A method for improving catalytic degradation of pet plastic by cutinase

Abstract

The application discloses a method for improving catalytic degradation of PET plastic by cutinase, and the method comprises the following steps: preparing a high-molecular condensed phase, collecting a concentrated phase of the condensed phase by centrifugation, adding LCC-ICCG-218Y enzyme solution and PET solid to the concentrated phase, and oscillating to make the concentrated phase uniformly distributed in the reaction system, starting the reaction under oil bath at 40-72 DEG C, collecting the reaction liquid, and characterizing the degradation product of the reaction liquid by ultraviolet. Compared with a phosphate buffer solution, the degradation rate of PET plastic at 40 DEG C and 50 DEG C is finally doubled. The method establishes a simple method for improving enzymatic hydrolysis of PET plastic, and only by adjusting the reaction microenvironment of the enzyme, without complex protein modification and enzyme immobilization means, the method has certain application value.

Description

Technical Field

[0001] This invention belongs to the field of enzyme reaction microenvironment regulation, and specifically relates to a method for improving the catalytic degradation of PET plastic by keratinase. Background Technology

[0002] Polyethylene terephthalate (PET) is a common plastic, renowned for its high mechanical properties, good processability, and excellent chemical stability, and is often used as a raw material for various packaging bottles. However, precisely because of its high-temperature resistance, chemical corrosion resistance, and difficulty in biodegradation, PET plastic has caused serious environmental problems. Finding a proper way to dispose of plastic waste is urgent. Compared with traditional physical recycling and chemical depolymerization, enzymatic depolymerization processes are milder, require no hazardous chemicals or expensive equipment, and the degradation products can be recycled, avoiding secondary pollution, making it considered an ideal method for PET depolymerization. A thermostable cutinase (LCC) derived from leaf and branch compost can cleave ester bonds, decomposing PET into monomers terephthalic acid (TPA) and ethylene glycol (EG). Jeffrey D. Martell et al. discovered that the H218Y mutant of LCC-ICCG can improve the binding of the enzyme to the PET substrate and enhance the depolymerization kinetics of PET.

[0003] Proteins, as complex biological macromolecules, play a crucial role in catalyzing metabolism due to their high efficiency and specificity. Enzymes are easily inactivated under harsh conditions, such as high temperatures, polar solvents, extreme pH values, and proteases. Because many factors influence enzyme catalytic activity, various methods are needed to enhance it. Currently, commonly used regulatory techniques include carrier immobilization modification, chemical covalent modification, and protein engineering. Carrier immobilization modification ensures high enzyme loading, but may also lead to changes in enzyme function due to its fragility; chemical covalent modification requires high enzyme tolerance to the reaction environment; protein engineering requires rational protein design and regulation of enzyme active sites or substrate / product channel structures to improve enzyme activity, presenting significant technical barriers. Summary of the Invention

[0004] The present invention aims to overcome the shortcomings of existing technologies in enzyme-catalyzed degradation of PET plastics and to develop a method that can improve the catalytic degradation of PET plastics by keratinase.

[0005] The technical solution of the present invention to achieve the above objectives is as follows:

[0006] A method for improving the degradation of PET plastic catalyzed by keratinase includes the following steps:

[0007] S1. Prepare the condensed phase of the polymer system. The viscosity of the system is controlled by KCl solution, and the concentrated phase of the condensed phase is collected by centrifugation.

[0008] S2. LCC-ICCG-218Y enzyme solution was prepared by microbial induced expression, and the expressed product was purified by affinity chromatography.

[0009] S3. Use a punch to prepare small round pieces of solid PET, and then process them with scissors.

[0010] S4. Add the LCC-ICCG-218Y enzyme solution and PET discs to the concentrated phase, mix the system evenly using a shaker to obtain a mixture, and heat to carry out the reaction.

[0011] S5. Collect the reaction solution and dilute it with KCl solution;

[0012] S6. Take the supernatant and detect the product by ultraviolet absorption.

[0013] Furthermore, the polymeric condensed phase component is two of polylysine, polyglutamic acid, polydiallyldimethylammonium chloride, and sodium polyacrylate; and is composed of a molar ratio of 40:100, 70:100, or 100:100.

[0014] Furthermore, the concentrated phase has a component concentration of 50-150 mM, a pH value of 5-10, and contains 0.05-0.1 mol / L phosphate buffer.

[0015] Furthermore, the concentration of the KCl solution is 1 mol / L.

[0016] Furthermore, the enzyme concentration in the mixture is 6 nmol / L-2 μmol / L.

[0017] Furthermore, the PET solid is a sheet-like film with a crystallinity of 4-7% and a thickness of 0.25 mm.

[0018] Furthermore, the mass-to-volume ratio of the PET solid to the concentrated phase is 2-20 g / L.

[0019] Furthermore, the reaction temperature is 40-72 ℃; the reaction stirring speed is 0-600 rpm.

[0020] Furthermore, the enzyme solution is one of keratinase LCC, and its mutants ICCG and 218Y;

[0021] Preferably, the enzyme solution is LCC-ICCG-218Y, which is produced by microbial induction of LCC-ICCG-218Y protein, and then purified to obtain an aqueous solution of LCC-ICCG-218Y enzyme.

[0022] The sequence of LCC-WT (WT) in the present invention is as follows:

[0023] MSNPYQRGPNPTRSALTADGPFSVATYTVSRLSVSGFGGGVIYYPTGTSLTFGGIAMSPGYTADASSLAWLGRRLASHGFVVLVINTNSRFDYPDSRASQLSAALNYLRTSSPSAVRARLDANRLAVAGHSMGGGGTLRIAEQNPSLKAAVPLTPWHTDKTFNTSVPVLIVGAEADTVAPVSQHAIPFYQNLPSTTPKVYVELDNASHFAPNSNNAAISVYTISWMKLWVDNDTRYRQFLCNVNDPALSDFRTNNRHCQLEHHHHHH

[0024] The sequence of LCC-ICCG (ICCG) is as follows:

[0025] MSNPYQRGPNPTRSALTADGPFSVATYTVSRLSVSGFGGGVIYYPTGTSLTFGGIAMSPGYTADASSLAWLGRRLASHGFVVLVINTNSRFDGPDSRASQLSAALNYLRTSSPSAVRARLDANRLAVAGHSMGGGGTLRIAEQNPSLKAAVPLTPWHTDKTFNTSVPVLIVGAEADTVAPVSQHAIPFYQNLPSTTPKVYVELCNASHIAPNSNNAAISVYTISWMKLWVDNDTRYRQFLCNVNDPALCDFRTNNRHCQLEHHHHHH

[0026] The sequence of LCC-ICCG-218Y is as follows:

[0027] MSNPYQRGPNPTRSALTADGPFSVATYTVSRLSVSGFGGGVIYYPTGTSLTFGGIAMSPGYTADASSLAWLGRRLASHGFVVLVINTNSRFDGPDSRASQLSAALNYLRTSSPSAVRARLDANRLAVAGHSMGGGGTLRIAEQNPSLKAAVPLTPWHTDKTFNTSVPVLIVGAEADTVAPVSQYAIPFYQNLPSTTPKVYVELCNASHIAPNSNNAAISVYTISWMKLWVDNDTRYRQFLCNVNDPALCDFRTNNRHCQLEHHHHHH.

[0028] The present invention has the following advantages and effects compared with the prior art:

[0029] 1. The present invention only requires adjustment of the type and molar ratio of the condensed phase and the concentrated phase, as well as the reaction temperature and stirring speed. It is simple to operate, has mild reaction conditions, and low equipment requirements.

[0030] 2. For the enzyme-catalyzed degradation reaction of PET plastics, the condensed phase concentrated phase reaction environment provided by this invention has a faster reaction rate than that of phosphate buffer solution. Attached Figure Description

[0031] Figure 1 This is a kinetic curve of enzyme degradation of PET in a concentrated phase and phosphate environment at 40℃;

[0032] Figure 2 This is a kinetic curve of enzyme degradation of PET in a concentrated phase and phosphate environment at 50℃;

[0033] Figure 3 The effect of different concentrations on the enzyme-catalyzed degradation of PET plastic;

[0034] Figure 4 This describes the effect of different concentrations on the enzyme-catalyzed degradation of PET plastic. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to specific embodiments, but the implementation of the present invention is not limited thereto. For process parameters not specifically noted, conventional techniques can be referred to.

[0036] Example 1

[0037] Step 1: Extraction of the concentrated phase from the condensed phase, including the following steps:

[0038] Take component a (see table below) with a concentration of 400 mM and a pH of 8, and component b (see table below) with a concentration of 400 mM and a pH of 8, and mix them at a molar ratio of 40:100, 70:100, or 100:100. Simultaneously add 1 mol / L KCl solution to adjust the viscosity of the system. Centrifuge at 8000 x g for 4 min, and completely remove the supernatant to obtain the concentrated phase. The above concentrated phase contains 0.05-0.1 M phosphate buffer.

[0039]

[0040] Step 2: Pretreatment of the PET substrate, including the following steps:

[0041] Amorphous PET solids purchased from Goodfellow were prepared into circular sheets with a diameter of approximately 0.03-0.8 cm using a punch, and then processed with scissors. The PET solids were sheet-like films with a crystallinity of approximately 4-7% and a thickness of 0.25 mm.

[0042] Step 3: Expression, purification, and identification of keratinase (LCC-ICCG-218Y)

[0043] Take 1-2 μL of the recombinant plasmid containing the LCC-ICCG-218Y gene and add it to 100 μL of E. coli BL21(DE3) competent cells thawed on ice. Gently mix and incubate on ice for 15-20 minutes. Then, place the cells in a 42°C water bath for 90 seconds, followed immediately by an ice bath for 5 minutes. Add 1 mL of LB medium (antibiotic-free) and incubate with shaking for 1 hour (37°C, 170-250 rpm). After incubation, spread 100-300 μL of the plasmid onto LB agar containing kanamycin (50 μg / mL) and incubate overnight at 37°C. The resulting single colony is E. coli BL21(DE3). pET 28b(+) LCC-ICCG-218Y. Pick single colonies and incubate them in LB medium containing kanamycin (50 μg / mL) for 10-16 hours. When the OD value reaches 0.6-0.8, inoculate the bacterial suspension at a ratio of 1:100 into LB medium containing kanamycin (50 μg / mL), and expand the culture at 37℃ and 170-250 rpm until the OD600 of the bacterial suspension is 0.6-0.8. After cooling to room temperature, add IPTG to a final concentration of 0.2 mmol / L and incubate at 18℃ and 225 rpm for 20 hours to induce expression. The induced bacterial cells were collected by centrifuging at 6000 rpm for 20 min at 4 °C using a high-speed refrigerated centrifuge. The cells were resuspended in tirs-HCl buffer (pH 7.5-8) and sonicated for 30 min at 140-220 W for 5 s on and 5.5 s off. The cells were then centrifuged at 15000 x g for 30 min at 4 °C using a high-speed refrigerated centrifuge. The supernatant was obtained as the crude enzyme solution, which was then purified by nickel affinity chromatography to obtain the LCC-ICCG-218Y enzyme solution.

[0044] Step 4, the method for LCC-ICCG-218Y enzyme-catalyzed degradation of PET plastic, includes the following specific steps:

[0045] Add 0.1M phosphate buffer (pH=8) prepared with ultrapure water, 2.4 mg of PET discs (0.25 mm thick), and 0.1 μmol / L LCC-ICCG-218Y enzyme solution to a 1.5 ml glass bottle. Mix thoroughly to obtain the reaction solution. Incubate in an oil bath at 50°C and 600 rpm for 7 days. Collect the reaction solution periodically and detect the degradation products using UV light.

[0046] Step 5: Calculation of degradation products, including the following steps:

[0047] The concentration of terephthalic acid (TPA) was calculated based on its absorbance values ​​at 240-260 nm, and a standard curve was plotted for TPA. Meanwhile, in the reference, a mass-to-volume ratio of PET to the reaction solution of 2 g / L resulted in a complete conversion to 10.4 mM TPA; this concentration was used as the 100% PET degradation rate to characterize the degradation effect.

[0048] Example 2 Enzymatic degradation of PET plastic in a concentrated phase and phosphate buffer solution environment at 40°C

[0049] A concentrated phase of LCC-ICCG-218Y enzyme solution (40:100 molar ratio) was mixed thoroughly with 2-20 mg of PET discs (0.25 mm thick) to obtain a homogeneous solution. The concentration of LCC-ICCG-218Y enzyme in this solution was 0.1 μmol / L. The solution was incubated in an oil bath at 40°C and 600 rpm for 1-18 days. The reaction solution was collected periodically, and the absorbance of the product was measured using UV light. The results are as follows: Figure 1 After 18 days of reaction, the degradation rate of the concentrated phase was nearly twice that of the buffer phase, at 65.5±3.92% and 37.23±0.72%, respectively.

[0050]

[0051] Example 3 Enzymatic degradation of PET plastic in a concentrated phase and phosphate buffer solution environment at 50°C

[0052] A concentrated phase (40:100 molar ratio, see table below), LCC-ICCG-218Y enzyme solution, and 2.4 mg of PET discs with a thickness of 0.25 mm were mixed thoroughly to obtain a mixture. The concentration of LCC-ICCG-218Y enzyme in the above mixture was 0.1 μmol / L. The mixture was incubated in an oil bath at 50℃ and 600 rpm for 5 days. The reaction solution was collected at intervals, and the absorbance of the product was detected by UV. The results are as follows. Figure 2After 5 days of reaction, the degradation rates of the concentrated phase and phosphate buffer were 95±2.41% and 42.27±7.21%, respectively, indicating that the concentrated phase significantly improved the degradation effect of keratinase LCC-ICCG-218Y on PET plastic.

[0053]

[0054] Example 4: Effect of different concentrations on enzyme-catalyzed degradation of PET plastic

[0055] The concentrated phase (see table below), LCC-ICCG-218Y enzyme solution, and 2.4 mg of PET disc with a thickness of 0.25 mm were mixed thoroughly to obtain a mixture. The concentration of LCC-ICCG-218Y enzyme in the above mixture was 0.1 μmol / L. The mixture was incubated in an oil bath at 50℃ and 600 rpm for 7 days. The reaction solution was collected at intervals, and the absorbance of the product was detected by UV. The results are as follows. Figure 3 As observed, the degradation rate of PET plastic catalyzed by enzymes varies in different concentration phases.

[0056]

[0057] Example 5: Effect of different concentration ratios on enzyme-catalyzed degradation of PET plastic

[0058] A 400 mM aqueous solution of polydienepropylene dimethyl ammonium chloride with a pH of 8 was mixed with a 400 mM aqueous solution of sodium polyacrylate with a pH of 8 at a molar ratio of 40:100, 70:100, or 100:100. A 2.4 mg PET disc with a thickness of 0.25 mm was then mixed thoroughly to obtain a mixture. The concentration of LCC-ICCG-218Y enzyme in the above mixture was 0.1 μmol / L. The mixture was incubated in an oil bath at 50°C and 600 rpm for 5 days. The reaction solution was collected at regular intervals, and the absorbance of the product was detected by UV light. The results are as follows: Figure 4 The reaction was carried out at 50℃ for 5 days. The effect of enzyme-catalyzed degradation of PET plastic varied depending on the concentration ratio of the concentrated phase.

[0059]

[0060] The above embodiments of the present invention are merely examples for clearly illustrating the present invention and are not intended to limit the implementation of the present invention. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all possible implementations here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for improving the catalytic degradation of PET plastic by keratinase, characterized in that, Includes the following steps: S1. Prepare a polymeric condensate phase. The viscosity of the system is controlled by KCl solution, and the concentrated phase of the condensate phase is collected by centrifugation. The components of the polymeric condensate phase are two of polylysine, polyglutamic acid, polydiallyldimethylammonium chloride, and sodium polyacrylate. The components of the polymeric condensate phase are composed of molar ratios of 40:100, 70:100, or 100:

100. The concentration of the components in the concentrated phase is 50-150 mM, the pH value is 5-10, and it contains 0.05-0.1 mol / L phosphate buffer. S2. LCC-ICCG-218Y enzyme was produced by microbial induction expression. The supernatant after ultrasonic disruption and centrifugation was purified by affinity chromatography to obtain pure enzyme solution. S3. Cut the solid PET into small pieces; S4. Add the LCC-ICCG-218Y enzyme solution and PET flakes to the concentrated phase. The sequence of the LCC-ICCG-218Y enzyme is shown in SEQ ID NO.

3. Mix the system thoroughly using a shaker to obtain a mixture, and heat to carry out the reaction. The concentration of LCC-ICCG-218Y enzyme in the mixture is 6 nmol / L - 2 μmol / L. S5. Collect the above reaction solution and dilute it with KCl solution; S6. Take the supernatant and detect the product by ultraviolet absorption.

2. The method for improving the keratinase-catalyzed degradation of PET plastic according to claim 1, characterized in that, In S1, the concentration of KCl is 0.5-3 mol / L.

3. The method for improving the keratinase-catalyzed degradation of PET plastic according to claim 1, characterized in that, In S1, the centrifugation speed and time are 5000-9000 x g and 4-8 min.

4. The method for improving the catalytic degradation of PET plastic by keratinase according to claim 1, characterized in that, In S4, the PET solid in the mixture is a sheet-like film with a crystallinity of 4%-7% and a thickness of 0.1-0.3 mm. The mass-to-volume ratio of PET solid to concentrated phase is 2-20 g / L.

5. The method for improving the keratinase-catalyzed degradation of PET plastic according to claim 1, characterized in that, In S4, the reaction temperature in the mixture is 40-72℃; the stirring speed is 0-600 rpm; and the reaction time is 1-18 days.

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