Preparation method of psychrophilic protease PETase

Abstract

The invention discloses a preparation method of psychrophilic protease PETase. The method comprises the following steps: mining and designing a PETase candidate sequence set; carrying out enzyme activity determination on each candidate enzyme in the PETase candidate sequence set by taking a derivative of a PET oligomer BHET as a substrate to obtain enzyme activity determination data; and screening the PETase candidate sequence set based on the enzyme activity determination data to obtain the target psychrophilic protease PETase. Excavated design PETase is combined with a derivative of PET oligomer BHET as a substrate for enzyme activity determination to obtain enzyme activity determination data for statistical analysis and screening of target psychrophilic protease PETase, accurate screening of high-novelty and high-activity PET hydrolase can be realized, and functional enzyme discovery efficiency is greatly improved.

Description

Technical Field

[0001] This invention relates to the field of PET degradation, and more specifically to a method for preparing PETase, a cold-loving protease capable of biodegrading PET. Background Technology

[0002] Plastic products are widely used in daily life due to their portability, durability, and low cost, with a global annual production exceeding 335 million tons. However, because plastics do not decompose naturally, billions of tons of plastic waste end up in landfills, floating on waste islands in the ocean, and dispersed as microplastics (MPs), causing serious global pollution.

[0003] Microplastics (MPs) are plastic particles with a diameter of less than 5 mm. Based on their origin, microplastics can be divided into primary microplastics and secondary microplastics. Primary microplastics are plastics manufactured directly in tiny forms, while secondary microplastics are tiny particles formed from the decomposition of large pieces of plastic waste in the environment. Despite their small size, microplastics are already prevalent even in the most remote marine habitats.

[0004] PET (polyethylene terephthalate) is a specific type of plastic commonly used in the manufacture of beverage bottles, food packaging, and textile fibers (such as polyester fibers) due to its low cost, portability, durability, and gas barrier properties. When PET products break down into fragments smaller than 5 mm in the environment, these PET fragments become part of microplastics. Large quantities of PET are introduced into the environment through its production and disposal; PET waste, with its high durability and moisture resistance, inevitably causes serious environmental pollution.

[0005] Currently, the main methods for recycling PET waste include physical (landfill, incineration, etc.), chemical (pyrolysis, etc.) and biological recycling (microbial enzymatic methods). However, physical and chemical recycling of plastics are costly and pollute the environment, leading to the accumulation of PET in the global ecosystem. Biological recycling technology is currently used less and has considerable room for development. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing a cryophilic protease PETase, which can accurately obtain a cryophilic protease PETase with high novelty and high activity, making the biodegradation process of PET waste more efficient.

[0007] The technical solution of the present invention is: to provide a method for preparing the cryophilic protease PETase, the method comprising:

[0008] Mining and designing PETase candidate sequence sets;

[0009] Enzyme activity data were obtained by measuring the enzyme activity of each candidate enzyme in the PETase candidate sequence set using a derivative of the PET oligomer BHET as a substrate.

[0010] Based on the enzyme activity assay data, the candidate sequence set of PETase was screened to obtain the target psychrophilic protease PETase.

[0011] In one embodiment of the above-described method for preparing a cryotropic protease PETase, the step of mining and designing a PETase candidate sequence set includes:

[0012] Potential homologous sequences were obtained based on existing PETase samples and HMMER;

[0013] Screening the potential homologous sequences to obtain the first PETase candidate sequence; and / or,

[0014] Relevant sequences were obtained based on existing PETase samples and hhblits;

[0015] A second PETase candidate sequence was obtained based on the aforementioned related sequences;

[0016] A PETase candidate sequence set is generated based on the first PETase candidate sequence and / or the second PETase candidate sequence.

[0017] In one embodiment of the above-described method for preparing a cryotropic protease PETase, the step of screening potential homologous sequences to obtain a first PETase candidate sequence includes:

[0018] Calculate the evolutionary distance between each potential homologous sequence and the sequence of the existing PETase sample;

[0019] PETase candidate enzymes are obtained by screening the potential homologous sequences whose evolutionary distance is greater than a first preset threshold;

[0020] The first PETase candidate sequence was obtained by screening the PETase candidate enzymes based on the number of catalytic triplets and the number of disulfide bonds.

[0021] In one embodiment of the above-described method for preparing a cryotropic protease PETase, obtaining a second PETase candidate sequence based on the related sequence includes:

[0022] The adjusted sequence is obtained by adjusting the relevant sequences based on the progen model;

[0023] Based on the DEDAL screening, a second PETase candidate sequence with homology to the existing PETase sample within a preset range is obtained.

[0024] In one embodiment of the above-described method for preparing a cryotropic protease PETase, the step of obtaining enzyme activity data by measuring the enzyme activity of each candidate enzyme in the PETase candidate sequence set using a derivative of the PET oligomer BHET as a substrate includes:

[0025] Obtain enzyme solutions for each candidate enzyme in the PETase candidate sequence set;

[0026] Enzyme activity data were obtained by preparing reaction systems based on the derivatives of the PET oligomer BHET and the enzyme solutions of each candidate enzyme at different temperatures.

[0027] In one embodiment of the above-described method for preparing a cold-loving protease PETase, an expression vector is constructed to obtain the transformed strains of each candidate enzyme in the PETase candidate sequence set;

[0028] The transformed strain was fermented and then lysed to obtain an enzyme solution for each candidate enzyme.

[0029] In one embodiment of the above-described method for preparing a cryophilic protease PETase, the method further includes:

[0030] The target cryotropic protease PETase was optimized based on ESM-1V technology and a saturation site-directed mutagenesis strategy.

[0031] In one embodiment of the above-described method for preparing a cryotropic protease PETase, the optimization of the target cryotropic protease PETase based on ESM-1V technology and a saturation site-directed mutagenesis strategy to obtain an optimized cryotropic protease PETase includes:

[0032] The first beneficial mutant was obtained by screening the target psychrophilic protease PETase according to preset rules;

[0033] The second beneficial mutant was obtained by screening the first beneficial mutant using the ESM-1V model; and / or,

[0034] A third beneficial mutant was obtained by screening the first beneficial mutant based on molecular docking.

[0035] Calculate the residual activity of the second beneficial mutant and / or the third beneficial mutant at 0°C;

[0036] Based on the residual activity of the second beneficial mutant and / or the third beneficial mutant at 0°C, a fourth beneficial mutant is obtained by screening the second beneficial mutant and / or the third beneficial mutant.

[0037] The fourth beneficial mutant obtained by recombining the above-mentioned mutants yielded an optimized psychrophilic protease, PETase.

[0038] In one embodiment of the above-described method for preparing a cryotropic protease PETase, the screening of the second beneficial mutant based on the ESM-1V model includes:

[0039] The mutation site and mutation type information of the first beneficial mutant were obtained based on gene sequencing.

[0040] Based on the ESM-1V model and the mutation site information and mutation type information, a second beneficial mutant with an impact on protein folding energy less than a second preset threshold was selected.

[0041] In one embodiment of the above-described method for preparing a cryotropic protease PETase, the step of obtaining a third beneficial mutant based on molecular docking screening of the first beneficial mutant includes:

[0042] Based on molecular docking screening, a third beneficial mutant was identified in the first beneficial mutant whose distance between BHET-OH and the catalytic triplet was shortened.

[0043] The advantages of this invention are: it provides a method for preparing a cryophilic protease PETase, which involves mining and designing PETase and using a derivative of the PET oligomer BHET as a substrate to obtain enzyme activity data for statistical analysis to screen out the target cryophilic protease PETase. This method can achieve precise screening of highly novel and highly active PET hydrolases, and significantly improve the efficiency of functional enzyme discovery. Attached Figure Description

[0044] The present invention will be further described below with reference to the accompanying drawings and embodiments:

[0045] Figure 1 This is a flowchart illustrating a method for preparing the cryophilic protease PETase according to one embodiment of this application;

[0046] Figure 2 This is a schematic diagram of the reaction pathway of BHET-OH being hydrolyzed and cleaved by PETase in the method for preparing the cold-loving protease PETase provided in one embodiment of this application;

[0047] Figure 3 This is a flowchart illustrating the preparation and application of the cryophilic protease PETase in Example 1 of this application;

[0048] Figure 4 The flowchart of the steps for mining and designing a PETase candidate sequence set in the PETase method provided in Embodiment 1 of this application is as follows:

[0049] Figure 5This is a schematic diagram of the synthetic route of BHET-OH, the substrate for enzyme activity determination in the PETase method provided in Example 1 of this application;

[0050] Figure 6 The NMR spectrum of BHET-OH, the substrate for enzyme activity determination in the PETase method provided in Example 1 of this application;

[0051] Figure 7 This is a schematic diagram of the TPA-OH standard curve in the PETase method provided in Example 1 of this application;

[0052] Figure 8 This is a schematic diagram showing the enzyme activity assay results of 20 candidate enzymes in the PETase method provided in Example 1 of this application.

[0053] Figure 9 This is a schematic diagram of the kinetic monitoring results after the enzyme activity determination of PET-10 and PET-12 enzymes in the PETase method for enzyme activity verification and screening provided in Example 1 of this application. Detailed Implementation

[0054] As mentioned in the background section, China's PET (petrol oxide) production remained relatively stable at around 56 million tons between 2013 and 2023. However, in terms of annual recycling, China only produced about 3 million tons, a significant gap compared to developed economies. This indicates substantial potential for the future development of the environmental protection industry in China. This has also greatly promoted the further industrial production of its monomers, terephthalic acid (TPA) and ethylene glycol (EG), both derived from petroleum.

[0055] The degradation of plastics typically depends on their chemical structure and the conditions required for degradation. For PET (polyethylene terephthalate), degradation mechanisms include, but are not limited to, thermal degradation, hydrolysis, and biodegradation. PET undergoes degradation at high temperatures, leading to polymer chain breakage and a reduction in molecular weight. Furthermore, high temperatures promote oxidation, causing material deterioration. Under relatively low temperatures, PET exhibits high chemical stability and is not prone to significant degradation. However, biodegradable PET or other modified PET materials can gradually degrade under specific environmental conditions (such as composting) through the action of microorganisms. Standard commercial PET, however, does not readily undergo significant chemical degradation at low temperatures. Psychrophilic enzymes maintain their activity at low temperatures primarily due to specific structural features that allow them to retain their function. Several key structural features include: ① Enhanced flexibility: Cold-adapted enzymes typically possess greater molecular flexibility, meaning their protein chains are looser and more prone to vibration and movement. This increased flexibility helps the enzyme maintain its three-dimensional conformation at low temperatures and prevents it from stiffening and losing activity like ordinary enzymes. ② Fewer hydrophobic interactions: Cold-adaptive enzymes may have fewer hydrophobic cores, reducing tight packing within the protein and allowing them to maintain activity at low temperatures. ③ More surface charge: Cold-adaptive enzymes may have more charged amino acid residues on their surface, increasing solubility and stability and preventing the formation of insoluble aggregates at low temperatures. ④ Dynamic side chains: The amino acid side chains of cold-adaptive enzymes may be more dynamic, allowing them to better adapt to changes in the low-temperature environment. ⑤ Enhanced hydration: Cold-adaptive enzymes may have more polar residues, enhancing the stability of the hydration layer and helping the enzyme maintain activity at low temperatures. Through genetic engineering, cold-adaptive enzymes can be immobilized on the cell surface of microorganisms, thereby improving the contact efficiency between the enzyme and the plastic substrate and accelerating the degradation rate. Some microorganisms have evolved the ability to survive in low-temperature environments, secreting specific enzymes to degrade complex organic matter. These microorganisms and their secreted enzymes are crucial for degrading plastics in cold environments. However, the currently used cryophilic protease PETase is relatively limited and singular.

[0056] To address the aforementioned issues, this application creatively proposes a method for preparing a cryophilic protease PETase. By designing and mining PETase, combining it with a derivative of the PET oligomer BHET as a substrate, and obtaining enzyme activity data through enzyme activity assay, the target cryophilic protease PETase can be screened through statistical analysis. This method enables precise screening of highly novel and highly active PET hydrolases, significantly improving the efficiency of functional enzyme discovery.

[0057] Specifically, this application provides a method for preparing the cryophilic protease PETase, referring to... Figure 1 As shown, the method includes:

[0058] S110, Mining and designing PETase candidate sequence sets.

[0059] S120. Using the derivative of the PET oligomer BHET as a substrate, the enzyme activity of each candidate enzyme in the PETase candidate sequence set was measured to obtain enzyme activity data.

[0060] S130. Based on enzyme activity assay data, a candidate sequence set of PETase was screened to obtain the target psychrophilic protease PETase.

[0061] Specifically, the principle of enzyme activity assay is as follows: In the PETase enzyme activity assay, BHET-OH is selected as the reaction substrate. BHET-OH has a hydroxyl group at its aromatic ring, which can avoid interference from the Fenton reaction and subsequent non-specific free radical oxidation products in the hydrolysis reaction mixture. (Refer to...) Figure 2 As shown, BHET-OH is hydrolyzed and cleaved by PETase, releasing the fluorescent product TPA-OH. Therefore, continuous monitoring of the fluorescent TPA-OH can reflect the hydrolytic activity of PETase. TPA-OH exhibits fluorescence upon excitation at a wavelength of 328 nm. The change in fluorescence intensity over a certain period of time can indicate the enzyme activity. Specifically, the stability and feasibility of the high-throughput screening system can be evaluated by measuring the fluorescence intensity of wild-type PETases (i.e., candidate enzymes in the PETase candidate sequence set) in a whole 96-well plate and calculating the residual activity.

[0062] Preferably, the screening criteria are: residual activity between 30% and 40%, and standard deviation below 20%.

[0063] In some embodiments, the PETase candidate sequence set for mining design includes at least one of a first mining design method and a second mining design method.

[0064] The first excavation design method includes:

[0065] Potential homologous sequences were obtained based on existing PETase samples and HMMER;

[0066] The first PETase candidate sequence is obtained by screening the potential homologous sequences.

[0067] Specifically, using existing PETases as existing samples, the HMMER tool was used to search metagenomic databases, identifying a total of 4399 candidate sequences. Subsequently, the first PETase candidate sequence was obtained from these candidate sequences.

[0068] The second excavation design method includes:

[0069] Relevant sequences were obtained based on existing PETase samples and hhblits;

[0070] A second PETase candidate sequence was obtained based on the relevant sequence.

[0071] For example, based on existing PETase samples, known PETase sequences were obtained. Using the hhblits tool with parameters n=3 and B=100000, 28397 homologous sequences, i.e. related sequences, were successfully captured using 16 CPU cores. Based on these related sequences, a second PETase candidate sequence was obtained.

[0072] A PETase candidate sequence set is generated based on the first PETase candidate sequence and / or the second PETase candidate sequence.

[0073] Specifically, when the PETase candidate sequence set for mining design includes the first mining design method, the PETase candidate sequence set contains the first PETase candidate sequence; when the PETase candidate sequence set for mining design includes the second mining design method, the PETase candidate sequence set contains the second PETase candidate sequence; when the PETase candidate sequence set for mining design includes both the first mining design method and the second mining design method, the PETase candidate sequence set contains both the first PETase candidate sequence and the second PETase candidate sequence.

[0074] Preferably, the first mining design method for obtaining the first PETase candidate sequence by screening the potential homologous sequences includes:

[0075] Calculate the evolutionary distance between each potential homologous sequence and the sequence of the existing PETase sample;

[0076] PETase candidate enzymes are obtained by screening the potential homologous sequences whose evolutionary distance is greater than a first preset threshold;

[0077] The first PETase candidate sequence was obtained by screening the PETase candidate enzymes based on the number of catalytic triplets and the number of disulfide bonds.

[0078] Specifically, by calculating the evolutionary distance between the 4399 candidate sequences identified and existing PETase samples, firstly, multiple sequence alignment was performed between the reference sequences of existing PETase samples and the 4399 candidate sequences mined by HMMER. Then, distance models (such as p-distance or Kimu Ra models) were used to calculate the difference rate between each candidate sequence and the reference sequence of existing PETase samples, screening out highly diverse sequences with a difference rate >50%. Next, the screened sequences were ranked based on the integrity of the catalytic triplet and the number of disulfide bonds, prioritizing candidate enzymes with strong structural stability and intact catalytic sites. This process ensures enzyme novelty through distant homology screening and combines key structural features to predict and enhance functional activity potential. Sequences with an evolutionary distance greater than 50 were selected. The difference rate (e.g., pd istance) between each sequence and the reference sequence of existing PETase samples was calculated. Distant homologous sequences with a difference rate >50% were retained to ensure functional novelty. The number of catalytic triples (key to activity) and disulfide bonds (stability indicators) in the remaining sequences were counted and sorted in descending order of total number, prioritizing candidates with high activity and high stability. Patent conflict sequences and structurally abnormal sequences were removed, ultimately resulting in 20 high-potential candidate enzymes. This ensured sufficient diversity among the selected sequences. Furthermore, the number of catalytic triples and disulfide bonds in each sequence was counted and sorted from highest to lowest based on the quantity of these two features, aiming to further explore their functions and characteristics in subsequent studies.

[0079] Preferably, the second mining design method for obtaining the second PETase candidate sequence based on the relevant sequence includes:

[0080] The adjusted sequence is obtained by adjusting the relevant sequences based on the progen model;

[0081] Based on the DEDAL screening, a second PETase candidate sequence with homology to the existing PETase sample within a preset range is obtained.

[0082] For example, the progen2-base model was fine-tuned using eight NVIDIA V100 GPUs, with a learning rate of 2.3e-06 and a weight decay of 0.015. The fine-tuned model was used to adjust relevant sequences, generating 10,000 new PETase sequences, i.e., adjusted sequences. Considering that the DEDAL tool improves performance by two to three times in long-range homology detection and is superior in distinguishing long-range homology, the DEDAL tool was used to identify sequences with high homology detection logits ranging from 84 to 109 from the generated adjusted sequences; these were designated as second PETase candidate sequences.

[0083] In some embodiments, obtaining enzyme activity data by measuring the enzyme activity of each candidate enzyme in the PETase candidate sequence set using a derivative of the PET oligomer BHET as a substrate includes:

[0084] Obtain enzyme solutions for each candidate enzyme in the PETase candidate sequence set;

[0085] Enzyme activity data were obtained by preparing reaction systems based on the derivatives of the PET oligomer BHET and the enzyme solutions of each candidate enzyme at different temperatures.

[0086] Specifically, the candidate enzyme is cultured in shake flasks and then lysed to obtain the enzyme solution. The prepared reaction system is: enzyme solution + buffer + BHET-OH, a derivative based on the PET oligomer BHET. For example, the shadow system consists of 50 μl enzyme solution + 50 μl buffer + 100 μl substrate, where the substrate is BHET-OH.

[0087] In some embodiments, expression vectors are constructed to obtain transformed strains for each candidate enzyme in the PETase candidate sequence set;

[0088] The transformed strain was fermented and then lysed to obtain an enzyme solution for each candidate enzyme.

[0089] Expression vectors are DNA tools that carry target genes and enable their expression in host cells. The construction steps are as follows: ① Select a suitable restriction endonuclease (such as EcoRI or BamHI) to cut the target gene and the multiple cloning site of the expression vector in the candidate enzyme, respectively, so that the two produce complementary sticky ends (or blunt ends); ② Use DNA ligase to ligate the two into a recombinant expression vector; ③ Introduce the recombinant vector into competent cells; ④ After resistance screening and enzyme digestion verification, obtain positive recombinant strains containing the target gene.

[0090] In some embodiments, the method further includes:

[0091] The target cryotropic protease PETase was optimized based on ESM-1V technology and a saturation site-directed mutagenesis strategy.

[0092] The ESM-1V technology used in the embodiments provided in this application trains an ESM-1V language model with 98 million sequences, which can establish a mutation effect prediction platform that does not require training or multiple sequence alignment (MSA). A single mutation scan takes only 10 seconds, and the prediction accuracy surpasses that of models such as UniRep / TAPE. It can overcome the industry pain points of high computational cost and complex process of traditional methods.

[0093] Optimizing the screened target psychrophilic protease using ESM-1V technology and a saturated site-directed mutagenesis strategy can significantly enhance its activity at low temperatures. This will make the biodegradation process of PET waste more efficient, especially in cold regions where traditional high-temperature treatment methods are difficult to implement.

[0094] In some embodiments, optimizing the target cryotropic protease PETase using ESM-1V technology and a saturation site-directed mutagenesis strategy to obtain an optimized cryotropic protease PETase includes:

[0095] The first beneficial mutant was obtained by screening the target psychrophilic protease PETase according to preset rules;

[0096] The second beneficial mutant was obtained by screening the first beneficial mutant using the ESM-1V model; and / or,

[0097] A third beneficial mutant was obtained by screening the first beneficial mutant based on molecular docking.

[0098] Calculate the residual activity of the second beneficial mutant and / or the third beneficial mutant at 0°C;

[0099] Based on the residual activity of the second beneficial mutant and / or the third beneficial mutant at 0°C, a fourth beneficial mutant is obtained by screening the second beneficial mutant and / or the third beneficial mutant.

[0100] The fourth beneficial mutant obtained by recombining the above-mentioned mutants yielded an optimized psychrophilic protease, PETase.

[0101] Specifically, firstly, degenerate primers were designed to perform site-directed saturation mutations of the amino acids at the junctions to charged amino acids. Then, the plasmid containing the mutant library was transformed into *E. coli* BL21(DE3) and cultured on LB agar plates. After single colonies grew, they were transferred to 96-well plates for protein expression. Subsequently, large-scale screening of mutants was conducted based on pre-defined rules to obtain the first beneficial mutant. The pre-defined rules refer to a high-throughput screening system, which can specifically employ the aforementioned screening method of evolutionary distance calculation and structural feature statistical analysis, i.e., the method in the first mining design method of screening potential homologous sequences to obtain the first PETase candidate sequence, and obtaining the second PETase candidate sequence based on the related sequences.

[0102] Then, based on the ESM-1V model, the first beneficial mutant is screened to obtain the second beneficial mutant, and the second beneficial mutant is screened based on its residual activity at 0℃ to obtain the fourth beneficial mutant; or, based on molecular docking, the first beneficial mutant is screened to obtain the third beneficial mutant, and the third beneficial mutant is screened based on its residual activity at 0℃ to obtain the fourth beneficial mutant; or, based on the ESM-1V model, the first beneficial mutant is screened to obtain the second beneficial mutant, and based on molecular docking, the first beneficial mutant is screened to obtain the third beneficial mutant, and the second and third beneficial mutants are screened based on their residual activity at 0℃ to obtain the fourth beneficial mutant.

[0103] After screening to obtain the fourth beneficial mutant, the fourth beneficial mutant was recombined to obtain the optimized psychrophilic protease PETase.

[0104] Recombining single-site beneficial mutants yields the optimal recombinant mutant. Based on the mutant analysis and grouping results from the previous stage, selecting recombinant combinations of beneficial mutants with high activity at low temperatures can improve the effect of degrading PET at low temperatures.

[0105] In some embodiments, the screening of the second beneficial mutant based on the ESM-1V model includes:

[0106] The mutation site and mutation type information of the first beneficial mutant were obtained based on gene sequencing.

[0107] Based on the ESM-1V model and the mutation site information and mutation type information, a second beneficial mutant with an impact on protein folding energy less than a second preset threshold was selected.

[0108] ESM-1V is a transformer language model with 650 million parameters, designed specifically for predicting the effects of protein mutations. Trained on 98 million diverse protein sequences, it uses only sequence data without relying on any functional experimental measurements. ESM-1V demonstrates exceptional zero-shot inference capabilities, outperforming other models on 30 out of 41 deep mutation scanning tasks, achieving state-of-the-art performance without additional training. The model is computationally efficient, requiring an average of only 10 seconds to label a deep mutation scan. ESM-1V achieves outstanding results on multiple benchmarks using a probabilistic scoring method with masked margins, demonstrating its potential for understanding and designing proteins.

[0109] Specifically, after the first beneficial mutant is obtained through screening, its gene sequencing is performed to determine the specific mutation site and mutation type. Then, a comprehensive analysis of the position of the mutated amino acid and the mutation type is performed on all the sequenced first beneficial mutants, and the position of the mutation in the protein structure (such as catalytic pocket / protein surface / hydrophobic core) is recorded. Based on the ESM-1V model, the first beneficial mutant is screened to obtain the second beneficial mutant.

[0110] Then, the trained ESM-1V model was used to predict the effect of mutations on protein folding energy (ΔΔG), and mutants with ΔΔG < 0 (stabilization) were screened. Mutants were grouped according to the degree of activity enhancement at low temperatures (e.g., high-gain group: >200% wild-type; medium-gain group: 100-200%), and common mutational characteristics were traced back. The enhancement of the target trait was determined by factors such as whether the amino acid is charged, the size of the amino acid side chain, the hydrophobicity of the amino acid, or the formation of hydrogen bonds. The enzyme activity data of the screened mutants were analyzed, the residual activity of the mutants was calculated, and the activity levels were compared to obtain a second beneficial mutant.

[0111] In some embodiments, obtaining a third beneficial mutant by screening the first beneficial mutant based on molecular docking includes:

[0112] Based on molecular docking screening, a third beneficial mutant was identified in the first beneficial mutant whose distance between BHET-OH and the catalytic triplet was shortened.

[0113] Specifically, a third beneficial mutant with a shortened distance between BHET-OH and the catalytic triplet was obtained through flexible conformational screening using MD simulation. Molecular docking (e.g., AutoDock Via) was then used to verify whether the mutation optimized substrate binding (e.g., shortened distance between BHET-OH and the catalytic triplet).

[0114] The following specific examples illustrate the preparation method of the psychrophilic protease PETase provided in this application.

[0115] Example 1: This example provides a method for preparing the cold-loving protease PETase, referring to... Figure 3 As shown, the method includes:

[0116] I. Reference Figure 4 As shown, using existing PETase samples, the HMMER and HHblits models were employed to discover and design PET hydrolases:

[0117] (1) The HMMER tool was used to search the metagenomic database, and a total of 4399 candidate sequences were identified. Then, the evolutionary distance between these candidate sequences and PETase was calculated. First, multiple sequence alignment was performed between the reference sequence of the existing PETase sample and the 4399 candidate sequences mined by HMMER. Then, the sequence difference rate between each candidate sequence and PETase was calculated using a distance model (such as p-distance or Kimu ra model), and high-diversity sequences with a difference rate >50% were screened. Finally, the screened sequences were ranked based on the integrity of the catalytic triplet and the number of disulfide bonds, and candidate enzymes with strong structural stability and intact catalytic sites were given priority. Sequences with an evolutionary distance greater than 50 were selected. The sequence difference rate (e.g., pd i stance) between each sequence and known PETases was calculated, and distant homologous sequences with a difference rate >50% were retained to ensure functional novelty. The number of catalytic triples (key to activity) and disulfide bonds (stability indicators) in the remaining sequences were counted and sorted in descending order of total number, prioritizing candidates with high activity and high stability. Patent conflict sequences (e.g., sequences with conflicting patents) and structurally anomalous sequences (e.g., sequences containing multiple disulfide bonds) were removed, resulting in 20 high-potential candidate enzymes for subsequent experiments. This ensured sufficient diversity among the selected sequences. Furthermore, the number of catalytic triples and disulfide bonds in each sequence was counted and sorted from highest to lowest based on the quantity of these two characteristics.

[0118] (2) Based on the reference sequences of existing PETase samples, the hhblits tool, with parameters n=3 and B=100000, successfully captured 28397 homologous sequences using 16 CPU cores. The progen2-base model was then fine-tuned using 8 NVIDIA V100 GPUs, with a learning rate of 2.3e-06 and a weight decay of 0.015. The fine-tuned model generated 10000 new PETase sequences. Considering that the DEDAL tool improves performance by two to three times in long-range homology detection and is superior in distinguishing long-range homology, the DEDAL tool was selected to identify sequences with high homology detection logits from the generated sequences, ranging from 84 to 109.

[0119] The sequences selected by combining the two methods above are used to obtain the PETase candidate sequence set.

[0120] II. Establishment of a high-throughput screening system for PET enzyme activity assay:

[0121] To achieve the goal of high-throughput screening, the candidate enzymes successfully expressed from the newly discovered and designed PETase candidate sequence set were incubated at different temperatures for a certain period of time, and their enzyme activity was measured.

[0122] The substrate for enzyme activity assay is BHET-OH. The synthesis method of BHET-OH is as follows: Figure 5 As shown: 1.82 g substrate acid, 15 ml ethylene glycol, 0.5 ml sulfuric acid, reacted at 200 °C for 48 h. The final NMR spectrum of BHET-OH is shown below. Figure 6 As shown.

[0123] BHET-OH was hydrolyzed and cleaved by the candidate PETase, releasing the fluorescent product TPA-OH. The excitation wavelength of TPA-OH was 320 nm, and the emission wavelength was 400 nm. The standard curve of TPA-OH was also measured. Figure 7 As shown, the stability and feasibility of the high-throughput screening system were evaluated by measuring the fluorescence intensity of the wild-type PET enzyme (the candidate enzyme) in a whole 96-well plate and calculating the residual activity. The residual activity was between 30% and 40% with a standard deviation of less than 20%.

[0124] Twenty candidate enzymes were labeled PET1, PET2, ..., PET20. Preliminary validation of these 20 enzymes was performed by culturing them in 96-well plates. PETase and Depo PETase served as controls, and PET-22b was used as an empty vector. The reaction system for enzyme activity assays consisted of 50 μl enzyme solution + 50 μl buffer + 100 μl substrate. The validation results for the 20 enzymes are as follows: Figure 8 As shown, PET-10 and PET-12 can be further verified.

[0125] PETase, Depo PETase, PET-10, PET-12, and PET-22b were cultured in shake flasks, with PETase and Depo PETase serving as controls, PET-10 and PET-12 as candidate enzymes, and PET-22b as an empty vector. The resulting enzyme solutions were incubated at 0℃, 10℃, 20℃, 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, and 90℃ for 2 hours before enzyme activity assays. The reaction system consisted of 50 μl enzyme solution + 50 μl buffer + 100 μl substrate (BHET-OH). Kinetics were monitored for 15 minutes. The results are shown below. Figure 9 As shown, PET-10 exhibits good activity within the range of 0-30℃, with the highest activity at 0℃. A PETase that significantly enhances the activity of the target enzyme at low temperatures was successfully prepared.

[0126] III. Establishment and screening of high-throughput mutant libraries, and evaluation of mutants using ESM-1V technology.

[0127] Construction and Screening of High-Quality Mutant Libraries: High-quality mutant libraries were constructed using site-directed saturation mutagenesis. First, degenerate primers were designed to perform site-directed saturation mutagenesis of the junction amino acids to charged amino acids. Then, the plasmid containing the mutant library was transformed into *E. coli* BL21(DE3) and cultured on LB agar plates. After single colonies grew, they were transferred to 96-well plates for protein expression. Subsequently, a high-throughput screening system was used for large-scale screening of the mutants. Using wild-type PETase as a baseline (100% activity), the relative residual activity of the mutants was required to be greater than that of the control group (background values ​​of the empty vector PET-22b were excluded). Enzyme activity was quantified by the rate of change in fluorescence intensity (ΔF / min) over 15 minutes using the fluorescent product TPA-OH (excitation / emission wavelengths: 328 nm / 400 nm) released after hydrolysis of the BHET-OH substrate.

[0128] After identifying beneficial mutants, their genes were sequenced to determine specific mutation sites and types. A comprehensive analysis of the mutated amino acid positions and mutation types was then performed on all sequenced beneficial mutants, recording the mutation's location in the protein structure (e.g., catalytic pocket / protein surface / hydrophobic core). The ESM-1V model was used to predict the impact of mutations on protein folding energy (ΔΔG), and mutations with ΔΔG < 0 (stabilization) were screened. Molecular docking (e.g., AutoDock Vina) was used to verify whether the mutation optimized substrate binding (e.g., shortening the distance between BHET-OH and the catalytic triplet). Mutants were grouped according to the degree of activity enhancement at low temperatures (e.g., high-gain group: >200% wild-type; medium-gain group: 100-200%), and common mutation characteristics were traced back. This indicated whether the enhancement of the target trait was determined by factors such as amino acid charge, amino acid side chain size, amino acid hydrophobicity, or hydrogen bond formation. The enzyme activity data of the screened mutants were analyzed, the residual activity of the mutants was calculated, and the activity levels were compared.

[0129] The optimal recombinant mutant was obtained by recombinizing single-site beneficial mutants. Based on the mutant analysis and grouping results from the previous stage, recombinant combinations of beneficial mutants with high activity at low temperatures were selected to improve the effect of degrading PET at low temperatures.

[0130] Finally, the optimal mutant can be enzymatically characterized and its molecular mechanism analyzed.

[0131] The method for preparing the psychrophilic protease PETase provided in this application has the following beneficial effects: 1. Improved degradation efficiency: By using the HMMER and HHblits models to discover and design PET hydrolases, and by employing ESM-1V technology and a saturated site-directed mutagenesis strategy to optimize and modify wild-type PETase, the activity of the target enzyme at low temperatures can be significantly improved. This will make the biodegradation process of PET waste, especially in cold regions where traditional high-temperature treatment methods are difficult to implement, more efficient.

[0132] 2. Reduced energy consumption and costs: The improved PETase developed in this study can operate under relatively mild or even low-temperature conditions, thereby greatly reducing energy consumption and processing costs.

[0133] 3. Reduce environmental pollution: The improved PETase can more effectively decompose PET waste, which helps to alleviate the global pollution problem caused by plastic waste, and is of great significance for the treatment of microplastic pollution.

[0134] 4. Expanding Application Scope: Enhancing the adaptability of microorganisms and the possibility of large-scale production of cold-adaptive biodegradable agents. This means that the new technology is expected to be applied to more fields, such as agricultural film recycling and marine plastic pollution control, providing solutions for plastic pollution in different scenarios.

[0135] In summary, this study has achieved effective modification of PETase through technological innovation, which not only improves its activity under low-temperature conditions, but also provides a low-cost and high-efficiency plastic waste treatment solution for the environmental protection industry, and has significant economic and social value.

[0136] Of course, the above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be used to limit the scope of protection of the present invention. All modifications made according to the spirit and essence of the main technical solution of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a cold-loving protease PETase, characterized in that, The method includes: Mining and designing PETase candidate sequence sets; Enzyme activity data were obtained by measuring the enzyme activity of each candidate enzyme in the PETase candidate sequence set using a derivative of the PET oligomer BHET as a substrate. Based on the enzyme activity assay data, the candidate sequence set of PETase was screened to obtain the target psychrophilic protease PETase.

2. The method for preparing a cryophilic protease PETase according to claim 1, characterized in that, The PETase candidate sequence set for mining design includes: Potential homologous sequences were obtained based on existing PETase samples and HMMER; Screening the potential homologous sequences to obtain the first PETase candidate sequence; and / or, Relevant sequences were obtained based on existing PETase samples and hhblits; A second PETase candidate sequence was obtained based on the aforementioned related sequences; A PETase candidate sequence set is generated based on the first PETase candidate sequence and / or the second PETase candidate sequence.

3. The method for preparing a cryophilic protease PETase according to claim 2, characterized in that, The step of screening the potential homologous sequences to obtain the first PETase candidate sequence includes: Calculate the evolutionary distance between each potential homologous sequence and the sequence of the existing PETase sample; PETase candidate enzymes are obtained by screening the potential homologous sequences whose evolutionary distance is greater than a first preset threshold; The first PETase candidate sequence was obtained by screening the PETase candidate enzymes based on the number of catalytic triplets and the number of disulfide bonds.

4. The method for preparing a cryophilic protease PETase according to claim 2, characterized in that, The step of obtaining the second PETase candidate sequence based on the relevant sequence includes: The adjusted sequence is obtained by adjusting the relevant sequences based on the progen model; Based on the DEDAL screening, a second PETase candidate sequence with homology to the existing PETase sample within a preset range is obtained.

5. The method for preparing a cryophilic protease PETase according to claim 1, characterized in that, The enzyme activity assay data obtained by using a derivative of the PET oligomer BHET as a substrate to measure the enzyme activity of each candidate enzyme in the PETase candidate sequence set includes: Obtain enzyme solutions for each candidate enzyme in the PETase candidate sequence set; Enzyme activity data were obtained by preparing reaction systems based on the derivatives of the PET oligomer BHET and the enzyme solutions of each candidate enzyme at different temperatures.

6. The method for preparing a cryophilic protease PETase according to claim 5, characterized in that, Expression vectors were constructed to obtain transforming strains for each candidate enzyme in the PETase candidate sequence set; The transformed strain was fermented and then lysed to obtain an enzyme solution for each candidate enzyme.

7. The method for preparing a cryophilic protease PETase according to any one of claims 1-6, characterized in that, The method further includes: The target cryotropic protease PETase was optimized based on ESM-1V technology and a saturation site-directed mutagenesis strategy.

8. The method for preparing a cryophilic protease PETase according to claim 7, characterized in that, The optimization of the target cryotropic protease PETase based on ESM-1V technology and saturation site-directed mutagenesis strategy to obtain the optimized cryotropic protease PETase includes: The first beneficial mutant was obtained by screening the target psychrophilic protease PETase according to preset rules; The second beneficial mutant was obtained by screening the first beneficial mutant using the ESM-1V model; and / or, A third beneficial mutant was obtained by screening the first beneficial mutant based on molecular docking. Calculate the residual activity of the second beneficial mutant and / or the third beneficial mutant at 0°C; Based on the residual activity of the second beneficial mutant and / or the third beneficial mutant at 0°C, a fourth beneficial mutant is obtained by screening the second beneficial mutant and / or the third beneficial mutant. The fourth beneficial mutant obtained by recombining the above-mentioned mutants yielded an optimized psychrophilic protease, PETase.

9. The method for preparing a cryophilic protease PETase according to claim 8, characterized in that, The screening of the second beneficial mutant based on the ESM-1V model includes: The mutation site and mutation type information of the first beneficial mutant were obtained based on gene sequencing. Based on the ESM-1V model and the mutation site information and mutation type information, a second beneficial mutant with an impact on protein folding energy less than a second preset threshold was selected.

10. The method for preparing a cryophilic protease PETase according to claim 8, characterized in that, The process of obtaining a third beneficial mutant by screening the first beneficial mutant based on molecular docking includes: Based on molecular docking screening, a third beneficial mutant was identified in the first beneficial mutant whose distance between BHET-OH and the catalytic triplet was shortened.

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