PLA-degrading lipase and application thereof

Abstract

This invention discloses a PLA-degrading lipase and its applications. Firstly, using lipase PaPlaA as a template, the ancestral enzyme ASR190 was constructed through ancestral sequence reconstruction, and its amino acid sequence is shown in SEQ ID NO. 1. Based on the obtained ancestral enzyme ASR190, this invention further obtained lipase N20-ASR190 by truncating its unstable N-terminal region and performing rational mutations through domain analysis. Experimental results show that the ancestral enzyme ASR190 has advantages such as high catalytic efficiency and good thermal stability. Compared with ancestral enzyme ASR190, lipase N20-ASR190 exhibits further enhanced enzyme activity while maintaining excellent thermal stability. The application of ancestral enzyme ASR190 in the biodegradation of polylactic acid demonstrates highly efficient degradation ability of PLA nonwoven fabrics. When combined with protease nprT612 in a two-step, staged process, the degradation rate can be further improved, showing application potential in industrial composting, high-temperature continuous reaction, and other scenarios.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and specifically relates to a PLA-degrading lipase and its applications. Background Technology

[0002] Polylactic acid (PLA) is a class of biodegradable polyester materials made from renewable resources (such as corn starch and lactic acid fermentation products). It possesses good mechanical properties and biocompatibility, and is widely used in packaging materials, disposable products, agricultural films, and biomedical materials. Although PLA is classified as a "biodegradable plastic," its actual degradation process is highly dependent on environmental conditions, typically requiring relatively high temperatures (≥58℃) and high humidity to significantly break the ester bonds. In natural environments such as soil and oceans, the degradation process of PLA is very slow, leading to its accumulation in the environment and even exhibiting similar durability to traditional plastics in some applications. Therefore, improving the actual biodegradability of PLA is a significant challenge in the field of biodegradable materials.

[0003] Currently, the main pathways for achieving efficient PLA degradation include chemical hydrolysis, thermal treatment, photocatalysis, and enzymatic degradation. Among these, enzymatic degradation is considered the most promising strategy for industrial application due to its mild conditions, high selectivity, and lack of secondary pollution. Several enzymes, such as proteases, cutinases, lipases, and esterases, have been proven to catalyze chain scission reactions in polyester polymers. However, these natural enzymes generally suffer from insufficient thermal stability and rapid inactivation above 50°C, limiting their application in practical conditions such as industrial composting, high-temperature and humid heat treatment, and hot-pressing pre-reaction.

[0004] While classic studies on PLA degradation have primarily focused on keratinases and some polyester hydrolases, recent research has shown that some lipases also possess the potential for polyester chain scission. However, lipases have mainly evolved to work with small molecule ester substrates, and their narrow active sites and hydrophobic substrate channels make it difficult for high-molecular-weight PLA segments to effectively enter the catalytic site. Furthermore, lipases generally have limited thermostability, with most exhibiting structural loosening and activity decay at 50-60℃. Currently, there is a lack of systematic research in the published literature on the use of lipases for PLA degradation; their structural basis, substrate recognition patterns, and actual degradation efficiencies remain unclear, which to some extent restricts the engineering development of lipases as potential PLA degradation tools. To improve enzyme stability and catalytic performance, various enzyme engineering strategies have emerged in recent years, including site-directed mutagenesis, semi-rational design, computer-aided design, and high-throughput screening. However, these strategies all rely on the structural framework of the modern sequence itself, and due to the inherent folding stability limitations, it is usually difficult to achieve a significant leap in stability.

[0005] Ancestral sequence reconstruction (ASR) technology infers the amino acid sequences of ancient enzymes through phylogenetic analysis, resulting in reconstructed enzymes that generally possess higher structural compactness, a more stable hydrophobic core, and enhanced folding energy, thus offering significant advantages in the development of thermostable enzymes. Previous studies have demonstrated that ASR technology can significantly improve the stability and engineering potential of many industrial enzymes, such as esterases, peptidases, nitrile hydrolases, and xylanases. However, the development of highly efficient PLA-degrading lipases based on ASR is still rarely reported. Therefore, there is an urgent need to construct a novel PLA-degrading enzyme that combines high thermal stability with high catalytic efficiency to overcome the temperature limitations and insufficient activity of traditional PLA-degrading lipases, providing an effective biocatalytic material for the rapid degradation of PLA under high-temperature environments. Summary of the Invention

[0006] To address the technical problems of insufficient thermostability, weak substrate recognition ability, and limited degradation efficiency in high-temperature industrial composting environments of existing PLA-degrading enzymes, the present invention aims to provide a PLA-degrading lipase and its application. Through ancestral sequence reconstruction and structural optimization strategies, a novel lipase was constructed, specifically the ancestral enzyme ASR190 and its N-terminal truncated derivative lipase N20-ASR190, which achieved efficient biodegradation of PLA.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of the present invention provides a PLA-degrading lipase, wherein the PLA-degrading lipase is the ancestral enzyme ASR190, and its amino acid sequence is shown in SEQ ID NO.1.

[0008] Preferably, the ASR190 is obtained by... Paenibacillus amylolyticus The ancestral sequence of the derived lipase PaPlaA was reconstructed.

[0009] A second aspect of the present invention provides a PLA-degrading lipase, wherein the polylactic acid-degrading lipase is a lipase N20-ASR190 derived from the N-terminal truncated mutation of the aforementioned ancestral lipase ASR190, and its amino acid sequence is shown in SEQ ID NO. 2.

[0010] A third aspect of the present invention provides the application of the aforementioned ancestral enzyme ASR190 or lipase N20-ASR190 in PLA degradation.

[0011] Preferably, the PLA degradation specifically refers to the degradation of PLA nonwoven fabric, and the conditions for the degradation of PLA nonwoven fabric by the ancestral enzyme ASR190 are: total enzyme activity of 200-500 U, pH of the reaction system of 8.6-9.0, and temperature of 90-95℃.

[0012] A fourth aspect of the present invention provides a complex enzyme preparation composed of the aforementioned ancestral enzyme ASR190 and protease nprT612.

[0013] The fifth aspect of the present invention provides the application of the above-mentioned complex enzyme preparation in PLA degradation.

[0014] Preferably, the PLA degradation specifically refers to the degradation of PLA nonwoven fabric. The PLA nonwoven fabric is degraded using a two-step, staged method with a compound enzyme preparation. The degradation conditions are: total enzyme activity of 200-500U and pH of 8.6-9.0.

[0015] Preferably, the degradation of PLA nonwoven fabric using the above-mentioned compound enzyme preparation is carried out in a two-step, staged manner, including the following steps: a) First stage: React with protease nprT612 at 40-50℃ for 4-6 hours; b) Second stage: The reaction is carried out at 90-95℃ using the ancestral enzyme ASR190 for 4-6 hours.

[0016] Beneficial effects (1) The present invention firstly uses Paenibacillus amylolyticus Using the derived lipase PaPlaA as a template, its ancestral sequence was deduced through phylogenetic tree construction and maximum likelihood method. Then, the ancestral sequence was reconstructed to construct a polylactic acid-degrading lipase, specifically the ancestral enzyme ASR190, whose amino acid sequence is shown in SEQ ID NO. 1. This ancestral enzyme ASR190 possesses a more compact folded structure and a stable hydrophobic core, thus significantly improving its overall thermal stability. Compared with the wild-type enzyme, this ancestral enzyme ASR190 exhibits advantages such as high catalytic efficiency and good thermal stability. Experimental results show that the enzyme activity of the ancestral enzyme ASR190 is 3.0 times that of wild-type PaPlaA. Compared to wild-type PaPlaA (16.6 min), its half-life at 50℃ is significantly extended to 255 min, and it maintains highly efficient catalytic activity even under sustained high-temperature environments.

[0017] Building upon the ancestral enzyme ASR190, this invention further truncated its unstable N-terminal region through domain analysis and rational mutation, yielding the lipase N20-ASR190. This truncated enzyme molecule exhibits a more compact fold and increased substrate channel openness, resulting in higher catalytic efficiency. Experimental results show that compared to the ancestral enzyme ASR190, the lipase N20-ASR190 demonstrates approximately 2.3 times higher enzyme activity while maintaining excellent thermal stability. This effectively overcomes the problem of traditional polylactic acid degrading enzymes failing to balance catalytic activity and stability at high temperatures.

[0018] (3) The ancestral enzyme ASR190 was applied to the biodegradation of polylactic acid. The experimental results showed that the ancestral enzyme ASR190 has a high degradation ability for PLA nonwoven fabric. The degradation rate of the single enzyme can reach 21.2% in 9 hours. When it is combined with the protease nprT612 in a two-step process, the degradation rate can be further increased to 84.2%, showing its application potential in industrial composting, high-temperature continuous reaction and other scenarios.

[0019] This invention, through ancestor sequence reconstruction and structural optimization strategies, successfully overcomes the technical limitations of insufficient heat resistance and low polymer substrate recognition ability of natural lipases, providing a new enzyme preparation solution for the efficient and green degradation of PLA-type biodegradable plastics. It has both significant theoretical innovation and promising prospects for practical industrial application. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The following are: (a) the constructed phylogenetic tree of PLA degrading enzymes; (b) the multiple sequence alignment results of the PaPlaA ancestral enzyme; and (c) the three-dimensional protein structure alignment results. Figure 2 SDS-PAGE detection of protein expression of ancestral enzyme ASR190 and lipase N20-ASR190; Lane M: protein marker, Lane 1: ASR136 (25.2 kDa), Lane 2: N20-ASR190 (19.4 kDa), Lane 3: ASR190 (21.7 kDa). Figure 3 Comparison of enzyme activities of ancestral enzyme ASR19 and wild-type PaPlaA (a) and residual enzyme activity curve at 50℃ (b). Figure 4 The results show the comparison of relative enzyme activity and thermostability of lipase N20-ASR190, which is derived from wild-type PaPlaA and the ancestral enzyme ASR190 by N-terminal truncated derivation mutation. Figure 5 The results of three-dimensional structural analysis of lipase N20-ASR190; Figure 6 The effects of (a) pH, (b) temperature, (c) enzyme dosage, and (d) reaction time on the degradation of PLA nonwoven fabric by the ancestral enzyme ASR190; and (e) the effect of the combination of ancestral enzyme ASR190 / nprT612 on the degradation of PLA nonwoven fabric. Detailed Implementation

[0022] In the following description, specific details such as particular system architectures and techniques are set forth for illustrative purposes and not for limitation, in order to provide a thorough understanding of the embodiments of the invention. However, those skilled in the art will understand that the invention may be implemented in other embodiments without these specific details.

[0023] Example 1 1. Experimental Methods 1.1 Evolutionary Landscape Construction and Ancestral Sequence Reconstruction Strains with high polylactic acid (PLA) degradation capabilities were selected as cluster centers. The 132 sequences used for constructing the evolutionary landscape of PaPlaA lipase were obtained from the NCBI database (https: / / www.ncbi.nlm.nih.gov / ) and the Uniprot database (https: / / www.uniprot.org / ). Multiple sequence alignment of the above PLA degrading enzyme amino acid sequences was performed using MEGA-X. Subsequently, based on the Maximum Likelihood algorithm and the JTT matrix model, the evolutionary relationships among these sequences were analyzed, and a phylogenetic tree was constructed to describe the evolutionary landscape of the PLA degrading enzyme. The obtained phylogenetic tree file was enhanced using the iTOL online tree visualization tool.

[0024] Based on the above multiple sequence alignment results and phylogenetic tree, ancestral sequences of PLA degrading enzymes were reconstructed using PAML 4.9j and EasyCodeML software packages. Key evolutionary nodes were reconstructed to obtain the amino acid sequences of the ancestral enzymes. Then, based on the *E. coli* codon optimization table, the gene sequences of these ancestral lipases were obtained for subsequent gene expression. Through ancestral sequence reconstruction, candidate ancestral enzyme gene sequences were successfully obtained, named PaPlaA ancestral enzyme ASR190, and its inferred amino acid sequence is shown in SEQ ID NO.1. Comparative analysis showed that PaPlaA ancestral enzyme ASR190 shared approximately 75.62% homology with the wild-type (template enzyme) PaPlaA, but conserved substitutions were found at key positions in several domains.

[0025] 1.2 Methods for determining the enzyme activity and half-life of lipases (a) Determination of lipase activity p-Nitrophenol octanoate (pNPO) is a synthetically produced lipase substrate. PaPlaA, an enzyme that degrades pNPO, catalyzes its hydrolysis to release p-nitrophenol (pNP), which, under alkaline conditions (pH > 10), exists as phenolate ions (p-NP). -It exists in a stable yellow form and can be measured colorimetrically at a wavelength of 405 nm. The sodium carbonate solution has a pH of approximately 11.0, which rapidly raises the pH of the reaction system, preventing lipase from further hydrolyzing the substrate (pNPO) and simultaneously stabilizing the generated pNP. Therefore, lipase activity can be calculated by monitoring the rate of pNP formation. One unit of enzyme activity (1 U) is defined as the amount of enzyme that produces 1 µmol of p-nitrophenol per minute under certain conditions.

[0026] The specific steps are as follows: Take an appropriate amount of crude PLA degrading enzyme solution, dilute it, and set aside. Add 850 μL of Tris-HCl buffer and 50 μL of pNPO substrate sequentially to a 1.5 mL centrifuge tube. Preheat in a metal bath at 40℃ and 1500 rpm for 5 min. Then add 100 μL of enzyme solution to form a 1 mL system and react in a metal bath at 40℃ and 1500 rpm for 5 min. Immediately after the reaction is complete, add 200 μL of 0.1 M Na2CO3 solution to terminate the reaction, and then dilute at 12000 × 10⁻⁶. g Centrifuge for 1 min, then detect OD using an ELISA reader. 405 nm .

[0027] (b) Using half-life to characterize the thermal stability of enzymes The specific steps are as follows: The crude PLA degrading enzyme solution is incubated at the temperature required to determine the half-life, and samples are taken at fixed time intervals to measure residual enzyme activity. The half-life of the PLA degrading enzyme is defined as the time required for 50% reduction of initial enzyme activity. The half-life at this temperature is calculated using a first-order inactivation function. All measurements are repeated three times.

[0028] 1.3 Construction of heterologous expression system for PLA degrading enzyme Using PCR molecular biology techniques, to carry PLA-degrading enzymes PaPlaA Using the plasmid of the ancestral gene as a template, the ancestral enzyme gene fragment was cloned and amplified using pre-designed primers ASR190-F (5'-GGATCCATGAAAAAACTGAAACTG-3') and ASR190-R (5'-TTATTCAATCAGCGCGGTTTTAATC-3'). Six histidine sequences (-CACCACCACCACCACCACCAC-) were added to the end of the sequence to facilitate subsequent enzyme isolation and purification.

[0029] Using the pET-28a(+) empty plasmid preserved in our laboratory as a template, primers were designed to amplify the linearized vector in reverse. The PCR products after the reaction were verified by agarose gel electrophoresis. Bands of the correct length were excised, recovered using a kit method, and the DNA concentration was determined. The linearized vector was then subjected to… DpnI. After digestion of the enzyme template, the plasmid is cyclized with the target fragment by the homologous recombinase Exnase II, purified, and then transformed into... E. coli JM109 competent cells were plated on solid LB agar plates and cultured at 37°C until transformants appeared. Using colonies as templates, colony PCR was performed using verification primers to verify the transformation of the recombinant plasmid. PCR products with correct bands were further sequenced for verification. DNAMAN 8.0 software was used for comparative analysis of the sequencing results, and the recombinant plasmid was finally extracted. Subsequently, using E. coli BL21(DE3) as the expression host, the successfully constructed recombinant plasmid was transformed into competent cells via heat shock. The transformed strains were first pre-cultured in 10 mL of LB medium containing the corresponding antibiotic at 37°C and 220 rpm for 8–10 h on a shaker. The culture was then expanded to 50 mL of TB medium containing the corresponding antibiotic at a 2% inoculum and cultured under the same conditions until OD (October Expiratory Time). 600 Values ​​were 0.4-0.8 (logarithmic growth phase), with the addition of isopropyl- at a final concentration of 0.5 mM. β Induction was performed using β-D-thiogalactoside (IPTG). After culturing at 30℃ and 220 rpm for 24 h, the bacterial cells were collected by centrifugation to construct the lipase recombinant expression plasmid Pa-ASR190, which was then stored at 4℃.

[0030] 1.4 Construction and transformation of recombinant plasmids containing lipase mutants Three-dimensional structural analysis of ASR190 revealed a flexible, random coiled structure of approximately 30 amino acids in its N-terminal region. This region may affect the enzyme's structural stability and catalytic performance. To further optimize enzymatic performance, the flexible N-terminal region was truncated, successfully constructing a mutant plasmid with a 20-amino acid truncated N-terminus. This mutant, named N20-ASR190, is a derivative enzyme obtained by deleting amino acid residues 2-21 from the N-terminus of the ASR190 amino acid sequence. Its amino acid sequence is shown in SEQ ID NO.2.

[0031] The specific construction method is as follows: Using the successfully constructed lipase recombinant expression plasmid Pa-ASR190 as a template, one-step reverse PCR amplification of the whole plasmid was performed using pre-designed primers N20ASR190-F (5'-CGCGGATCCATGGGCATTGGCCAGAGCACCAAAG-3') and N20ASR190-R (5'-CTGGCCAATGCCCATGGATCCGCGACCCATTTGCTG-3'), with the first 20 amino acids of the lipase DNA coding sequence being directionally truncated. After PCR, the size of the amplified product was verified by nucleic acid gel electrophoresis, and the target band was excised and purified. The purified product was then used... DpnThe enzyme I was used for digestion to remove the methylated template plasmid. After the digestion reaction was completed, the reaction system was cooled on ice, and 10 μL of the digestion product was transformed into the cloning host Escherichia coli JM109. After the transformants grew, bacteria were selected for sequencing verification. After successful verification, the plasmid was extracted and transformed into Escherichia coli BL21(DE3) for recombinant expression of the mutant lipase.

[0032] 1.5 SDS-PAGE analysis of lipase A suitable amount of recombinant *E. coli* fermentation broth produced by enzyme production for 24 h was collected and centrifuged at 6000 × g for 10 min at 4 °C. The cell pellet was washed and resuspended with PBS buffer (100 mM, pH 7.2), and cell lysis was promoted by sonication. Sonication was performed at 4 °C for 5 min (10 s on / off cycle) until the solution was clear, avoiding protein inactivation during the process. Subsequently, the cell lysate was separated by centrifugation at 12000 × g for 1 min at 4 °C. The cell lysate supernatant and lysate were collected, and 40 μL of the sample was added to 5× protein gel loading buffer and heated at 100 °C for 10 min to ensure complete protein denaturation. Finally, the heated protein gel sample was analyzed by SDS-PAGE, and after Coomassie brilliant blue staining and destaining, the protein bands were observed.

[0033] 1.6 Three-dimensional structural analysis AlphaFold2, a high-precision protein structure prediction tool, has achieved a revolutionary breakthrough in the field of protein engineering. This toolkit, deployed and installed on a cloud computing server, could be directly used for the 3D structure prediction of the initial PLA-degrading enzyme in this experiment. The predicted 3D structures were evaluated using the SAVES v6.1 online server (https: / / saves.mbi.ucla.edu / ). Ramachandran diagram analysis (PROCHECK) was used to assess the dihedral angles of the main chain, visually determining whether the spatial arrangement of amino acid residues in the model conformed to the geometric characteristics of natural proteins; more than 90% of the residues should fall within the dominant region. The overall quality factor of the protein was analyzed using the crystallographic evaluation tool ERRAT; a score exceeding 90 indicated a reasonable model. In subsequent studies, the structure prediction of various PLA-degrading enzyme mutants can be modeled using SWISS-MODEL (https: / / swissmodel.expasy.org / ) based on the 3D structure of the template protein. The spatial conformation, active pocket size, and hydrophobicity of its catalytic triplet were visualized and analyzed using PyMOL software (https: / / pymol.org / 2 / ), thereby assessing its potential for binding to PLA substrates at the structural level.

[0034] The substrate was molecularly docked with the PLA degrading enzyme ASR190. The SDF file of the substrate compound structure was obtained from the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ) and converted to PDB format using Open Babel software. AutoDock v4.2.6 was used for docking, and the Lamarckian genetic algorithm was used as the search engine. During molecular docking, the substrate was used as the ligand and configured as flexible, while the PLA degrading enzyme protein was used as the acceptor and configured as rigid. The resulting enzyme-substrate complex structure will be used in subsequent studies.

[0035] 2. Experimental Results Based on the systematic collection and multiple sequence alignment of PaPlaA and its homologous lipase sequences, a corresponding phylogenetic tree was constructed, and probabilistic inferences were made about the ancestral sequences of key evolutionary nodes (e.g., Figure 1 Based on sequence conservation analysis and structural feasibility assessment, an ancestral sequence with high posterior probability (ASR190) was selected for subsequent experimental validation. This ancestral PaPlaA variant maintains high sequence similarity to wild-type PaPlaA, but exhibits conserved amino acid substitution characteristics at several dispersed sites that differ from the modern sequence. These differential sites are mainly distributed within secondary structures and structural connection regions, rather than directly around the catalytic triplet, suggesting that they are more likely to affect overall conformational stability than the catalytic mechanism itself.

[0036] Depend on Figure 2 The results showed that the soluble recombinant protein ASR190 with a molecular weight of approximately 21.6 kDa (its amino acid sequence is shown in SEQ ID NO. 1) was successfully expressed in Escherichia coli by SDS-PAGE analysis.

[0037] Depend on Figure 3 The results showed that the crude enzyme solution of the ancestral enzyme ASR190 was obtained by determining its enzyme activity and stability using method 1.2. Its enzyme activity reached 7.45 U / mL, which is 3.0 times that of wild-type PaPlaA (2.50 U / mL). In the thermostability test at 50℃, its half-life was extended to 255 min, which is more than 15 times that of wild-type PaPlaA (half-life of 16 min).

[0038] Depend on Figure 4The results showed that after purification, the crude enzyme solution of the derived lipase N20-ASR190 was tested for enzyme activity and stability using method 1.2. The enzyme activity reached 17.14 U / mL, which is 6.8 times that of wild-type PaPlaA (2.50 U / mL). In the thermostability test at 50℃, its half-life was extended to 557 min, which is more than 33 times longer than that of wild-type PaPlaA (16 min).

[0039] Depend on Figure 5 The results showed that lipase N20-ASR190 retained the typical folded structure of an α / β hydrolase, with a stable spatial framework composed of a central β-sheet and an outer α-helix. The catalytic triplet was located in the conserved active pocket region. Compared to the original enzyme, the N-terminal truncation did not disrupt the overall spatial conformation of the enzyme, and the structure of the active center region remained stable. Simultaneously, the removal of the flexible random coil region at the N-terminus made the enzyme surface structure more compact, helping to reduce structural redundancy and improve overall structural stability. The spatial configuration of the substrate binding pocket and channel regions did not show significant collapse, still providing binding and entry channels for hydrophobic polyester substrates such as PLA.

[0040] 3. Application of lipase in PLA degradation and optimization of degradation conditions PLA nonwoven fabrics were pretreated as follows: They were ultrasonically cleaned with deionized water for 20 min to remove surface impurities, dried in a 60℃ oven, and several 50 mg portions of the PLA sample were accurately weighed for later use. During the degradation reaction, the PLA sample was added to 50 mL of diluted enzyme solution containing different total enzyme activities, and the mixture was kept in a water bath with magnetic stirring. After the reaction, the system was treated at 105℃ for 10 min to completely inactivate the enzyme, followed by centrifugation (8,000 r / min, 10 min). Finally, the supernatant was subjected to acid-base titration analysis. The degraded PLA residue was washed with water, dried, and weighed.

[0041] First, the degradation conditions of the ancestral enzyme ASR190 were investigated: 50 mg of PLA sample was added to 50 mL of diluted enzyme solution containing different total enzyme activities, and the mixture was incubated in a water bath with magnetic stirring. The effects of pH (7-10.0), temperature (30-90℃), total enzyme activity (100-1000 U), and reaction time (3-12 h) on the degradation rate were investigated. The results are shown in [Figure number missing]. Figure 6 (ad).

[0042] Depend on Figure 6(ad) The results showed that the optimal degradation conditions for the ancestral enzyme ASR190 were: pH 8.6-9.0, temperature 90-95℃, total enzyme activity 200-500 U, and reaction time 9h. Under the above degradation conditions, the ancestral enzyme ASR190 showed a high efficiency in degrading PLA nonwoven fabrics, with a degradation rate of up to 21.2%.

[0043] To further improve the degradation rate, this invention also explored the effect of a composite enzyme preparation obtained by combining the aforementioned ancestral enzyme ASR190 with other enzymes on the degradation rate of PLA nonwoven fabric. Here, the ancestral enzyme ASR190 was combined with the protease nprT612, and a two-step, staged degradation method was used to degrade the PLA nonwoven fabric, including: First stage: Pretreatment with protease nprT612 for 4-6 hours at 40-50℃ and pH 8.6-9.0; (b) Without changing the system, heat to 90-95℃ and add the ancestral enzyme ASR190 (total enzyme activity 200-500 U), and continue the reaction at the same temperature for 4-6 h. Results are shown below. Figure 6 .

[0044] Depend on Figure 6 (e) The results show that the degradation rate of PLA nonwoven fabric can be significantly improved by using a two-step compounding method, up to 84.2%. The above results demonstrate the superiority of the two-step treatment strategy of the compound enzyme preparation.

[0045] This invention is not limited to the specific embodiments described above. Any modifications made by those skilled in the art based on the above concept without creative effort are within the scope of protection of this invention.

Claims

1. A PLA-degrading lipase, characterized in that, The PLA-degrading lipase is either the ancestral enzyme ASR190 or the lipase N20-ASR190, the amino acid sequence of which is shown in SEQ ID NO.1; and the amino acid sequence of which is shown in SEQ ID NO.

2.

2. The PLA-degrading lipase according to claim 1, characterized in that, The ASR190, through... Paenibacillus amylolyticus The ancestral sequence of the derived lipase PaPlaA was reconstructed.

3. The PLA-degrading lipase according to claim 1, characterized in that, The lipase N20-ASR190 was derived by truncating the N-terminal flexible region of the ancestral enzyme ASR190.

4. A compound enzyme preparation, characterized in that, It is composed of the ancestral enzyme ASR190 as described in claim 1 and the protease nprT612.

5. The application of the PLA-degrading lipase as described in claim 1 in PLA degradation, characterized in that, The PLA-degrading lipase is the ancestral enzyme ASR190.

6. The application according to claim 5, characterized in that, The PLA degradation specifically refers to the degradation of PLA nonwoven fabric. The conditions for the degradation of PLA nonwoven fabric by the ancestral enzyme ASR190 are: total enzyme activity of 200-500 U, pH of the reaction system of 8.6-9.0, and temperature of 90-95℃.

7. The application of the compound enzyme preparation as described in claim 4 in PLA degradation.

8. The application according to claim 7, characterized in that, The PLA degradation specifically refers to the degradation of PLA nonwoven fabric. The degradation of PLA nonwoven fabric is carried out in a two-step, staged manner using a compound enzyme preparation. The degradation conditions are: total enzyme activity of 200-500 U and pH of the reaction system of 8.6-9.

0.

9. The application according to claim 8, characterized in that, The degradation of PLA nonwoven fabric using a compound enzyme preparation is carried out in a two-step, staged manner, including the following steps: a) First stage: React with protease nprT612 for 4-6 hours at 40-50℃; b) Second stage: React at 90-95℃ using the ancestral enzyme ASR190 for 4-6 hours.

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