction (mL).
Wherein, specific enzyme activity = protease activity (U/mL)/protein concentration (mg/mL).
The specific enzyme activity of PETase is detected to be 33U/mg, and the specific enzyme activity of Aes72 is detected to be 67U/mg.
Wherein, the preparation method of PETase refers to A bacterium (cat) grades and libraries poly (ethylene terephthalate), SCIENCE,351 (6278), 1196-1199.
Among them, reference is made to patent EP 3587570A1 for the preparation of Aes72.
Phosphate buffer (PB buffer) used in the examples of the present invention, 50mM phosphate buffer, pH =7.74.
In the High Performance Liquid Chromatography (HPLC) in the embodiment of the present invention, an instrument used is a high performance liquid chromatograph (Agilent Technologies), and a specific detection method is as follows:
liquid chromatography column: agilent 5HC-C18 (2) 150X 4.6mm;
detection wavelength: 240nm;
column temperature: 30 ℃;
flow rate: 1mL/min;
sample introduction amount: 10 mu L of the solution;
the mobile phase is water and acetonitrile respectively;
gradient elution: 0-5 min, the volume fraction of water is 90%, and the volume fraction of acetonitrile is 10%; 5-14 min, the volume fraction of water is changed from 90% to 35%, and the volume fraction of acetonitrile is changed from 10% to 65%.
Example 1: mass loss measurement of depolymerized polyurethane plastic films
Weighing 1.5g of PBA-PU powder, dissolving in 20mL of dichloromethane solution, and ultrasonically dissolving at 25 ℃ for 1h, wherein the ultrasonic frequency is 40Hz; and after the ultrasonic treatment is finished, pouring the mixed solution into a glass flat plate with the diameter of 9cm, placing the glass flat plate in a fume hood for naturally airing, forming a film, and cutting the film into a film with the thickness of 1cm multiplied by 1cm to be used as a degradation substrate.
Four sets of experiments were designed, control (CK), aes72, PETase and PETase + Aes72. Wherein weighed PBA-PU films (1 cm. Times.1cm, 20-25 mg) were added to 5mL of 50mM PB buffer (pH = 7.74) for each set of experiments. Wherein, no enzyme is added in CK group, 3.7mg of Aese 72 is added in Aes72 group, 0.1mg of PETAse is added in PETAse group, 0.1mg of PETAse + Aese 72 is added in PETAse group, and 3.7mg of Aese 72 is added in PETAse + Aese 72 group.
Then, the four groups of reaction solutions are placed in a shaking table at 37 ℃ and 200rpm for reaction for 48 hours, the film is taken out, washed by pure water, placed at 30 ℃ for drying and weighed, and the weight loss before and after the reaction is calculated by weighing, and the result is shown in figure 2. FIG. 1 is a schematic diagram of PETase and Aes72 dual-enzyme degradation of PBA-PU.
After the reaction, the reaction solution supernatant was centrifuged at 12000rpm for 2min, and the supernatant was stored at 4 ℃ for further use.
As can be seen from FIG. 2, the mass loss results show that the mass loss of the Aes72 group is lower, indicating that the exonuclease activity of the enzyme on PBA-PU is weaker and the activity ratio on high polymer is lower; PETase has higher quality loss, which shows that the enzyme has stronger exonuclease activity to PBA-PU and can effectively hydrolyze high polymer. The quality loss of the PETase + Aes72 group is higher than that of the PETase group and the Aes72 group, which shows that the double enzymes have a synergistic effect on the degradation of the PBA-PU and improves the degradation rate of the PBA-PU.
Example 2: FTIR analysis of polyurethane plastic films after reaction
FTIR analysis was performed on the four reacted PBA-PU films of example 1, and the FTIR was measured by Nicolet iN10 type infrared spectrometer with wave number ranging from 4000 to 500cm -1 The number of scans was 4, and a horizontal attenuated total reflection accessory was provided, and the result is shown in fig. 3.
As can be seen from FIG. 3, the Aes72 group is 1725cm -1 Characteristic peak signal of ester bond (C = O) and 1260cm -1 The characteristic peak signals of the amido bond (C-N) are weakened but the weakening degree is lower, which indicates that the enzyme has certain activity on ester bonds and carbamate bonds but lower activity; PETase group at 1725cm -1 The characteristic peak signal of ester bond (C = O) of (A) was very reduced, but at 1260cm -1 The characteristic peak signal of the amido bond (C-N) is hardly weakened, which shows that PETase can only hydrolyze ester bonds and has stronger hydrolytic capability but can not hydrolyze urethane bonds; the PETase + Aes72 group was at 1725cm -1 Characteristic peak of ester bond (C = O) and 1260cm -1 The characteristic peak signals of amide bond (C-N) are weaker than those of PETase group and Aes72 group, which indicates that the double enzymes hydrolyze ester bond and carbamate bond on PBA-PU more completely, and further proves that the double enzymes have synergistic effect on PBA-PU degradation.
Example 3: HPLC analysis of the supernatant after the reaction
Four groups of reacted supernatants from example 1 were analyzed by HPLC using 1260Infinity ii high performance liquid chromatography (Agilent Technologies) using the following specific detection method:
liquid chromatography column: agilent 5HC-C18 (2) 150X 4.6mm;
detection wavelength: 240nm;
column temperature: 30 ℃;
flow rate: 1mL/min;
sample introduction amount: 10 mu L of the solution;
the mobile phase is water and acetonitrile respectively;
gradient elution: 0-5 min, the volume fraction of water is 90%, and the volume fraction of acetonitrile is 10%; 5-14 min, the volume fraction of water is changed from 90% to 35%, and the volume fraction of acetonitrile is changed from 10% to 65%. The specific detection results are shown in fig. 4.
The MDA content in the supernatant represents a degree of complete depolymerization, since MDA is only produced if both ester and urethane bonds are hydrolyzed. As can be seen from the experimental results of fig. 4, the PETase group did not produce MDA because PETase could hydrolyze only ester bonds and did not hydrolyze urethane bonds, while the Aes72 group produced MDA in a trace amount because Aes72 could hydrolyze both urethane bonds and ester bonds, but the efficiency was not high and the yield was low. The MDA yield of the double enzyme (PETase + Aes72 group) is obviously higher than that of the single enzyme, which indicates that the double enzyme system can depolymerize PBA-PU more completely.
Preparing MDA standard yeast: 19.8mg MDA was weighed out and dissolved in10 mL ethanol to prepare a 10mM stock solution. It was diluted to 1mM, 0.5mM, 0.4mM, 0.3mM, 0.2mM, 0.1mM, 0.05mM of standard sample, respectively. The peak areas corresponding to the different concentrations were analyzed by HPLC to prepare a standard curve of MDA concentration, which is shown in fig. 5.
And (3) MDA content determination: the peak areas determined by HPLC for the four experiments were substituted into the standard to calculate the corresponding MDA concentrations, and the results are shown in fig. 6.
As can be seen from fig. 4, fig. 5, and fig. 6, the Aes72 group produced 0.025mM MDA by HPLC of the four groups of experimental supernatants, indicating that Aes72 has a certain hydrolysis ability of PBA-PU, and can hydrolyze ester bonds and urethane bonds, but has a weak hydrolysis ability; the PETase group does not produce MDA, which indicates that the PETase has no degradation capability on urethane bonds and cannot be further hydrolyzed to produce MDA. The PETase + Aes72 group generates MDA with the thickness of 0.1875mM, which shows that the PETase + Aes72 in the double-enzyme system can simultaneously and efficiently hydrolyze ester bonds and urethane bonds, thereby generating a large amount of monomer substances MDA, and proving that the double-enzyme system can more completely depolymerize thermoplastic polyurethane.
The present invention provides a method and a concept for a method of depolymerizing thermoplastic polyester polyurethane plastic completely with two enzymes, and a method and a way for implementing the method are many, and the above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and embellishments can be made without departing from the principle of the present invention, and these modifications and embellishments should be regarded as the protection scope of the present invention. All the components not specified in the present embodiment can be realized by the prior art.
Claims (10)
1. A method for double-enzyme complete depolymerization of thermoplastic polyester polyurethane plastic is characterized by comprising the following steps:
(1) Dissolving polyurethane plastic in an organic solvent, and dissolving the polyurethane plastic in an ultrasonic manner to obtain a first mixed solution; pouring the first mixed solution into a glass flat plate, and naturally airing to obtain a polyurethane plastic film;
(2) And (2) dissolving esterase and the polyurethane plastic film obtained in the step (1) in a phosphate buffer solution to carry out depolymerization reaction.
2. The method according to claim 1, wherein in step (1), the polyurethane plastic is a polyester polyurethane.
3. The method of claim 2, wherein in step (1), the polyurethane plastic is PBA-PU.
4. The method according to claim 1, wherein in step (1), the organic solvent is dichloromethane; the mass volume ratio of the polyurethane plastic to the organic solvent is 1.5-2 g:20mL.
5. The method according to claim 1, wherein in the step (1), the ultrasonic dissolution is carried out at an ultrasonic frequency of 40Hz and at an ultrasonic temperature of 25-30 ℃.
6. The method according to claim 1, wherein in step (2), the esterase is PETase and Aes72; in the esterase, the mass ratio of PETase to Aes72 is 1:37 to 1:40.
7. the method according to claim 6, wherein in the step (2), the specific enzyme activity of the PETase is 33-40U/mg; the specific enzyme activity of the Aes72 is 67-75U/mg.
8. The method according to claim 1, wherein in the step (2), the mass-to-volume ratio of the esterase to the phosphate buffer is 3.8-4.1 mg:5mL.
9. The method according to claim 1, wherein in the step (2), the mass-to-volume ratio of the polyurethane plastic film to the phosphate buffer is 3-6 mg:1mL.
10. The method according to claim 1, wherein in the step (2), the depolymerization reaction is carried out at a temperature of 37 to 40 ℃ for 24 to 48 hours.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310054434.9A CN115896193A (en) | 2023-02-03 | 2023-02-03 | Method for complete depolymerization of thermoplastic polyester type polyurethane plastic by double enzymes |
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| CN116814712A (en) * | 2023-06-25 | 2023-09-29 | 南京工业大学 | Method for producing amine monomer by degrading polyurethane through chemical biological method |
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| CN116814712A (en) * | 2023-06-25 | 2023-09-29 | 南京工业大学 | Method for producing amine monomer by degrading polyurethane through chemical biological method |
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