A novel polyester model compound, its preparation method and application

Abstract

The application provides a novel polyester model compound and a preparation method and application thereof. The compound is 2-hydroxy terephthalic acid bis-hydroxyethyl ester; the application is that the compound is used as a substrate, and a polyester degradation enzyme to be detected is used for degradation reaction, and the activity of the polyester depolymerase is detected quickly and simply by using a fluorescence microplate reader. The application is suitable for high-throughput screening of a polyester depolymerase mutant library, and is helpful to realize new enzyme mining and directional evolution of the polyester depolymerase, so as to provide infinite possibilities for optimization of the performance of the polyester depolymerase.

Description

Technical Field

[0001] This invention belongs to the field of chemical and enzyme engineering technology, and relates to polyester model compounds, their preparation methods and applications. Background Technology

[0002] Polyester depolymerases, such as PETase, keratinase, and lipase, have become a hot topic in the study of microbial degradation of plastics in recent years. These enzymes can depolymerize polyethylene terephthalate (PET) into components such as mono(2-hydroxyethyl) terephthalic acid (MHET), bis(2-hydroxyethyl) terephthalic acid (BHET), ethylene glycol (EG), and terephthalic acid (TPA), showing great promise for industrial applications. However, these polyester depolymerases still have many shortcomings in terms of catalytic efficiency and thermal stability, which cannot fully meet the needs of industrial applications. There is still a need to explore new polyester depolymerases or improve the activity of existing ones. However, the current lack of high-throughput screening methods severely limits the discovery and modification of polyester depolymerases.

[0003] Currently, the analysis of polyester depolymerase activity mainly relies on high-performance liquid chromatography (HPLC) to detect MHET and TPA, degradation products of PET or PET oligomers. HPLC has high detection sensitivity, good separation effect, and accurate and reliable results, but it has a limited number of analyses and is time-consuming, making it unsuitable for screening mutant libraries containing thousands of clones. Summary of the Invention

[0004] The purpose of this invention is to provide a novel polyester model compound, its preparation method, and its application.

[0005] This invention discovers a novel compound, named BHET-OH, with the English name bis(2-hydroxyethyl) 2-hydroxyterephthalate and the chemical formula C. 12 H 14 O7, its chemical structural formula is shown below: Based on this, the present invention provides a polyester-like compound, specifically 2-hydroxy terephthalate dihydroxyethyl ester with the following structure: The present invention also provides a method for preparing the compound, which uses 2-hydroxyterephthalic acid as a substrate and sulfuric acid as a catalyst, and reacts in the presence of ethylene glycol to obtain the compound.

[0006] Preferably, the reaction further includes removing the ethylene glycol by vacuum distillation, followed by column chromatography to obtain a pure compound.

[0007] More preferably, the reaction is carried out under stirring conditions, preferably by magnetic stirring and reflux at 180-220°C for 10-75 hours.

[0008] The present invention also provides the application of the compound in the detection of the activity and / or stability of polyester degrading enzymes.

[0009] In a specific embodiment, the compound is used as a substrate, and the polyester degrading enzyme to be tested is used to degrade it. Kinetics are employed to detect the activity of the polyester degrading enzyme to be tested; preferably, the polyester degrading enzyme is... Is If the PETase or lipase is present, its hydrolytic activity is detected by kinetic fluorescence at 30°C; or if the polyester degrading enzyme is a keratinase LCC, its hydrolytic activity is detected by kinetic fluorescence at 40°C. The kinetic detection conditions are: detection wavelength λ... Ex =320nm, λ Em =420nm, detection time 1 hour, measurement once every 1 minute.

[0010] Alternatively, it is preferably used for screening thermostability mutants of polyester degrading enzymes.

[0011] More specifically, the wild-type polyester depolymerase and the polyester degrading enzyme mutant to be screened were degraded at a set temperature, and the thermal stability was compared to obtain the thermally stable polyester degrading enzyme mutant.

[0012] Specifically, when determining the thermal stability of the mutant, the pure enzyme solution is heated to a set temperature and held for a set time, then cooled, and the remaining enzyme activity is determined using the compound as a substrate. The enzyme activity without heat treatment is defined as 100% to compare the thermal stability of the mutant.

[0013] In a more specific embodiment, the set temperature is a certain temperature of the wild type of polyester depolymerase, for example, heating to 44.5 °C for 30 min, cooling to 4 °C for 10 min, and then using the compound as a substrate to determine the remaining enzyme activity.

[0014] This invention provides a highly efficient and simple screening method for polyester depolymerases in directed evolution using a novel compound, BHET-OH. This compound, BHET-OH, is similar to the PET oligomer BHET and can be catalyzed by polyester depolymerases to produce polymers with a molecular weight of λ. Ex =320nm, λ EmThe final product, 2-hydroxyterephthalic acid (2-HOTP), exhibits stable fluorescence at 420 nm. Using BHET-OH as a model substrate, it produces a fluorescent product after catalysis by polyester depolymerase. Therefore, a fluorescent microplate reader can be used to rapidly and easily detect the activity of polyester depolymerase, making it suitable for high-throughput screening of polyester depolymerase mutant libraries. This facilitates the discovery and directed evolution of new polyester depolymerases, thus providing unlimited possibilities for optimizing polyester depolymerase performance. Attached Figure Description

[0015] Figure 1 Chemical synthesis route of BHET-OH.

[0016] Figure 2 BHET-OH 1 H-NMR spectrum.

[0017] Figure 3 The following describes the construction of a method for detecting polyester depolymerase hydrolysis activity using BHET-OH as a model substrate. In the diagram, a represents the UV-Vis absorption spectra of BHET-OH and 2-HOTP; b represents the fluorescence excitation spectra of BHET-OH and 2-HOTP at different ratios; c represents the fluorescence emission spectra of BHET-OH and 2-HOTP at different ratios; and d represents the fluorescence detection standard curve.

[0018] Figure 4 Schematic diagram of the mechanism of BHET-OH fluorescence method.

[0019] Figure 5 The method for detecting the hydrolytic activity of polyester depolymerase using BHET-OH as a model substrate was optimized. In the figure, a is the kinetic fluorescence intensity curve of enzymatic hydrolysis with 1.25 mM BHET-OH; b is the kinetic fluorescence intensity curve of enzymatic hydrolysis with 2.5 mM BHET-OH under different enzyme concentrations.

[0020] Figure 6 BHET-OH was used as the substrate to determine the hydrolytic activity of different polyester depolymerases. In the figures, a is the fluorescence intensity curve for the kinetic detection of the pET22b-PETase reaction system; b is the fluorescence intensity curve for the kinetic detection of the pET22b-LCC keratinase reaction system; c is the fluorescence intensity curve for the kinetic detection of the lipase (Candida albicans) reaction system; and d is the enzyme activity expressed as the fluorescence intensity slope value (Flu / s).

[0021] Figure 7 Determination of the standard deviation of fluorescence screening in 96-well plates. Detailed Implementation

[0022] Example 1: Synthesis, purification and identification of BHET-OH The reaction route is as follows Figure 1 As shown, 2-hydroxyterephthalic acid, concentrated sulfuric acid, and excess ethylene glycol were added to a three-necked round-bottom flask, and the mixture was refluxed at 200°C for 48 hours with magnetic stirring. The mixture was then subjected to vacuum distillation to remove the ethylene glycol, followed by column chromatography to obtain dihydroxyethyl 2-hydroxyterephthalate (BHET-OH). The structure of product BHET-OH was identified using 400 MHz NMR. 1 H NMR (400 MHz, DMSO- d 6) δ 7.93 (d, J = 8.2 Hz, 1H), 7.55 (d, J =1.6 Hz, 1H), 7.51 (dd, J = 8.2, 1.6 Hz, 1H), 4.96 (s, 1H), 4.41 – 4.24 (m, 4H), 3.79 – 3.66 (m, 4H), see attached NMR spectrum. Figure 2 The S-shaped line represents the integral.

[0023] The table below shows several conditions under which BHET-OH can be synthesized.

[0024] Example 2: Construction of a method for detecting polyester depolymerase activity using BHET-OH as a substrate The substrate BHET-OH and the target product 2-hydroxyterephthalic acid (2-HOTP) were scanned in UV wavelength using a Tecan microplate reader, and the UV spectra were obtained as follows: Figure 3 As shown in Figure a, BHET-OH and 2-HOTP exhibit distinct UV absorption peaks at 320 nm. Using 320 nm as the fluorescence excitation wavelength, full-wavelength emission scans of BHET-OH and 2-HOTP with different ratios were performed, yielding the emission spectra shown below. Figure 3 As shown in Figure c, both BHET-OH and 2-HOTP exhibit fluorescence. The maximum emission wavelength of BHET-OH is 475 nm, and that of 2-HOTP is 420 nm. The product 2-HOTP shows a high fluorescence value at 400 nm, while the substrate BHET-OH shows virtually no fluorescence background at this wavelength. Therefore, 400 nm was determined to be the optimal emission wavelength. A full-wavelength excitation scan was performed at the optimal emission wavelength of 400 nm, resulting in the excitation spectrum shown below. Figure 3 As shown in Figure b, 320nm was determined to be the optimal excitation wavelength.

[0025] Plot the proportion of 2-HOTP in the mixed solution on the x-axis, λ Ex =320nm, λ EmThe standard curve for this fluorescence detection method is obtained by using the fluorescence intensity at 400 nm as the ordinate. Figure 3 As shown in Figure d, the concentration of 2-HOTP in the reaction system exhibits a good linear relationship with the fluorescence intensity (Rd). 2 =0.9994), indicating that this method can be tried for the detection of polyester depolymerase activity.

[0026] Example 3: Using BHET-OH as a substrate to detect the hydrolytic activity of different polyester depolymerases The activity of different polyester depolymerases was detected using BHET-OH as a substrate, and the reaction mechanism was as follows: Figure 4 As shown, polyester hydrolase hydrolyzes BHET-OH to give the product 2-HOTP. 2-HOTP is then processed in λ... Ex =320 nm, λ Em The reaction exhibits significant fluorescence at 400 nm, and the activity of the polyester hydrolase can be characterized by detecting the rate of increase in fluorescence value.

[0027] First, the source was tested. Ideonella sakaiensis The hydrolytic activity of PETase (corresponding to the amino acid sequence in SEQ ID No. 1) against 1.25 mM BHET-OH was determined using a 200 µL reaction system as follows: 100 µL of crude PETase expression supernatant was added to 100 mM pH 8 sodium phosphate buffer, followed by 50 µL of 5 mM BHET-OH (dissolved in DMSO). Two control groups were set up, one without enzyme and containing equal volumes of empty vector pET22b supernatant and reaction buffer, respectively. After mixing, the reaction mixture was placed in a Tecan microplate reader with the following parameters set: detection temperature 30 ℃; λ Ex =320 nm, λ Em =400 nm; detection time 4 h, measured once every 2 min. The corresponding curve was obtained by plotting reaction time (s) on the x-axis and fluorescence value (Flu) on the y-axis, as shown in the figure. Figure 5As shown in Figure a, after 4 hours of reaction, the fluorescence value of the pET22b-PETase hydrolysis system increased by approximately 600, while the fluorescence value of the control group increased by approximately 200, indicating high background interference and excessively long reaction time. To improve the detection sensitivity of this fluorescence method and shorten the reaction time, the reaction system was optimized. First, the substrate concentration was increased, with the final concentration of BHET-OH in the reaction system adjusted from 1.25 mM to 2.5 mM. The organic solvent DMSO in the reaction system was removed to reduce its impact on enzyme activity. BHET-OH was replaced with a sodium salt to increase its solubility in aqueous solution, accelerate the enzymatic hydrolysis rate, and shorten the fluorescence detection time. The sodium salt preparation method was optimized to use equimolar amounts of Na2CO3 and ddH2O for dissolution. The effect of different amounts of crude enzyme solution in the reaction system was also tested. The 200 µL reaction system was as follows: 50 µL or 100 µL of PETase crude enzyme solution supernatant was added to 50 µL of 10 mM BHET-OH dissolved in Na2CO3 and ddH2O (final concentration: 2.5 mM). The reaction volume was then brought to 200 µL with 100 mM pH 8 sodium phosphate buffer. The detection time was changed to 1 h, measured once every 1 min, and other parameters were the same as above. The results are as follows. Figure 5 As shown in Figure b, under the conditions of 2.5 mM BHET-OH (dissolved in equimolar amounts of Na2CO3 and ddH2O) and 100 µL of enzyme, the fluorescence value of the reaction system increased by approximately 2000 within 1 hour. The control group showed virtually no background interference. Under these conditions, the fluorescence method exhibited the best sensitivity and detection effect.

[0028] The optimized method described above was applied to the activity detection of different polyester depolymerases, including pET22b-PETase, pET22b-LCC keratinase derived from Leaf-branch compost (corresponding to the amino acid sequence in SEQ ID No. 2), and commercial lipase (Candida Lipase, MREDA, 50,000 U / g). The reactions were carried out in 96-well fluorescent plates. The reaction system (200 µL) for PETase and LCC was as follows: 100 µL of crude enzyme expression supernatant was added to 50 µL of 100 mM pH 8 sodium phosphate buffer, and finally 50 µL of 10 mM BHET-OH dissolved in an equimolar mass of Na2CO3 and ddH2O (final concentration: 2.5 mM) was added. The reaction system (200 µL) for lipase was as follows: 50 µL of 10 mM BHET-OH dissolved in an equimolar mass of Na2CO3 and ddH2O (final concentration: 2.5 mM) was added to 150 µL of lipase (dissolved in ddH2O, final concentration: 75 U). Two control groups were set up, with no enzyme added to the control groups, and equal volumes of empty vector pET22b supernatant and reaction buffer added to the control groups, respectively. After mixing, the reaction was placed in a Tecan microplate reader with the following parameters set: the detection temperature for PETase and lipase was 30 ℃, and the detection temperature for LCC was 40 ℃; λ Ex =320nm, λ Em =400nm; detection time 1 h, measured once every 1 min. A curve was obtained by plotting reaction time (s) on the x-axis and fluorescence value (Flu) on the y-axis. The activity of the crude enzyme solution was characterized by calculating the slope (Flu / s) within the initial linear range of the curve. For example... Figure 6 As shown in a, b, and c, the fluorescence values ​​of pET22b-PETase, pET22b-LCC, and lipase increased with increasing reaction time, while the supernatant of the empty vector pET22b and the control group with only buffer showed almost no fluorescence background. Figure 6 In the figure, d represents enzyme activity expressed as fluorescence intensity slope (Flu / s). The enzyme activities of LCC and lipase are 0.07 (Flu / s), and the enzyme activity of PETase is 0.39 (Flu / s), indicating that BHET-OH can be widely used for the determination of the hydrolytic activity of polyester depolymerase.

[0029] Example 4: Screening and identification of a polyester depolymerase mutant library using BHET-OH as a substrate. The constructed screening method was applied to screen for the activity and thermal stability of random mutant libraries of the polyester depolymerase PETase, such as... Figure 7As shown, the standard deviation (STDEV) of this method for screening PETase96-well plates was 2.39%. After two rounds of screening and combinations of mutation sites, three mutants with improved activity and thermostability were obtained, and the results are shown in Table 1.

[0030] The hydrolysis activity assay was performed as follows: 40 µL of 0.5 mg / mL polyester depolymerase wild-type and different mutants were added to 110 µL of 100 mM pH 8 NaH2PO4-Na2HPO4 buffer. Finally, 50 µL of 10 mM BHET-OH dissolved in equimolar amounts of Na2CO3 and ddH2O (final concentration: 2.5 mM) was added. After mixing, the mixture was placed in a Tecan microplate reader with the following parameters set: detection temperature 30 ℃; λ Ex =320nm, λ Em =400nm; detection time 1 h, 1 min per measurement. The reaction time (s) was plotted on the x-axis and the fluorescence value (Flu) on the y-axis to obtain the corresponding curve. The activity of the crude enzyme solution was characterized by calculating the slope (Flu / s) within the initial linear range of the curve.

[0031] The method for determining thermal stability is as follows: 45 µL of 0.5 mg / mL polyester hydrolase wild-type and different mutants were incubated at 44.5 ℃ for 30 min, cooled at 4 ℃ for 10 min, and then 40 µL was placed in a 96-well fluorescent plate. 110 µL of 100 mM pH 8 NaH2PO4-Na2HPO4 buffer was added, followed by 50 µL of 10 mM BHET-OH dissolved in equimolar amounts of Na2CO3 and ddH2O (final concentration: 2.5 mM). After mixing, the plate was placed in a Tecan microplate reader with the parameters set as described above. A curve was obtained by plotting reaction time (s) on the x-axis and fluorescence value (Flu) on the y-axis. The residual enzyme activity was characterized by calculating the slope (Flu / s) within the initial linear range of the curve. The enzyme activity after heat treatment was defined as 100% to compare the thermal stability of the mutants.

[0032] Table 1. Relative activities and thermal stability of wild-type polyester hydrolase and mutants using BHET-OH as a substrate. WT 100 4.47 N246D 121 33.94 Q119R 123 10.10 N246D / Q119R 140 41.56 Note: Uppercase letters in the table indicate amino acid substitutions, using the following nomenclature: original amino acid (wild type), position (i.e., position in SEQ ID No. 1), substituted amino acid. Accordingly, for example, if glutamine is used to replace the original arginine at position 119 of SEQ ID No. 1, it is named "Q119R". <110> Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences <120>A Novel Polyester Model Compound and Preparation Method and Use Thereof <160>2 <170>PatentIn version 3.5 <210> 1 <211>290 <212> PRT <213>Ideonella sakaiensis <400>1 MNFPRASRLMQAAVLGGLMAVSAAATAQTNPYARGPNPTAASLEASAGPFTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPSSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPSLKAAAPQAPWDSSTNFSSVTVPTLIFACENDSIAPVNSSALPIYDSMSRNAKQFLEINGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTRVSDFRTANCS 290 <210> 2 <211>290 <212> PRT <213>Leaf-branch compost <400>2 MSNPYQRGPNPTRSALTADGPFSVATYTVSRLSVSGFGGGVIYYPTGTSLTFGGIAMSPGYTADASSLAWLGRRLASHGFVVLVINTNSRFDYPDSRASQLSAALNYLRTSSPSAVRARLDANRLAVAGHSMGGGGTLRIAEQNPSLKAAVPLTPWHTDKTFNTSVPVLIVGAEADTVAPVSQHAIPFYQNLPSTTPKVYVELDNASHFAPNSNNAAISVYTISWMKLWVDNDTRYRQFLCNVNDPALSDFRTNNRHCQ 259

Claims

1. The application of a polyester-structure-similar compound in the detection of the activity and / or stability of polyester-degrading enzymes; The polyester-like compound is 2-hydroxytetraethyl terephthalate with the structure shown below: 。 2. The application as described in claim 1, characterized in that, Using compounds with structures similar to those of polyester as substrates, The polyester was degraded using the test polyester degrading enzyme, and the activity of the test polyester degrading enzyme was calculated by kinetic analysis.

3. The application as described in claim 2, characterized in that, If the polyester degrading enzyme is IsPETase or lipase, its hydrolytic activity is detected by kinetic fluorescence at 30°C; or if the polyester degrading enzyme is keratinase LCC, its hydrolytic activity is detected by kinetic fluorescence at 40°C. The kinetic detection conditions are: detection wavelength λ... Ex =320nm, λ Em =420nm, detection time 1 hour, measurement once every 1 minute.

4. The application as described in claim 1, characterized in that, It is used for screening thermostability mutants of polyester degrading enzymes.

5. The application as described in claim 4, characterized in that, Wild-type polyester depolymerase and the polyester degrading enzyme mutant to be screened were used to degrade compounds with similar polyester structures at a set temperature. The thermal stability was compared to obtain the thermally stable polyester degrading enzyme mutant.

6. The application as described in claim 5, characterized in that, When determining the thermal stability of the polyester degrading enzyme mutant, the pure enzyme solution was heated to a set temperature and held for a set time, then cooled, and the remaining enzyme activity was determined using a compound with a similar polyester structure as a substrate. The enzyme activity without heat treatment was defined as 100% to compare the thermal stability of the polyester degrading enzyme mutant.

7. The application as described in claim 5 or 6, characterized in that, The set temperature is achieved by heating the wild-type polyester depolymerase or the polyester degrading enzyme mutant to be screened to 44.5°C for 30 min, cooling it to 4°C for 10 min, and then using a compound with a similar polyester structure as a substrate to determine the remaining enzyme activity.

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