Method for strengthening cutinase hydrolysis of water dispersible polyester by using surfactant as additive

Abstract

The invention discloses a method for promoting cutinase to hydrolyze water dispersible polyester by using a surfactant as an additive, and belongs to the technical field of biological enzyme preparations and the technical field of textile assistants. The method for hydrolyzing the water dispersible polyester comprises the following steps: dissolving surfactants such as bis-rhamnolipid, polyethylene glycol, lauryl betaine and the like in a Tris-HCl buffer solution to form a surfactant solution, and adjusting the pH value by using hydrochloric acid; and then adding a water dispersion polyester solution and cutinase for hydrolysis reaction, after the reaction is stopped, centrifugally collecting an emulsion, and freeze-drying the emulsion. The method can improve the stability of cutinase and increase the release amount of the water dispersible polyester hydrolysate, and is simple to operate, safe, environment-friendly and low in energy consumption.

Description

Technical Field

[0001] The present invention relates to a method for enhancing the hydrolysis of water-dispersed polyester by a surfactant as an additive, belonging to the technical fields of biological enzyme preparations and textile auxiliaries. Background Art

[0002] Polyester is a major part of synthetic fibers and has received extensive attention due to its smooth handfeel, easy washing, quick drying, high strength, resistance to chemical erosion, etc. To improve the mechanical properties, processing properties, appearance, and handfeel of polyester fibers, sizing agents are coated on the fiber surface. However, due to the high crystallinity, high orientation, and high hydrophobicity of polyester fibers, it is particularly difficult to treat polyester fibers with modified traditional polyvinyl alcohol and starch sizing agents. Currently, the sizing of polyester fibers mainly relies on polyacrylates, which have a relatively high application cost. Water-dispersed polyester (WPET) has developed rapidly in recent years due to its excellent dispersibility, adhesiveness, and convenience, and occupies an important position in the textile, coating, and packaging industries. Especially in the textile industry, WPET has become the fourth major type of textile sizing agent. When used for coating the surface of polyester fibers, WPET exhibits excellent adhesion and durability. However, WPET often produces a large amount of polluted wastewater during application processing, resulting in serious environmental pollution problems.

[0003] Currently, the wastewater treatment methods for water-dispersed polyester mainly rely on traditional chemical methods, such as alkaline removal methods. However, these methods are often accompanied by high corrosion and the use of toxic solvents, causing more negative impacts on the environment. Therefore, it is crucial to explore green and environmentally friendly means to repair the pollution of WPET.

[0004] As an environmentally friendly and efficient alternative technology, the biological enzyme method has shown great potential. Cutinase, as a biological enzyme with a wide substrate adaptability, has unique catalytic characteristics and can hydrolyze various polyester compounds. However, when cutinase is used for the hydrolysis of water-dispersed polyester, the enzyme is easily inactivated in an aqueous medium, and the hydrolysis rate is relatively slow. The molecular structure of water-dispersed polyester has a certain complexity, and its crystalline regions and intermolecular forces make it difficult for cutinase to quickly and effectively bind to it and initiate the hydrolysis reaction. This results in a large amount of time being required to achieve a certain degree of hydrolysis in the actual production process, greatly reducing the production efficiency and limiting the actual large-scale industrial application of this technology. Summary of the Invention

[0005] [Technical Problem]

[0006] When cutinase is used for the hydrolysis of water-dispersed polyester, the enzyme is easily inactivated in the aqueous medium, resulting in poor hydrolysis effect. Moreover, the molecular structure complexity of water-dispersed polyester makes it difficult for cutinase to quickly and effectively bind to it and initiate the hydrolysis reaction. This leads to a large amount of time consumption to achieve a certain degree of hydrolysis in the actual production process, greatly reducing the production efficiency.

[0007] [Technical Solution]

[0008] In order to solve the above technical problems, the purpose of the present invention is to provide a method for strengthening the hydrolysis of water-dispersed polyester by cutinase using a surfactant as an additive. This method uses the biosurfactant rhamnolipid (RL), the non-ionic surfactant (PEG300), and the zwitterionic surfactant lauryl betaine (BET), which not only effectively strengthens the action of enzyme hydrolysis of water-dispersed polyester but also improves the stability of the enzyme; the method is simple to operate, safe, environmentally friendly, and has low energy consumption.

[0009] In order to achieve the above purpose, the provided technical solution is as follows:

[0010] The present invention provides a method for strengthening the hydrolysis of water-dispersed polyester by cutinase using a surfactant as an additive, and the method includes the following:

[0011] Place the water-dispersed polyester solution in the surfactant solution, add cutinase for hydrolysis reaction; after the reaction ends, inactivate the enzyme, centrifuge, take the lower layer emulsion for washing and drying, and that's it.

[0012] In one embodiment, the surfactant solution is obtained by dissolving the surfactant in Tris-HCl buffer, mixing well, and adjusting the pH.

[0013] In one embodiment, the temperature in the Tris-HCl buffer is 25 - 35 °C, the concentration is 10 mmol / L, and the pH is 9 - 10.

[0014] In one embodiment, the pH is adjusted to 9 - 10, preferably 9.

[0015] In one embodiment, the surfactant is any one or more of biosurfactants, non-ionic surfactants, and zwitterionic surfactants.

[0016] In one embodiment, the biosurfactant includes any one or more of phosphate esters, proteins, glycolipids, cholesterol derivatives, etc.

[0017] In one embodiment, the non-ionic surfactant includes any one or more of polyoxyethylene type, polyol type, alkanolamide type, polyether type, amine oxide type, etc.

[0018] In one embodiment, the zwitterionic surfactant includes one or more of amino acid type, betaine type, sulfate type, sulfonate type, and phosphate type.

[0019] In one embodiment, the sugar esters include any one of sophorolipid and rhamnolipid; the rhamnolipid includes mono-rhamnolipid and di-rhamnolipid.

[0020] In one embodiment, the betaine type is anhydrous betaine and / or lauryl betaine (BET).

[0021] In one embodiment, the polyol type includes any one or more of glycerol mono- and di-fatty acid esters, pentaerythritol fatty acid esters, and polyethylene glycol (PEG).

[0022] In one embodiment, the molecular weight of the PEG is 200 - 4000; preferably, the molecular weight of the PEG is 200 - 1000; more preferably, the molecular weight of the PEG is 300, i.e., PEG300.

[0023] In one embodiment, the surfactant is a zwitterionic surfactant.

[0024] In one embodiment, the surfactant is any one of PEG300, di-rhamnolipid, and lauryl betaine (BET).

[0025] In one embodiment, the concentration of the surfactant solution is 0.0625 - 12%, w / v, g / mL; preferably 2 - 12%, w / v, g / mL; more preferably 4 - 8%, w / v, g / mL.

[0026] In one embodiment, the concentration of the RL solution is 6%, w / v, g / mL; the concentration of the PEG300 solution is 6%, w / v, g / mL; the concentration of the BET solution is 8%, w / v, g / mL.

[0027] In one embodiment, for the aqueous dispersion polyester solution: dissolve the aqueous dispersion polyester in a constant temperature oil bath at a bath ratio of 1:20 - 30 and 90 - 95 °C for 3 - 5 h, and remove solid impurities with a 0.22 μm filter membrane. The retained filtrate is the aqueous dispersion polyester solution.

[0028] In one embodiment, the aqueous dispersion polyester solution is placed in the surfactant solution, and the concentration of the aqueous dispersion polyester in the mixed solution is 5 - 10 g / L.

[0029] In one embodiment, the cutinase is derived from Humicola insolen and has an enzyme activity of 1800 U.

[0030] In one embodiment, the enzyme concentration of the cutinase relative to the surfactant solution is 50 - 225 U / mL; preferably 175 U / mL.

[0031] In one embodiment, the hydrolysis reaction conditions are as follows: temperature 30 - 80°C, constant temperature shaker 120 - 200 rpm; time 3 - 18 h; preferably 40°C, 120 rpm, 12 h.

[0032] In one embodiment, the centrifugation parameters are: 10000 - 15000 r / min, 5 - 10 min.

[0033] In one embodiment, the washing refers to washing the lower layer emulsion with ethanol and deionized water.

[0034] In one embodiment, the drying is freeze-drying, freezing at -18 to -20°C for 24 - 36 h, and then transferring to a freeze-dryer at -60 to -80°C for drying for 48 - 60 h.

[0035] The second object of the present invention is to provide a water-dispersible polyester obtained by the treatment of the above-mentioned method.

[0036] The third object of the present invention is to provide an application of the above-mentioned method in the treatment of water pollution by water-dispersible polyester.

[0037] The fourth object of the present invention is to provide an application of the above-mentioned water-dispersible polyester in the preparation of slurries and textile processing.

[0038] Advantages of the present invention:

[0039] (1) The present invention uses RL, PEG300, and BET as additives to assist the enzymatic hydrolysis of water-dispersible polyester. The process is simple, low-cost, biodegradable, safe, and environmentally friendly.

[0040] (2) The present invention uses RL, PEG300, and BET, which can improve the activity and thermal stability of cutinase, promote the cutinase to catalyze the cleavage of the ester bond of water-dispersible polyester, and generate substances such as 5-sodium sulfoisophthalate (5-SSIPA), terephthalic acid (TPA), mono(2-hydroxyethyl) phthalate (MHET), and bis(2-hydroxyethyl) terephthalate (BHET). Through the synergistic effect of RL, PEG300, and BET with cutinase on the enzymatic hydrolysis of water-dispersible polyester, the release amount of enzymatic hydrolysis products is increased by 70.22%, 68.44%, and 99.85% compared with that without additives. Description of the drawings

[0041] Figure 1FTIR test result diagrams of WPET obtained in Example 1 and Comparative Example 1; (a) is Comparative Example 1, FTIR of WPET hydrolyzed by cutinase; (b) is Example 1, FTIR of WPET hydrolyzed by adding 6% RL, 6% PEG300 and 8% BET

[0042] Figure 2 TG test result diagrams of WPET obtained in Example 1 and Comparative Example 1; (a) is Comparative Example 1, TG of WPET hydrolyzed by cutinase; (b) is Example 1, TG of WPET hydrolyzed by adding 6% RL, 6% PEG300 and 8% BET cutinase

[0043] Figure 3 DTG test result diagrams of WPET in Example 1 and Comparative Example 1; (a) is Comparative Example 1, DTG of WPET hydrolyzed by cutinase; (b) is Example 1, DTG of WPET hydrolyzed by adding 6% RL, 6% PEG300 and 8% BET cutinase

[0044] Figure 4 Effect diagram of adding 6% RL, 6% PEG300 and 8% BET on the molecular weight of cutinase in Example 3; SDS-PAGE: M - standard protein; 1 - cutinase; 2 - 6% RL + cutinase; 3 - 6% PEG300 + cutinase and 8% BET + cutinase

[0045] Figure 5 Effect diagram of adding 6% RL, 6% PEG300 and 8% BET on the Zeta potential of the reaction system in Example 4

[0046] Figure 6 Effect diagrams of adding 6% RL, 6% PEG300 and 8% BET on the pH stability and temperature stability of cutinase in Example 5; (a) is the pH stability result diagram; (b) is the temperature stability result diagram

[0047] Figure 7 Effect diagram of adding 6% RL, 6% PEG300 and 8% BET on the secondary structure of cutinase in Example 6

[0048] Figure 8 Effect diagram of adding 6% RL, 6% PEG300 and 8% BET on the fluorescence intensity of cutinase in Example 7 Detailed implementation mode

[0049] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope protected by the present invention. The following specific embodiments further describe the present invention.

[0050] Raw materials used in the embodiments and comparative examples of the present invention:

[0051] The molecular weight of the water-dispersible polyester is: 12000 g / mol;

[0052] Rhamnolipid RL is a dirhamnolipid;

[0053] Cutinase, derived from Humicola insolen, the preparation method refers to the existing patent document CN 108753671A. The specific process of fermenting and producing cutinase includes:

[0054] 1. Seed culture: The E. coli BL21(DE3) / pET-20b(+) / hic strain obtained in CN 108753671A was inoculated into the seed medium and cultured using a constant temperature shaker at a temperature of 37 °C, a rotation speed of 200 rpm, and cultured for 8 h.

[0055] 2. Fermentation for enzyme production: The seed liquid was inoculated into a 3.6 L fermenter at an inoculation amount of 10% for fermentation. The rotation speed in the fermenter was controlled at 300 rpm, the ventilation rate was 1.5 vvm, the dissolved oxygen in the fermentation broth was maintained at 30%, the temperature was controlled at 37 °C, and 25% (v / v) ammonia water was added dropwise to control the pH at 7.0. After the initial glycerol was consumed, the dissolved oxygen rose to 80 - 100%, and the batch fermentation culture ended; fed-batch fermentation was carried out by adding the feed medium in an exponential feeding manner; when the cell concentration OD600 reached 75, the temperature was lowered to 30 °C, and lactose solution was added at a constant rate of 0.4 - 1.2 g·L -1 ·h -1 Lactose solution was added dropwise at a constant rate for induction, the dissolved oxygen was maintained at 30%, the pH was controlled at about 7.0, and induction was carried out for about 8 h, and the fermentation supernatant was collected by centrifugation.

[0056] Its nucleotide sequence and amino acid sequence are disclosed in Table 1 of Patent CN 113338044A, and the enzyme activity is 1800 U.

[0057] The test methods involved in the present invention:

[0058] 1. Qualitative and quantitative analysis of hydrolysis products by high performance liquid chromatography (HPLC)

[0059] An Agilent 1260 series high performance liquid chromatograph was used, equipped with an Ultimate XB-C18 (4.6×250mm, 5μm) chromatographic column and a UV detector. The elution program used was as follows: the mobile phase was 1% glacial acetic acid: methanol = 35:65, the injection volume was 20 μL, the flow rate was 0.5 mL / min, the chromatographic column was maintained at about 35 °C, and detection was carried out at a wavelength of 240 nm.

[0060] The hydrolysis products of the water-dispersed polyester were 5-sodium sulfoisophthalate (5-SSIPA), terephthalic acid (TPA), mono(2-hydroxyethyl) phthalate (MHET), and bis(2-hydroxyethyl) terephthalate (BHET), respectively. Standard solutions of 5-SSIPA, TPA, MHET, and BHET with concentrations of 0.3125 mg / L, 0.625 mg / L, 1.25 mg / L, 2.5 mg / L, 5 mg / L, and 10 mg / L were prepared with methanol. The standard curve between the peak area (Y-axis) and the standard product concentration (X-axis) was plotted through HPLC testing.

[0061] Y 5-SSIPA = 379507.04534X + 201439.50746 (R 2 = 0.999)

[0062] Y TPA = 189907.37202X - 3470.5301 (R 2 = 0.999)

[0063] Y MHET = 188903.04451X + 25718.06956 (R 2 = 0.999)

[0064] Y BHET = 153764.31265X - 31960.64503 (R 2 = 0.999)

[0065] After the reaction of cutinase with the water-dispersed polyester was completed, the enzyme was inactivated at 100 °C for 30 min. The reaction system was centrifuged at 10000 r / min for 5 min, and the supernatant was filtered through a hydrophilic filter membrane (0.1 μm). The peak areas of the hydrolysis products of the water-dispersed polyester at 223 nm and 240 nm were measured by HPLC. The obtained peak area values were substituted into the standard curve to obtain the concentrations C 5-SSIPA 、C TPA 、C MHET 、C BHET . Furthermore, the total product release was calculated as = C 5-SSIPA + C TPA + CMHET +C BHET 。

[0066] 2. Zeta Potential Test and Analysis

[0067] The electrostatic interaction of the reaction system was measured by Zeta potential.

[0068] 3. Fluorescence Spectrum Test

[0069] The fluorescence spectrum of the sample was collected using a Hitachi F-7000 fluorescence spectrometer. The fluorescence excitation wavelength was set at 280 nm, and the emission wavelength was 300 - 500 nm. The instrument settings were as follows: excitation slit 5 nm, emission slit 10 nm, photomultiplier tube voltage 700 V, and scanning speed 240 nm / min.

[0070] 4. Circular Dichroism (CD) Test

[0071] At room temperature, a quartz cuvette with a 1-mm optical path was used to measure the spectral curve from 190 to 250 nm at a scanning speed of 50 nm / min and a bandwidth of 1 nm.

[0072] 5. Chemical Composition Test of Water-Dispersed Polyester

[0073] A Fourier transform infrared (FTIR) spectrophotometer was used to collect the surface chemical property spectra of WPET before and after enzymatic hydrolysis in the wavenumber range of 400 - 4000 cm -1 and analyze the changes in chemical bonds and functional groups.

[0074] 6. Thermal Stability Test of Water-Dispersed Polyester

[0075] The pyrolysis characteristics and thermal stability of WPET before and after enzymatic hydrolysis were studied using a thermogravimetric analyzer in an N2 atmosphere. The initial temperature was set at 25 °C, and the temperature was gradually increased to 800 °C at a heating rate of 10.0 °C / min.

[0076] 7. Method for Measuring Enzyme Activity

[0077] The product of the hydrolysis of p-nitrophenyl butyrate (pNPB) by cutinase is p-nitrophenol (pNP), which is yellow in an alkaline environment and has a strong absorption peak at 405 nm. Therefore, the amount of pNP produced was determined by continuously measuring the change in absorbance at this wavelength using a spectrophotometer, and the enzyme activity of cutinase was calculated. The total volume for testing enzyme activity was 1.5 mL, including 1440 μL of 10 mmol / L Tris-HCl buffer (pH 8), 30 μL of 50 mmol / L pNPB, and 30 μL of appropriately diluted enzyme solution. Definition of enzyme activity: At 37 °C, the amount of enzyme that catalyzes the hydrolysis of p-nitrophenyl butyrate to produce 1 μmol of p-nitrophenol per minute is defined as one enzyme activity unit.

[0078] Example 1

[0079] A method for enhancing the hydrolysis of hydrophobically modified polyesters by cutinase using a surfactant as an additive, comprising the following steps:

[0080] (1) Dissolution treatment of hydrophobically modified polyesters

[0081] Add hydrophobically modified polyesters according to a bath ratio of 1:10 and treat at 95 °C for 3 h; remove solid impurities with a hydrophilic filter membrane with a diameter of 45 mm and a pore size of 0.22 μm, and reserve the filtered liquid for use;

[0082] (2) Preparation of surfactant solutions

[0083] Mix RL, PEG300, and BET with 10 mmol / L Tris-HCl respectively, stir and dissolve at 30 °C, shake evenly, and adjust the pH to 9 with sodium hydroxide to obtain RL, PEG300, and BET solutions; the concentrations of RL, PEG300, and BET in the solutions are 6%, 6%, and 8%, w / v, g / mL respectively;

[0084] (3) Enzymatic hydrolysis

[0085] Put the filtered liquid of hydrophobically modified polyesters after the dissolution treatment in step (1) into the RL, PEG300, and BET solutions prepared in step (2), the WPET concentration is 5 g / L, then add cutinase, the enzyme concentration is 175 U / mL, react in a constant temperature shaker at 40 °C and 120 rpm for 12 h. After the reaction, inactivate the enzyme at 100 °C, centrifuge the reaction solution at 10000 r / min for 5 min to obtain the lower layer emulsion, wash the lower layer emulsion with ethanol and deionized water, and freeze-dry it.

[0086] Example 2

[0087] The difference from Example 1 is only that the RL, PEG300, and BET solutions in step (2) are replaced with non-ionic surfactant PEG200, PEG1000, and PEG4000 solutions with a concentration of 6% w / v, g / mL, and other parameters and conditions are the same as those in Example 1.

[0088] WPET PEG200 PEG1000 PEG4000 Total product release amount (mg / L) 1793.21 1820.40 1052.64

[0089] Comparative Example 1

[0090] The difference from Example 1 is only that step (2) is omitted, and the RL, PEG300, and BET solutions in step (3) are replaced with 10 mmol / L Tris-HCl buffer solution with pH 9, that is, directly perform enzymatic hydrolysis on the filtered liquid of hydrophobically modified polyesters after the dissolution treatment in step (1), and other parameters and conditions are the same as those in Example 1.

[0091] Comparative Example 2

[0092] It is only different from Example 1 in that the cutinase in step (3) is omitted, and other parameters and conditions are the same as those in Example 1.

[0093] Performance measurement

[0094] 1. The supernatant after centrifugation of the reaction solutions of the enzyme-hydrolyzed aqueous-dispersed polyesters obtained in Example 1 and Comparative Examples 1 and 2 was tested, and the test results are as follows:

[0095] Table 1. Results of the release amount of the enzyme-hydrolyzed aqueous-dispersed polyester products obtained in Example 1 and Comparative Examples 1 and 2

[0096]

[0097] As can be seen from Table 1, the addition of RL, PEG300, and BET significantly increased the release amount of the WPET hydrolysis products. Compared with pure enzymatic hydrolysis, the release amounts of the hydrolysis products increased by 70.22%, 68.44%, and 99.85%. From the results of Comparative Example 2, it can be seen that the solution containing RL, PEG300, and BET has little effect on the hydrolysis effect of WPET. It is mainly the combined action of RL, PEG300, and BET with the enzyme that increases the hydrolysis yield.

[0098] 2. Perform performance tests on the WPET obtained in Example 1 and Comparative Example 1

[0099] The results of exploring the chemical structure changes caused by the enzymatic hydrolysis of WPET using Fourier transform infrared spectroscopy are as Figure 1 shown, Figure 1 are the FTIR spectra of the WPET after being treated in Example 1 and Comparative Example 1, where Figure (a) is for Comparative Example 1 and Figure (b) is for Example 1; as can be seen from the figure, the content of ester bonds on the surface of the WPET after enzymatic hydrolysis in Figure (a) and (b) decreases.

[0100] The results of thermogravimetric analysis tests are as Figure 2 shown, Figure 2 in which (a) is for Comparative Example 1 and (b) is for Example 1; from Figure 2 it can be seen that: the thermogravimetric curves of the WPET after being enzymatically hydrolyzed alone and the WPET after being enzymatically hydrolyzed with the addition of RL and PEG300 are similar. However, the residual mass after thermal decomposition is lower than that without enzymatic hydrolysis. In the BET enzymatic hydrolysis system, there are two thermal weight loss stages at 250 °C and 399 °C. After being treated by the method of the present invention, the thermal weight loss of the WPET is greater.

[0101] Figure 3The DTG graphs after enzymatic hydrolysis in Example 1 and Comparative Example 1 further analyze the changes in thermal properties of WPET after enzymatic hydrolysis based on TG. The maximum thermogravimetric loss temperature after enzymatic hydrolysis moves to low temperature. When RL and PEG300 are introduced, the maximum thermogravimetric loss of WPET occurs at around 399°C. When BET is introduced, there are two thermogravimetric loss stages at 250°C and 399°C.

[0102] Comparative Example 3

[0103] The only difference from Example 1 is that the RL, PEG300 and BET solutions in step (2) are replaced by 0.1% w / v, g / mL; 1% w / v, g / mL cationic surfactant (CTAB) and 0.1% w / v, g / mL; 1% w / v, g / mL anionic surfactant (SDS) solution, and the other parameters and conditions are the same as those in Example 1.

[0104]

[0105] Example 2 Effect of RL, PEG300 and BET concentrations on the amount of enzymatic hydrolysis product released

[0106] The only difference from Example 1 is that the concentrations (w / v, g / mL) of RL, PEG300 and BET in step (3) of Example 1 are adjusted to 1%, w / v; 2%, w / v; 4%, w / v; 6%, w / v; 8%, w / v; 10%, w / v; 12%, w / v, respectively; the other parameters and conditions are the same as those in Example 1.

[0107] 1. The obtained reaction solution of enzymatic hydrolysis of WPET was tested, and the test results were as follows:

[0108] Table 4. Results of the release of WPET products from the enzymatic hydrolysis of surfactants at different concentrations in Example 4

[0109]

[0110] As can be seen from Table 4, 1-6% RL and PEG300 and 1-8% BET concentrations increase the release of enzymatic hydrolyzed polyester products. When the RL and PEG300 concentrations exceed 6% and the BET concentration exceeds 8%, it is not conducive to enzymatic hydrolysis because excessively high RL, PEG300 and BET concentrations may destroy the enzyme conformation and cause enzyme inactivation.

[0111] 2. Effects of RL, PEG300 and BET on the molecular weight of cutinase

[0112] Figure 4 To analyze the molecular weight of cutinase using SDS-PAGE gel electrophoresis. Figure 4As can be seen from the electrophoresis diagram, the molecular weight of cutinase is 20 kDa, and the addition of 6% RL, 6% PEG300, and 8% BET has no effect on the molecular weight of cutinase.

[0113] 3. Effects of RL, PEG300, and BET on the Zeta potential of the reaction system

[0114] Figure 5 The effects of the addition of RL, PEG300, and BET on the Zeta potential of the reaction system were analyzed. After adding 6% RL, 6% PEG300, and 8% BET, the absolute value of the zeta potential of cutinase decreased, indicating that the addition of the three surfactants reduced the electrostatic repulsion of WPET and could all promote hydrolysis.

[0115] 4. Effects of RL, PEG300, and BET on enzyme activity and stability

[0116] pH stability: At 40 °C, a certain amount of enzyme solution was incubated in buffer solutions with pH values ranging from 5.0 to 10.0 for 12 h, and then the enzyme activity was measured. The enzyme activity at 0 h was defined as 100%, and the relative enzyme activity of the residual enzyme activity in the enzyme solution was calculated as a percentage.

[0117] Temperature stability: Cutinase was placed in Tris-HCl buffer with a pH of 8 and solutions of 6% w / v RL, 6% w / v PEG300, and 8% w / v BET, respectively, and incubated at 40 °C for 72 h. The enzyme activity at 0 h was defined as 100%, and the relative enzyme activity of the residual enzyme activity in the enzyme solution was calculated as a percentage.

[0118] As Figure 6 shown, a shows the effects of RL, PEG300, and BET on the pH stability of cutinase, and b shows the effects of RL, PEG300, and BET on the temperature stability of cutinase. Compared with the pure enzyme, the relative enzyme activities after introducing the surfactants increased by 4.07%, 5.89%, and 11.05%, respectively. Under acidic conditions, the introduction of surfactants could not significantly improve the enzyme activity. The effects of surfactants on the temperature stability of cutinase are as Figure 6 shown in b. In the absence of surfactants, at a temperature of 40 °C, after incubating for 72 h, the enzyme activity of cutinase decreased by nearly 43.02%. However, adding surfactants to the cutinase solution before incubation could reduce this loss. Under incubation at 40 °C for 72 h, the relative enzyme activities were maintained at 60.27%, 60.48%, and 66.38%, respectively, which were increased by 3.29%, 3.50%, and 9.40% compared with cutinase without adding surfactants.

[0119] 5. Effects of RL, PEG300, and BET on the secondary structure of the enzyme

[0120] To further determine the interaction between the surfactant and cutinase, as Figure 7 shown. Different spectral regions show different structures of the enzyme protein. The β-sheet structure of cutinase shows a negative band in the CD spectral region of 210-220 nm. In addition, the troughs around 222 nm and 208 nm also show the presence of the α-helix structure. The introduction of the surfactant reduces the content of random coils, indicating that the structure of the enzyme becomes more regular, and the enzyme activity and stability increase.

[0121] 6. Effects of RL, PEG300, and BET on the fluorescence intensity of the enzyme

[0122] After adding RL, PEG300, and BET, the fluorescence intensity decreases, and the position of the maximum fluorescence emission peak shifts slightly blue, as Figure 8 shown. These results directly confirm that the exposure of the chromophore in cutinase decreases. Further analysis shows that the interaction between the surfactant and cutinase makes the tertiary structure of the enzyme protein more compact. On the other hand, the addition of the surfactant causes changes in the viscosity and polarity of the reaction system. The viscosity of the microenvironment of cutinase increases and the polarity decreases, resulting in a slight decrease in fluorescence and a slight blue shift of the fluorescence emission peak.

[0123] The embodiments provided above are not intended to limit the scope covered by the present invention, nor are the described steps intended to limit their execution order. Obvious improvements made by those skilled in the art to the present invention in combination with the existing common general knowledge also fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for using a surfactant as an additive to enhance the hydrolysis of water-dispersible polyester by cutinase, the method comprising the following steps: The water-dispersible polyester solution is placed in a surfactant solution, and cutinase is added to carry out a hydrolysis reaction; after the reaction is completed, the enzyme is inactivated, centrifuged, and the lower layer of emulsion is taken out for washing and drying; The surfactant is any one or more of a biosurfactant, a nonionic surfactant and a zwitterionic surfactant.

2. The method according to claim 1, characterized in that The biosurfactant includes any one or more of phosphates, proteins, sugar esters, and cholesterols; the nonionic surfactant includes any one or more of polyoxyethylene type, polyol type, alkanolamide type, polyether type, and amine oxide type; the zwitterionic surfactant includes one or more of amino acid type, betaine type, sulfate type, sulfonate type, and phosphate type.

3. The method according to claim 1, characterized in that The surfactant is a zwitterionic surfactant.

4. The method according to claim 1, characterized in that: The concentration of the surfactant solution is 0.0625-12%, w / v, g / mL.

5. The method according to claim 2, characterized in that: The sugar esters include any one of sophorose esters and rhamnose esters; the rhamnose esters include monorhamnose esters and disrhamnose esters; the betaine type is anhydrous betaine and / or lauryl betaine; the polyol type includes any one or more of glycerol mono- and di-fatty acid esters, pentaerythritol fatty acid esters, and polyethylene glycol.

6. The method according to claim 1, characterized in that The enzyme concentration of the cutinase relative to the surfactant solution is 50-225 U / mL.

7. The method according to claim 1, characterized in that The surfactant is any one of PEG300, disorhamnosyl ester and lauryl betaine.

8. The water-dispersible polyester obtained by the method according to any one of claims 1 to 7.

9. Use of the method according to any one of claims 1 to 7 in the treatment of water pollution caused by water-dispersible polyester.

10. Use of the water-dispersible polyester according to claim 8 in slurry preparation and textile processing.

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