CN-121991920-A - Alkali-resistant polylactic acid depolymerizing enzyme and application thereof in degradation of polylactic acid plastics

Abstract

The invention discloses an alkali-resistant polylactic acid depolymerase and application thereof in degradation of polylactic acid plastics, and belongs to the technical fields of bioengineering and environmental biology, wherein the amino acid sequence of the alkali-resistant polylactic acid depolymerase E01 is shown as SEQ ID NO. 2, and the nucleotide sequence of a coding gene is shown as SEQ ID NO. 1. The enzyme is derived from metagenomic mining, has sequence similarity of less than 25% with the known PLA depolymerizing enzyme PAM, has an optimal reaction temperature of 50 ℃ and an optimal pH of 10.0, and exhibits excellent stability in the alkaline range of pH 8.0 to 11.0 and under 50 ℃. Degradation experiments prove that E01 has remarkable degradation capability on various commercial PLA products (including films, straws and cutlery boxes), and obvious lactic acid monomer release and material surface erosion can be observed after the treatment for 48 hours under the condition of 50℃, pH and 10.0. The invention provides a novel enzyme preparation with important application potential for biological recovery treatment of PLA plastic waste.

Inventors

• ZHOU JIE
• Wang Qingsui
• JIANG MIN
• DONG WEILIANG
• XIE BIN

Assignees

• 南京工业大学

Dates

Publication Date

20260508

Application Date

20251205

Claims (9)

1. 1. An alkali-resistant polylactic acid depolymerase, which is characterized in that the amino acid sequence is shown as SEQ ID NO. 2.
2. 2. An isolated polynucleotide encoding the alkaline-resistant polylactic acid depolymerase of claim 1.
3. 3. The polynucleotide according to claim 2, wherein the nucleotide sequence is shown in SEQ ID NO. 1.
4. 4. A recombinant expression vector comprising the polynucleotide of claim 2 or 3.
5. 5. A recombinant cell comprising the recombinant expression vector of claim 4.
6. 6. The method for producing an alkali-resistant polylactic acid depolymerase according to claim 1, wherein the recombinant cell according to claim 5 is cultured, induced to express and isolated and purified from a culture supernatant to obtain the alkali-resistant polylactic acid depolymerase.
7. 7. The use of the alkali-resistant polylactic acid depolymerase as defined in claim 1 for degrading polylactic acid plastics or recovering lactic acid monomers.
8. 8. The method according to claim 7, wherein the enzyme is directly added into the solution containing the polylactic acid plastic to be degraded for degradation or recovery reaction, wherein the enzyme is used in an amount of 1-10% of the total volume, and the degradation time is 12-36 hours.
9. 9. The use according to claim 8, wherein the degradation or recovery reaction is carried out under alkaline conditions of 50-70° C, pH 9.0.0-11.0.

Description

Alkali-resistant polylactic acid depolymerizing enzyme and application thereof in degradation of polylactic acid plastics Technical Field The invention belongs to the technical field of industrial enzyme preparations and biodegradation, and particularly relates to novel polylactic acid depolymerizing enzyme E01 with alkali resistance, a coding gene thereof, a preparation method and application thereof in PLA plastic biodegradation and lactic acid recovery. Background Polylactic acid (PLA) is widely used in packaging, textile, medical and other fields as a bio-based degradable plastic derived from renewable resources. PLA, however, degrades slowly in the natural environment, and if disposed of improperly, it can also cause "white pollution". Chemical hydrolysis of PLA generally requires high temperature or strong acid/alkali conditions, is energy-intensive and is prone to environmental pollution. In contrast, enzymatic degradation has the advantages of mild conditions, environmental friendliness, recoverable products and the like. At present, the reported PLA depolymerases (such as protease K, PLA depolymerase PAM, etc.) have still limited resources and there are common application bottlenecks such as poor thermal stability of proteinase K at the optimum pH (10.0) and low degradation efficiency of many enzymes to high crystallinity or practical PLA preparations. More importantly, PLA depolymerizases capable of maintaining high activity under alkaline conditions are particularly scarce, limiting the possibilities of their co-action in alkaline industrial wastewater treatment or with other alkaline processes (such as washing). Therefore, the excavation of novel PLA depolymerases with novel catalytic properties (in particular alkali resistance) from nature is of great importance for the promotion of the development of bioremediation technology of PLA waste. Disclosure of Invention The invention aims to overcome the defects of the prior art, provide a depolymerizing enzyme E01 which has novel sequence, excellent alkali resistance and high-efficiency degradation capability on actual PLA products, and provide a new candidate enzyme for the application of the depolymerizing enzyme E01 in industrial biodegradation. The technical scheme of the invention is as follows: An alkali-resistant polylactic acid depolymerase E01 having an amino acid sequence as shown below: 1) An amino acid sequence shown as SEQ ID NO.2, or 2) The amino acid sequence shown as SEQ ID NO.2 is substituted, deleted and/or added with one or more amino acids without changing the function, or 3) An amino acid sequence derived from 2) and functionally identical to 2). Preferably, the alkali-resistant polylactic acid depolymerase E01 consists of 291 amino acid residues as shown in SEQ ID NO.2, and has a predicted molecular weight of 32.36 kDa and a theoretical isoelectric point pI of 4.98. An isolated polynucleotide encoding the polylactic acid depolymerase described above. Preferably, it has the nucleotide sequence: 1) Nucleotide sequence shown as SEQ ID NO.1, or 2) The nucleotide sequence shown in SEQ ID No.1 is substituted, deleted and/or added with one or more nucleotides and codes for the same functional protein, or 3) Nucleotide sequence which has 75%,80%,85%,88%,90% or more than 95% homology with the sequence shown in SEQ ID NO.1 and codes for the same functional protein. The protein nucleotide obtained by the present invention can be easily mutated by a person of ordinary skill in the art using a known method, for example, by site-directed mutagenesis and rational modification of a gene nucleotide sequence. Those nucleotide sequences which have been artificially modified and which have a degree of similarity of 75% or more with the nucleotide sequence encoding the protein isolated according to the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the nucleotide sequence of the present invention as long as the encoded enzyme has activity. A recombinant expression vector comprising the polynucleotide described above. Preferably, the vector is pET-26b (+). A recombinant cell comprising the recombinant expression vector, preferably the host cell of the recombinant cell is e.coli BL21 (DE 3). A method for producing alkali-resistant polylactic acid depolymerase E01 comprises culturing the recombinant cells, inducing the expression of target proteins, and purifying the recombinant cells from the culture by steps of cell disruption, centrifugation, nickel ion affinity chromatography and the like to obtain high-purity E01 enzyme. The alkali-resistant polylactic acid depolymerizing enzyme E01 is applied to degrading PLA plastics or recovering lactic acid monomers. The application comprises: and dissolving PLA plastic to be degraded in a buffer solution, adding alkali-resistant polylactic acid depolymerizing enzyme E01 for degradation, degrading the PLA plastic into lactic acid monomers, and recover