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CN-122016743-A - Method for measuring PLA degradation degree and application thereof

CN122016743ACN 122016743 ACN122016743 ACN 122016743ACN-122016743-A

Abstract

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

Inventors

  • LI XIUJUAN
  • HU MENG
  • QIAO YICHEN
  • WANG MINGHUI
  • LI MOYAN
  • ZHANG LEI

Assignees

  • 南京师范大学
  • 江苏海洋大学

Dates

Publication Date
20260512
Application Date
20260129

Claims (10)

  1. 1. A method for determining the degradation degree of PLA, comprising the steps of: (1) Preparing a PLA-FDL film, wherein the PLA-FDL film is a PLA film embedded with fluorescein dilaurate; (2) And mixing PLA hydrolase with the PLA-FDL film to react, and measuring the fluorescence value of the product in the reaction process.
  2. 2. The method according to claim 1, wherein in the step (1), the PLA-FDL film is produced by mixing PLA, the fluorescein dilaurate and a solvent to obtain a mixed solution, and drying the mixed solution to form a film.
  3. 3. The assay according to claim 2, wherein 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 film is 0.25-1 wt%o.
  4. 4. The method of claim 2, wherein the mixing comprises mixing the PLA and the solvent prior to adding the fluorescein dilaurate; Preferably, the drying conditions at least comprise a temperature of 45-55 ℃ and a time of 1-3 hours.
  5. 5. The method according to claim 2, wherein the step of drying the mixed solution to form a film comprises coating the mixed solution in micropores of a substrate, and drying the mixed solution to form a film; Preferably, the coating amount of the mixture liquid in the individual microwells is 30 to 50. Mu.L.
  6. 6. The method according to claim 5, wherein in the step (2), the PLA hydrolase is mixed with the PLA-FDL film as 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 in each of the microwells is 20 to 200. Mu.L.
  7. 7. The assay according to any one of claims 1 to 6 wherein in step (2) the PLA hydrolase is selected from at least one of PAM FLI , proteinase K, aquaysin-1 and Protein T, preferably PAM FLI .
  8. 8. The method according to any one of claims 1 to 6, wherein in the step (2), the conditions of the mixing reaction include at least a temperature of 40 to 50℃and a time of 18 to 48 hours.
  9. 9. Use of the assay of any one of claims 1 to 8 in screening for PLA hydrolase activity.
  10. 10. Use of the assay of any one of claims 1 to 8 to evaluate the degradability of PLA in different environments.

Description

Method for measuring PLA degradation degree and application thereof Technical Field The invention relates to the technical field of polymer degradation, in particular to a method for measuring PLA degradation degree and application thereof. Background Polylactic acid (PLA) is receiving attention as one of the most representative bio-based degradable plastics worldwide due to its renewable sources, good biocompatibility and compostability properties. With the rapid increase of the dosage of the polylactic acid composite material in the fields of packaging materials, agricultural mulching films, disposable products, biomedicine and the like, the sustainable treatment of PLA waste is an important subject in the field of current environmental materials. Although PLA can be effectively degraded under the condition of industrial composting, the characteristic of slow degradation under the normal temperature natural environment limits the exertion of the green performance of the full life cycle, so that the development of the efficient PLA biodegradation technology has important practical significance. At present, PLA biodegradation research is mainly developed around two directions, namely, firstly, high-efficiency PLA degrading microorganisms are excavated and modified, and secondly, PLA degrading enzymes with practical values are developed. In microbial terms, in addition to common actinomycetes, some bacterial strains (such as Bacillus brevis Brevibacillus brevis) have been demonstrated to have PLA degrading ability in recent years. Transcriptomics studies show that these microorganisms achieve attachment, depolymerization and product utilization of PLA by modulating genes related to biofilm formation, hydrolase secretion, and metabolic transport systems. In terms of enzymology, various hydrolases such as protease, esterase and cutinase are reported to be capable of degrading PLA, wherein certain proteases with excellent thermal stability (such as ProteinT FLTIER variants) are successfully modified by protein engineering and are embedded into PLA matrix to prepare self-degradable materials, so that the method has good application prospect. However, both microbial resource development and enzyme preparation optimization face a common technical bottleneck, namely, lack of an efficient and rapid degradation activity evaluation method, such as complicated monitoring method flow, dependence on complex pretreatment, incapability of real-time monitoring and the like. Disclosure of Invention The invention aims to solve the problems that a PLA degradation monitoring method in the prior art is complex in flow, depends on complex pretreatment and cannot be monitored in real time, and provides a PLA degradation degree-based measuring method and application. The determination method can realize real-time and nondestructive monitoring of the PLA degradation process, dynamically reflect the degradation dynamics of enzymes or microorganisms, has simple flow and simple and convenient operation, and has remarkable application prospect. In order to achieve the above object, a first aspect of the present invention provides a method for determining the degradation degree of PLA, comprising the steps of: (1) Preparing a PLA-FDL film, wherein the PLA-FDL film is a PLA film embedded with fluorescein dilaurate; (2) And mixing PLA hydrolase with the PLA-FDL film to react, and measuring the fluorescence value of the product in the reaction process. Preferably, in the step (1), the preparation process of the PLA-FDL film comprises the steps of mixing PLA, the fluorescein dilaurate and a solvent to obtain a mixed solution, and drying the mixed solution to form a film. Preferably, the solvent is at least one selected from hexafluoroisopropanol, dimethyl sulfoxide and acetone, and more preferably hexafluoroisopropanol. Preferably, the content of fluorescein dilaurate in the PLA-FDL film is 0.25-1 wt%o. Preferably, the mixing includes mixing the PLA and the solvent prior to adding the fluorescein dilaurate. Preferably, the drying conditions at least comprise a temperature of 45-55 ℃ and a time of 1-3 hours. Preferably, the process of drying the mixed solution to form a film comprises the steps of coating the mixed solution in micropores of a substrate and drying the mixed solution to form the film. Preferably, the coating amount of the mixture liquid in the individual microwells is 30 to 50. Mu.L. Preferably, in step (2), the PLA hydrolase is mixed with the PLA-FDL film in an enzymatic liquid. Preferably, the concentration of PLA hydrolase in the enzyme solution is 0.15-0.2. Mu.M. Preferably, the amount of enzyme solution in each of the microwells is 20 to 200. Mu.L. Preferably, in step (2), the PLA hydrolase is selected from at least one of PAM FLI, protein K, aquaysin-1 and Protein T, preferably PAM FLI. Preferably, in the step (2), the condition of the mixing reaction at least comprises the temperature of 40-50 ℃ and the time of 18-36h. A