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CN-120984662-B - Novel PVC degrading enzyme excavation and application thereof

CN120984662BCN 120984662 BCN120984662 BCN 120984662BCN-120984662-B

Abstract

The invention discloses excavation and application of a novel PVC degrading enzyme, and belongs to the field of degradation of environmental pollutant plastics. The invention utilizes macro proteome technology to screen out new PVC plastic degrading enzyme based on different responses of PVC degrading flora to artificial polymer and natural polymer, and further characterizes the catalytic activity of the enzyme. The invention provides a new effective strategy for screening PVC degrading enzyme from the soil microbial community of the alpine meadow in Qinghai-Tibet plateau so as to expand the resource library of the plastic degrading enzyme for resource recovery and bioremediation.

Inventors

  • HAN HUAWEN
  • JIANG YUCHAO
  • LI XIANGJIE
  • FU BAOTONG

Assignees

  • 兰州大学

Dates

Publication Date
20260508
Application Date
20250704

Claims (10)

  1. 1. The application of the phosphoserine phosphatase SerB in degrading plastics is characterized in that the plastics is PVC, and the amino acid sequence of the phosphoserine phosphatase SerB is shown as SEQ ID No. 2.
  2. 2. Use according to claim 1, characterized in that the phosphoserine phosphatase SerB shown in SEQ ID No.2 is added to a PVC-containing system and reacted for at least 7 days.
  3. 3. The use according to claim 2, wherein the system comprises at least 4 mg-mL -1 PVC and at least 50 μg-mL -1 SerB.
  4. 4. The use according to claim 3, wherein the system further comprises 20-50 mmol/L HEPES, 5-10 mmol/L MgSO 4 .
  5. 5. The use according to claim 4, wherein the pH of the system is 7 to 8.
  6. 6. The use according to claim 5, wherein the reaction conditions are 25-45 ℃.
  7. 7. The method according to claim 6, wherein the PVC has a number average molecular weight Mn of 50 to 70kDa, a weight average molecular weight Mw of 100 to 120 kDa and a Z average molecular weight Mz of 150 to 170 kDa.
  8. 8. The use according to claim 7, wherein the PVC comprises a PVC film or PVC particles.
  9. 9. The use according to any one of claims 1 to 8, wherein PVC is added to the reaction system at a final concentration of 5mg mL -1 and reacted at 25 ℃ for at least 7 days, said reaction system containing 50% HEPES at a final concentration of 50 mmol/L, 5% mmol/L MgSO 4 、50 μg·mL -1 SerB.
  10. 10. A method for increasing the surface roughness of a PVC film, characterized in that the PVC film is added to a solution containing phosphoserine phosphatase SerB as shown in SEQ ID No.2 and reacted for at least 7 days.

Description

Novel PVC degrading enzyme excavation and application thereof Technical Field The invention relates to excavation and application of novel PVC degrading enzyme, belonging to the field of degradation of environmental pollutant plastics. Background The use amount of plastics in the fields of daily life, agricultural production and chemical industry is continuously rising. There is a need for efficient, environmentally friendly solutions to address the plastic contamination problem. The production of polyvinyl chloride (PVC) is 10% of the global plastic production, next to polyethylene (PE, 29.7%) and polypropylene (PP, 19.3%). Its high molecular weight characteristics, stable covalent bond structure and hydrophobic character lead to difficult degradation in natural environment. In addition, millions of micro plastic particles, phthalic acid ester, bisphenol A and other additives can be released in the PVC pipe cutting process, and potential threats are formed to the ecosystem and human health. Microbial degradation of plastic waste is considered as an environmentally friendly alternative to conventional disposal methods such as incineration and landfills. Currently, more than 430 microorganisms among 20 bacteria have been demonstrated to have the ability to degrade various plastics such as PE, polystyrene (PS), polyethylene terephthalate (PET), polyurethane (PUR), and the like, including Gordonia, sphingomonas (Novosphingabium), bacillus, and the like. However, only a few microorganisms, such as Klebsiella pneumoniae (Klebsiella sp.) EMBL-1 and degrading flora EF1, are able to perform growth metabolism with PVC as the sole carbon source. The existing researches prove that part of the microorganisms can degrade PVC, the analysis of degradation paths is still focused on multiple groups of chemical predictions, and related PVC degrading enzyme gene excavation and action mechanisms are not yet deeply explored. In view of the above, the development of novel PVC degrading enzyme becomes a key problem to be solved in the current plastic degradation field, and has important significance for enriching the existing plastic degradation related gene library and promoting the development of plastic biodegradation technology. Disclosure of Invention The invention provides a novel PVC degrading enzyme SerB. The invention also provides a gene for encoding the PVC degrading enzyme SerB. The invention also provides a recombinant microorganism expressing the PVC degrading enzyme SerB. In one embodiment, the microorganism includes, but is not limited to, E.coli. In one embodiment, the recombinant microorganism is a host E.coli BL21 (DE 3) and pET-28a (+) is an expression vector. The invention also provides a method for screening PVC plastic degrading enzyme from expression difference protein of synthetic polymer and natural polymer by PVC degrading flora (DC), comprising the following steps: The method comprises the steps of (1) sequencing a macro-proteome of PVC-DC and lignon-DC, (2) constructing a plastic degradation related gene library, (3) screening candidate enzymes through gene biological information and phylogenetic tree thereof, (4) constructing and purifying target proteins, and (5) determining protein functions through appearance and detection of degradation intermediate products. In one embodiment, the method is to culture recombinant E.coli expressing PVC degrading enzyme in LB medium to OD 600 > 0.6, adding IPTG to a final concentration of 0.4mM, and culturing at 18℃overnight. In one embodiment, the culturing is at 37℃and 180-200 rpm. The invention also provides application of the PVC degrading enzyme obtained by screening in plastic degradation. In one embodiment, the phosphoserine phosphatase SerB shown in SEQ ID No.2 is added to a PVC-containing system and reacted for at least 7 days. In one embodiment, the system contains at least 4 mg-mL -1 PVC and at least 50 μg-mL -1 SerB. In one embodiment, the system further comprises 20-50 mmol/L HEPES and 5-10 mmol/L MgSO 4. In one embodiment, the reaction pH is 7 to 8. In one embodiment, the reaction pH is 7.5. In one embodiment, the reaction conditions are 25 to 45 ℃. In one embodiment, the PVC has a weight average molecular weight (Mw) of 50 to 70kDa, a number average molecular weight (Mn) of 100 to 120kDa, and a Z average molecular weight (Mz) of 150 to 170kDa. In one embodiment, the PVC comprises a PVC film or PVC particles. In one embodiment, PVC is added to the reaction system containing 50mmol/L HEPES and 5mmol/L MgSO 4,50μg·mL-1 SerB at a final concentration of 5 mg/mL -1 and reacted at 25℃for at least 7 days. The invention also provides a method for increasing the surface roughness of the PVC film, which comprises the step of adding the PVC film into a solution containing phosphoserine phosphatase SerB shown in SEQ ID No.2, and reacting for at least 7 days. The beneficial effects are that: (1) The invention provides a PVC degrading enzyme screening method, whic