Alkali-resistant polylactic acid depolymerases and application thereof in degradation of polylactic acid plastics

Abstract

The invention discloses alkali-resistant polylactic acid depolymerases and application thereof in degradation of polylactic acid plastics, and belongs to the technical field of bioengineering and environmental biology, an amino acid sequence of the alkali-resistant polylactic acid depolymerases E01 is shown as SEQ ID NO: 2, and a nucleotide sequence of a coding gene of the alkali-resistant polylactic acid depolymerases E01 is shown as SEQ ID NO: 1. The enzyme is derived from metagenome mining, the sequence similarity of the enzyme and the known PLA depolymerases PAM is lower than 25%, the optimal reaction temperature of E01 is 50 DEG C, the optimal pH is 10.0, and the enzyme shows excellent stability within the alkaline range of pH 8.0-11.0 and under the condition of 50 DEG C. Degradation experiments prove that E01 has remarkable degradation capacity on various commercially available PLA products (including films, straws and meal boxes), and obvious lactic acid monomer release and material surface erosion can be observed after the PLA products are treated for 48 hours under the conditions that the temperature is 50 DEG C and the pH value is 10.0. The invention provides a novel enzyme preparation with important application potential for biological recovery treatment of PLA plastic waste.

Description

Technical Field

[0001] This invention belongs to the field of industrial enzyme preparations and biodegradation technology, specifically relating to a novel polylactic acid depolymerase E01 with alkali resistance, its encoding gene, preparation method, and its application in the biodegradation of PLA plastics and lactic acid recovery. Background Technology

[0002] Polylactic acid (PLA), a bio-based biodegradable plastic derived from renewable resources, is widely used in packaging, textiles, and medical fields. However, PLA degrades slowly in the natural environment, and its waste, if improperly disposed of, can also cause "white pollution." Chemical hydrolysis of PLA typically requires high temperatures or strong acid / alkali conditions, resulting in high energy consumption and environmental pollution. In contrast, enzymatic degradation offers advantages such as milder conditions, environmental friendliness, and recyclable products.

[0003] Currently, the reported resources of PLA depolymerases (such as proteinase K and PLA depolymerase PAM) are still limited, and several application bottlenecks exist: for example, proteinase K has poor thermal stability at its optimal pH (10.0); many enzymes have low degradation efficiency for high-crystallinity or actual PLA products. More importantly, PLA depolymerases that can maintain high activity under alkaline conditions are particularly scarce, which limits their potential for use in alkaline industrial wastewater treatment or in synergistic effects with other alkaline processes (such as washing). Therefore, discovering novel PLA depolymerases with novel catalytic properties (especially alkali resistance) from nature is of great significance for promoting the development of biorecycling technologies for PLA waste. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a depolymerase E01 with a novel sequence, excellent alkali resistance, and high efficiency in degrading actual PLA products, thus providing a new candidate enzyme for its application in industrial biodegradation.

[0005] The technical solution of the present invention is as follows: An alkali-resistant polylactic acid depolymerase E01 has the following amino acid sequence: 1) The amino acid sequence as shown in SEQ ID NO.2; or 2) Substitution, deletion, and / or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO.2 without altering its function; or 3) An amino acid sequence derived from 2) and having the same function as 2).

[0006] Preferably, the alkali-resistant polylactic acid depolymerase E01 consists of 291 amino acid residues as shown in SEQ ID NO.2, with a predicted molecular weight of 32.36 kDa and a theoretical isoelectric point pI of 4.98.

[0007] An isolated polynucleotide encoding the aforementioned polylactic acid depolymerase.

[0008] Preferably, it has the following nucleotide sequence: 1) A nucleotide sequence as shown in SEQ ID NO.1; or 2) A nucleotide sequence encoding a protein with the same function, but with one or more nucleotides substituted, deleted, and / or added to the sequence shown in SEQ ID NO.1; or 3) A nucleotide sequence that has 75%, 80%, 85%, 88%, 90% or more homology with the sequence shown in SEQ ID NO.1 and encodes the same functional protein.

[0009] Those skilled in the art can easily mutate the protein nucleotides obtained in this invention using known methods, such as site-directed mutagenesis and rational modification of gene nucleotide sequences. Those artificially modified nucleotide sequences that have a 75% or higher similarity to the nucleotide sequences encoding the proteins obtained in this invention, provided the encoded enzyme is active, are all derived from and equivalent to the nucleotide sequences of this invention.

[0010] A recombinant expression vector comprising the aforementioned polynucleotides.

[0011] Preferably, the carrier is pET-26b(+).

[0012] A recombinant cell comprising the recombinant expression vector, preferably wherein the host cell of the recombinant cell is Escherichia coli BL21(DE3).

[0013] A method for producing alkali-resistant polylactic acid depolymerase E01 includes culturing the recombinant cells, inducing the expression of the target protein, and then purifying the E01 enzyme from the culture by steps such as cell disruption, centrifugation, and nickel ion affinity chromatography.

[0014] Application of the alkali-resistant polylactic acid depolymerase E01 in the degradation of PLA plastics or the recycling of lactic acid monomers.

[0015] The applications include: The PLA plastic to be degraded was dissolved in a buffer solution, and alkali-resistant polylactic acid depolymerase E01 was added for degradation to degrade the PLA plastic into lactic acid monomers, which were then recovered.

[0016] The amount of polylactic acid depolymerase E01 used is 1-10% of the total volume, and the degradation time is 12-36 hours.

[0017] The buffer solution is Tris-HCl buffer.

[0018] Preferably, the degradation is carried out at 50-70°C and pH 9.0-11.0.

[0019] Beneficial effects:

[0020] 1. Novel sequence: The E01 gene was obtained through metagenomics technology. Its amino acid sequence similarity with the known PLA depolymerase PAM is only 24.7%, which is far below the 40% similarity threshold usually accepted for homologous proteins. It belongs to a completely new member of the PLA depolymerase family.

[0021] 2. Unique alkali resistance and thermal stability: E01 has an optimal reaction pH of 10.0 and can maintain more than 70% of its relative enzyme activity within a wide pH range of 8.0-11.0, clearly defined as an alkali-resistant depolymerase. Furthermore, it retains more than 80% of its activity after incubation at 50°C for 1 hour, exhibiting good medium-temperature thermal stability, making it suitable for various industrial applications.

[0022] 3. Efficient soluble expression and convenient purification: By using the pET-26b(+) vector and the E. coli BL21(DE3) host system, large-scale soluble expression of E01 was achieved under low-temperature induction conditions. High-purity protein could be obtained by one-step purification using simple Ni²⁺-NTA affinity chromatography, laying the foundation for its large-scale preparation.

[0023] 4. Strong ability to degrade real PLA waste: Degradation experiments have confirmed that E01 can not only degrade standard PLA films, but also show a significant degradation effect on commercially available high-crystallinity PLA products (such as disposable straws and lunch boxes), indicating that it has the potential to treat actual PLA waste and has broad application prospects. Attached Figure Description

[0024] Figure 1 : Multiple sequence alignment diagram of E01 with typical serine hydrolases; Figure 2 Phylogenetic tree analysis of E01; Figure 3 SDS-PAGE images of E01 expression and purification in E. coli; Figure 4 : The optimal temperature and pH for E01; Figure 5 Molecular docking and catalytic pocket binding analysis of E01 protease to PLA Figure 6 Degradation effect of E01 on different PLA products; Figure 7E01 verifies the degradation ability of commercial PLA plastic particles. Detailed Implementation

[0025] The present invention will now be described in detail with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0026] Example 1: Mining and Bioinformatics Analysis of the E01 Gene Using HMMER 3.3.2 software, the MGnify metagenomic database was mined using the serine hydrolase signature profile (Pfam: PF03959) as a probe. Candidate sequences with an E value less than 1e-10 were screened and optimized to obtain the target gene sequence, named E01 (SEQ ID NO:1). The molecular weight of its encoded protein (SEQ ID NO:2) was predicted to be 32.36 kDa, and its isoelectric point pI was 4.98, using the ExPASy ProtParam tool. Multiple sequence alignment using Clustal Omega showed that E01 contains the conserved catalytic triplet (Ser-Asp-His) of the serine hydrolase family and substrate-binding pocket characteristics (…). Figure 1 Using MEGA 11 software, a phylogenetic tree was constructed using the neighbor-joining method. The results showed that E01 is distantly related to known PLA depolymerases (such as PAM and proteinase K), forming an independent branch. Figure 2 This confirms the novelty of its sequence.

[0027] The optimized nucleic acid sequence (SEQ ID NO. 1) is as follows: atgggccatagcaacatggatcatagcgaaatggatcatagcaacatggaaaacccggtgatggataaaccgctgccgaacaccaaagatcagaaagaagtgaaacagcaggaatttagcaacgcggtgagcgattggcataaccaggatattaaaggcagcgatgtgaaagtggcggtgctggataccggcattgataaagataacaaagatctgatttatgtgaaaggcgtgaactttgtgggcgataacaaagataactatgatgatgataacggccatggcaccaaaattaccggcattattggcgcgcgcgaaaacgattttaacctgctgggcattgcgccgaacagcgatctgtatattgcgaaagtggcggataaaaacggcgcggtgcaggtggaaaacctgattaaaggcattaactgggcgattaacgaagatgtgcagattattaacattagcctggaactgccggaagatcataaaaaactgcataccgcgattaaaaaagcgcatcagaaaggcattgtggtgattgcgagcagcggcaacattaaatttccgggcgataaacagctgagctatccgggcagctatagcgaagtgattaacgtgggcatgctgaacattgcgggcgaaatttatagccaggcggcggcggataaaaaagtggatgtgtatgcgccgggcgaagatatttttagcctgtatctgaacgataaaatgaccctggataccggcgtgagctatgcgaccgcgtataccagcggctatgcggcgctgctgattaacaactatcagaaacagagcgaagattatgatattaaaaaaattaaaggcgaactgcagaactatctgaaaccgaccaaa Optimized protein sequence (SEQ ID NO .2):MGHSNMDHSEMDHSNMENPVMDKPLPNTKDQKEVKQQEFSNAVSDWHNQDIKGSDVKVAVLDTGIDKDNKDLIYVKGVNFVGDNKDNYDDDNGHGTKITGIIGARENDFNLLGIAPNSDLYIAKVADKNGAVQVENLIKGINW AINEDVQIINISLELPEDHKKLHTAIKKAHQKGIVVIASSGNIKFPGDKQLSYPGSYSEVINVGMLNIAGEIYSQAAADKKVDVYAPGEDIFSLYLNDKMTLDTGVSYATAYTSGYAALLINNYQKQSEDYDIKKIKGELQNYLKPTK Example 2: Cloning, expression, and purification of the E01 gene The optimized E01 gene (SEQ ID NO:1) was synthesized by GenScript Biotech Ltd., and NdeI and XhoI restriction sites were introduced at its 5' and 3' ends, respectively. The target gene was cloned into the pET-26b(+) expression vector by restriction endonuclease digestion and T4 DNA ligase ligation to construct the recombinant plasmid pET-26b-E01. The correctly identified recombinant plasmid was then heat-shocked and transformed into *E. coli* BL21(DE3) competent cells.

[0028] Single colonies were picked and inoculated into LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C with shaking at 200 rpm until the OD600 reached approximately 0.6-0.8. IPTG was added to a final concentration of 0.5 mM, and expression was induced at 18°C ​​for 20 hours. After induction, the cells were collected by centrifugation at 4°C and 8000×g for 10 minutes. The cells were resuspended in lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0), sonicated, and then centrifuged at 4°C and 12000×g for 30 minutes. The supernatant was collected.

[0029] The supernatant was passed through a Ni²⁺-NTA affinity chromatography column pre-equilibrated with equilibration buffer (50 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, pH 8.0). Impurities were washed with wash buffers containing 40 mM and 60 mM imidazole, respectively, and the target protein was eluted with elution buffer containing 250 mM imidazole. The eluted fractions were collected and analyzed by SDS-PAGE. The results are shown below. Figure 3 As shown, a single, clear protein band was obtained at approximately 32 kDa, consistent with the predicted molecular weight, with a purity higher than 95%.

[0030] Example 3: Enzymatic Properties Analysis of E01 Using p-nitrophenol octanoate as a substrate, the relative enzyme activity of E01 was determined under different temperature (30-70℃) and pH (7.0-11.0) conditions, with the highest enzyme activity being 100%.

[0031] 3.1 Optimal temperature and thermal stability: such as Figure 4 As shown, the optimal reaction temperature for E01 is 50℃. Residual enzyme activity was measured after incubation at 50℃ for different times (0-120 minutes). The results showed that its half-life was approximately 75 minutes, and the relative enzyme activity remained above 80% after 1 hour of incubation, indicating good thermal stability at the operating temperature.

[0032] 3.2 Optimal pH and acid-base stability: such as Figure 4 As shown, E01 exhibits the highest enzyme activity at pH 10.0. Its relative enzyme activity remains above 70% across a wide pH range of 8.0 to 11.0, demonstrating its significant alkali resistance.

[0033] 3.3 Effects of Metal Ions and Chemical Reagents: Different metal ions or chemical reagents (1 mM each) were added to the standard reaction system. The results showed that Mg²⁺ and Ca²⁺ had a slight activating effect on enzyme activity (105%-115%), while Co²⁺, Cu²⁺, and Zn²⁺ exhibited strong inhibitory effects (relative enzyme activity <40%). 1% (v / v) methanol and ethanol had little effect on enzyme activity (retention rate >85%), but SDS completely inhibited enzyme activity.

[0034] E01 hydrolase homology modeling The active site and substrate-binding region of E01 exhibit a unique surface channel structure. Molecular docking analysis revealed that the substrate (lactic acid oligomer) forms hydrogen bonds with Lys97, Tyr251, Asn185, Ser250, and Ser182 in the E01 protein, hydrophobic interactions with leucine residues Leu156 and tyrosine residue Tyr251, and C-H interactions with His94.

[0035] This special surface channel structure can specifically accommodate polypeptide substrates or bind substrates at least six lactic acid monomers in polylactic acid, providing precise binding sites for efficient substrate-enzyme binding and subsequent catalytic reactions, thus ensuring the high efficiency and specificity of the enzyme catalysis process.

[0036] Example 4: Verification of E01's ability to degrade PLA products 4.1 Substrate Preparation: Select three types of PLA products: PLA plastic film (0.02 mm thick) and discarded PLA disposable straws and food containers (cut into 1 cm × 1 cm pieces). All substrates should be cleaned with anhydrous ethanol and dried before use.

[0037] 4.2 Degradation reaction: 20 μg of purified E01 enzyme and 50 mg of PLA substrate were added to 50 mM Gly-NaOH buffer (pH 10.0), and the reaction was carried out at 50℃ and 200 rpm for 48 hours with shaking. A control group without enzyme solution was set up.

[0038] 4.3 Degradation effect evaluation: Lactic acid release detection: After filtering the reaction solution through a 0.22 μm filter membrane, the concentration of lactic acid monomers was determined by high-performance liquid chromatography (HPLC). Results are as follows: Figure 6 The results showed that, compared with the control group, the E01 treatment group had a significantly increased amount of lactic acid release, especially on the degradation effect on the finished PLA film material.

[0039] Example 5 Performance evaluation under real-world plastic depolymerization scenarios To verify the actual degradation ability of E01 on highly crystalline commercial PLA products, we conducted a large-scale degradation experiment. In a 100 mL reaction system, using 1 g of PLA particles (model: 3260HP) as substrate, under optimal reaction conditions (60°C, pH 8.0), a depolymerization reaction was carried out for 48 hours using 3% B01 (high-temperature resistant polylactic acid depolymerase), proteinase K (PK), and E01, respectively.

[0040] After the reaction is complete, it can be seen that ( Figure 7 The E01-treated group showed very high solution transparency and a significant reduction in solid PLA residue at the bottom of the bottle. This phenomenon directly indicates that, under the same conditions, the E01 depolymerase provided by this invention can more effectively depolymerize solid PLA plastic into soluble small molecule products, highlighting its great value in practical industrial applications.

[0041] Conclusion: This invention provides a novel alkali-resistant polylactic acid depolymerase E01 with a novel sequence, easy industrial production, and high efficiency in degrading various real PLA wastes, showing great application potential in the bio-recycling of PLA plastics and environmental pollution control.

Claims

1. An alkali-resistant polylactic acid depolymerase, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

2.

2. An isolated polynucleotide, characterized in that, Its encoding is the alkali-resistant polylactic acid depolymerase as described in claim 1.

3. The polynucleotide according to claim 2, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:

1.

4. A recombinant expression vector, characterized in that, It contains the polynucleotide as described in claim 2 or 3.

5. A recombinant cell, characterized in that, It comprises the recombinant expression vector as described in claim 4.

6. The method for preparing the alkali-resistant polylactic acid depolymerase according to claim 1, characterized in that, The recombinant cells of claim 5 are cultured, and the expression of the alkali-resistant polylactic acid depolymerase is induced and purified from the culture supernatant.

7. The application of the alkali-resistant polylactic acid depolymerase of claim 1 in the degradation of polylactic acid plastics or the recycling of lactic acid monomers.

8. The application according to claim 7, characterized in that, The enzyme is directly added to the solution containing polylactic acid plastic to be degraded for degradation or recycling reaction; the amount of enzyme used is 1-10% of the total volume, and the degradation time is 12-36 hours.

9. The application according to claim 8, characterized in that, The degradation or recycling reaction is carried out under alkaline conditions at 50-70°C and pH 9.0-11.0.

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