Laccase derived from bacteria and application of laccase in degradation of polyurethane plastics
By using Bacillus subtilis bacteria laccase CotA to hydrolyze the ester bonds and urethane bonds in polyester type polyurethane plastics, the problem of limited enzyme types in the prior art is solved, and the effect of efficient degradation of polyester type polyurethane plastics is achieved.
Patent Information
- Application Number
- CN202510382929.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-28
- Publication Date
- 2025-07-01
AI Technical Summary
In the prior art, there are limited types of enzymes used to degrade polyurethane plastics, especially fewer enzymes that directly degrade carbamate bonds, resulting in low degradation efficiency.
The bacterial laccase CotA derived from Bacillus subtilis is used to achieve efficient degradation by hydrolyzing the ester bonds and urethane bonds in polyester-type polyurethane plastics.
Bacterial laccase CotA can significantly degrade polyester polyurethane plastics, thinning and transparent films, increasing pores on the surface of foam, significant mass loss, reduced molecular weight, weakened characteristic peaks, cracks and holes on the surface, proving efficient hydrolysis of ester bonds and urethane bonds.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of biological treatment, and relates to a laccase derived from bacteria and its application in degrading polyurethane plastics. Background Art
[0002] The information provided in this section is only background information related to the present disclosure, and it is not necessarily prior art.
[0003] PU is a common type of plastic with very wide uses, including insulation barriers in construction (as a main component of automotive interior parts), adhesives, and foams in seats and bedding. They are mainly used to resist the erosion of environmental factors, such as corrosion by microorganisms, reduction of wear, decomposition by water, or damage by ultraviolet rays. The extensive use of a large number of PU products has generated a lot of waste plastics, and due to the lack of reasonable recycling and treatment, it has caused serious damage and threat to the ecosystem. So far, using microorganisms to treat polyurethane waste is considered the most environmentally friendly method and is also one of the research hotspots for treating polyurethane waste. Since urethane bonds, ester bonds, amide bonds, peptide bonds, etc. are commonly present in industrially used PU plastics, there are few reported enzymes with PU degradation. And the specific degradation mechanism varies due to different PU types, the characteristics of the strains themselves, and environmental conditions, etc. So far, these determined PU hydrolases are mainly polyester-type hydrolases, which can only hydrolyze the ester bonds at the soft end of PU, and there are very few enzymes that can directly degrade urethane bonds, so the degradation efficiency is low.
[0004] Laccase (EC 1.10.3.2) is a polyphenol oxidase that can catalytically oxidize phenolic and aromatic amine compounds while reducing molecular oxygen to water. Bacterial laccase has applications in fields such as dye degradation, the paper industry, electrochemistry, and biosensors. This study found the role of bacterial laccase in the field of PU plastic degradation, which can effectively hydrolyze the urethane bonds in PU. This discovery enriches the resource library of PU-degrading enzymes. Summary of the Invention
[0005] Object of the Invention: The technical problem to be solved by the present invention is to provide a laccase derived from bacteria and its application in degrading polyurethane plastics in view of the deficiencies of the prior art.
[0006] To solve the above technical problems, the present invention discloses the following technical solutions:
[0007] In a first aspect, the present invention discloses the application of a laccase CotA derived from bacteria in depolymerizing polyester-type polyurethane (PU) plastics.
[0008] In a second aspect, the present invention discloses a method for depolymerizing polyester-type polyurethane plastics, which is to add a laccase CotA derived from bacteria to polyester-type polyurethane plastics.
[0009] In the above first aspect and second aspect,
[0010] In some embodiments, the laccase CotA is laccase CotA derived from Bacillus subtilis 168, and its amino acid sequence is shown as SEQ ID NO.1.
[0011] In some embodiments, the specific enzyme activity of the laccase CotA is 6 - 10 U / mg, such as 7 U / mg, 8 U / mg, 9 U / mg. In some embodiments, the method for measuring the enzyme activity of the laccase CotA is as follows: The enzyme activity measurement reaction system is 3 mL: 10 μL of enzyme solution, 150 μL of 10 mM ABTS, 2.75 mL of 50 mM phosphate buffer solution (pH 4.0). After reacting at room temperature for 3 min, a boiling water bath is carried out for 5 min, and the absorbance is detected at OD 420 . The specific enzyme activity = protease activity (U / mL) / protein concentration (mg / mL).
[0012] In some embodiments, the polyester - type polyurethane plastics include thermoplastic polyester - type polyurethane films (thermoplastic PBA - PU films) and thermosetting PU foams.
[0013] In some embodiments, the dosage ratio of the laccase to the polyester - type polyurethane plastics is 0.8 - 1.2 U: 50 mg, such as 1.0 U: 50 mg.
[0014] In some embodiments, the depolymerization system further includes a buffer ion mediator. The buffer is preferably sodium acetate buffer with a pH of 3.0 - 5.0, and the ion mediator is preferably 2,2'-azino - bis(3 - ethylbenzothiazoline - 6 - sulfonic acid) (ABTS).
[0015] In some embodiments, the mass - to - volume ratio of the polyester - type polyurethane plastics to the buffer is 8 - 12 mg / mL, such as 10 mg / mL. In some embodiments, in the reaction system, the final concentration of the ion mediator is 0.01 - 0.03 mM, such as 0.02 mM.
[0016] In some embodiments, the reaction temperature for depolymerization is 25 - 35 °C, and the reaction time is 2 - 10 days.
[0017] The laccase CotA derived from bacteria provided by the present invention has the ability to efficiently catalyze the hydrolysis of ester bonds and urethane bonds in PU plastics and can be used for depolymerizing polyester - type polyurethane plastics. Specifically, for example: Under the condition of 30 °C, during a 10 - day depolymerization cycle, apparently, the PBA - PU film becomes significantly thinner and transparent, and the surface pores increase ( Figure 2 A), and the surface of the PU foam significantly turns yellow and the pores become larger (Figure 2 B), the apparent depolymerization effect is significant. The mass loss of the PBA-PU film is 15.08% ( Figure 3 ). GPC shows that the molecular weights M n , M w and M z of the PBA-PU film decrease by 24.11%, 19.43%, and 41.11% respectively after depolymerization ( Figure 4 ). FTIR shows that after the depolymerization of the PBA-PU film and the PU foam, the characteristic peak intensities of the carbonyl group (C=O) corresponding to around 1721 cm -1 and the urethane group (-C-NH-) corresponding to around 1267 cm -1 and 1532 cm -1 are significantly weakened ( Figure 5 A and 5B). SEM characterization shows that the surfaces of the PBA-PU film and the PU foam become rough after depolymerization, and cracks and holes appear ( Figure 6 A and 6B). It can be seen that the technical solution provided by the present invention solves the problems in the prior art that the types of enzymes with the ability to degrade PU are limited, especially the enzymes that directly degrade urethane bonds are less.
[0018] Beneficial effects:
[0019] The bacterial laccase CotA used in the present invention can effectively degrade different types of polyester-based PU substrates (thermoplastic PBA-PU film and thermosetting PU foam), mainly by acting on the ester bonds and urethane bonds in PU for hydrolysis, providing an efficient depolymerization element and theoretical guidance for the enzymatic depolymerization technology of PU plastics. Description of the drawings
[0020] The following further specifically describes the present invention in conjunction with the drawings and specific embodiments, and the above and / or other advantages of the present invention will become clearer.
[0021] Figure 1 It is the SDS-PAGE electrophoresis diagram of laccase CotA.
[0022] Figure 2 It is the physical diagram of laccase CotA before and after depolymerizing polyester-based plastics.
[0023] Figure 3 It is the mass loss of laccase CotA on the PBA-PU film before and after depolymerization.
[0024] Figure 4 It is the GPC characterization of laccase CotA on the PBA-PU film before and after depolymerization.
[0025] Figure 5FTIR characterization of polyester plastics before and after depolymerization by laccase CotA. A and B are thermoplastic PBA-PU film diagrams, and C and D are thermosetting PU foam diagrams.
[0026] Figure 6 SEM characterization of polyester plastics before and after depolymerization by laccase CotA. A is a thermoplastic PBA-PU film diagram, and B is a thermosetting PU foam diagram. Specific implementation mode
[0027] According to the following embodiments, the present invention can be better understood. However, those skilled in the art can easily understand that the content described in the embodiments is only used to illustrate the present invention and should not and will not limit the present invention detailed in the claims.
[0028] In the following embodiments, the experimental methods are all conventional methods unless otherwise specified; the reagents and materials can be obtained from commercial channels unless otherwise specified.
[0029] Heterologous Expression and Protein Purification of Laccase CotA
[0030] The protein expression strain pET-28a(+)-cotA of laccase CotA was constructed in the previous laboratory (Yue Zhangetal. 2018. Surface Display of Bacterial Laccase CotAon Escherichia coli Cellsand its Application in Industrial Dye Decolorization). It was fermented intracellularly by Escherichia coli. The medium used was LB medium, and the resistance used was kanamycin sulfate. After induced expression, through purification treatment and detected by SDS-PAGE, the results are as Figure 1 shown. In the 200 mM imidazole eluent (pH 8.0), the protein band is single and the band size is 65.0 kDa, and the theoretical protein size is about 65.0 kDa.
[0031] Streak the pET-28a(+)-cotA bacterial solution on an LB plate containing kanamycin sulfate resistance. After culturing on a solid plate for 12 h, pick a single colony and inoculate it into an LB liquid medium containing kanamycin sulfate resistance. After culturing at 37 °C for 12 h, add the Escherichia coli bacterial solution cultured in LB medium to the LB liquid medium at an inoculation amount of 1% (v / v) and culture at 37 °C for 2.5 h. Then, wait for OD 600Between 0.6 - 0.8, IPTG with a final concentration of 0.1 mM was added. After inducing at 18 °C for 24 h, the bacterial solution was transferred to a centrifuge tube and centrifuged at 8000 rpm for 10 min. Then, the supernatant was discarded and the cells were retained. After the cells were ultrasonically disrupted, the supernatant was collected by centrifugation, which was the crude enzyme solution. After purification through a sterile filter membrane, the pure enzyme could be obtained.
[0032] Among them, the formula of LB liquid medium is: 10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast extract.
[0033] Preparation Method of PBA-PU Film
[0034]
[0035] Synthesis raw material PBA - PU: 120 g of poly(butylene adipate) diol (PBA, MW: 2000) was dissolved in 60 mL of DMSO solution under nitrogen protection. A DMSO solution (80 mL) containing 18 g of MDI was slowly added dropwise to the reaction solution within 1 h. The reaction was stirred at room temperature for 1 h, and then slowly heated to 80 - 85 °C and kept warm for 12 h until the reaction solution became viscous. Immediately, the reaction solution was transferred to a beaker. After naturally cooling to room temperature, it was freeze-dried at -80 °C for 48 h. The residual DMSO was soaked in deionized water for 12 h, washed several times, and the white solid was collected after filtration, which was PBA - PU. 1H NMR (400 MHz, DMSO) δ 9.39 (s, 1H), 8.45 (s, 1H), 7.27 - 7.25 (m, 4H), 7.03 (m, 2H), 4.64 - 4.56 (br s, 1H), 3.94 (s, 2H), 3.64 (t, J = 6.0 Hz, 2H), 2.67 (t, J = 7.5 Hz, 2H), 2.33 (t, J = 7.5 Hz, 2H), 2.21 - 2.20 (m, 2H), 1.53 (quin, J = 7.0 Hz, 2H), 1.45 (quin, J = 7.0 Hz, 2H).
[0036] Preparing the raw material into a film: 1.5 g of PBA - PU was dissolved in 20 mL of dichloromethane. After stirring on a magnetic stirrer until dissolved, it was sonicated for 30 min and then poured onto a clean glass petri dish and volatilized in a fume hood to obtain a PBA - PU film.
[0037] Enzyme Activity Assay Method
[0038] The enzyme activity was determined by the ABTS method. The total volume of the reaction system was 3 mL, which included: 10 μL of crude enzyme solution, 150 μL of 10 mM ABTS solution (Sigma), and 2.75 mL of 50 mM phosphate buffer (100 mM, pH 4.0). The blank control replaced the enzyme solution with an equal volume of buffer. The reaction system was initiated in a constant temperature water bath at 30 ± 0.5 °C. After reacting precisely for 3 min, it was immediately transferred to a boiling water bath to terminate the reaction for 5 min. After the sample cooled to room temperature, the absorbance was measured at 420 nm using an ultraviolet-visible spectrophotometer (Infinite M, Tecan). Each experiment was set with three replicates.
[0039]
[0040] Where: ΔA420 = A2 of the experimental group - A1 of the blank group
[0041] 0.1844 = Molar extinction coefficient of ABTS (L·μmol-1·cm-1)
[0042] D = Dilution factor of the enzyme solution
[0043] t = Catalytic reaction time (seconds), t = 180 in this experiment
[0044] 60 = Time conversion coefficient (seconds → minutes)
[0045] Specific enzyme activity = Enzyme activity (U / mL) / Protein concentration (mg / mL)
[0046] The specific enzyme activity of laccase CotA was detected to be 8.3819 U / mg.
[0047] Example 1: Degradation of different polyester-based plastic substrates by laccase CotA
[0048] 0.125 mg of laccase CotA and ABTS with a final concentration of 0.02 mM were added to 5 mL of sodium acetate buffer containing 50 mg ± 5 mg of PBA-PU film. The reaction was carried out at 30 °C and pH 4.0 for 10 d. After the reaction of laccase CotA degrading the PBA-PU film ended, the film was recovered, rinsed, dried to a constant weight at 30 °C, and the mass loss rate was calculated. At the same time, no laccase CotA was added as a comparison. The results show that Figure 2 as can be seen from A, after laccase CotA degraded the PBA-PU film for 10 d, it can be found from the physical picture that the film became transparent. From Figure 3 it can be seen that its mass loss rate reached 15.08%.
[0049] Add 0.125 mg of laccase CotA and ABTS with a final concentration of 0.02 mM to 5 mL of sodium acetate buffer containing 50 mg ± 5 mg of PU foam, and react at 30 °C and pH 4.0 for 10 d. After the reaction of laccase CotA degrading PU foam, recover and rinse the film, and dry it at 60 °C to constant weight. At the same time, use the absence of laccase CotA as a control. The results show that Figure 2 As can be seen from Figure B, after 10 d of degradation of PU foam by laccase CotA, it can be found from the physical picture that the surface of the foam becomes significantly yellowish and the pores become larger.
[0050] Example 2: Characterization of the Degraded Polyester-Type Plastic Substrate by Laccase CotA
[0051] After laccase CotA depolymerizes the PBA-PU film and PU foam in Example 1, analyze the changes in the material properties of the degraded polyester-type PU by GPC, FTIR, and SEM.
[0052] As can be seen from Figure 4 Figure B, after the degraded PBA-PU film is characterized by GPC, the number-average molecular weight (M n n), weight-average molecular weight (M w w), and z-average molecular weight (M z z) of the PBA-PU film all decrease, by 24.11%, 19.43%, and 41.11% respectively. At the same time, as can be seen from the Figure 4 effluent curve in Figure A, as the retention time of the substance increases, the signal intensity of the depolymerized film is significantly lower than that of the control without enzyme, indicating that the molecular weight of the depolymerized film is smaller than that of the control group, and the PBA-PU film undergoes depolymerization.
[0053] As can be seen from Figure 5 Figures A and 5C, FTIR analysis shows that there is a stretching vibration peak of free imino (-NH) at 3419 cm -1 . 1721 cm -1 is the stretching vibration peak of the carbonyl group (C=O), and 1267 cm -1 is the bending vibration peak of the urethane bond (-C-NH-). After the polyester-type plastic is depolymerized, the signal peak at the free imino (3419 cm -1 ) is enhanced, the signal at the characteristic peak of the urethane bond (1267 cm -1 ) is weakened, and the signal at the characteristic peak of the ester bond (1721 cm -1 ) is significantly weakened, indicating that the urethane bond breaks and free imino is generated during the depolymerization process. As can be seen from Figure 5 Figures B and 5C, the absorbance of each characteristic peak shows that at the ester bond (1721 cm -1 ) and the urethane bond (1267 cm -1) absorbance decreased, indicating that laccase CotA hydrolyzes ester bonds and carbamate bonds when depolymerizing polyester plastics. This result further proves that the hydrolysis sites of laccase CotA when depolymerizing polyester plastics are ester bonds and carbamate bonds.
[0054] Depend on Figure 6 It can be seen that after the degraded polyester plastic was characterized by SEM, at magnifications of 500× and 1000×, it can be clearly observed that the surface of the depolymerized polyester plastic became rough and had cracks and holes compared with the control group, indicating that both were severely attacked by enzymes.
[0055] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.
Claims
1. Application of a bacterial laccase CotA in depolymerization of polyester polyurethane plastics.
2. The use according to claim 1, characterized in that: The amino acid sequence of the laccase CotA is shown in SEQ ID NO.1; preferably, the specific enzyme activity of the laccase CotA is 6-10 U / mg.
3. The use according to claim 1, characterized in that: The polyester polyurethane plastics include thermoplastic polyester polyurethane films and thermosetting polyurethane foams.
4. The use according to claim 1, characterized in that: The dosage ratio of the laccase and the polyester type polyurethane plastic is 0.8-1.2U:50mg.
5. The use according to claim 1, characterized in that: The depolymerization system further comprises a buffer and an ion mediator, wherein the buffer is preferably a sodium acetate buffer at pH 3.0-5.0, and the ion mediator is preferably 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid; preferably, the mass volume ratio of the polyester polyurethane plastic to the buffer is 8-12 mg / mL; preferably, in the reaction system, the final concentration of the ion mediator is 0.01-0.03 mM; Preferably, the reaction temperature of the depolymerization is 25-35°C, and the reaction time is 2-10 days.
6. A method for depolymerizing polyester polyurethane plastics, characterized in that: The bacterial laccase CotA was added to polyester-type polyurethane plastic.
7. The method according to claim 6, characterized in that The amino acid sequence of the laccase CotA is shown in SEQ ID NO.1; preferably, the specific enzyme activity of the laccase CotA is 6-10 U / mg.
8. The method according to claim 6, characterized in that The polyester polyurethane plastic includes thermoplastic polyester polyurethane film and thermosetting PU foam.
9. The method according to claim 6, characterized in that The dosage ratio of the laccase and the polyester type polyurethane plastic is 0.8-1.2U:50mg.
10. The method according to claim 6, characterized in that The depolymerization system further comprises a buffer and an ion mediator, wherein the buffer is preferably a sodium acetate buffer at pH 3.0-5.0, and the ion mediator is preferably 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid; preferably, the mass volume ratio of the polyester polyurethane plastic to the buffer is 8-12 mg / mL; preferably, in the reaction system, the final concentration of the ion mediator is 0.01-0.03 mM; Preferably, the reaction temperature of the depolymerization is 25-35°C, and the reaction time is 2-10 days.