A method for high throughput screening of polyester-degrading enzymes and compounds used therein

Abstract

The application discloses a method for high-throughput screening of polyester-degrading enzymes and a compound used in the method. The compound is 3-hydroxy-4-((2-hydroxyethoxy)carbonyl)benzoic acid, namely MHET-OH. Experiments prove that the activity and thermal stability of a polyester-degrading enzyme mutant V5 pure enzyme obtained by using the MHET-OH provided by the application to high-throughput screen the polyester-degrading enzyme mutant are obviously improved. Therefore, the high-throughput screening of the polyester-degrading enzyme by using the MHET-OH has the characteristics of high accuracy and strong reliability. The application has important application value.

Description

Technical Field

[0001] This invention belongs to the field of biological and chemical synthesis technology, specifically relating to a method for high-throughput screening of polyester degrading enzymes and the compounds used therein. Background Technology

[0002] Polyethylene terephthalate (PET) and other polyesters are among the most widely used macromolecular polymers, commonly used in packaging, textiles, construction, and electrical appliances, such as beverage bottles, films, and engineering plastics. However, PET is resistant to natural degradation, and traditional treatment methods have drawbacks such as secondary pollution and severe environmental impact. Therefore, PET accumulates in large quantities in nature, making it a significant source of white pollution.

[0003] In recent years, biodegradation methods based on microorganisms and enzymes have attracted widespread attention from researchers due to their environmental friendliness. Various polyester-degrading enzymes, such as cutinase, esterase, and lipase, have been discovered, which can degrade PET into PET oligomers or monomers under certain conditions. For example, Yoshida et al. discovered the superbug *Ideonella sakaiensis*, which can "eat" PET, and isolated and identified a novel polyester-degrading enzyme—PETase. This enzyme can specifically degrade PET into MHET and monomers TPA and EG at 30°C, showing great application potential. Several laboratories have resolved the crystal structure of PETase and designed and modified the active pocket of PETase based on rational design methods, obtaining a large number of mutants with improved performance (i.e., enhanced enzyme catalytic activity and thermal stability). However, the full evolution of non-surface regions or regions far from the active site of PETase has not yet been reported, possibly due to the lack of efficient screening methods.

[0004] p-Nitrophenyl acetate (4-pNPA) can be hydrolyzed by esterases to p-nitrophenol and acetic acid, with p-nitrophenol exhibiting a characteristic absorbance at 405 nm. Based on this, 4-pNPA, a model substrate for esterases, was used to screen for PETases. However, the significant difference in molecular structure between 4-pNPA and PETase affects the screening efficiency and accuracy. Currently, the lack of novel and efficient screening methods for polyester (e.g., PET) degrading enzymes hinders their development. Summary of the Invention

[0005] The purpose of this invention is to provide a new high-throughput screening method for polyester degrading enzymes, which is applicable to the screening of polyester degrading enzymes such as lipase, keratinase or PET hydrolase and their mutants.

[0006] This invention first provides a compound that can be used for high-throughput screening of polyester degrading enzymes—3-hydroxy-4-((2-hydroxyethoxy)carbonyl)benzoic acid, i.e., MHET-OH, whose structural formula is shown in formula (I):

[0007]

[0008] This invention also protects a method for preparing MHET-OH, which may include the following steps:

[0009] (1) Mix 2-hydroxyterephthalic acid, concentrated sulfuric acid and ethylene glycol; then reflux at 150°C or above (e.g., 150°C, 200°C) for 8 hours or more (e.g., 8 hours, 48 ​​hours);

[0010] (2) After completing step (1), remove the ethylene glycol to obtain a mixture;

[0011] (3) After completing step (2), the mixture is separated by column chromatography to obtain MHET-OH.

[0012] In the above method, the removal of ethylene glycol can be achieved by vacuum distillation.

[0013] The application of MHET-OH in screening polyester degrading enzymes also falls within the scope of protection of this invention.

[0014] In the above applications, the polyester degrading enzyme exhibits improved enzymatic catalytic activity and / or increased thermal stability compared to the wild-type PETase enzyme. The amino acid sequence of the wild-type PETase enzyme may be as shown in SEQ ID NO:1.

[0015] This invention also protects a method for screening polyester-degrading enzymes, which may include the following steps:

[0016] (a1) The polyester degrading enzyme, buffer, and MHET-OH were mixed to obtain mixture 1; then the rate of increase of fluorescence value of mixture 1 was detected, which is A1;

[0017] (a2) Prepare the polyester degrading enzyme to be screened; then mix the polyester degrading enzyme to be screened, buffer, and MHET-OH to obtain mixture 2; then detect the rate of increase of fluorescence value of mixture 2, which is A. x ;

[0018] (a3) Compare A x With A1, if A x If the value is greater than A1, then the polyester degrading enzyme to be screened has higher or suspected higher polyester degradation activity than the polyester degrading enzyme.

[0019] In the above method, the polyester degrading enzyme can be a lipase, a keratinase, or a PET hydrolase.

[0020] In the above method, the excitation wavelength for detecting the increase rate of fluorescence value of mixture 1 can be 310-340nm (e.g., 310-320nm, 320-340nm, 310nm, 320nm, or 340nm), and the emission wavelength can be 370-445nm (e.g., 370-400nm, 400-445nm, 370nm, 400nm, or 445nm). The conditions for detecting the fluorescence value of mixture 2 can be the same as those for detecting the fluorescence value of mixture 1.

[0021] This invention also protects a method for screening polyester-degrading enzymes with improved thermal stability, which may include the following steps:

[0022] (b1) Polyester-degrading enzyme was prepared, and a portion of the enzyme was heated. Then, equal amounts of the heated and unheated enzymes were added to MHET-OH solution to obtain mixtures 3 and 4. The product amounts of mixtures 3 and 4 were then measured, and the enzyme activity was calculated, denoted as A. 热 Calculate A with A0. 热 The ratio to A0;

[0023] (b2) Prepare the polyester degrading enzyme to be screened. Take a portion of the polyester degrading enzyme to be screened and heat it. The heating conditions and time are exactly the same as those in step (b1). Add equal amounts of the heated and unheated polyester degrading enzyme to be screened into MHET-OH solution to obtain mixtures 5 and 6 respectively. Then, detect the amount of product in mixtures 5 and 6 respectively and calculate the enzyme activity, which is denoted as B. 热 Calculate B with B0. 热 The ratio to B0;

[0024] (b3) Compare B 热 / B0 and A 热 / A0, if B 热 / B0 is greater than A 热 If / A0, then the polyester degrading enzyme to be screened has higher or suspected higher thermal stability than the polyester degrading enzyme.

[0025] In the above method, the excitation wavelength for detecting the product amounts of mixtures 3 and 4 can be 310-340 nm (e.g., 310-320 nm, 320-340 nm, 310 nm, 320 nm, or 340 nm), and the emission wavelength can be 370-445 nm (e.g., 370-400 nm, 400-445 nm, 370 nm, 400 nm, or 445 nm). The conditions for detecting the product amounts of mixtures 5 and 6 can be the same as those for detecting the product amounts of mixtures 3 and 4.

[0026] In the above method, the heating conditions and time can be 50℃-55℃ (e.g., 50℃-52℃, 52℃-55℃, 50℃, 52℃ or 55℃) for more than 5 minutes (e.g., 5 minutes or 10 minutes).

[0027] In any of the methods described above, the polyester degrading enzyme may be PETase (i.e., wild-type PETase). The polyester degrading enzyme to be screened may be a PETase mutant.

[0028] The amino acid sequence of any of the above-described PETase wild-type enzymes may be as shown in SEQ ID NO:1.

[0029] The present invention also protects an MHET-OH derivative, which can be obtained by one or more esterification reactions of the MHET-OH.

[0030] Experiments have shown that using the MHET-OH high-throughput screening method provided by this invention to screen polyester degrading enzyme mutants significantly improves the activity and thermostability of the obtained pure enzyme V5. This demonstrates that the MHET-OH high-throughput screening method for polyester degrading enzymes offers high accuracy and reliability. This invention has significant application value. Attached Figure Description

[0031] Figure 1 The MHET-OH synthesized in step one of Example 1 1 H-NMR spectrum.

[0032] Figure 2 The scan results are for the excitation wavelength in Example 2.

[0033] Figure 3 The result is the scanning result of the emission wavelength in Example 2.

[0034] Figure 4 This is the standard curve plotted in Example 2.

[0035] Figure 5 The results are from the hydrolysis test of the polyester degrading enzyme PETase-WT in Example 3.

[0036] Figure 6 The results of the keratinase hydrolysis test for polyester degradation enzyme in Example 3 are shown.

[0037] Figure 7 The results are from the lipase hydrolysis test in Example 3.

[0038] Figure 8 The results show the detection of the products of PET powder degradation by the polyester degrading enzyme mutant V5 in Example 4. Detailed Implementation

[0039] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0041] Example 1: Synthesis and Identification of 3-Hydroxy-4-((2-hydroxyethoxy)carbonyl)benzoic acid –MHET-OH

[0042] 1. Add 1.82g of 2-hydroxyterephthalic acid, 0.5ml of concentrated sulfuric acid and 50ml of ethylene glycol to a round-bottom flask and mix well; then reflux at 200℃ for 48h with magnetic stirring.

[0043] 2. After completing step 1, perform vacuum distillation (to remove ethylene glycol) to obtain a mixture.

[0044] 3. After completing step 2, the mixture is separated by column chromatography to obtain 3-hydroxy-4-((2-hydroxyethoxy)carbonyl)benzoic acid, i.e., MHET-OH.

[0045] 4. One-dimensional NMR spectra were acquired using a 400MHz NMR spectrometer to identify MHET-OH.

[0046] MHET-OH 1 The H-NMR spectrum is shown below. Figure 1 .

[0047] The final structural formula of MHET-OH is shown in formula (I):

[0048]

[0049] Example 2: Determination of excitation and emission wavelengths and plotting of standard curves

[0050] 1. Preparation of MHET-OH with hydrolysis product 2-hydroxyterephthalic acid

[0051] (1) Dissolve 0.0182g of 2-hydroxyterephthalic acid (TPA-OH) in 10mL of pH8.0, 100mM PB buffer solution and dissolve completely to obtain a TPA-OH solution with a concentration of 10mM.

[0052] (2) Dissolve 0.0229g MHET-OH in 10ml pH8.0, 100mM PB buffer solution and dissolve completely to obtain a 10mM MHET-OH solution.

[0053] 2. Wavelength determination

[0054] Mix 50 μL of the test solution (TPA-OH solution or MHET-OH solution), 100 μL of pH 8.0, 200 mMPB buffer, and 50 μL of distilled water, and then perform a full wavelength scan to determine the excitation and emission wavelengths.

[0055] The excitation wavelength scanning results are shown below. Figure 2 .

[0056] The results of the emission wavelength scan are shown below. Figure 3 .

[0057] The results showed that the strongest fluorescence signals of TPA-OH and MHET-OH were detected at an excitation wavelength of 320 nm. When the emission wavelength was between 370 and 450 nm, the fluorescence signal of TPA-OH was higher than that of MHET-OH. Specifically, when the excitation wavelength was 320 nm and the emission wavelength was 400 nm, the fluorescence value of TPA-OH was 3.3 times that of MHET-OH.

[0058] 3. Drawing the standard curve

[0059] (1) MHET-OH and TPA-OH were mixed in ratios of 5:0, 4:1, 3:2, 2:3 and 1:4 to obtain a mixed solution. The final concentrations of TPA-OH in the mixed solution were 0 mM, 0.5 mM, 1 mM, 1.5 mM and 2 mM, respectively, and the corresponding final concentrations of MHET-OH were 2.5 mM, 2 mM, 1.5 mM, 1 mM and 0.5 mM, respectively; the total concentration of MHET-OH and TPA-OH was 2.5 mM.

[0060] (2) Detect the fluorescence signal value of the mixture obtained in step (1) under the conditions of excitation wavelength of 320nm and emission wavelength of 400nm.

[0061] A standard curve was plotted with the final concentration of TPA-OH on the x-axis and the fluorescence value on the y-axis.

[0062] Standard curve see Figure 4 The results showed that the fluorescence value increased with the increase of TPA-OH concentration, and the increasing trend was linear, indicating that the detection conditions (Ex = 320 nm, Em = 400 nm) are suitable for the detection of TPA-OH.

[0063] Example 3: Activity detection of polyester degrading enzyme

[0064] 1. Preparation of crude enzyme solution for polyester degradation

[0065] E. coli BL21-Gold(DE3) containing the plasmid was streaked onto plates and incubated overnight at 37°C to grow single colonies. These single colonies were then inoculated into 3 ml of LB broth and cultured at 37°C and 220 rpm for 12 h. Finally, a 1% (v / v) inoculum was added to 15 ml of LB broth and cultured at 37°C and 220 rpm until OD (Organic Dysplasia) was reached. 600nm The concentration was increased to approximately 0.8. Then, IPTG was added to a concentration of 0.1 mM in the system. Expression was induced at 20°C and 220 rpm for 48 h. After centrifugation at 4°C and 4000 rpm for 30 min, the supernatant was collected (the supernatant is the EV supernatant or crude polyester degrading enzyme solution).

[0066] When the plasmid is pET-22b(+), the collected supernatant is the EV supernatant.

[0067] When the plasmid is pET-22b-PETase, the collected supernatant is the crude wild-type PETase enzyme solution, i.e., the crude PETase-WT enzyme solution. The pET-22b-PETase plasmid is a recombinant plasmid obtained by replacing the small DNA fragment between the restriction endonucleases BstBⅠ and XhoⅠ in the pET-22b(+) plasmid with the PETase gene, a polyester-degrading enzyme derived from Ideonella sakaiensis (see Science (New York, NY), 2016, 351(6278):1196-9.). The pET-22b-PETase plasmid expresses PETase-WT (or the wild-type PETase enzyme) shown in SEQ ID NO:1.

[0068] When the plasmid is pET-22b-LCC-ICCG, the collected supernatant is the crude keratinase LCC-ICCG enzyme solution. The plasmid pET-22b-LCC-ICCG is a recombinant plasmid obtained by replacing a small DNA fragment between the restriction endonucleases NdeⅠ and XhoⅠ of plasmid pET-22b(+) with the leaf-branch compost cutinase mutant ICCG gene (see Nature, 2020, 580(7802):216-219). The plasmid pET-22b-LCC-ICCG expresses the keratinase LCC-ICCG shown in SEQ ID NO:2.

[0069] 2. PETase-WT hydrolysis test

[0070] (1) Mix 30 μL of the crude PETase-WT enzyme solution or EV supernatant prepared in step 1 with 120 μL of pH 8.0, 100 mM phosphate buffer and MHET-OH to obtain a mixture; the concentration of MHET-OH in the mixture is 2.5 mM.

[0071] (2) Detect the fluorescence signal value of the mixture obtained in step (1) under the conditions of excitation wavelength of 320nm and emission wavelength of 400nm.

[0072] Some test results can be found Figure 5 (EV is EV supernatant, WT is PETase-WT crude enzyme solution). The results showed that the fluorescence value of EV supernatant did not increase during the enzymatic reaction, indicating that no product was generated and background interference was negligible; while the fluorescence value of PETase-WT crude enzyme solution increased linearly, indicating that PETase-WT can degrade MHET-OH to produce TPA-OH.

[0073] 3. Keratinase hydrolysis test

[0074] (1) Mix 30 μL of the crude keratinase LCC-ICCG solution or EV supernatant prepared in step 1 with 120 μL of pH 8.0, 100 mM phosphate buffer and MHET-OH to obtain a mixture; the concentration of MHET-OH in the mixture is 2.5 mM.

[0075] (2) Detect the fluorescence signal value of the mixture obtained in step (1) under the conditions of excitation wavelength of 320nm and emission wavelength of 400nm.

[0076] Test results are shown Figure 6 (EV is the EV supernatant, and LCC-ICCG is the crude keratinase LCC-ICCG solution). The results showed that during the reaction, the fluorescence value of the EV supernatant did not increase, indicating that background interference was negligible; while the fluorescence value of the crude keratinase LCC-ICCG solution increased linearly, indicating that the crude keratinase LCC-ICCG solution degraded MHET-OH, and the concentration of the product TPA-OH increased linearly with reaction time.

[0077] 4. Lipase hydrolysis test

[0078] Experimental group:

[0079] 1. Mix 10 mg of lipase Novozym 435 (Novozymes, BAN118), 120 μL of pH 8.0, 100 mM phosphate buffer, and MHET-OH to obtain a mixed system; the concentration of MHET-OH in the mixed system is 2.5 mM.

[0080] 2. After completing step 1, take the mixture and detect the fluorescence signal under the conditions of excitation wavelength of 320nm and emission wavelength of 400nm.

[0081] Control group:

[0082] The lipase Novozym 435 in the experimental group was replaced with ultrapure water, while all other steps remained unchanged, serving as the control group.

[0083] Test results are shown Figure 7 The results showed that no increase in fluorescence value occurred in the control group, while the fluorescence value of the experimental group increased significantly, indicating that the product 2-hydroxyterephthalic acid (TPA-OH) was detected in the experimental group.

[0084] Therefore, MHET-OH can be hydrolyzed by lipase Novozym 435 to produce the product 2-hydroxyterephthalic acid (TPA-OH).

[0085] Example 4: Application of MHET-OH in high-throughput screening of polyester degrading enzyme mutants

[0086] 1. Preparation and culture of a mutant library of polyester degrading enzymes

[0087] (1) Primers were designed and synthesized based on the bases of the sites to be mutated, and then PCR amplification was performed using plasmid pET-22b-PETase as a template to obtain PCR products.

[0088] PCR program: (1) Pre-denaturation: 95℃, 2min; (2) Denaturation: 95℃, 30s; Annealing: Tm-5℃, 30s; Extension: 72℃, 6min; Repeat this step 15 times; (3) Final extension: 72℃, 10min.

[0089] (2) After completing step (1), the PCR products were subjected to agarose gel electrophoresis to verify whether there were bands; then, the digestive enzyme Dpn1 was added at a ratio of 2% for digestion.

[0090] (3) The digested PCR products were electroporated into E. coli BL21-Gold(DE3) competent cells and cultured to obtain single clones. Single clones were picked with sterile toothpicks and placed into 96-well microplates containing 150 μL of LB liquid medium containing 100 ng / μL ampicillin. After culturing in a shaker (37℃, 220 rpm) for 14 h, they were transferred to V-bottom 96-well plates containing 200 μL of LB liquid medium containing 100 ng / μL ampicillin using a replicator. After culturing in a shaker (37℃, 800 rpm) for 4 h, 0.1 mM IPTG was added and the plates were induced to express for 48 h in a shaker (20℃, 800 rpm). The plates were then centrifuged at 4℃ (4000 rpm, 40 min) to collect the crude enzyme solution.

[0091] 2. Screening of polyester degrading enzyme mutant libraries

[0092] Enzyme catalytic activity is expressed as relative enzyme activity. The specific detection method is as follows: 30 μL of crude enzyme solution is mixed thoroughly with 120 μL of pH 8, 100 mM PB buffer, and then 50 μL of MHET-OH (final concentration 2.5 mM) is added. The mixture is then quickly placed in a microplate reader (Infinite M200, TECAN, Switzerland) and the fluorescence signal is detected over 60 minutes at an excitation wavelength of 320 nm and an emission wavelength of 400 nm (fluorescence values ​​are read every 5 minutes). Enzyme activity is calculated as the increase in fluorescence value (Flu / min) of the PETase-WT crude enzyme solution reaction system per unit time, denoted as A. WT The increase in fluorescence value of the mutant is denoted as A. V1、2、3….. The method for calculating relative enzyme activity is A. WT、V1、2、3…. / A WT Select mutants with a relative enzyme activity of 20% or higher for further screening and identification in step 3 using shake flasks.

[0093] Thermal stability is reflected by residual enzyme activity. The specific detection method is as follows: 30 μL of the crude enzyme solution prepared in step 1 is mixed with 120 μL of pH 8, 100 mM PB buffer. The mixture is then heated at 50°C for a certain period in a microplate shaker. 50 μL of MHET-OH (final concentration 2.5 mM) is added and mixed thoroughly. The product amount is measured and the enzyme activity (i.e., the activity after heating) is calculated under the conditions of Ex = 320 nm and Em = 400 nm. The enzyme activity of PETase-WT after heating is denoted as B. WT The enzyme activity of the mutant after heating is denoted as B. V1、2、3… The method for calculating residual enzyme activity is as follows: B WT / A WT Or B V1、2、3… / A V1、2、3…Select mutants with residual enzyme activity of 10% or more for further screening and identification in step 3 using shake flasks.

[0094] Eight beneficial mutants (increased enzyme catalytic activity and / or increased thermal stability) were obtained, namely eight polyester degrading enzyme mutants, named V1-V8 in sequence.

[0095] 3. Shake-flask rescreening and identification of polyester degrading enzyme mutants

[0096] Eight polyester degrading enzyme mutants obtained through screening were inoculated into 3 ml of LB liquid medium and cultured at 37°C and 220 rpm for 12 h; then, they were inoculated into 15 ml of LB liquid medium at a 1% (v / v) inoculation rate and cultured at 37°C and 220 rpm until OD reached. 600nm The concentration was approximately 0.8. IPTG was then added to a concentration of 0.1 mM, and expression was induced at 20°C and 220 rpm for 48 h. The mixture was then centrifuged at 4°C and 4000 rpm for 30 min, and the supernatant was collected (this supernatant is the crude enzyme solution of the polyester degrading enzyme mutant). The activity and post-heating activity of the polyester degrading enzyme mutants were then detected according to the method in step 2, and the relative enzyme activity and residual enzyme activity were calculated according to the calculation method in step 2. Subsequently, the eight polyester degrading enzyme mutants were identified by sequencing. The mutation sites and mutated amino acid types (mutation sites correspond to the sequence shown in SEQ ID No: 1), relative enzyme activity, heating conditions, and residual enzyme activity of the eight polyester degrading enzyme mutants are shown in Table 1.

[0097] Table 1

[0098]

[0099] 4. Identification of polyester degrading enzyme mutant V5

[0100] (1) Preparation of PET powder

[0101] PET particles (ALDRICH, 25038-59-9) were ground and filtered through a 0.6 μm sieve. PET powder with a diameter less than 0.6 μm was collected for subsequent reactions.

[0102] (2) Purification of polyester degrading enzyme mutant V5 (i.e., obtaining pure polyester degrading enzyme mutant V5)

[0103] (2-1) Pretreatment

[0104] Take 500 mL of crude enzyme solution of polyester degrading enzyme mutant V5 and concentrate it to 100 mL using a concentration pump; filter the concentrate using a 0.22 μm filter membrane to obtain the sample.

[0105] (2-2) Column Equilibrium

[0106] Flush the nickel column with Lysis buffer until the baseline stabilizes (20 column volumes).

[0107] (2-3) Sample loading

[0108] The sample obtained in step (2-1) was injected into the nickel column by a pump, so that the sample was adsorbed onto the nickel column; then, 10 column volumes were equilibrated with Lysis buffer.

[0109] (2-4) Washing miscellaneous items

[0110] Wash with 30 mM imidazole (i.e., 12% Elution buffer), and after the impurity protein peak appears and stabilizes, continue to equilibrate with Lysis buffer for 10 column volumes.

[0111] (2-5) Elute the target protein

[0112] The target protein was collected by eluting with an Elution buffer gradient for 40 min and the elution peaks were collected to obtain the pure enzyme of the polyester degrading enzyme mutant V5.

[0113] (2-6) Verification

[0114] The purity of the polyester degrading enzyme mutant V5 was verified by SDS-PAGE.

[0115] (2-7) Desalination

[0116] Desalination and collection were carried out using a gravity column and in accordance with the instructions.

[0117] Following the steps described above, replace the crude enzyme solution of polyester degrading enzyme mutant V5 with crude enzyme solution of PETase-WT, while keeping all other steps unchanged, to obtain pure PETase-WT enzyme.

[0118] (3) Detection of products from the degradation of PET powder by polyester degrading enzyme mutant V5

[0119] (3-1) The pure enzyme of polyester degrading enzyme mutant V5 with a concentration of 10 μg / mL was mixed with pH 8.0 and 10 mM PB buffer. Then, 6 mg of PET powder was used as substrate and the mixture was placed in a shaking reactor (1000 rpm) and reacted at 37°C or 50°C for 3 days. 120 μL of the reaction solution was taken out every 24 h.

[0120] (3-2) The reaction solution obtained in step (3-1) was placed in a water bath at 100°C and heated for 10 min (to terminate the reaction), and filtered through a 0.22 μm membrane (to remove impurities) to obtain the reaction product.

[0121] (3-3) The reaction products were detected by HPLC (Ultimate 3000) at 25℃ and 260mM. The mobile phase consisted of 70% buffer A (pH 7.0, 20mM phosphate buffer) and 30% buffer B (100% acetonitrile); the flow rate was 0.8mL / min.

[0122] Following the steps described above, replace the pure enzyme V5 of the polyester degrading enzyme with the pure enzyme PETase-WT, while keeping all other steps unchanged.

[0123] Test results are shown Figure 8 The results showed that, at 37℃, the total product release of the polyester degrading enzyme mutant V5 purified enzyme increased by approximately 3.3 times on the third day compared to PETase-WT; at 50℃, the total product release of the polyester degrading enzyme mutant V5 purified enzyme increased by approximately 12.9 times on the third day compared to PETase-WT. This indicates that the activity and thermostability of the polyester degrading enzyme mutant V5 purified enzyme were significantly improved, demonstrating that high-throughput screening based on MHET-OH has the advantages of high accuracy and reliability.

[0124] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims. <110> Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences <120> A method for high-throughput screening of polyester-degrading enzymes and the compounds used therein <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 297 <212> PRT <213> Artificial sequence <400> 1 Asn Phe Pro Arg Ala Ser Arg Leu Met Gln Ala Ala Val Leu Gly Gly 1 5 10 15 Leu Met Ala Val Ser Ala Ala Ala Thr Ala Gln Thr Asn Pro Tyr Ala 20 25 30 Arg Gly Pro Asn Pro Thr Ala Ala Ser Leu Glu Ala Ser Ala Gly Pro 35 40 45 Phe Thr Val Arg Ser Phe Thr Val Ser Arg Pro Ser Gly Tyr Gly Ala 50 55 60 Gly Thr Val Tyr Tyr Pro Thr Asn Ala Gly Gly Thr Val Gly Ala Ile 65 70 75 80 Ala Ile Val Pro Gly Tyr Thr Ala Arg Gln Ser Ser Ile Lys Trp Trp 85 90 95 Gly Pro Arg Leu Ala Ser His Gly Phe Val Val Ile Thr Ile Asp Thr 100 105 110 Asn Ser Thr Leu Asp Gln Pro Ser Ser Arg Ser Ser Gln Gln Met Ala 115 120 125 Ala Leu Arg Gln Val Ala Ser Leu Asn Gly Thr Ser Ser Ser Pro Ile 130 135 140 Tyr Gly Lys Val Asp Thr Ala Arg Met Gly Val Met Gly Trp Ser Met 145 150 155 160 Gly Gly Gly Gly Ser Leu Ile Ser Ala Ala Asn Asn Pro Ser Leu Lys 165 170 175 Ala Ala Ala Pro Gln Ala Pro Trp Asp Ser Ser Thr Asn Phe Ser Ser 180 185 190 Val Thr Val Pro Thr Leu Ile Phe Ala Cys Glu Asn Asp Ser Ile Ala 195 200 205 Pro Val Asn Ser Ser Ala Leu Pro Ile Tyr Asp Ser Met Ser Arg Asn 210 215 220 Ala Lys Gln Phe Leu Glu Ile Asn Gly Gly Ser His Ser Cys Ala Asn 225 230 235 240 Ser Gly Asn Ser Asn Gln Ala Leu Ile Gly Lys Lys Gly Val Ala Trp 245 250 255 Met Lys Arg Phe Met Asp Asn Asp Thr Arg Tyr Ser Thr Phe Ala Cys 260 265 270 Glu Asn Pro Asn Ser Thr Arg Val Ser Asp Phe Arg Thr Ala Asn Cys 275 280 285 Ser Leu Glu His His His His His His 290 295 <210> 2 <211> 297 <212> PRT <213> Artificial sequence <400> 2 Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala Gln Pro Ala Met Ala Met Asp Ile Gly Ile Asn Ser Asp Pro Ser 20 25 30 Asn Pro Tyr Gln Arg Gly Pro Asn Pro Thr Arg Ser Ala Leu Thr Ala 35 40 45 Asp Gly Pro Phe Ser Val Ala Thr Tyr Thr Val Ser Arg Leu Ser Val 50 55 60 Ser Gly Phe Gly Gly Gly Val Ile Tyr Tyr Pro Thr Gly Thr Ser Leu 65 70 75 80 Thr Phe Gly Gly Ile Ala Met Ser Pro Gly Tyr Thr Ala Asp Ala Ser 85 90 95 Ser Leu Ala Trp Leu Gly Arg Arg Leu Ala Ser His Gly Phe Val Val 100 105 110 Leu Val Ile Asn Thr Asn Ser Arg Phe Asp Gly Pro Asp Ser Arg Ala 115 120 125 Ser Gln Leu Ser Ala Ala Leu Asn Tyr Leu Arg Thr Ser Ser Pro Ser 130 135 140 Ala Val Arg Ala Arg Leu Asp Ala Asn Arg Leu Ala Val Ala Gly His 145 150 155 160 Ser Met Gly Gly Gly Gly Thr Leu Arg Ile Ala Glu Gln Asn Pro Ser 165 170 175 Leu Lys Ala Ala Val Pro Leu Thr Pro Trp His Thr Asp Lys Thr Phe 180 185 190 Asn Thr Ser Val Pro Val Leu Ile Val Gly Ala Glu Ala Asp Thr Val 195 200 205 Ala Pro Val Ser Gln His Ala Ile Pro Phe Tyr Gln Asn Leu Pro Ser 210 215 220 Thr Thr Pro Lys Val Tyr Val Glu Leu Cys Asn Ala Ser His Ile Ala 225 230 235 240 Pro Asn Ser Asn Asn Ala Ala Ile Ser Val Tyr Thr Ile Ser Trp Met 245 250 255 Lys Leu Trp Val Asp Asn Asp Thr Arg Tyr Arg Gln Phe Leu Cys Asn 260 265 270 Val Asn Asp Pro Ala Leu Cys Asp Phe Arg Thr Asn Asn Arg His Cys 275 280 285 Gln Leu Glu His His His His His His 290 295

Claims

1. Application of 3-hydroxy-4-((2-hydroxyethoxy)carbonyl)benzoic acid in screening polyester-degrading enzymes with improved catalytic activity and / or improved thermal stability; The 3-hydroxy-4-((2-hydroxyethoxy)carbonyl)benzoic acid, i.e. MHET-OH, has the structural formula shown in formula (I):

2. A method for screening polyester-degrading enzymes with enhanced catalytic activity, comprising the following steps: (a1) The polyester degrading enzyme, buffer solution and MHET-OH as described in claim 1 are mixed to obtain mixture 1; then the rate of increase of fluorescence value of mixture 1 is detected, which is A1; (a2) Prepare the polyester degrading enzyme to be screened; then mix the polyester degrading enzyme to be screened, buffer solution, and MHET-OH as described in claim 1 to obtain mixture 2; then detect the rate of increase of fluorescence value of mixture 2, which is A. x ; (a3) Compare A x With A1, if A x If the value is greater than A1, then the polyester degrading enzyme to be screened has higher or suspected higher polyester degradation activity than the polyester degrading enzyme.

3. The method according to claim 2, characterized in that: The polyester-degrading enzyme is a lipase, keratinase, or PET hydrolase.

4. The method according to claim 2, characterized in that: The excitation wavelength for detecting the increase rate of fluorescence value of the mixture 1 is 310-340 nm, and the emission wavelength is 370-445 nm; the conditions for detecting the fluorescence value of the mixture 2 are the same as those for detecting the fluorescence value of the mixture 1.

5. A method for screening polyester-degrading enzymes with improved thermal stability, comprising the following steps: (b1) A polyester degrading enzyme was prepared, and a portion of the polyester degrading enzyme was heated. Then, equal amounts of the heated and unheated polyester degrading enzymes were added to the MHET-OH solution described in claim 1, respectively, to obtain mixtures 3 and 4. The product amounts of mixtures 3 and 4 were then measured and the enzyme activities were calculated, denoted as A. 热 Calculate A with A0. 热 The ratio to A0; (b2) Prepare the polyester degrading enzyme to be screened. Take a portion of the polyester degrading enzyme to be screened and heat it. The heating conditions and time are exactly the same as those in step (b1). Add equal amounts of the heated and unheated polyester degrading enzyme to be screened to the MHET-OH solution described in claim 1 to obtain mixtures 5 and 6 respectively. Then, detect the product amount of mixtures 5 and 6 respectively and calculate the enzyme activity, which is denoted as B. 热 Calculate B with B0. 热 The ratio to B0; (b3) Compare B 热 / B0 and A 热 / A0, if B 热 / B0 is greater than A 热 If / A0, then the polyester degrading enzyme to be screened has higher or suspected higher thermal stability than the polyester degrading enzyme.

6. The method according to claim 5, characterized in that: The excitation wavelength for detecting the product amounts of mixtures 3 and 4 is 310-340 nm, and the emission wavelength is 370-445 nm; the conditions for detecting the product amounts of mixtures 5 and 6 are the same as those for detecting the product amounts of mixtures 3 and 4.

7. The method according to any one of claims 2-6, characterized in that: The polyester-degrading enzyme is PETase.

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