PLA depolymerases and mutants and application thereof

Abstract

The invention discloses PLA (polylactic acid) depolymerases as well as mutants and application thereof. The amino acid sequence of the PLA depolymerases is shown as SEQ ID NO: 1; the mutation site of the mutant comprises at least one of the 144th amino acid residue and the 183th amino acid residue, and the 144th amino acid residue is mutated into glutamic acid; and the 183th amino acid residue is mutated into alanine. The mutant provided by the invention has high catalytic activity, high thermal stability and excellent high substrate resistance, and can achieve an efficient depolymerization capability of degrading more than 90% of a 100g / L real PLA substrate in 48 hours.

Description

Technical Field

[0001] This invention belongs to the field of biocatalysis and genetic engineering, specifically relating to a PLA depolymerase, its mutants, and its applications. Background Technology

[0002] Polylactic acid (PLA) is a type of bio-based plastic derived from renewable resources, but its degradation rate is extremely slow under natural environmental and home composting conditions, still forming microplastics, which limits its value in the circular economy system. Compared to the rapid development of enzymatic recycling of PET plastics, PLA depolymerases with high efficiency in depolymerization at high temperatures and high substrate concentrations are still scarce. Most of the more than 40 PLAases reported have problems such as low activity, insufficient thermal stability, or poor resistance to high-load reactions. Summary of the Invention

[0003] The first objective of this invention is to provide a PLA depolymerase mutant, the amino acid sequence of which is shown in SEQ ID NO: 1; the mutation site of the mutant includes at least one of the amino acid residues at position 144 and position 183;

[0004] Among them, the 144th amino acid residue is mutated to glutamic acid;

[0005] The 183rd amino acid residue is mutated to alanine.

[0006] In some embodiments of the present invention, the mutation site of the mutant further includes the 66th amino acid residue; the 66th amino acid residue is mutated to histidine.

[0007] In some embodiments of the present invention, the mutation sites of the mutant further include amino acid residues at positions 109 and 164;

[0008] Among them, the 109th amino acid residue is mutated to phenylalanine;

[0009] The 164th amino acid residue is mutated to lysine.

[0010] The present invention further provides a gene encoding the above-mentioned PLA depolymerase mutant.

[0011] The present invention further provides a recombinant vector containing the above-mentioned genes.

[0012] In some embodiments of the present invention, pET-26b is selected as the gene expression vector for constructing recombinant vectors.

[0013] The present invention further provides a recombinant strain containing the above-mentioned genes.

[0014] In some embodiments of the present invention, the host bacterium of the recombinant strain is Escherichia coli BL21(DE3).

[0015] The present invention further provides the application of the above-mentioned PLA depolymerase and its mutants in PLA depolymerization.

[0016] In some embodiments of the present invention, the PLA depolymerase or mutant is added to a reaction system containing PLA substrate to depolymerize PLA.

[0017] In some embodiments of the present invention, the PLA depolymerase or mutant is added to the reaction system containing PLA substrate in the form of crude enzyme solution or pure enzyme.

[0018] In some embodiments of the present invention, the PLA depolymerase and its mutants depolymerize PLA under alkaline conditions at 50-70°C.

[0019] In some embodiments of the present invention, the amount of pure enzyme, including PLA depolymerase and its mutants, added to the PLA depolymerization system is 2–5‰ of the substrate mass.

[0020] This invention, based on systematic structural comparison and activity screening of known PLA depolymerases, has for the first time discovered a novel serine protease (hereinafter referred to as SP-01) with significantly superior natural activity compared to existing PLA depolymerases. Furthermore, its amino acid sequence similarity to the reported PLA depolymerase PAM is only 52.24%. Based on SP-01, this invention constructed a mutant with significantly improved catalytic efficiency and thermal stability. The optimal mutant achieved 90% efficient depolymerization at 60–70 °C and a substrate loading of 100 g / L PLA. This PLA depolymerase mutant can be widely applied to the biorecycling of waste PLA plastics and the rapid degradation of PLA products. Attached Figure Description

[0021] Figure 1 Comparison of lactic acid monomer concentrations obtained from the depolymerization of ground PLA solid powder by wild-type protease SP-01 and its representative multi-point mutants X2, X3 and X4.

[0022] Figure 2 The results show the thermostability of wild-type protease SP-01 and its mutants X2, X3 and X4.

[0023] Figure 3 This study compares the degradation capabilities of mutant X4, wild-type protease SP-01, and commercial proteinase K on ground pipette powder.

[0024] Figure 4 The results show the change in lactic acid monomer concentration over time obtained from PLA pipette degradation in a 1L system by mutant X4. Detailed Implementation

[0025] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0026] The raw materials and equipment used in the specific embodiments of the present invention are all known products, obtained by purchasing commercially available products.

[0027] To enhance the industrial application value of PLA depolymerase, this invention uses SP-01, a serine protease obtained through big data mining, as the starting enzyme, synthesizes its encoding gene, and expresses and purifies it in *E. coli*. Through analysis of the enzyme's three-dimensional structure and substrate complex model, combined with machine learning prediction and molecular dynamics simulation, site-directed and combinatorial mutagenesis is performed on amino acids surrounding the active site that are involved in substrate recognition and structural stability, thereby improving the enzyme's catalytic activity and thermal stability towards PLA.

[0028] Example 1: Construction of wild-type PLA depolymerase recombinant plasmid and its mutant recombinant plasmid

[0029] 1. In this embodiment, SP-01 was used as a template to perform site-directed mutagenesis to construct a mutant.

[0030] The amino acid sequence of SP-01 is shown in SEQ ID NO: 1, and the coding sequence is shown in SEQ ID NO: 2. To facilitate efficient expression in E. coli, the coding sequence was codon-optimized and synthesized by a commercial company.

[0031] 2. Design of mutation sites.

[0032] (1) Screening of single-point mutation sites;

[0033] Based on the crystal structure of SP-01, a deep learning model was used to score the microenvironmental adaptability of each residue in the holoenzyme, screening out several "non-preferred residues." Simultaneously, an SP-01–PLA tetramer (4LA) complex model was constructed and molecular dynamics simulations were performed to identify pocket residues that maintain frequent contact with the substrate (e.g., distance ≤ 5 Å). Combining machine learning results, molecular dynamics analysis, and sequence conservation (PSSM) information, several candidate mutation sites were finally selected, including stability-related sites Q66, Q144, and S183, as well as S107, S109, T137, A164, and L221 near the catalytic pocket.

[0034] Site-directed mutagenesis primers were designed targeting the aforementioned sites. Point mutations were performed on pET-26b(+)-SP-01 using overlap extension PCR or a commercial mutagenesis kit to obtain single-point mutants, including but not limited to: Q66, S86, T113, S109, G135, Q149, S183, A164, I209, N247, and K268. The amplified products were digested with DpnI to remove the template plasmid, transformed into E. coli DH5α, and single clones were selected for sequencing to confirm the correct mutation, yielding recombinant plasmids expressing the corresponding single-point mutants.

[0035] (2) To further obtain enzymes with both high activity and high thermostability, this invention, based on the results of single-point mutation screening, combines multiple mutation sites with positive effects to construct multi-point mutants. Preferred combinations include, but are not limited to:

[0036] Substitutions involving at least two sites;

[0037] Substitutions involving at least three sites;

[0038] Substitutions involving four or more sites;

[0039] The combined mutations were constructed using stepwise superposition PCR or by continuing point mutations using the previous round of mutant plasmids as templates. All recombinant plasmids were verified by sequencing to ensure that their mutation sites were correct.

[0040] The mutant of the present invention may also be a variant in which, while maintaining the above-mentioned key mutation sites, the remaining amino acids are allowed to undergo conserved substitutions, and the sequence corresponding to the position of SEQ ID NO: 1 has more than 85% (preferably more than 90%, or more than 95%, or more than 99%) homology, and still has significant PLA depolymerase activity.

[0041] 3. Construction of recombinant plasmids;

[0042] The codon-optimized SP-01 gene was inserted into the multiple cloning site of plasmid pET-26b(+) via Nco I and Xho I restriction endonuclease sites. The ligation product was transformed into E. coli DH5α. After single clones were picked, identified, and sequenced to confirm the correct insertion sequence, the recombinant plasmid pET-26b(+)-SP-01 expressing wild-type SP-01 was obtained. This plasmid was then transformed into E. coli BL21(DE3) competent cells for subsequent protein expression.

[0043] The recombinant plasmids and recombinant expression cells of the mutants were constructed in the same manner as the original enzymes.

[0044] Subsequent examples use mutants X2 (Q144E / S183A) with two site mutations, mutant X3 (Q144E / S183A / Q66H) with three site mutations, and mutant X4 (Q144E / S183A / S109F / A164K) with four site mutations to further compare the performance of wild-type enzymes and mutants.

[0045] Example 2: Purification of wild-type and mutant PLA depolymerases, PLA powder depolymerization test and thermal stability test

[0046] (1) Obtaining purified enzymes;

[0047] Using pET-26b(+) as the expression vector, the recombinant plasmids obtained in Example 1 were transformed into E. coli BL21(DE3) competent cells.

[0048] 1. Protein expression conditions

[0049] Transformants were inoculated into 50 mL of LB broth containing kanamycin and cultured at 37°C with shaking until OD reached. 600 Add IPTG at a final concentration of 0.1 mM to induce induction, and continue low-temperature shaking culture at 18℃ for 20-24 h.

[0050] 2. Crude enzyme preparation

[0051] Collect bacterial cells by centrifuging at 8000–9000 rpm for 10 min at 4℃, resuspend in 50 mM Tris-HCl buffer (pH 9.0, containing 300 mM NaCl), disrupt cells by sonication, centrifuge at 12000 rpm for 20 min, and take the supernatant as crude enzyme solution.

[0052] 3. Purification

[0053] The supernatant was filtered through a 0.22 μm filter and loaded onto a Ni-NTA affinity chromatography column. Elution was performed using a gradient of elution buffers containing 20–300 mM imidazole (50 mM Tris-HCl, 300 mM NaCl), and the elution peak containing the target protein was collected. The solution was concentrated via ultrafiltration centrifuge tubes and dialyzed into 50 mM Tris-HCl (pH 9.0) buffer, then stored at 4°C for later use.

[0054] (2) Comparison of enzyme activity between wild-type enzyme and mutant.

[0055] To verify the differences between wild-type PLA depolymerase and PLA depolymerase mutants, this example initially used commercial substrates to determine changes in the depolymerization ability of the mutants.

[0056] The reaction system using PLA powder as a substrate (taking 1 mL as an example) includes: 50 mM Tris-HCl buffer, pH 9.0; PLA powder (3260HP PLA, NatureWorks, Mw approximately 80,000) 10 g / L; purified enzyme protein, with an enzyme addition amount of 3 mg·g⁻¹ PLA, and the lactate monomer concentration was determined after shaking in a metal bath at 60℃ for 48 h.

[0057] Figure 1 This study compares the depolymerization ability of wild-type enzyme (SP-01, hereinafter referred to as WT) and three representative mutants X2, X3, and X4 in the above reaction system. It can be seen that mutants X2, X3, and X4 all showed higher concentrations of lactic acid monomers than WT in depolymerization. Among them, mutant X3 exhibited the highest activity, showing a significant improvement over WT, indicating that the mutant combination has a significant positive contribution to catalytic efficiency.

[0058] (3) Comparison of the thermal stability of wild-type enzymes and mutants.

[0059] The method for testing the thermal stability of PLA depolymerase is as follows:

[0060] The thermostability of the enzyme was assessed using the soluble substrate p-nitrophenol octanoate (p-NPO). First, enzyme samples were pre-incubated at 70 °C for different time gradients (e.g., 0, 5, 10, 20, 30, 40, 60 min). An equal volume of sample was taken at each time point and immediately transferred to an ice bath for cooling. Subsequently, the residual specific activity was measured under standard reaction conditions (e.g., 50 mM Tris-HCl, pH 9.0, appropriate concentration of p-NPO, 40 °C, 5 min). The activity of the unheated sample (0 min) was recorded as 100%, and the relative activity at each time point was calculated. The relative activity-time data were imported into software such as GraphPad Prism, and a one-phase decay model was used for fitting to obtain the time corresponding to the residual activity decaying to 50%. This time (the time corresponding to the enzyme incubating at 70 °C until the residual activity decays to 50%) was defined as... .

[0061] Wild-type enzymes and mutants like Figure 2 As shown in the figure, this figure illustrates the residual activity of WT and the two mutants after heat treatment at 70°C. In contrast, mutant X3 exhibits the weakest thermal stability, while mutant X2 demonstrates the strongest heat resistance, maintaining higher enzyme activity at high temperatures, indicating that this mutant combination also significantly enhances the thermal stability of the wild-type protein.

[0062] Example 3: PLA depolymerization ability test of wild-type and mutant PLA depolymerases

[0063] This embodiment uses PLA pipettes (Kingfa Science & Technology, G200 biodegradable pipettes) as substrates to compare the PLA depolymerization ability of wild-type protease SP-01 (WT) and mutant X4. The test results, considering both PLA depolymerization activity and thermal stability, show that mutant X4 maintains high PLA depolymerization efficiency while also exhibiting good heat resistance, demonstrating more balanced and stable overall performance. This performance characteristic makes it more advantageous for applications under high temperature, long duration, and high solids content PLA substrate conditions, especially suitable for industrial scale-up or continuous reaction applications where enzyme stability is critical. Therefore, mutant X4 is preferred as the optimal mutant for pipette substrate depolymerization testing and scale-up application verification.

[0064] The degradation reaction system for the pipette substrate was as follows: Based on the PLA powder reaction system described in Example 2, the substrate was replaced with PLA pipettes. The pipettes were mechanically pulverized and then added to the reaction system. The substrate concentration was 10 g / L (1 mL system) or 100 g / L (1 L fermenter scale-up reaction). The reaction system used 50 mM Tris-HCl buffer (pH 9.0) and purified mutant X4 enzyme protein was added, with the enzyme addition amount remaining consistent with Example 2. The reaction was carried out at 60 °C for 48 h. To alleviate the acidification of the system caused by lactic acid generation during depolymerization, an excess of saturated calcium carbonate powder was added to the reaction system as a neutralizing agent to maintain the relative stability of the system pH during the reaction.

[0065] The product was determined by reacting the mixture in a metal bath at 60℃ for 48 h with shaking. After the reaction was complete, the mixture was centrifuged at 12000 r / min for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane. The concentration of lactic acid monomer was determined by HPLC.

[0066] The HPLC conditions can be referenced as follows:

[0067] Instrument: Agilent 1260 Series; Column: C18 reversed-phase column (4.6 mm × 250 mm, 5 μm); Mobile phase: methanol: 0.02 M KH2PO4 (pH 2.1, adjusted with phosphoric acid) = 5: 95 (v / v); Column temperature: 30℃; Flow rate: 0.5 mL / min; Detection wavelength: 210 nm; Injection volume: 10 μL.

[0068] By plotting a lactate standard curve, the amount of lactate produced in each sample was calculated to characterize the depolymerization activity of wild-type enzyme and PLA in each mutant.

[0069] Figure 3 The results of PLA depolymerization tests for the optimal mutant X4, using PLA pipettes as substrates, are presented. The experiment was conducted in a small system with a total reaction volume of 1 mL. Figure 3 The right side of the image shows the degradation effect of commercial proteinase K. It can be clearly seen that mutant X4 has a significantly higher depolymerization ability on PLA pipettes than WT and proteinase K.

[0070] Figure 4 This study demonstrates the depolymerization capability of the optimal mutant X4 for actual PLA products under scaled-up reaction conditions. The experiment was conducted in a bioreactor system with a total reaction volume of 1 L and a substrate concentration increased to 100 g / L to evaluate the catalytic performance and stability of mutant X4 under near-industrial application conditions. It can be seen that the lactic acid production continuously increases with increasing reaction time, ultimately reaching a depolymerization rate of approximately 90%. Compared to the results of the chemical alkaline method (4 M NaOH, 100℃, 3 h), enzymatic depolymerization achieves comparable depolymerization efficiency under milder conditions, further demonstrating the advantages of X4 in the treatment and large-scale application of real-world waste PLA.

Claims

1. A PLA depolymerase mutant, characterized in that, The amino acid sequence of the PLA depolymerase is shown in SEQ ID NO: 1; the mutation site of the mutant includes at least one of the amino acid residues at position 144 and position 183; Among them, the 144th amino acid residue is mutated to glutamic acid; The 183rd amino acid residue is mutated to alanine.

2. The mutant according to claim 1, characterized in that, The mutation site of the mutant also includes the 66th amino acid residue; the 66th amino acid residue is mutated to histidine.

3. The mutant according to claim 1, characterized in that, The mutation sites of the mutant also include amino acid residues at positions 109 and 164; Among them, the 109th amino acid residue is mutated to phenylalanine; The 164th amino acid residue is mutated to lysine.

4. A gene encoding the PLA depolymerase mutant according to any one of claims 1 to 3.

5. A recombinant vector comprising the gene of claim 4.

6. A recombinant strain comprising the gene of claim 4.

7. The application of the PLA depolymerase and its mutants according to any one of claims 1 to 3 in PLA depolymerization.

8. The application according to claim 7, characterized in that, The PLA depolymerase or mutant is added to a reaction system containing PLA substrate to depolymerize PLA.

9. The application according to claim 7, characterized in that, The PLA depolymerase and its mutants depolymerize PLA under alkaline conditions at 50-70℃.

10. The application according to claim 8, characterized in that, In the PLA depolymerization system, the amount of pure PLA depolymerase and its mutant added is 2–5‰ of the substrate mass.

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