Method for measuring degradation degree of PLA (polylactic acid) and application

Abstract

The invention relates to the technical field of polymer degradation, and discloses a PLA degradation degree determination method and application. The determination method comprises the following steps: (1) preparing a PLA-FDL film, wherein the PLA-FDL film is a PLA film embedded with fluorescein dilaurate; (2) carrying out mixed reaction on PLA hydrolase and the PLA-FDL membrane; and measuring the fluorescence value of the product in the reaction process. According to the determination method, real-time and nondestructive monitoring of the PLA degradation process can be realized, degradation kinetics of enzymes or microorganisms can be dynamically reflected, the process is simple, operation is simple and convenient, and the determination method has a remarkable application prospect.

Description

Technical Field

[0001] This invention relates to the field of polymer degradation technology, specifically to a method for determining the degree of PLA degradation and its application. Background Technology

[0002] Polylactic acid (PLA), as one of the most representative bio-based biodegradable plastics, has attracted widespread attention globally due to its renewable sources, good biocompatibility, and compostability. With its rapid growth in use in packaging materials, agricultural films, disposable products, and biomedicine, the sustainable management of PLA waste has become an important issue in the field of environmental materials. Although PLA can be effectively degraded under industrial composting conditions, its slow degradation under ambient temperature limits its overall green performance throughout its life cycle. Therefore, developing efficient PLA biodegradation technologies is of significant practical importance.

[0003] Currently, PLA biodegradation research mainly focuses on two directions: first, identifying and modifying highly efficient PLA-degrading microorganisms; and second, developing PLA-degrading enzymes with practical value. Regarding microorganisms, in addition to common actinomycetes, some bacterial strains (such as Bacillus brevis) have been observed in recent years. Brevibacillus brevis These microorganisms have also been shown to possess PLA-degrading capabilities. Transcriptomic studies have shown that these microorganisms achieve PLA attachment, depolymerization, and product utilization by regulating genes related to biofilm formation, hydrolytic enzyme secretion, and metabolic transport systems. In terms of enzymology, various hydrolases, including proteases, esterases, and keratinases, have been reported to degrade PLA, among which some thermostable proteases (such as Protein T) are particularly effective. FLTIER The variant has been successfully engineered through protein engineering and embedded in a PLA matrix to prepare self-degradable materials, showing promising application prospects.

[0004] However, both microbial resource development and enzyme preparation optimization face a common technical bottleneck: the lack of efficient and rapid methods for assessing degradation activity. For example, monitoring methods are cumbersome, rely on complex pretreatment, and cannot be monitored in real time. Summary of the Invention

[0005] The purpose of this invention is to overcome the problems of existing PLA degradation monitoring methods, such as cumbersome procedures, reliance on complex pretreatment, and inability to monitor in real time, and to provide a method and application for determining the degree of PLA degradation. This method can achieve real-time, non-destructive monitoring of the PLA degradation process, dynamically reflecting the degradation kinetics of enzymes or microorganisms, and is simple in procedure and easy to operate, showing significant application prospects.

[0006] To achieve the above objectives, the first aspect of the present invention provides a method for determining the degree of PLA degradation, comprising the following steps: (1) Prepare a PLA-FDL membrane, wherein the PLA-FDL membrane is a PLA membrane with fluorescein dilaurate embedded in it; (2) The PLA hydrolase and the PLA-FDL membrane are mixed and reacted; the fluorescence value of the product is measured during the reaction.

[0007] Preferably, in step (1), the preparation process of the PLA-FDL membrane includes: mixing PLA, the fluorescein dilaurate and a solvent to obtain a mixture, and drying the mixture into a membrane.

[0008] Preferably, the solvent is selected from at least one of hexafluoroisopropanol, dimethyl sulfoxide, and acetone, and more preferably hexafluoroisopropanol.

[0009] Preferably, the content of fluorescein dilaurate in the PLA-FDL membrane is 0.25-1 wt‰.

[0010] Preferably, the mixing process includes: first mixing the PLA and the solvent, and then adding the fluorescein dilaurate.

[0011] Preferably, the drying conditions include at least the following: a temperature of 45-55°C and a time of 1-3 hours.

[0012] Preferably, the process of drying the mixture into a film includes: coating the mixture into the micropores of the substrate and drying it into a film.

[0013] Preferably, the coating amount of the mixture in a single micropore is 30-50 μL.

[0014] Preferably, in step (2), the PLA hydrolase is mixed with the PLA-FDL membrane in the form of an enzyme solution.

[0015] Preferably, the concentration of PLA hydrolase in the enzyme solution is 0.15-0.2 μM.

[0016] Preferably, the amount of enzyme solution used in a single microwell is 20-200 μL.

[0017] Preferably, in step (2), the PLA hydrolase is selected from PAM. FLI At least one of Proteinase K, Aquaysin-1, and Protein T, preferably PAM FLI .

[0018] Preferably, in step (2), the conditions for the mixing reaction include at least: a temperature of 40-50°C and a time of 18-36h.

[0019] The second aspect of this invention provides the application of the determination method described in the first aspect in the screening of PLA hydrolase activity. The third aspect of the present invention provides the application of the determination method described in the first aspect above in evaluating the degradation ability of PLA in different environments.

[0020] The beneficial effects of the present invention through the above technical solution are as follows: The method for determining the degree of PLA degradation provided by this invention achieves highly uniform dispersion and low background leakage of fluorescein dilaurate in the PLA matrix through the combination of fluorescein dilaurate and PLA, overcoming the technical difficulty of poor binding between fluorescent probes and PLA. On this basis, a two-step synergistic mechanism of "PLA enzymatic hydrolysis-FDL release-fluorescence response" is established, which enables the fluorescence signal to be directly correlated with the actual degradation efficiency of PLA substrate, effectively connecting enzyme screening and practical application, and has significant application prospects. Attached Figure Description

[0021] Figure 1 This is a process flow diagram of a specific embodiment of the method for determining the degree of PLA degradation in this invention; Figure 2 These are graphs showing the activity measurement results of the films obtained in Example 1, Comparative Example 1, and Comparative Example 2 of this invention. Figure 3 This is a graph showing the fluorescence value test results of the catalytic PLA-FDL thin film hydrolysis process in Example 1 of this invention; Figure 4 These are Raman imaging test results of the PLA-FDL film obtained in Preparation Example 1, the PLA-FDL film obtained in Preparation Example 2, pure PLA, and pure FDL in this invention. Figure 5 These are the fluorescence value test results during the catalytic hydrolysis process of PLA-FDL thin films in Examples 1, 8, 9 and 10 of this invention; Figure 6 These are the fluorescence value test results during the catalytic hydrolysis process of PLA-FDL thin films in Examples 1-6 of this invention. Detailed Implementation

[0022] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0023] In a first aspect, the present invention provides a method for determining the degree of PLA degradation, comprising the following steps: (1) Prepare a PLA-FDL membrane, wherein the PLA-FDL membrane is a PLA membrane with fluorescein dilaurate (FDL) embedded in it; (2) The PLA hydrolase and the PLA-FDL membrane are mixed and reacted; the fluorescence value of the product is measured during the reaction.

[0024] During their research, the inventors of this invention unexpectedly discovered that the method for determining the degree of PLA degradation, through the combination of fluorescein dilaurate and PLA, achieved highly uniform dispersion of fluorescein dilaurate in the PLA matrix and low background leakage, overcoming the technical challenge of poor binding between fluorescent probes and PLA. Based on this, a two-step synergistic mechanism of "PLA enzymatic hydrolysis-FDL release-fluorescence response" was established, enabling the fluorescence signal to be directly correlated with the actual degradation efficiency of the PLA substrate, effectively bridging enzyme screening and practical application, and showing significant application prospects.

[0025] According to the present invention, preferably, in step (1), the preparation process of the PLA-FDL membrane includes: mixing PLA, the fluorescein dilaurate, and a solvent to obtain a mixture, and drying the mixture into a membrane. The inventors have found that, under this preferred embodiment, the preparation process of the PLA-FDL membrane is simple and can provide an encapsulation effect of PLA on FDL, thereby improving the accuracy of monitoring the PLA degradation process.

[0026] According to the present invention, the solvent is a solvent conventionally selected in the art that can dissolve PLA. In order to further improve the solvent's dissolution effect on PLA, preferably, the solvent is selected from at least one of hexafluoroisopropanol, dimethyl sulfoxide and acetone, and more preferably hexafluoroisopropanol.

[0027] In this invention, there is no particular limitation on the amount of solvent used, as long as it can achieve the effect of dissolving PLA. Preferably, the mass ratio of solvent to PLA is 150-200:1, which can be 150:1, 160:1, 170:1, 180:1, 190:1, 200:1, or any value between the two aforementioned values.

[0028] According to the present invention, preferably, the content of fluorescein dilaurate in the PLA-FDL membrane is 0.25-1 wt‰, which can be 0.25 wt‰, 0.5 wt‰, 0.75 wt‰, 1 wt‰, or any value between the aforementioned two values. The inventors have found that, under this preferred embodiment, by controlling the content of fluorescein dilaurate in the PLA-FDL membrane within the above range, the sensitivity and signal response intensity of PLA hydrolysis degree detection can be further improved, thus possessing the reliability as a real-time monitoring indicator.

[0029] According to the present invention, in order to further improve the accuracy of monitoring the PLA degradation process, preferably, the mixing process includes: first mixing the PLA and the solvent, and then adding the fluorescein dilaurate.

[0030] According to the present invention, in order to further improve the film-forming effect of PLA-FDL film, preferably, the drying conditions include at least: a temperature of 45-55°C, which can be 45°C, 50°C, 55°C, or any value between the two aforementioned values; and a time of 1-3h, which can be 1h, 2h, 3h, or any value between the two aforementioned values.

[0031] According to the present invention, preferably, the process of drying the mixture into a film includes: coating the mixture into the micropores of a substrate and drying it into a film.

[0032] In this invention, preferably, the substrate can be a 96-well plate or a 384-well plate. This preferred embodiment supports large-scale parallel detection, thereby significantly reducing the time required for a single detection cycle and simplifying operation. For example, when the measurement method uses a 96-well plate high-throughput format, it can support parallel detection of up to 2850 samples, drastically reducing the time required for a single detection cycle from several hours in traditional methods to real-time monitoring within 15 minutes to 24 hours, without requiring complex pretreatment, making operation extremely simple.

[0033] According to the present invention, preferably, the coating amount of the mixture in a single micropore is 30-50 μL, which can be 30 μL, 40 μL, 50 μL, or any value between the two aforementioned values.

[0034] According to the present invention, preferably, in step (2), the PLA hydrolase is mixed with the PLA-FDL membrane in the form of an enzyme solution.

[0035] According to the present invention, preferably, the concentration of PLA hydrolase in the enzyme solution is 0.15-0.2 μM, which can be 0.15 μM, 0.17 μM, 0.19 μM, 0.2 μM, or any value between the two aforementioned values.

[0036] According to the present invention, preferably, the amount of enzyme solution used in a single microwell is 20-200 μL, which can be 20 μL, 50 μL, 100 μL, 150 μL, 200 μL, or any value between the aforementioned two values. More preferably, the amount of PLA hydrolase used in a single microwell is 50-100 μL. The inventors have found that, under this preferred embodiment, while ensuring the detection sensitivity of the assay method, excessive reagent consumption and potential nonlinear interference can be effectively avoided, thereby improving the reliability and repeatability of the detection.

[0037] According to the present invention, preferably, in step (2), the PLA hydrolase is selected from PAM. FLI At least one of Proteinase K, Aquaysin-1, and Protein T. Among them, PAM... FLI Source Actinomadura keratinilytica T16-1; Proteinase K is derived from Parengyodontium album Aquaysin-1 originates from Thermus aquaticus Protein T originates from Thermus sp. strain Rt41A More preferably, the PLA hydrolase is PAM. FLI .

[0038] According to the present invention, in order to further improve the degradation effect of PLA hydrolase on PLA, preferably, in step (2), the conditions of the mixed reaction include at least: a temperature of 40-50℃, which can be 40℃, 45℃, 50℃, or any value between the two aforementioned values; and a time of 18-36h, which can be 18h, 24h, 30h, 36h, or any value between the two aforementioned values.

[0039] According to a particularly preferred embodiment of the present invention, a method for determining the degree of PLA degradation is provided, comprising the following steps: (1) Prepare PLA-FDL membrane, which is a PLA membrane with fluorescein dilaurate embedded in it; (2) React PLA hydrolase and PLA-FDL membrane at 40-50℃ for 18-36 h; measure the fluorescence value of the product during the reaction; In step (1), the preparation process of PLA-FDL membrane includes: first, mixing PLA and solvent, then adding fluorescein dilaurate to obtain a mixture, coating the mixture into the micropores of the substrate, and drying at 45-55℃ for 1-3 hours to form a film; the solvent is hexafluoroisopropanol; the coating amount of the mixture in a single micropore is 30-50 μL; the content of fluorescein dilaurate in PLA-FDL membrane is 0.25-1 wt‰; In step (2), PLA hydrolase is mixed with the PLA-FDL membrane in the form of an enzyme solution; the concentration of PLA hydrolase in the enzyme solution is 0.15-0.2 μM; the volume of enzyme solution used in a single microwell is 20-200 μL; the PLA hydrolase is PAM FLI .

[0040] Through the above-mentioned preferred embodiments, the method for determining the degree of PLA degradation can realize real-time, non-destructive monitoring of the PLA degradation process, dynamically reflect the degradation kinetics of enzymes or microorganisms, and has a simple process and easy operation, with significant application prospects.

[0041] For example, the process flow diagram of the PLA degradation degree determination method provided by the present invention is shown below. Figure 1 .

[0042] Secondly, the present invention provides the application of the determination method described in the first aspect in the screening of PLA hydrolase activity.

[0043] Thirdly, the present invention provides the application of the determination method described in the first aspect above in evaluating the degradation ability of PLA in different environments.

[0044] The present invention will be described in detail below through embodiments.

[0045] Unless otherwise specified, the experimental methods and equipment described in the following examples are conventional methods and equipment.

[0046] In the following examples, PLA was purchased from Shenzhen Guangyuan Plastic Chemical Co., Ltd.; PLA hydrolase PAM FLI The product was prepared by Beijing Qingke Biotechnology Co., Ltd. based on the description in the Nature journal article "An engineered enzyme embedded into PLA to make self-biodegradable plastic"; unless otherwise specified, other raw materials can be obtained commercially.

[0047] Preparation Example 1 Add 0.1g PLA powder to 10mL HFIP (hexafluoroisopropanol) and shake in a constant temperature shaker (200rpm) at 30℃ for 2h until PLA is completely dissolved to form a transparent PLA-HFIP solution. Dissolve FDL (fluorescein dilaurate) and PLA together in HFIP and continue shaking for 30min to ensure that FDL is uniformly dispersed in the PLA-HFIP solution to obtain a mixture in which the mass ratio of FDL to PLA is 1:999. Pipette 40μL of the mixture to the bottom of each well of a black 96-well plate and dry in an oven at 50℃ for 2h to evaporate the hexafluoroisopropanol solvent, thus obtaining a PLA-FDL film attached to the bottom of the well (the FDL content in the film is 1‰ by mass).

[0048] Preparation Example 2 PLA-FDL films were prepared according to the method of Preparation Example 1, except that the mass ratio of FDL to PLA was 1:1999, resulting in PLA-FDL films (the mass content of FDL in the films was 0.5‰).

[0049] Preparation Example 3 PLA-FDL films were prepared according to the method of Preparation Example 1, except that the mass ratio of FDL to PLA was 1:3030, resulting in PLA-FDL films (the mass content of FDL in the films was 0.33‰).

[0050] Preparation Example 4 PLA-FDL films were prepared according to the method of Preparation Example 1, except that the mass ratio of FDL to PLA was 1:3999, resulting in PLA-FDL films (the mass content of FDL in the films was 0.25‰).

[0051] Preparation Example 5 The film was prepared according to the method of Preparation Example 1, except that the fluorescein dilaurate was replaced with F to obtain the PLA-F film.

[0052] Preparation Example 6 The film was prepared according to the method of Preparation Example 1, except that fluorescein dilaurate was replaced with fluorescein diacetate (FDA) to obtain PLA-FDA film.

[0053] Example 1 The PLA-FDL membrane obtained in Preparation Example 1 was mixed with 50 μL of enzyme solution (the enzyme in the enzyme solution was PLA hydrolase PAM). FLI PAM in enzyme solution FLI The concentration was 0.17 μM. The PLA-FDL membrane was catalyzed for hydrolysis, and the reaction was carried out at 45 °C for 48 h. The fluorescence value FI during the reaction was recorded. 494 / 525nm .

[0054] Example 2 Example 1 was prepared by catalytic preparation of PLA-FDL membrane hydrolysis according to the method of Example 1, except that the amount of enzyme solution was replaced with 80 μL.

[0055] Example 3 Example 1 was prepared by catalytic hydrolysis of PLA-FDL membrane according to the method of Example 1, except that the amount of enzyme solution was replaced with 100 μL.

[0056] Example 4 Example 1 was prepared by catalytic preparation of PLA-FDL membrane hydrolysis according to the method of Example 1, except that the amount of enzyme solution was replaced with 150 μL.

[0057] Example 5 Example 1 was prepared by catalytic hydrolysis of PLA-FDL membrane according to the method of Example 1, except that the amount of enzyme solution was replaced with 200 μL.

[0058] Example 6 Example 1 was prepared by catalytic preparation of PLA-FDL membrane hydrolysis according to the method of Example 1, except that the amount of enzyme solution was replaced with 20 μL.

[0059] Example 7 The PLA-FDL membrane hydrolysate prepared in Example 1 was prepared by catalytic synthesis according to the method of Example 1, except that the PLA hydrolase PAM in the enzyme solution was used. FLI The concentration was replaced with 0.085 μM.

[0060] Example 8 The PLA-FDL thin film obtained in Example 2 was prepared by catalytic hydrolysis according to the method of Example 1.

[0061] Example 9 The PLA-FDL thin film obtained in Example 3 was prepared by catalytic hydrolysis according to the method of Example 1.

[0062] Example 10 The PLA-FDL thin film obtained in Example 4 was prepared by catalytic hydrolysis according to the method of Example 1.

[0063] Comparative Example 1 The PLA-F thin film obtained in Example 5 was prepared by catalytic hydrolysis according to the method of Example 1.

[0064] Comparative Example 2 The PLA-FDA thin film obtained in Example 6 was prepared by catalytic hydrolysis according to the method of Example 1.

[0065] Test Example 1 For the films obtained in Example 1, Comparative Example 1, and Comparative Example 2, 200 μL of 0.1 M tris-HCl buffer was added to each well and incubated for 48 h. Then, 150 μL of the solution was aspirated from each well onto a new plate for activity assay, i.e., the rate of change (slope) of fluorescence value was measured. Next, 50 μL of enzyme solution (containing PAM) was added. FLI The concentration was 0.17 μM), and the mixture was incubated for 24 h. Activity was measured by determining the rate of change (slope) of the fluorescence value. The results are shown in […]. Figure 2 The formula for calculating the rate of change (slope) of fluorescence value is shown in equation (1): Equation (1); EV stands for Empty vector, which does not contain the target gene.

[0066] The fluorescence value of the product in Example 1 was tested during the 24-hour incubation process described above. A blank control group was also set up, and the fluorescence value of pure FDL was tested during the 24-hour incubation process. The results are shown below. Figure 3 .

[0067] from Figure 2 It can be seen that, among the three encapsulation cases of PLA-F film (Comparative Example 1), PLA-FDA film (Comparative Example 2), and PLA-FDL film (Example 1), the PLA-FDL film has the lowest autofluorescence leakage value. Figure 3 It can be seen that the FDL (blank group) not encapsulated by PLA has extremely strong autofluorescence leakage, which proves that the FDL was successfully encapsulated by PLA. The autofluorescence leakage values ​​of the PLA-F and PLA-FDA encapsulation methods are much higher.

[0068] Test Example 2 Raman imaging tests were performed on the PLA-FDL film obtained in Preparation Example 1, the PLA-FDL film obtained in Preparation Example 2, pure PLA, and pure FDL. The results are shown in [Figure number missing]. Figure 4 .

[0069] Raman imaging is a microscopic analysis technique that combines Raman spectroscopy with spatial scanning, enabling two-dimensional or three-dimensional visualization and analysis of the chemical composition or structural distribution of a sample surface. Its basic principle is based on the Raman scattering effect: when a laser is focused on the sample surface, some photons interact inelastically with molecular vibrations, producing Raman scattered light with characteristic frequency shifts. The displacement information corresponds to the vibrational modes of intramolecular chemical bonds. During Raman imaging, the sample surface is scanned point-by-point by moving the sample stage or laser spot with high precision, and Raman spectra are acquired in each micrometer-scale region. Subsequently, data is integrated based on specific Raman characteristic peaks (such as intensity or shift) and spatial location information to generate images of compositional distribution, crystallinity, or stress state. This technique has advantages such as non-destructive operation and high spatial resolution (down to sub-micrometer levels), and is widely used in materials science, biomedicine, and pharmaceutical analysis.

[0070] from Figure 4 As can be seen, the blue area in image ① represents the PLA component, and the red area in image ② represents the FDL component; images ③ and ④ show the composite structure where FDL is successfully encapsulated by PLA. Because the sample is in powder form, there are microscopic morphological fluctuations on the surface, leading to inconsistent Raman responses in some areas (such as the green signal), a common phenomenon in powder samples. Furthermore, in ③ and ④, the green area indicates the coexistence of PLA and FDL, suggesting that the FDL may have reached a near-atomic level of dispersion. Since the spatial resolution limit of Raman spectroscopy is at the micrometer scale, even with extended acquisition time, it is difficult to completely distinguish between two highly blended substances spectrally. Therefore, Figure 4 The results show that FDL has been successfully encapsulated by PLA in PLA-FDL film.

[0071] Test Example 3 The fluorescence values ​​of the PLA-FDL thin film during the hydrolysis process in Examples 1, 8, 9, and 10 were tested and recorded. The results are shown in [Figure number missing]. Figure 5 .

[0072] The fluorescence values ​​of the PLA-FDL thin film during the hydrolysis process catalyzed in Examples 1-6 were tested and recorded. The results are shown in the table below. Figure 6 .

[0073] from Figure 5 and Figure 6 The results show that by monitoring the fluorescence intensity (FI) of PLA films with different FDL doping concentrations (0.25‰–1‰) during enzymatic digestion, it can be seen that... 494 / 525 The dynamic changes of the fluorescence signal were observed, and the correlation between the fluorescence signal and the degree of substrate hydrolysis was systematically evaluated. The results showed that throughout the reaction time, the fluorescence signal of each group... 494 / 525 All values ​​continuously increased with the hydrolysis process, exhibiting a good linear growth trend; simultaneously, at the same reaction time point, the fluorescence intensity significantly increased with the increase of FDL doping concentration. These data fully demonstrate that FI... 494 / 525 It can accurately and continuously reflect the degree of hydrolysis of PLA and has the reliability to serve as a real-time monitoring indicator.

[0074] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A method for determining the degree of PLA degradation, characterized in that, Includes the following steps: (1) Prepare a PLA-FDL membrane, wherein the PLA-FDL membrane is a PLA membrane with fluorescein dilaurate embedded in it; (2) The PLA hydrolase and the PLA-FDL membrane are mixed and reacted; the fluorescence value of the product is measured during the reaction.

2. The determination method according to claim 1, characterized in that, In step (1), the preparation process of the PLA-FDL membrane includes: mixing PLA, the fluorescein dilaurate and a solvent to obtain a mixture, and drying the mixture into a membrane.

3. The determination method according to claim 2, characterized in that, The solvent is selected from at least one of hexafluoroisopropanol, dimethyl sulfoxide and acetone, preferably hexafluoroisopropanol; Preferably, the content of fluorescein dilaurate in the PLA-FDL membrane is 0.25-1 wt‰.

4. The determination method according to claim 2, characterized in that, The mixing process includes: first mixing the PLA and the solvent, and then adding the fluorescein dilaurate; Preferably, the drying conditions include at least the following: a temperature of 45-55°C and a time of 1-3 hours.

5. The determination method according to claim 2, characterized in that, The process of drying the mixture to form a film includes: coating the mixture into the micropores of the substrate and drying it to form a film; Preferably, the coating amount of the mixture in a single micropore is 30-50 μL.

6. The determination method according to claim 5, characterized in that, In step (2), the PLA hydrolase is mixed with the PLA-FDL membrane in the form of an enzyme solution; Preferably, the concentration of PLA hydrolase in the enzyme solution is 0.15-0.2 μM; Preferably, the amount of enzyme solution used in a single microwell is 20-200 μL.

7. The determination method according to any one of claims 1 to 6, characterized in that, In step (2), the PLA hydrolase is selected from PAM. FLI At least one of Proteinase K, Aquaysin-1, and Protein T, preferably PAM FLI .

8. The determination method according to any one of claims 1 to 6, characterized in that, In step (2), the conditions for the mixing reaction include at least the following: temperature of 40-50℃ and time of 18-48h.

9. The application of the assay method according to any one of claims 1 to 8 in the screening of PLA hydrolase activity.

10. The application of the determination method according to any one of claims 1 to 8 in evaluating the degradation ability of PLA in different environments.

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