A high-efficiency plastic-degrading enzyme screening system based on metagenomic mining and yeast surface display and application thereof

Abstract

The application discloses a high-efficiency plastic-degrading enzyme screening system based on metagenome mining and yeast surface display and application thereof, relates to the field of synthetic biology and biological catalysis technology, and can realize the following effects: screening and excluding false positives through a PET microplastic substrate, realizing single-cell enzyme activity visualization through a microfluidic droplet, and coupling high-specificity screening and high-throughput detection in series in combination with flow high-throughput sorting, so that the target PET-degrading enzyme can be rapidly and efficiently enriched under the premise of ensuring screening accuracy, the technical problem that high substrate specificity and high throughput are difficult to be considered in the existing PET-degrading enzyme screening is solved, the screening efficiency and practicability of the PET-degrading enzyme are greatly improved, and the application has important significance for breaking through the technical bottleneck of PET biological recycling and realizing plastic closed-loop circulation.

Description

Technical Field

[0001] This invention relates to the fields of synthetic biology and biocatalysis, specifically to a highly efficient screening system for plastic-degrading enzymes based on metagenomic mining and yeast surface display, and its applications. Background Technology

[0002] With global annual plastic production exceeding 400 million tons, polyethylene terephthalate (PET) is widely used in beverage bottles, fibers, and films due to its excellent mechanical and barrier properties. However, waste PET is extremely difficult to degrade naturally in the environment, and microplastics have been widely detected in water, soil, and the atmosphere, becoming a focal issue threatening ecological security and human health. Existing plastic-degrading enzymes (such as PETase and cutinase) suffer from low activity, poor thermal stability, and difficulty in efficient expression. Traditional screening methods rely on culturable microorganisms, resulting in low throughput, long cycles, and an inability to cover potentially high-activity enzyme resources in difficult-to-culture bacterial communities. Furthermore, E. coli expression systems often lead to the formation of inclusion bodies in plastic-degrading enzymes, while eukaryotic modifications (such as disulfide bonds) are crucial for enzyme activity. Currently reported PET hydrolases are mainly derived from culturable strains such as Ideonella sakaiensis and Thermobifida fusca. However, their catalytic efficiency, thermal stability, and activity against high-crystallinity PET (hcPET) still cannot meet industrial demands. In addition, existing directed evolution or metagenomic screening methods mostly rely on 96-well plate culturable libraries, which have low throughput and insufficient coverage, making it difficult to discover potential high-activity enzyme resources in difficult-to-culture microorganisms.

[0003] On the other hand, in the fields of enzyme engineering, biocatalysis, and directed evolution, the synergistic optimization of screening throughput and substrate specificity has long been a technical bottleneck that is difficult to balance. Conventional screening often adopts a model of high-throughput coarse screening followed by low-throughput validation. First, a large library is rapidly enriched using alternative substrates and high-throughput sorting systems, and then candidate clones are validated one by one using real substrates. However, this model has obvious drawbacks. The fluorescent substrates or simulated substrates used in coarse screening differ significantly from real solid-state PET in terms of structure, state, and reaction characteristics, which easily leads to a large number of false positives. The subsequent low-throughput validation is labor-intensive, time-consuming, and inefficient. Moreover, some dominant enzymes with high activity to real substrates may be missed in the coarse screening stage due to incompatibility with alternative substrates, making it impossible to guarantee the reliability of screening results from the source. Ultimately, it is difficult to truly synergize screening throughput and substrate specificity, which restricts the efficient acquisition of high-activity and high-specificity enzymes. Furthermore, real solid-state substrates and high-throughput fluorescent detection systems are inherently incompatible in terms of substrate morphology, reaction system, and detection principle, resulting in a long-standing technical bottleneck that makes it difficult to balance screening throughput and substrate specificity. Summary of the Invention

[0004] This invention provides a highly efficient screening system for plastic-degrading enzymes based on metagenomic mining and yeast surface display, and its application. False positives are eliminated through screening with PET microplastic substrates, and single-cell enzyme activity is visualized using microfluidic droplets. Combined with high-throughput flow cytometry sorting, this system tandemly couples high-specificity screening with high-throughput detection, achieving rapid and efficient enrichment of target PET-degrading enzymes while ensuring screening accuracy. This solves the technical problem of balancing high substrate specificity and high throughput in existing PET-degrading enzyme screening, significantly improving the screening efficiency and practicality of PET-degrading enzymes.

[0005] This objective is achieved using the following technical solution:

[0006] In a first aspect, the present invention provides a highly efficient plastic-degrading enzyme screening system based on metagenomic mining and yeast surface display, comprising:

[0007] Metagenomic library construction module, used to construct cosmid libraries;

[0008] The functional screening module is used to transform the granular library into the host strain, spread it on the screening medium with PET microplastics as the sole carbon source, and screen for positive clones after culture.

[0009] The yeast surface display module is used to recombinantly express the target enzyme gene in a positive clone, so that the target enzyme is displayed on the surface of the yeast cell to obtain recombinant yeast cells.

[0010] The high-throughput sorting module is used to co-embed single recombinant yeast cells with PET fluorescent simulated substrates in microdroplets, generate fluorescence signals through enzymatic reactions, and obtain highly active recombinant yeast cells through fluorescence-activated cell sorting.

[0011] The secretion expression and activity verification module is used to isolate and purify highly active recombinant yeast cells to obtain monoclonal strains, induce secretion expression in the monoclonal strains, and obtain PET degrading enzymes through activity verification screening.

[0012] This method utilizes PET as the sole carbon source to achieve specific enrichment of PET-degraded positive clones while eliminating false positives caused by non-degraded clones. This approach thoroughly eliminates non-specific degradation and false positive clones at the source, ensuring high specificity of the screening results for the PET substrate. This provides a highly specific screening basis for subsequent high-throughput sorting. Positive clone cells are then co-encapsulated with PET fluorescent simulated substrates in microdroplets, forming independent, closed single-cell reaction units in each droplet. This avoids cross-interference between strains, allowing for enzymatic reactions and fluorescence signal generation at the single-cell level. The fluorescence intensity is positively correlated with enzyme activity, enabling visual detection of enzyme activity at the single-cell level. Flow cytometry sorting achieves high-throughput enrichment of highly active yeast strains, solving the problems of low efficiency and inability to rapidly enrich highly active strains in traditional specific screening. The functional primary screening module ensures high substrate specificity by targeting only PET, eliminating false positives. Simultaneously, the high-throughput sorting module enables ultra-high-throughput screening. These two modules complement each other, providing high throughput while maintaining high specificity, thus solving the technical problem of balancing high substrate specificity and high throughput in existing PET degrading enzyme screening methods.

[0013] Traditional PET degrading enzyme screening mainly relies on population-level detection methods such as the plate clear zone method and multi-well plate colorimetry. Traditional methods use cell populations as the detection target and cannot distinguish the true enzyme activity of individual cells. Highly active single cells are easily masked by a large number of low-activity cells, resulting in low sensitivity and difficulty in obtaining highly active clones. Extracellular secreted enzymes are prone to diffuse in the system, leading to false positive signals around non-target cells, making it impossible to accurately correspond to the relationship between cells and enzyme activity. Although screening using PET as the sole carbon source has high specificity, it has low throughput and long cycle. Ordinary high-throughput methods are easily interfered with by non-specific esterases and lipases, resulting in a high false positive rate. Conventional flow cytometry can only detect cell scattering signals, surface markers, or intracellular fluorescence, and cannot complete the in-situ catalysis and signal recognition of PET degrading enzymes at the single-cell level in a closed system, making it difficult to achieve true enzyme activity-guided high-throughput sorting.

[0014] This invention provides a single-cell closed reaction system using microfluidic droplets, allowing each microdroplet to independently encapsulate a single cell and a fluorescent substrate, forming a miniaturized, cross-contamination-free reaction unit. This avoids enzyme diffusion and signal crosstalk, ensuring that each cell corresponds to a single detectable signal. Simultaneously, the substrate structure mimics the chemical structure of PET, generating a specific response only to PET-degrading enzymes, eliminating non-specific interference from endogenous esterases and ensuring signal authenticity. The fluorescence intensity generated by the enzymatic reaction directly reflects the PET degradation capacity of a single cell, enabling visualization and quantification of enzyme activity. Finally, single-cell droplets are rapidly sorted based on the fluorescence signal. This invention is the first to couple microfluidic droplet technology, PET fluorescent simulated substrates, and flow cytometry (FACS). Relying on the dual specificity guarantee of the PET-specific screening in the functional screening module and the PET fluorescent simulated substrate in the high-throughput sorting module, it achieves high-throughput, high-sensitivity, and high-accuracy efficient screening of PET-degrading enzymes while ensuring high substrate specificity.

[0015] Furthermore, the coliform library is constructed from high molecular weight total DNA extracted from plastic-polluted environmental samples, and after end repair, it is inserted into the pCC2FOS coliform vector, transformed into host bacteria, and a metagenomic coliform library is obtained. This library directly obtains the genetic information of all microorganisms from the plastic-polluted environment without the need to isolate and culture microorganisms, fully covering the genetic information of environmental microorganisms. It can efficiently clone and express PET degradation-related genes and gene clusters, providing sufficient and reliable gene resources for subsequent high-specificity, high-throughput enzyme screening.

[0016] Furthermore, the host strain is a Pseudomonasputida ΔcutA1-deficient strain. Pseudomonasputida itself possesses an endogenous esterase gene, cutA1, which can cause non-specific hydrolysis of PET and ester substrates, leading to false positives. By using a ΔcutA1-deficient strain, the endogenous interfering enzyme is knocked out, avoiding the influence of its own enzyme activity on the screening results. This strain exhibits extremely low background activity against PET and polyester substrates. After transforming the metagenomic coccidial library into this deficient strain, only the PET-degrading enzyme encoded by the exogenously inserted metagenomic DNA can catalyze the production of reaction signals on PET substrates or fluorescent mimic substrates, significantly improving the specificity and accuracy of PET-degrading enzyme screening.

[0017] Furthermore, the PET microplastics are polyethylene terephthalate powder with a particle size of 50–80 μm and a crystallinity of 40–50%. They have a moderate specific surface area and good dispersibility, which can truly reflect the degradation ability of PET degrading enzymes. The crystallinity is close to that of commercial PET in the real environment, which can screen out highly active enzymes that have degradation activity on actual plastics, making the screening results accurate, with low false positives, and more in line with the needs of industrial applications.

[0018] Furthermore, the recombinant expression involves obtaining the target enzyme gene from a positive clone, cloning it into a fusion expression cassette of a yeast surface display vector, transforming yeast cells, and inducing expression to anchor the target enzyme to the surface of the yeast cell wall. This ensures that the fluorescent signal generated by the enzymatic reaction belongs only to that cell, solving the signal crossover and false positive problems caused by traditional secretory enzymes. Yeast surface display fixes the enzyme to the cell surface, making it easier for the substrate to contact the enzyme molecule, resulting in a faster and more direct enzymatic reaction. It eliminates the need for cell disruption and enzyme protein purification, simplifying the operation steps and adapting to high-throughput screening processes.

[0019] Furthermore, a flexible peptide linker (GGGGS)3 is inserted between the target enzyme and the anchoring protein on the yeast cell surface, and the molecular chaperone PDI1 is co-expressed. The flexible peptide linker (GGGGS)3 is a short peptide with a flexible structure that can form a spatial buffer region between the target enzyme and the anchoring protein, preventing folding interference and structural compression caused by spatial proximity between the two proteins. This ensures the enzyme maintains its native, relaxed, and active spatial conformation. PDI1 (protein disulfide isomerase) is a key molecular chaperone in yeast, capable of catalyzing the correct formation of disulfide bonds, assisting in protein folding, and repairing misfolded proteins. Misfolding significantly improves the correct folding rate and soluble expression level of exogenous proteins in the yeast expression system, increasing the number of active enzymes displayed on the cell surface. Through the synergistic effect of the flexible linker and the molecular chaperone PDI1, the flexible peptide linker (GGGGS)3 eliminates steric hindrance, ensuring the enzyme structure unfolds, while PDI1 assists in correct folding, improving the expression level and stability of active enzymes. The combined effect of these two factors significantly improves the display efficiency, catalytic activity, and structural stability of PET degrading enzymes on the yeast surface, ensuring a stronger and more stable enzyme activity signal, which is conducive to achieving high-sensitivity and high-throughput screening.

[0020] Furthermore, the PET fluorescent mimic substrate is a BODIPY-FL-labeled PET mimic substrate, and the BODIPY-FL-PET mimic has the chemical formula C 34 H 39 The BF2N3O8 oligomeric fluorescent substrate contains PET-characteristic oligomeric units, which can be specifically recognized and cleaved by PET degrading enzymes. After enzymatic cleavage, the fluorescence signal is significantly enhanced, resulting in high detection sensitivity and specificity. This substrate has good water solubility and strong photostability, and its excitation wavelength is matched with flow cytometry. It is non-toxic to yeast cells and can be adapted to microdroplet single-cell high-throughput screening systems to achieve efficient and accurate screening of highly active PET degrading enzymes.

[0021] Secondly, the present invention also provides a PET degrading enzyme mutant, which is obtained by screening using the efficient plastic degrading enzyme screening system based on metagenomic mining and yeast surface display described in the present invention.

[0022] Furthermore, the amino acid sequence of the PET degrading enzyme mutant contains two point mutations, S121P and D186H, relative to the wild-type PETase, as shown in SEQ ID NO:1.

[0023] Furthermore, the crystal structure of the PET degrading enzyme mutant shows that the S121P mutation introduces cis-proline knots, and the D186H mutation increases the hydrogen bond network of the active center, which together enhance local rigidity.

[0024] Thirdly, the present invention also provides a nucleic acid molecule encoding the PET degrading enzyme mutant, the nucleotide sequence of which is shown in SEQ ID NO:2. The nucleotide sequence is optimized according to the codon preference of Pichia pastoris, which can significantly improve the transcription and translation efficiency of the gene in Pichia pastoris, enhance the soluble expression level of the recombinant protein, and improve the stability of gene expression, thus facilitating the efficient heterologous expression of the PET degrading enzyme.

[0025] Fourthly, the present invention also provides the application of the system or the PET degrading enzyme mutant in the enzymatic depolymerization of polyurethane, polyolefin, polylactic acid, or polybutylene terephthalate waste.

[0026] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0027] 1. This invention discloses a high-efficiency plastic-degrading enzyme screening system based on metagenomic mining and yeast surface display, and its application. Using PET microplastic substrates for initial screening, it can effectively eliminate false-positive clones, significantly improving the specificity and accuracy of the screening system. Furthermore, it tandemly couples highly specific substrate screening with high-throughput activity detection at the single-cell level, efficiently completing the rapid enrichment and separation of highly active PET-degrading enzymes while ensuring the reliability of the screening results. This solves the technical problem of balancing high substrate specificity and high throughput in existing PET-degrading enzyme screening, greatly improving screening efficiency and accuracy.

[0028] 2. This invention discloses a high-efficiency plastic-degrading enzyme screening system based on metagenomic mining and yeast surface display, and its application. By encapsulating single cells and fluorescent substrates with microfluidic droplets, a micro-reaction unit without cross-contamination is formed, effectively avoiding enzyme diffusion and signal crosstalk. At the same time, a PET structure is used to simulate the fluorescent substrate, and the fluorescence intensity can directly reflect the PET degradation ability of single cells, realizing the visualization and accurate quantification of enzyme activity. Finally, through flow cytometry cell sorting (FACS), highly active PET-degrading enzymes can be rapidly and in high throughput enriched, significantly improving screening efficiency and sensitivity.

[0029] 3. This invention provides a high-efficiency plastic-degrading enzyme screening system based on metagenomic mining and yeast surface display, and its application. The PET-degrading enzyme mutant has excellent thermal stability, can maintain structural stability and catalytic activity at high temperatures, and has high degradation efficiency, thus possessing strong practicality and industrial application value. Attached Figure Description

[0030] The accompanying drawings, which are provided to further illustrate embodiments of the invention and constitute a part of this invention, are not intended to limit the scope of the invention.

[0031] Figure 1 This is a schematic diagram of the overall process of the screening system based on metagenomics and yeast display of the present invention;

[0032] Figure 2 This is a schematic diagram illustrating an example of a positive clone from the initial screening plate in an embodiment of the present invention;

[0033] Figure 3 This is a schematic diagram of the yeast surface display structure and microfluidic screening principle in an embodiment of the present invention;

[0034] Figure 4 This is a comprehensive diagram illustrating the research and characterization of the PET-degrading enzyme mutant (PETase-X1-M3) in this invention embodiment;

[0035] Figure 5 This is a schematic diagram of the enzymatic degradation and monomer recycling process of the PET degrading enzyme mutant (PETase-X1-M3) in an embodiment of the present invention;

[0036] Figure 6 This is a schematic diagram of the polyurethane (PU) foam biodegradation and monomer recycling process in an embodiment of the present invention;

[0037] Figure 7 This is a schematic diagram illustrating metagenomic library quality verification in an embodiment of the present invention;

[0038] Figure 8 This is a schematic diagram illustrating the verification of yeast surface display efficiency in an embodiment of the present invention;

[0039] Figure 9 This is a schematic diagram illustrating the verification of the specificity of microfluidic droplets in an embodiment of the present invention; Detailed Implementation

[0040] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be noted that, where there is no conflict, the embodiments of the present invention and the features thereof can be combined with each other.

[0041] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and therefore the scope of protection of the invention is not limited to the specific embodiments disclosed below.

[0042] Example 1

[0043] A highly efficient screening system for plastic-degrading enzymes based on metagenomic mining and yeast surface display, such as Figure 1 The diagram shows the overall process of the screening system based on metagenomics and yeast display according to the present invention.

[0044] The overall process of the screening system based on metagenomics and yeast display includes:

[0045] Metagenomic library construction, such as Figure 1 -A is shown.

[0046] High molecular weight total DNA (HMW DNA) was extracted from environmental samples contaminated with plastic. Metagenomic DNA extraction was performed using the MP Biomedicals FastDNA SPIN Kit for Soil. The lysis conditions were 0.5 mm glass beads, 6.0 m / s, 45 s × 2 lysis cycles, with a major band >50 kb and a total amount ≥10 μg. After end repair, the DNA was inserted into the pCC2FOS cosmid vector and transformed into the host bacterium (E. coli). The average insert fragment was 35–45 kb, and the library size was ≥1 × 10⁻⁶. 5 Cloned metagenomic viscous library.

[0047] Functional primary screening / PET filter, such as Figure 1 -B is shown.

[0048] The viscous library was electroporated to transform the Pseudomonas putida ΔcutA1 defective strain and coated onto M9 solid plates (M9-PET Plates) using PET microplastics as the sole carbon source. The PET microplastics were polyethylene terephthalate powder with a particle size of 50–80 μm and a crystallinity of 40–50%, obtained from waste beverage bottles through cryogenic grinding and differential scanning calorimetry screening. The functional screening plates contained 0.5–1.0 mmol / L of microplastic suspension with a particle size of 50–80 μm. After incubation at 30 ℃ for 48–72 h, a hydrolysis halo diameter ≥2 mm was considered positive. Bacterial colonies that formed hydrolysis halos on the plates were picked to obtain positive clones containing candidate degradative enzyme genes.

[0049] Yeast Surface Display System, such as Figure 1 -C is shown.

[0050] The target enzyme (PETase enzyme) gene was cloned into the pYD1-Aga2p fusion expression vector. A flexible linker peptide (GGGGS)3 was inserted between Aga2p (a yeast cell wall anchoring protein) and the target enzyme (PETase enzyme), and the molecular chaperone PDI1 was co-expressed to promote the correct formation of disulfide bonds by the displayed enzyme and improve extracellular esterase activity. The recombinant expression vector was transformed into Saccharomyces cerevisiae EBY100 and induced for expression for 20 h in SG-Trp medium with 2% galactose at 20 ℃ and 220 rpm. The positive display rate was ≥95% by flow cytometry detection with anti-FLAG antibody. The target enzyme was anchored to the surface of the yeast cell wall via Aga2p under galactose induction, forming reusable recombinant yeast cells with the surface displaying the target enzyme (PETase enzyme).

[0051] Microfluidic & FACS Screening, such as Figure 1 -D is shown.

[0052] Single yeast cells were co-encapsulated with BODIPY-FL-PET mimic substrate in 35 μm fluorinated oil droplets. Using fluorinated oil HFE-7500 containing 2% PFPE-PEG-PFPE surfactant, single-cell droplets with a diameter of 30-40 μm were generated (droplet generation), with a flux ≥2×10⁻⁶. 5 droplets / min. The BODIPY-FL-PET simulated substrate has the chemical formula C. 34 H 39 A fluorescent oligomer substrate of BF2N3O8, upon ester bond cleavage, releases a fluorophore with an Ex / Em ratio of 488 / 520 nm, which can be used for real-time quantification of enzyme activity. This substrate was synthesized via the Schmidt glycosylation-deacetylation-HPLC purification route, with a yield ≥40%. ESI-MS [MH] - It is 771.3 ± 0.1.

[0053] After incubation at 30–37 °C for 1–3 h, the embedded droplets were sorted by flow cytometry, with the top 0.1% of droplets in terms of fluorescence halo intensity (High-Throughput Sorting (FACS)) achieving a concentration ≥1×10⁻⁶. 7 High-throughput screening of mutants per day; the sorted droplets were demulsified using 1H,1H,2H,2H-perfluorohexanol at a volume ratio of 1:1, vortexed for 30 s, with a recovery rate of ≥80%; after demulsification, the bacterial culture was diluted and spread to obtain positive yeast clones.

[0054] Production and Validation, such as Figure 1 -E is shown.

[0055] The target enzyme gene from a positive yeast clone was subcloned into an expression vector and transformed into Pichia pastoris X-33 host cells. The host cells could be Pichia pastoris X-33, GS115, SMD1168, or Escherichia coli BL21 (DE3). The fermentation scale was 0.1-10 m³. 3 .

[0056] Secretory expression was carried out under the strong promoter of AOX1 and the α-factor signal peptide. High-density fermentation was first fed with glycerol until the cell wet weight was 180-200 g / L, and then methanol was switched to induce fermentation. The methanol flow rate gradient was 1-5 mL / min, the dissolved oxygen (DO) was controlled at 20-30%, and fermentation was induced at 25 ℃ for 96 h. The supernatant enzyme yield was ≥200 mg / L.

[0057] The fermentation broth was heat-treated at 65 °C for 30 min, centrifuged, filtered through a 0.22 μm filter membrane, subjected to Ni-NTA affinity chromatography (Ni-NTA purification), and ultrafiltration to obtain a recombinant enzyme with a purity ≥95% (SDS-PAGE optical density scan) and a specific activity ≥18 U / mg. The recombinant enzyme exhibited a 72 h mass loss rate ≥5% on high-crystallinity PET film, and kcat / Km ≥0.5 s. -1 ·mM -1 .

[0058] Example 2

[0059] This embodiment details the specific implementation steps of a high-efficiency plastic-degrading enzyme screening system based on metagenomic mining and yeast surface display.

[0060] (1) Sample collection and pretreatment

[0061] 500 mL of leachate was collected from a landfill in East China and transported at 4℃. 0.22 Polyethersulfone membrane was used for negative pressure enrichment of bacterial cells. DNA protectant was added (final volume 1%). (-Mercaptoethanol), flash-frozen in liquid nitrogen and stored at -80°C.

[0062] (2) Extraction of high molecular weight DNA

[0063] The MP Biomedicals FastDNA SPIN Kit for Soil (116560200) was used with the following optimizations:

[0064] ① Add 0.5 mm glass beads, vibrate at 6.0 m / s for 45 s using FastPrep-24, and repeat twice;

[0065] ② Pyrolysis temperature 65 ℃, 10 min;

[0066] ③ Pulsed-field electrophoresis (Bio-Rad CHEF-Mapper) confirmed a main band >50 kb, a total volume of approximately 12 μg, and OD. 260 / 280 =1.81, OD 260 / 230 =2.05.

[0067] (3) Construction of the Metagenomic Mixed Plasmids Library

[0068] 1 HMW-DNA was end-repaired using the CopyControl Fosmid Kit (Epicentre), ligated to pCC2FOS (Eco72I dephosphorylation), and electroporated into E. coli EPI300-T1R. The library size was 1.2 × 10⁻⁶. 5 CFU; 24 clones were randomly selected, and NotI digestion showed an average insert size of 41 kb and an empty vector rate of 2.4%.

[0069] (4) Primary screening of PET as the sole carbon source

[0070] ENLARGED VIEW: Primary Screening Positive Clone Example is an enlarged view of the primary screening positive clone example. The library mixed plasmid was electroporated and transformed into Pseudomonas putida KT2440 (ΔcutA1), plated on M9-PET plates (0.5 mmol / L PET microplastics, particle size 50-80 μm, crystallinity 45%), and incubated at 30 ℃ for 72 h. 32 clones that formed transparent hydrolysis halo were selected, and plasmids were extracted. EcoRI restriction enzyme fingerprints were merged into 7 non-replicated clones (PET1-PET7).

[0071] (5) Construction of yeast surface display carrier

[0072] like Figure 3As shown, YEAST DISPLAY is a yeast surface display. Taking PET5 as an example, primers PET5-F / PET5-R were designed to amplify the 1.8 kb esterase gene. After double digestion with NheI / NotI, the gene was inserted downstream of pYD1-Aga2p, allowing PET5 esterase and Aga2p to fuse via a linker, maintaining the enzyme's spatial conformation and activity, forming the Aga2p-PET5 fusion anchor structure, thus obtaining pYD1-PET5. The gene was then electroporated into S. cerevisiae EBY100 (Saccharomyces cerevisiae strain EBY100) and incubated on SD-Trp-glucose plates at 30 ℃ for 48 h. The primers are shown in Table 1.

[0073] Table 1. List of PET5 primers

[0074]

[0075] Induced Display

[0076] Monoclonal antibodies were inoculated into SD-Trp-glucose overnight, then transferred to SG-Trp-galactose (2%) at A600=0.5 and induced at 20 ℃ and 220 rpm for 20 h. Flow cytometry was used to detect anti-FLAG-FITC markers, with a positive rate of 96% and an MFI increase of 120-fold.

[0077] (6) Microfluidic Droplet Generation

[0078] Mobile phase (Fluorinated Oil Phase): Fluorinated oil HFE-7500 + 2% PFPE-PEG-PFPE; Aqueous phase: 10 6 cells / mL yeast (Aqueous Phase 1: Single Yeast Cells (10 6 cells / mL) + 200 μM BODIPY-FL-PET mimic (Aqueous Phase 2: BODIPY-FL-PET Substrate (200 μM)) (synthesized in our laboratory, C 34 H 39 BF2N3O8); chip flow rate 6 μL / h, generating single-cell droplets with a diameter of 35 μm, droplet generation flux (FlowRate: 2 × 10⁻⁶). 5 droplets / min).

[0079] (7) FACS sorting and recovery

[0080] Incubation at 37 ℃ for 2 h allowed the enzymatic reaction to generate a signal from the fluorescent substrate. The MoFlo Astrios EQ (488 nm excitation, 525 / 50 nm collection) was used, where fluorescence signal intensity was positively correlated with enzyme activity (signal intensity ≈ enzyme activity), with stronger signals indicating higher enzyme activity. Fluorescence-activated cell sorting (FACS Sorting) was performed using a MoFlo Astrios EQ flow cytometer. A 488 nm laser excited the fluorophore within the droplet, generating a fluorescence signal. The detector captured and quantified the fluorescence intensity, setting the collection gate P2 to the top 0.1% of fluorescence intensity, collecting only the clonal droplets with the highest enzyme activity. During sorting, a deflector plate deflected the target droplet into the collection tube (high-activity) through electrostatic interaction. Mutants), and the remaining low-activity or inactive droplets enter the waste liquid pipe. The screening throughput of this system can reach ≥1×10⁻⁶. 7 The mutants / day ratio demonstrates the advantages of high-throughput screening. Approximately 300 droplets were recovered, and 1H,1H,2H,2H-perfluorohexanol was added to break the emulsion. After dilution, the mixture was spread on SD-Trp plates, ultimately yielding 28 single clones.

[0081] (8) Activity retest

[0082] After 28 clones were secreted and expressed according to Example 3, 0.1 μM crude enzyme was reacted with an hcPET membrane (1 cm × 1 cm, approximately 5 mg) at pH 8.0 and 30 °C for 72 h. GPC was used to determine the Mn reduction rate; 9 clones showed a Mn reduction >5%, with 5E8 (named PETase-X1-M3) showing a 12.3% reduction (kcat / Km = 0.68 s). -1 mg -1 L, which is 8.8 times higher than the wild type.

[0083] (9) Construction of expression strains

[0084] like Figure 4As shown, the PETase-X1-M3 gene (SEQ ID NO:1, S121P / D186H) was subcloned into the expression vector pPICZαA, with nucleus as the cell nucleus. After linearization, it was electroporated into Pichia pastoris X-33 cells, screened with YPDS+Zeocin 100 mg / L, and the copy number was confirmed to be ≈6 by qPCR.

[0085] (10) High-Density Fermentation

[0086] ① Seeds: 250 mL BMGY, 30 ℃, 250 rpm for 16 h, A600=6.0;

[0087] ② Batch: 2.5 L of basal salt culture medium was packaged in 5 L (5L Scale-up Fermentation) containers, sterilized at 121 ℃, and 200 mL of seed culture was inoculated. The initial stirring was 600 rpm, aeration was 1.0 vvm, and pH was 5.0 (adjusted with NH4OH).

[0088] ③ Glycerol feed: When DO > 30%, add 50% glycerol (containing PTM1) until the wet weight is 180 g / L;

[0089] ④ Methanol Induction: 100% methanol (containing PTM1) gradient 1→5 mL / min, DO 20–30%, 25 ℃, 96 h; container volume 2.3 L, total protein in supernatant 2.4 g, target band percentage 28% (SDS-PAGE optical density scan).

[0090] (11) Purification and Purity Verification (The Result)

[0091] The fermentation supernatant (crude supernatant, untreated enzyme-containing supernatant after fermentation) was heat-treated at 65°C for 30 min (after 65°C heat treatment to remove heat-labile proteins) → centrifuged at 8000×g → filtered at 0.22 μm → purified via Ni-NTA 6FF (50 mL column, equilibration buffer 20 mM Tris-HCl pH 8.0 + 300 mM NaCl + 20 mM imidazole, eluent + 250 mM imidazole) to obtain Purified PETase-X1-M3 via Ni-NTA (PETase-X1-M3 purified by Ni-NTA affinity chromatography) → ultrafiltration (20 mM Tris-HCl pH 8.0 + 150 mM NaCl) to finally obtain 280 mg of pure enzyme with a specific activity of 18.7 U / mg (pNP-butyrate, 30 The protein was measured at ℃. The purity was determined by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis, used to separate proteins and determine purity; protein molecular weight is measured in kDa) and RP-HPLC (high performance liquid chromatography, for precise quantification of protein purity). The HPLC peak showed Purity > 95%, with Absorbance (mAU) as the ordinate and Retention Time (min) as the abscissa.

[0092] (12) Enzymatic Characterization

[0093] Optimum temperature: 65 ℃;

[0094] Optimum pH: 8.5 (Tris-HCl);

[0095] Thermal stability: half-life at 65 °C t½ = 125 min (wild type 15 min);

[0096] For hcPET film, kcat / Km = 0.68 s -1 mg -1 L, optimal pH 8.0–8.5, optimal temperature 60–65 ℃.

[0097] Temperature (°C) represents the reaction temperature on the x-axis, and Relative Activity (%) is the relative enzyme activity (with the highest enzyme activity being 100%) on the y-axis. Figure 4It can be seen that the optimal temperature for this mutant is 65 ℃, and it retains approximately 25% of its relative enzyme activity at 20 ℃. With pH on the x-axis and relative enzyme activity on the y-axis, the curve peaks around pH 8.5, indicating that the enzyme exhibits the highest activity under weakly alkaline conditions.

[0098] Incubation Time (min) is plotted on the x-axis, and Residual Activity (%) is plotted on the y-axis, representing residual enzyme activity (the percentage of remaining activity compared to the unincubated sample). The solid line represents the PET degrading enzyme mutant (PETase-X1-M3), and the dashed line represents wild-type PETase. Figure 4 It can be seen that the mutant has a half-life of 125 minutes at 65 °C, meaning that it still retains 50% of its enzyme activity after 125 minutes of incubation. The wild type has a much shorter half-life, indicating that the mutant has significantly improved thermal stability.

[0099] The PETase-X1-M3 mutant of this invention was successfully fermented and purified at high density in Pichia pastoris X-33, achieving a purity >95%. Enzymatic characterization showed that the mutant's optimal temperature was 65 °C, and it retained approximately 25% relative enzyme activity at 20 °C; the optimal pH was approximately 8.5, with the highest catalytic activity under weakly alkaline conditions; in the 65 °C thermostability test, the mutant had a half-life of 125 min, significantly better than the wild type, demonstrating excellent high-temperature tolerance. Furthermore, the enzyme exhibited a ≥5% mass loss rate on highly crystalline PET films after 72 h, and a catalytic efficiency kcat / Km ≥0.5 s. -1 ・mM -1 This demonstrates its potential for efficient and stable application in real-world PET degradation scenarios.

[0100] Example 3

[0101] This embodiment describes a PET-degrading enzyme mutant, PETase-X1-M3, obtained using a highly efficient plastic-degrading enzyme screening system based on metagenomic mining and yeast surface display according to the present invention. Its amino acid sequence is shown in SEQ ID NO:1.

[0102] The sequence characteristics of SEQ ID NO:1 are as follows:

[0103] (1) Sequence length: 289 amino acids

[0104] (2) Sequence type: amino acid sequence

[0105] (3) Chain type: single chain

[0106] (4) Topology: Linear

[0107] SEQ ID NO:1: (Amino acid sequence)

[0108] MNFPRASRLMQAAVLGGLMAVSAAATAQTNPYARGPNPTAASLEASAGPFTVRSFTVSPSSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNDS LKAAAPQAPWDSSTNFSSVTVPTLIFACENDSIAPVNSSALPIYDSMSRNAKQFLEINGGSHSCANSGNSNQALIGKKGVAMKRFMDNDTRYSTFACENPNSTRVSDFRTANCS

[0109] The PET-degrading enzyme mutant PETase-X1-M3 contains two point mutations, S121P and D186H, compared to the wild-type PETase. These mutations occur only at position 121 (serine → proline, S121P) and position 186 (aspartic acid → histidine, D186H), with no other amino acid deletions, insertions, or substitutions. The total length is 289 amino acids. In its crystal structure, the S121P mutation introduces a cis-proline knot, and the D186H mutation increases the hydrogen bond network at the active site, both enhancing local rigidity. The PET-degrading enzyme mutant PETase-X1-M3 has a half-life ≥120 min at 65 °C and a 72 h mass loss rate ≥12% for waste PET bottle flakes with 45% crystallinity.

[0110] The polynucleotide sequence encoding the above-mentioned PET degrading enzyme mutant PETase-X1-M3 is shown in SEQ ID NO:2.

[0111] The sequence characteristics of SEQ ID NO:2 are as follows:

[0112] (1) Sequence length: 867 nucleotides

[0113] (2) Sequence type: Polynucleotide (DNA sequence)

[0114] (3) Chain type: double chain

[0115] (4) Topology: Linear

[0116] SEQ ID NO:2: (Polynucleotide sequence)

[0117] ATGAACTTCCCACGCGCCTCCAGACTGATGCAGGCTGCTGTTCTGGGCGGCTTGATGGCTGTGAGCGCTGCTGCTACCGCTCAGACGAACCCTTACGCGCGCGGCCCGAACCCGACCGCTGCTTCACTGGAGGCTTCGGCTGGCCCGTTCACCGTTCGCTCGTTCACCGTCTCGCGCCCCTCCGGCTACGGCGCTGGCTCGACCGTCTATTATCCCACCAACGCTGGCGGCTACCGTGGCTGCTATCGCTATCGTCCCAGGCTACACCGCGCGCCAGTCGTCGATCAAGTGGTGGGGCCCCCGCTTGCCTCTCACGGCTTCGTGGTGATCACCGATACCAACAGCACCTTGGACCAGCCCTCCAGCCGCTCGTCGCAGCAGATGGCTGCTCTGCGCCAGGTGGCTTCGCTGAACGGCACCTCGTCGTCGCCGATCTACGGCAAGGTGGACACCGCGCGCATGGGCGTGATGGGCTGGTCGATGGGCGGCGGCGGCAGCCTGATCAGCGCTGCTAACAACGACAGCCTGAAGGCTGCTGCTCCGCAGGCACCCTGGGACAGCAGCACCAACTTCTCGTCGGTGACCGTCCCACCTCTGATCTTCGCTTGCGAGGACAACAGCGATGCTCCGGTGAACAGCAGCGCTCTGCCGATCTACGACAGCATGAGCCGCAACGCTAAGCAGTTCCTGGAGATCAACGGCGGCTCCCACAGCTGCGCTAACTCCGGCAACAGCAACCAGGCTCTGATCGGCAAAAAGGGCGTGGCTTGGATGAAGCGCTTCATGGACAACGACACCCGCTACAGCACCTTCGCTTGCGAGGACAACCCGAACAGCACCCGCGTGAGCGACTTCCGCACCGCTAACTGCTCG

[0118] The full-length polynucleotide sequence is 867 bp. The nucleotide sequence is optimized according to the codon preference of Pichia pastoris, with CAI≥0.85 and GC content of 47–52%. It can significantly improve the transcription and translation efficiency of the gene in Pichia pastoris, enhance the soluble expression level of recombinant protein, and improve the stability of gene expression, which is conducive to achieving efficient heterologous expression of PET degrading enzyme.

[0119] Example 4

[0120] This embodiment describes the enzymatic degradation process of the PET degrading enzyme mutant, such as... Figure 5 The diagram shown illustrates the enzymatic degradation and monomer recycling process of PET degrading enzyme mutants, including:

[0121] (1) Pretreatment of PET bottle flakes

[0122] Commercially available colorless carbonated beverage bottles (labels and caps removed) were collected, rinsed three times with deionized water, and dried in a 65 ℃ oven to constant weight (water content ≤0.2%). They were then cut into 1 cm × 1 cm square slices with a thickness of 250 ± 20 μm using a hydraulic shearing machine. The crystallinity was determined by differential scanning calorimetry (DSC, Q2000, TA Instruments) to be 45 ± 1%, and the density was 1.38 g / cm³. -3 .

[0123] (2) Scale-up of 1L glass reactor

[0124] Add to a 1L jacketed glass reactor, which has a marine-blade impeller:

[0125] 50 g of PET sheets (5% w / v);

[0126] 900 mL of 20 mM Tris-HCl pH 8.0 buffer solution;

[0127] 150 mM NaCl;

[0128] 0.05% (w / v) Tween-80.

[0129] After premixing, equilibrate at 30 °C for 30 min, then add 2 mg / mL of purified PETase-X1-M3 from Example 3 to a final concentration. -1(Total enzyme amount 2 g). The impeller was a boat-type impeller with a diameter of 70 mm and a rotation speed of 150 rpm. DO was maintained at >80% air saturation. 2 mL samples were taken every 12 hours during the 72-hour reaction period, immediately filtered through a 0.22 μm filter, and the terephthalic acid (TPA) concentration was determined by HPLC.

[0130] (3) Online TPA quantification

[0131] Instrument: Waters e2695 high performance liquid chromatograph, UV detector λ = 254 nm;

[0132] Column: XBridge C18 4.6×250 mm, 5 μm;

[0133] Mobile phase: 20 mM phosphate pH 2.5 : acetonitrile = 85 : 15, isocratic elution, flow rate 1.0 mL / min -1 Column temperature 30 ℃.

[0134] Standard curve: TPA 0.1–5 mM (r = 0.9998), LOD 0.02 mM, LOQ 0.05 mM. Results: TPA concentration at 72 h was 1.83 ± 0.04 mM (n=3), equivalent to a tablet mass loss of 11.9 ± 0.3%, consistent with the decrease in GPC number-average molecular weight (Mn) (45 kDa → 28 kDa).

[0135] (4) Scale-up of 10 L stainless steel fermentation tank (10L Automated Bioreactor)

[0136] The total volume of the tank is 15 L, with a working volume of 10 L. It is equipped with a pH-Stat automatic control system to maintain pH stability in the reaction system (NaOH 2 M titration) and dissolved oxygen control (DO control (Dissolved Oxygen control)) in a cascade (stirring 100-600 rpm, aeration 0.5-1.5 vvm). 500 g of PET sheets are added, and other conditions are the same as in 4.2. After 72 h of enzymatic degradation, the material is transferred to the tank, centrifuged to remove residual sheet base, and the supernatant is decolorized with activated carbon (1% w / v, 60 ℃, 30 min), filtered through a plate and frame filter, and acidified to pH 3.0 (pH 3.0 Acidification & Crystallization) (6 M HCl). It is then allowed to stand at 4 ℃ for 12 h for crystallization. Vacuum filtration and vacuum drying at 60 °C for 8 h yielded 31.2 g of white terephthalic acid (TPA) crystals with a purity of 98.7% (HPLC area normalized) and a recovery rate of 90.1% (relative to theoretical yield). The mother liquor was recycled, resulting in a crystallization yield decrease of <5%.

[0137] (5) Crystal characterization

[0138] DSC: Melting point 425 ℃ (literature value 427 ℃); FT-IR: 1680 cm⁻¹ -1 Carboxyl group C=O stretching, compared with standard spectra Figure 1 To; 1 ¹H-NMR (400 MHz, DMSO-d⁶) δ 8.06 (s, 4H), no impurity signal; elemental analysis: C 57.8%, H 3.6% (theoretical C 57.7%, H 3.6%). The crystal can be directly used for re-polymerization to synthesize new PET.

[0139] Example 5

[0140] This embodiment verifies the degradation performance of polyurethane (PU) foam and its recovery. Figure 6 As shown, the PETase-X1-M3 mutant was used to verify its degradation activity on polyurethane plastics containing ester bonds, demonstrating its broad-spectrum plastic degradation potential.

[0141] (1) Substrate Preparation

[0142] Flexible polyurethane foam (density 30 kg m³) -3 Cut the sample (with an average pore size of 600 μm) into 5 mm × 5 mm × 5 mm cubes, wash three times with deionized water, and dry at 60 ℃ to constant weight. Weigh 5 g (approximately 167 cm³) 3 Place it in a 500 mL reaction flask.

[0143] (2) Enzymatic reaction

[0144] Buffer: 50 mM PBS pH 7.5, Ca 2+ 1 mM, Mg 2+ 1 mM, 0.02% NaN3 for antibacterial effect; temperature 40 ℃; stirring 150 rpm; enzyme dosage: fermentation supernatant from Example 2 (esterase activity 18.7 U mg) -1 Final concentration 0.5 mg / mL -1 (Total enzyme amount 250 mg). Total system volume 500 mL.

[0145] A 1 mL sample was taken every 12 h, filtered through a 0.22 μm filter, and the concentrations of adipic acid (ADA) and 4,4'-methylenediphenylamine (MDA) were determined by HPLC (High Performance Liquid Chromatography). Chromatographic conditions: A = 20 mM KH₂PO₄, pH 2.8; B = acetonitrile; gradient 0-15 min 20% B → 80% B; flow rate 1 mL / min. -1 UV 210 nm. The x-axis represents Retention Time (HPLC elution time), and the y-axis represents Absorbance (signal intensity). Results (Degradation Evidence (Micro & Macro)): ADA concentration reached 1.24 mM at 48 h, MDA 0.31 mM. Final Recovery Results: Monomer Recovery 88% (molar basis); Mass Loss 21%. SEM (SEM-style erosion) showed that from 0 h to 48 h, the foam changed from an initial state with intact pore walls to degradation, resulting in pore wall erosion and breakage.

[0146] (3) Scale-up verification

[0147] 300 g of PU foam was added to a 5 L reactor (working volume 3 L), and the other conditions were the same as in 5.2. After 48 h, the ADA concentration was 1.21 mM and the recovery rate was 85%, proving that the system can be linearly scaled up.

[0148] Example 6

[0149] This example describes the immobilization preparation and reusability study of the PETase-X1-M3 mutant PET degrading enzyme.

[0150] (1) Carrier pretreatment

[0151] Epoxy resin LX-1000EP (Xi'an Lanxiao, particle size 150–300 μm, epoxy value 0.8 mmol g⁻¹) was washed three times with 0.1 M PBS pH 7.5 and dried for later use.

[0152] (2) Immobilization

[0153] The purified PETase-X1-M3 from Example 3 was dissolved in the same buffer solution at a protein concentration of 2 mg / mL⁻¹, and mixed with the wet carrier at a ratio of 1:10 (w / w). Immobilization was carried out at 25 °C and 150 rpm for 12 h. After removal, the enzyme was washed three times with 1 M NaCl to remove non-covalent adsorption, yielding the immobilized enzyme with 72% activity recovery and a loading of 45 mg / g. -1 Carrier.

[0154] (3) Batch cycle

[0155] Add the following to a 50 mL reaction tube: 2 g immobilized enzyme, 1 g hcPET film, and 40 mL of 20 mM Tris-HCl (pH 8.0). React at 30 ℃ and 150 rpm for 12 h as one cycle. After each cycle, filter to recover the carrier and immediately add new substrate. Determine the initial and residual enzyme activity after each cycle (pNP-butyrate method). Results: After 20 cycles, the residual activity was 71%, the PET mass loss was stable at 5–6% per cycle, and there was no significant carrier breakage.

[0156] (4) Storage stability

[0157] Immobilized enzymes dried at 4 ℃ for 90 days showed an activity decrease of <8%; dried at 25 ℃ for 30 days showed an activity decrease of <15%, meeting the requirements for industrial reuse.

[0158] Example 7

[0159] This embodiment is for the quality assessment of the metagenomic library of the present invention. Figure 7 As shown, the inserted fragment size, empty vector rate, and functional coverage of the constructed library are used to verify the library's performance and ensure that the library meets the requirements for efficiently mining novel PET degrading enzymes.

[0160] (1) Pulsed field electrophoresis (PFGE): 20 fosmid vector clones were randomly selected, digested with NotI and subjected to CHEF-Mapper electrophoresis. The average insert size was 41.2 ± 1.8 kb, which is consistent with the fosmid vector capacity of 2.1%.

[0161] (2) Functional coverage: The library mixed plasmid was transformed into P. putida ΔcutA1, plated on M9-PET plates, and after 72 h, the positive rate of the hydrolysis zone quality was 0.26% (32 / 1.2×10⁻⁶). 5 The result is consistent with the theoretical estimate (≥0.2%), indicating that the library coverage meets the mining requirements.

[0162] Example 8

[0163] This embodiment is a test of yeast surface display efficiency. Figure 8 As shown, the yeast display system constructed in this invention is used to verify the display performance of the PET degrading enzyme mutant PETase-X1-M3.

[0164] The anti-FLAG-FITC (ITC-Flag Antibody Binding) fluorescence-activated cell sorting analysis (FACSAnalysi) was used to determine the positive rate of 96.3% after 20 h of induction. The mean fluorescence intensity (MFI) was 122-fold higher than that of the control. Mutant was a mutant yeast sample containing PETase-X1-M3, and Blank Control was a blank control yeast sample (which did not show the target enzyme). Cryo-electron microscopy showed that the fusion protein was uniformly distributed on the outer side of the cell wall with a density of ≈2×10⁻⁶. 4 The display efficiency of molecules / cells is >95%, demonstrating that the yeast display system of this invention has a high-efficiency and stable ability to display PETase mutants, laying the foundation for subsequent high-throughput screening.

[0165] Example 9

[0166] This example demonstrates the specificity verification of microfluidic-fluorescent substrates, such as... Figure 9As shown, this was used to verify the specificity of the BODIPY-FL-PET mimic probe for recognizing PET ester bonds, ensuring that high-throughput screening only enriches real PET degrading enzymes.

[0167] Yeast cells containing PETase-X1-M3 were encapsulated in aqueous droplets (aqueous droplets, microfluidic units encapsulating cells and substrates) with either BODIPY-FL-PET mimic (PET mimic substrate channel, Ex / Em 488 / 520 nm) or p-nitrophenylbutyrate (a common ester bond substrate, used as a control). Fluorescent probes were excited using a laser beam. At the same concentration, the positive sorting signal / background ratio (S / N) for the PET mimic was 18.2, and for the pNP-butyrate, it was 2.1. S / N = 18.2 / 2.1 represent the signal-to-noise ratio of the PET mimic substrate and p-nitrophenylbutyrate, respectively, reflecting the signal intensity / background interference, demonstrating that the substrate exhibits high specificity for PET ester bonds.

[0168] Example 10

[0169] This embodiment is for the detection of enzymatic parameters of the mutant PETase-X1-M3. By comparing it with wild-type PETase, the thermal stability, catalytic efficiency and high crystallinity PET degradation ability of the mutant of the present invention are verified.

[0170] The PETase-X1-M3 prepared and purified in Example 2 and wild-type PETase were dissolved in 20 mM Tris-HCl pH 8.0 buffer, and the protein concentration was adjusted to 0.1 μM. They were then stored at 4 °C for later use.

[0171] (1) Thermal stability at 65 ℃ ( ) detection

[0172] Take 1 mL of enzyme solution and place it in a 65 ℃ constant temperature water bath. Take 100 μL samples at 0, 5, 15, 30, 60, 90, 120, and 150 min respectively, and immediately place them in an ice bath to terminate thermal inactivation. The remaining enzyme activity is determined by the pNP-butyrate method: react the sample with 5 mM pNP-butyrate (dissolved in 20 mM Tris-HCl pH 8.0) at 37 ℃ for 10 min, and measure the absorbance at 405 nm. With the initial enzyme activity as 100%, calculate the enzyme activity residual rate, and fit the time when the enzyme activity drops to 50% of the initial value as the 65 ℃ half-life t½.

[0173] (2) Catalytic efficiency ( ) detection

[0174] Using highly crystalline PET (hcPET) powder as a substrate, a substrate concentration gradient of 0.1–2.0 mg / mL was established. Under conditions of pH 8.5 and 65 ℃, the amount of carboxyl equivalents released per unit time was recorded using the pH-stat method (maintaining a constant pH of 8.5), and the results were calculated. .

[0175] 4. 72 h hcPET mass loss rate test

[0176] Add the following to a 50 mL reaction tube: 0.1 μM purified enzyme, hcPET membrane (1 cm × 1 cm, approximately 5 mg), and 20 mL of 20 mM Tris-HCl pH 8.5. React at 65 ℃ and 150 rpm for 72 h. After the reaction, remove the membrane, wash it thoroughly with 0.1 M NaOH and deionized water, dry it at 60 ℃ to constant weight, weigh the remaining mass, and calculate the PET mass loss rate.

[0177] 5. Optimal temperature detection

[0178] Under pH 8.5 conditions, a temperature gradient of 30-75 ℃ was set, and enzyme activity was measured using pNP-butyrate as a substrate. The temperature corresponding to the highest enzyme activity was taken as the optimum temperature.

[0179] 6. Optimal pH detection

[0180] At 65 °C, a pH gradient of 6.0–9.5 was set (using 20 mM MES, Tris-HCl, and Glycine-NaOH buffer, respectively), and enzyme activity was measured using pNP-butyrate as the substrate. The pH corresponding to the highest enzyme activity was taken as the optimal pH.

[0181] The test results for each parameter are shown in Table 2:

[0182] Table 2. Enzymatic parameters of mutant PETase-X1-M3

[0183]

[0184] Example 11

[0185] This example demonstrates the scaled-up determination of the purity of degradation products.

[0186] After decolorization with activated carbon and acidification crystallization, the 10 L reaction solution showed a TPA crystal purity of 98.7% (HPLC area normalized), a melting point of 425.2 ℃ (DSC), and elemental analysis, ¹H-NMR, and FT-IR results consistent with the standard. The heavy metal content was <0.5 ppm, meeting the requirements for food-grade repolymerization.

[0187] Example 12

[0188] This example demonstrates the cyclic stability testing of immobilized enzymes.

[0189] Immobilized PETase-X1-M3 (LX-1000EP carrier) was continuously operated for 20 batches under the conditions of 5% hcPET, 30 ℃, and 12 h / batch, with 71% remaining activity, carrier breakage rate <2% (laser particle size analyzer), PET weight loss per batch maintained at 5-6%, and RSD <5%.

[0190] Example 13

[0191] This embodiment uses a traditional 96-well plate culturable library screening method for comparison with the metagenomic screening method of this invention.

[0192] The same batch of leachate sample from Example 2 was diluted and plated onto LB agar plates, yielding 1.2 × 10⁻⁶ culturable colonies. 4 CFU was initially screened using pNP-butyrate as a substrate, yielding 0.8% positive clones; further screening with PET membranes yielded 0 active clones. Compared to metagenomic methods (0.26% positive and ultimately obtaining highly active mutants), the traditional method has two orders of magnitude lower coverage and failed to capture difficult-to-culture strains.

[0193] Example 14

[0194] In this embodiment, an intracellular expression system without yeast surface display was used as a control.

[0195] The PET5 gene was directly inserted into the pYES2 intracellular expression vector. After induction, the enzyme activity in the supernatant of the lysed cells was 3.2 U / L. Under the same induction conditions, the enzyme activity of the whole cell catalyst on the surface of yeast reached 28 U / L (per unit volume of fermentation broth), and no lysis was required. The display strategy increased the detectable enzyme activity by 8.8 times.

[0196] Example 15

[0197] This embodiment uses a non-FACS sorting control.

[0198] After induction, cells were displayed without droplet-FACS. 200 clones were randomly selected for secondary screening in 96-well plates. The positive rate of Mn decrease >5% was only 1.5% (3 / 200). However, after enrichment by FACS, the positive rate increased to 35% (28 / 80), with an enrichment factor of 23 times, which significantly shortened the screening cycle.

[0199] Example 16

[0200] This embodiment uses a single mutation control.

[0201] The single mutant PETase-S121P containing only S121P was constructed. At 65 ℃, t½=45 min, which was lower than that of the double mutant (125 min), indicating that D186H made a synergistic contribution to thermal stability. Similarly, the weight loss of hcPET at 72 h was 8.2%, which was lower than that of the double mutant (12.3%), demonstrating that the combined mutation has a synergistic effect.

[0202] Example 17

[0203] This embodiment uses a traditional chemical alcoholysis process, which is compared with the enzymatic degradation of PET of the present invention.

[0204] Take 50 g of PET bottle flakes from the same batch as in Example 2 and use traditional ethylene glycol alcoholysis (197 ℃, 0.3 MPa, Zn(OAc) 20.5%, 2 h). The TPA yield is 90%, but the energy consumption is 2.8 times that of the enzymatic method (steam consumption), and the zinc-containing waste liquid generated requires additional treatment. The enzymatic method, at 30 ℃ and normal pressure, without organic solvents, reduces the carbon footprint by 60%, meeting the requirements of green manufacturing.

[0205] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0206] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A highly efficient screening system for plastic-degrading enzymes based on metagenomic mining and yeast surface display, characterized in that, include: Metagenomic library construction module, used to construct cosmid libraries; The functional screening module is used to transform the granule library into the host strain, spread it on the screening medium with PET microplastics as the sole carbon source, and screen to obtain positive clones after culture. The yeast surface display module is used to recombinantly express the target enzyme gene in a positive clone, so that the target enzyme is displayed on the surface of the yeast cell to obtain recombinant yeast cells. The high-throughput sorting module is used to co-embed single recombinant yeast cells with PET fluorescent simulated substrates in microdroplets, generate fluorescence signals through enzymatic reactions, and obtain highly active recombinant yeast cells through fluorescence-activated cell sorting. The secretion expression and activity verification module is used to isolate and purify highly active recombinant yeast cells to obtain monoclonal strains, induce secretion expression in the monoclonal strains, and obtain PET degrading enzymes through activity verification screening.

2. The efficient plastic-degrading enzyme screening system based on metagenomic mining and yeast surface display according to claim 1, characterized in that, The copoid library was obtained by extracting high molecular weight total DNA from plastic-polluted environmental samples, inserting it into the pCC2FOS copoid vector after end repair, and then transforming it into host bacteria.

3. The efficient plastic-degrading enzyme screening system based on metagenomic mining and yeast surface display according to claim 1, characterized in that, The host strain was the PseudomonasputidaΔcutA1 defective strain.

4. The efficient plastic-degrading enzyme screening system based on metagenomic mining and yeast surface display according to claim 1, wherein the PET microplastic is polyethylene terephthalate powder with a particle size of 50–80 μm and a crystallinity of 40–50%.

5. The efficient plastic-degrading enzyme screening system based on metagenomic mining and yeast surface display according to claim 1, characterized in that, The recombinant expression involves obtaining the target enzyme gene from a positive clone, cloning it into a fusion expression cassette of a yeast surface display vector, transforming yeast cells, and then inducing expression to anchor the target enzyme to the surface of the yeast cell wall.

6. The efficient plastic-degrading enzyme screening system based on metagenomic mining and yeast surface display according to claim 5, characterized in that, The target enzyme is inserted between the anchoring protein on the surface of yeast cells and the flexible peptide linker (GGGGS)3, and the molecular chaperone PDI1 is co-expressed.

7. The efficient plastic-degrading enzyme screening system based on metagenomic mining and yeast surface display according to claim 1, wherein the PET fluorescent mimic substrate is a BODIPY-FL labeled PET mimic substrate.

8. The PET-degrading enzyme mutant obtained by screening according to any one of the systems described in claims 1–7, characterized in that, The amino acid sequence of the PET degrading enzyme mutant contains two point mutations, S121P and D186H, relative to the wild-type PETase, and the amino acid sequence is shown in SEQ ID NO:

1.

9. A nucleic acid molecule encoding the PET degrading enzyme mutant of claim 8, wherein the nucleotide sequence is shown in SEQ ID NO:

2.

10. The use of the system of any one of claims 1–7 or the PET degrading enzyme mutant of any one of claims 8–9 in the enzymatic depolymerization of polyurethane or polyolefin or polylactic acid or polybutylene terephthalate waste.

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