Polypeptide and its use, method, composition or kit for preparing polypeptide

Abstract

The present application discloses a polypeptide and its use, a method for preparing the polypeptide, a composition or a kit. The amino acid sequence of the polypeptide is shown in SEQ ID NO: 1; the method comprises: culturing the recombinant cell described in the fourth aspect to obtain the polypeptide. The polypeptide of the present application has lysozyme activity, PETase activity and chitinase activity, and can be used as a trifunctional hydrolase that simultaneously degrades bacterial cell walls, hydrolyzes chitin and hydrolyzes PET; the polypeptide of the present application can be expressed in large quantities in a protein expression system, and thus, the method of the present application can be mass-produced and standardized; the composition or kit containing the above-mentioned polypeptide plays an important role in biological control, medical applications, waste treatment and environmental protection.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology, and specifically relates to polypeptides and uses thereof, methods, compositions or kits for preparing polypeptides, and more specifically to a trifunctional hydrolase of lysozyme, chitinase and PETase obtained by screening based on an artificial intelligence model and uses thereof. Background Art

[0002] Lysozyme, also known as muramidase or N-acetylmuramide glycanohydrlase, is an alkaline enzyme that can hydrolyze mucopolysaccharides in bacteria. Lysozyme mainly breaks down the β-1,4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the cell wall, decomposing the insoluble mucopolysaccharides in the cell wall into soluble glycopeptides, causing the cell wall to rupture and the contents to escape, thereby dissolving the bacteria. Lysozyme can also directly bind to negatively charged viral proteins, forming complexes with DNA, RNA, and apoproteins to inactivate the virus. The enzyme is widely present in many tissues of the human body, and is also found in egg whites of birds and poultry, tears, saliva, plasma, milk and other fluids of mammals, as well as microorganisms, with the highest content in egg white.

[0003] Chitin (also known as chitosan) is the second largest natural polymer compound after cellulose, which is formed by the polymerization of N-acetyl-D-glucosamine (GlcNAc). Chitinase is an enzyme that catalyzes the hydrolysis of chitin and can break the glycosidic bonds in chitin.

[0004] Polyethylene terephthalate (PET or PETE) is a common resin in life and can be divided into APET, RPET and PETG. PETase can degrade PET plastic into its monomer mono-2-hydroxyethyl terephthalic acid (MHET) molecule. MHET will be further degraded into hydroxyethyl terephthalic acid by MHETase in bacteria, and then decomposed into environmentally friendly terephthalic acid and ethylene glycol in water, which can be used by other bacteria and finally produce carbon dioxide and water.

[0005] At present, no hydrolase has been found that can dissolve bacterial cell walls, hydrolyze chitin and hydrolyze PET at the same time. Summary of the invention

[0006] The present application aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present application provides a trifunctional hydrolase of lysozyme, chitinase and PETase.

[0007] This application is completed based on the following findings of the inventors:

[0008] Lysozyme, chitinase and PETase can be used in many fields. Among them:

[0009] 1. Application of lysozyme

[0010] 1.1 Application in the medical field

[0011] Antibacterial effect: Lysozyme can destroy the peptidoglycan structure in the bacterial cell wall, causing the bacterial cell wall to rupture and the contents to leak out, thereby causing the bacteria to dissolve and die.

[0012] Antiviral effect: Lysozyme can exert its antiviral effect by binding to viral coat proteins, preventing the virus from adsorbing and invading host cells.

[0013] Tissue repair: Lysozyme can remove the debris of damaged tissues and cells, creating a good environment for the growth and repair of new cells.

[0014] Clinical application: Lysozyme is used to treat chronic rhinitis, acute and chronic pharyngitis, oral ulcers, chickenpox, herpes zoster and flat warts.

[0015] 1.2 Application in the food industry

[0016] Food preservation: Lysozyme can be used as a preservative by hydrolyzing bacterial cell walls, destroying the β-1,4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the cell wall, and decomposing the insoluble mucopolysaccharides in the cell wall into soluble glycopeptides, causing the cell wall to rupture and the contents to escape, thereby dissolving the bacteria.

[0017] Food preservation: Adding lysozyme to dairy products, meat products, cakes, sake, cooking wine and beverages can extend the shelf life of food.

[0018] 2. Application of Chitinase

[0019] 2.1 Biological control: Chitinase has a significant biological control effect in the prevention of plant fungal and pest diseases and mosquito-borne zoonotic diseases, and can effectively control plant fungal infections and pest diseases.

[0020] 2.2 Medical application: The products of chitin degradation by chitinase, such as N-acetylglucosamine oligosaccharides and chitosan oligosaccharides, have multiple physiological functions such as anti-fungal infection, anti-tumor, anti-infection and prevention and treatment of malaria.

[0021] 2.3 Waste treatment: In daily life and industrial production activities, a large amount of chitin waste is generated, such as crab shells, shrimp shells, etc. These wastes will cause serious pollution to the natural environment such as water bodies. Chitinase-producing microorganisms are widely used in the treatment of chitin waste, improving the utilization rate of waste and preventing water bodies and other environments from being polluted.

[0022] 2.4 Agricultural application: Chitinase can improve plant resistance, enhance plant disease resistance, and reduce losses caused by pests and diseases. At the same time, chitinase can also improve soil quality, increase soil fertility, promote plant growth and development, and increase crop yields.

[0023] 2.5 Food processing: Chitinase is used in the food processing industry to degrade chitin, produce biologically active oligosaccharides or monosaccharides, and improve the nutritional value and functionality of food.

[0024] 2.6 Environmental protection: Chitinase is used in the field of environmental protection to treat waste containing chitin, such as shrimp shells, crab shells, etc., to reduce environmental pollution and improve resource utilization.

[0025] 3. Application of PETase

[0026] 3.1 Degradation of PET plastics: PET enzyme can catalyze the hydrolysis reaction of PET plastics, decomposing them into terephthalic acid and ethylene glycol, thereby achieving biodegradation of PET plastics.

[0027] 3.2 Environmental protection and resource recycling: By degrading PET plastic, PET enzyme helps reduce the pollution of plastic waste to the environment and promotes the recycling of resources.

[0028] 3.3 Industrial application: PETase shows great potential in plastic recycling and biodegradation and can be used to treat PET waste in industrial production.

[0029] However, existing lysozymes, chitinases and PETases are all single-activity proteins, either with only lysozyme activity, only chitinase activity, or only PETase activity. If multiple protein functions need to be achieved, multiple enzyme proteins need to be used at the same time, which is costly and different enzyme proteins may affect each other.

[0030] Based on this, the present application designs a trifunctional hydrolase (referred to as trifunctional hydrolase) protein sequence of lysozyme, chitinase and PETase that are different from natural products based on artificial intelligence technology, and screens the designed protein sequence to obtain the trifunctional hydrolase of the present application. The trifunctional hydrolase can be prepared in a large-scale standardized protein expression system and has lysozyme activity, PETase activity and chitinase activity at the same time.

[0031] Therefore, in the first aspect of the present application, the present application proposes a polypeptide. According to an embodiment of the present application, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1. The polypeptide of the present application has lysozyme activity, PETase activity and chitinase activity, and can be used as a trifunctional hydrolase that simultaneously degrades bacterial cell walls, hydrolyzes chitin and hydrolyzes PET, and has excellent thermal stability.

[0032] APPPTLVVYWGQNGSEGTLAEACATGRYDLVNIAFLDVFGRGSPPPVITLDGHCDDPDDTGCPGLAALKSCQAKGIKVLLSIGGGSGGYGLSSPEDAKSVANYLWDNFLGGSSSRPLGDAVLDGIDLDIEKGGATGH WDDLARFLKAYSGVGRRVYLTAAPQCPFPDASLGPALDTGLFDYVWVQFYNNPPCQYSSGNINNLVKAWNQWTTSIPARVFLGLPAAPEAAGSGYIPPDVLTSQILPAVKTSAKYGGVMLWSRYYDELTGYSSKIKHHV (SEQ ID NO:1).

[0033] It should be noted that in the present application, "the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 1" means that the polypeptide has an amino acid sequence as shown in SEQ ID NO: 1, or a polypeptide has an amino acid sequence in a conservatively modified form as shown in SEQ ID NO: 1, or a polypeptide has an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, all of which are within the scope of protection of the present application.

[0034] According to an embodiment of the present application, the polypeptide has an amino acid sequence as shown in SEQ ID NO: 1 or a sequence having at least 90% identity thereto.

[0035] In the second aspect of the present application, the present application proposes a nucleic acid molecule. According to an embodiment of the present application, the nucleic acid molecule encodes the polypeptide described in the first aspect. The nucleic acid molecule of the present application can encode the polypeptide of the first aspect, and the polypeptide has lysozyme activity, PETase activity, and chitinase activity.

[0036] According to an embodiment of the present application, the nucleic acid molecule is DNA.

[0037] It should be noted that, for the nucleic acid molecules mentioned herein, those skilled in the art will understand that they actually include any one or two of the complementary double strands. For convenience, in this article, although only one strand is provided in most cases, the other strand complementary thereto is actually disclosed. In addition, the nucleic acid molecule sequence in this application includes a DNA form or an RNA form, and disclosing one of them means that the other is also disclosed.

[0038] In the third aspect of the present application, the present application proposes an expression vector. According to an embodiment of the present application, the expression vector carries the nucleic acid molecule described in the second aspect. When the above-mentioned nucleic acid molecule is connected to the expression vector, the above-mentioned nucleic acid molecule can be directly or indirectly connected to the control element on the expression vector, as long as these control elements can control the translation and expression of the above-mentioned nucleic acid molecule. Of course, these control elements can come directly from the expression vector itself, or they can be exogenous, that is, not from the expression vector itself. Of course, the above-mentioned nucleic acid molecule can be operably connected to the control element.

[0039] Herein, "operably linked" means connecting the exogenous gene to the expression vector so that the control elements in the expression vector, such as transcription control sequences and translation control sequences, etc., can play their intended functions of regulating the transcription and translation of the exogenous gene. Commonly used expression vectors can be, for example, plasmids, bacteriophages, etc. After the expression vectors according to some specific embodiments of the present application are introduced into suitable recipient cells, the expression of the aforementioned polypeptide can be effectively achieved under the mediation of the regulatory system, thereby achieving large-scale in vitro acquisition of the polypeptide.

[0040] In some specific embodiments of the present application, the expression vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus or a bacteriophage.

[0041] In some specific embodiments of the present application, the expression vector is a lentiviral vector.

[0042] In an optional embodiment of the present application, the expression vector is a plasmid expression vector.

[0043] In the fourth aspect of the present application, the present application proposes a recombinant cell. According to an embodiment of the present application, the recombinant cell carries the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect, or the recombinant cell expresses the polypeptide described in the first aspect. The recombinant cell can be used to effectively express the aforementioned polypeptide in the recombinant cell under suitable conditions.

[0044] According to an embodiment of the present application, the recombinant cell is obtained by introducing the expression vector described in the fourth aspect into a host cell.

[0045] It should be noted that the host cell of the present application is not particularly limited and can be a prokaryotic cell, a eukaryotic cell or a bacteriophage. The prokaryotic cell can be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc. The aforementioned eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, insect cells such as armyworm, plant cells such as tobacco, mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells, etc.

[0046] According to an embodiment of the present application, the host cell is a eukaryotic cell.

[0047] According to an embodiment of the present application, the host cell is a mammalian cell, including but not limited to BHK cells, CHO cells, NSO cells or COS cells, and does not include animal germ cells, fertilized eggs or embryonic stem cells.

[0048] It should be noted that the "suitable conditions" described in this application refer to conditions suitable for the expression of the polypeptides described in this application. It is easy for those skilled in the art to understand that conditions suitable for polypeptide expression include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy cell states, suitable cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for polypeptide expression according to the specific environment of the laboratory.

[0049] In the fifth aspect of the present application, the present application proposes a method for preparing a polypeptide. According to an embodiment of the present application, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1; the method comprises: culturing the recombinant cell described in the fourth aspect to obtain the polypeptide. The method of the present application can be scaled and standardized for production, and the obtained polypeptide has lysozyme activity, PETase activity, and chitinase activity.

[0050] In the sixth aspect of the present application, the present application proposes the use of the polypeptide described in the first aspect as a trifunctional hydrolase of lysozyme, chitinase and PETase. As can be seen from the foregoing, the polypeptide of the first aspect has lysozyme activity, PETase activity and chitinase activity, and has excellent thermal stability. Thus, the polypeptide of the present application can be used as a trifunctional hydrolase of lysozyme, PETase and chitinase, which can dissolve bacteria, degrade chitin and PET at the same time, and is used in environmental governance, industrial fields and agricultural fields; it can also be used for antifungal, antitumor, etc.

[0051] Herein, the "trifunctional hydrolase of lysozyme, chitinase and PETase" refers to a trifunctional hydrolase that simultaneously has the functions of lysing bacteria, degrading chitin, and degrading PET.

[0052] In the seventh aspect of the present application, the present application proposes a reagent or a kit. According to an embodiment of the present application, the reagent or the kit comprises the polypeptide described in the first aspect. As can be seen from the foregoing, the polypeptide of the first aspect has lysozyme activity, PETase activity, and chitinase activity. Thus, the composition reagent or the kit containing the above polypeptide can be used in environmental management, agriculture, and industry.

[0053] In the eighth aspect of the present application, the present application proposes a composition. According to an embodiment of the present application, the composition includes the polypeptide described in the first aspect. As can be seen from the foregoing, the polypeptide of the first aspect has lysozyme activity, PETase activity, and chitinase activity. Thus, the composition containing the above polypeptide can be used in environmental management, agriculture, industry, and medicine.

[0054] According to an embodiment of the present application, the composition includes but is not limited to a food composition, a pharmaceutical composition, and an agricultural composition.

[0055] According to an embodiment of the present application, the composition may be a pharmaceutical composition, and further the composition includes a pharmaceutically acceptable excipient.

[0056] In the ninth aspect of the present application, the present application proposes a use of the polypeptide of the first aspect in preparing a product, wherein the product is used for at least one of the following: dissolving bacteria, hydrolyzing chitin and / or hydrolyzing PET, treating waste, antifungal, and food preservation. As can be seen from the foregoing, the polypeptide of the first aspect has excellent lysozyme activity, PETase activity, and chitinase activity, and can be used in environmental management, agriculture, industry, and medicine. Thus, the product of the present application can be used for dissolving bacteria, hydrolyzing chitin and / or hydrolyzing PET, treating waste, antifungal, antiviral, etc.

[0057] In this article, "waste treatment" means that the polypeptide of the present application or a product containing the polypeptide of the present application can achieve the purpose of waste treatment by degrading items containing bacteria, chitin and / or PET.

[0058] According to the embodiments of the present application, the products include but are not limited to reagents, kits, and pharmaceutical compositions.

[0059] According to an embodiment of the present application, the product is selected from food preservatives, antibacterial agents, waste treatment agents, fungi inhibitors, and drugs.

[0060] In the tenth aspect of the present application, the present application proposes a method for hydrolyzing bacteria, chitin and / or hydrolyzing PET, or treating waste. According to an embodiment of the present application, the method comprises: contacting a sample or waste containing bacteria, chitin and / or PET using the polypeptide described in the first aspect, the polypeptide prepared by the method described in the fifth aspect, the reagent or kit described in the seventh aspect, or the composition described in the eighth aspect.

[0061] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0063] Figure 1 A flowchart for protein design in the examples of this application;

[0064] Figure 2 These are western blots bands showing the expression of the trifunctional hydrolase in the examples of this application. DETAILED DESCRIPTION

[0065] The embodiments of the present application are described in detail below. The embodiments described below are exemplary and are only used to explain the present application, and cannot be understood as limiting the present application.

[0066] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of this application, unless otherwise specified, the meaning of "plurality" is two or more.

[0067] In this document, the terms "include" or "comprising" are open expressions, that is, including the contents specified in the present application but not excluding other contents.

[0068] In this article, "conservatively modified forms of amino acid sequences" refer to amino acid modifications that do not significantly affect or change the lysozyme, PETase activity, chitinase activity comprising the amino acid sequence, including amino acid substitutions, additions and deletions. Modifications can be introduced into the polypeptides of the present application by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which the amino acid residues are replaced by amino acid residues with similar side chains. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (such as lysine, arginine, histidine), amino acids with acidic side chains (such as aspartic acid, glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (such as threonine, valine, isoleucine) and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine). Exemplarily, conservative modifications are in a number not exceeding 20% ​​of the total number, preferably not exceeding 10% of the total number.

[0069] As used herein, the terms "identity", "homology" or "similarity" are used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70:173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs that utilize these algorithms are also available, and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al. (1990) J. Mol. Biol. 215:403-410). et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0070] In this article, the term "having at least 90% sequence similarity" can be a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence similarity. The sequence identity described in the application can be measured using sequence analysis software. For example, the computer program BLAST, especially BLASTP or TBLASTN using default parameters is used. The amino acid sequences described in the application are all shown in a manner from N to C.

[0071] As used herein, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients comprising the polypeptide or derivative thereof and / or the mammal to be treated therewith.

[0072] In this article, the term "pharmaceutically acceptable excipient" may include any solvent, including but not limited to pharmaceutically acceptable stabilizers, diluents or other liquid excipients, etc., suitable for a specific target dosage form. In addition to any conventional excipients and the polypeptides, pharmaceutical compositions or drugs containing them in the present application, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, their use is also within the scope of consideration of the present application.

[0073] In addition to any conventional excipients, the use of excipients that are incompatible with the polypeptides, pharmaceutical compositions or drugs containing them of the present application, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, is also within the scope of consideration of the present application.

[0074] As used herein, the term "treatment" refers to the use of drugs to obtain the desired pharmacological and / or physiological effects. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as blocking the progression of the disease; or (c) alleviating the disease, such as alleviating symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug of a polypeptide or a derivative thereof to an individual to treat, cure, alleviate, improve, mitigate or inhibit an individual's disease, including but not limited to administering a drug containing a polypeptide described herein to an individual in need.

[0075] The scheme of the present application will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present application and should not be considered as limiting the scope of the present application. If no specific technology or conditions are indicated in the embodiments, the technology or conditions described in the literature in this area or the product specification are carried out. The reagents used or the instruments that do not indicate the manufacturer are all conventional products that can be obtained commercially.

[0076] Embodiment 1:

[0077] The schematic diagram of the polypeptide design and screening process of the present application is shown in Figure 1, which covers a series of steps from the generation of the protein sequence of the trifunctional hydrolase of lysozyme, chitinase and PETase (hereinafter referred to as the trifunctional hydrolase) to the screening and optimization.

[0078] See also Figure 1 The design and screening mainly involves generating trifunctional hydrolase protein sequences through a protein generation model (PGM), screening out target (quality-qualified) trifunctional hydrolase protein sequences from the generated trifunctional hydrolase protein sequences, synthesizing protein-coding DNA based on the screened target trifunctional hydrolase protein sequences, expressing them in a protein expression system, and expressing the trifunctional hydrolase ability and thermal stability of the protein through a wet-lab assay (Wet-labAssay).

[0079] 1. Generation of trifunctional hydrolase protein sequences

[0080] Exemplarily, first, a protein design agent (PDA) is used to generate structured prompt instructions that meet the needs of protein design, so as to provide the protein generation model with richer and more accurate prompt instructions to guide protein design. This step can also be performed by manually writing prompt instructions. Then, the protein generation model is initially trained (Initial Training) using data of mono-function enzymes (Mono-function Enzymes) and multi-function enzymes (Multi-function Enzymes). In addition, the protein generation model is fine-tuned (Fine-tuning) based on the data of specific enzymes (such as lysozyme, chitinase and PETase) to optimize the function of the protein generation model. Finally, after the protein generation model is trained, the protein generation model is used to generate (Generation) a trifunctional hydrolase protein sequence design (Protein Designs) according to the prompt instructions.

[0081] 2. Screening of target trifunctional hydrolase protein sequences

[0082] Exemplarily, the Unified Sequence Model (USM) is used to screen out target trifunctional hydrolase protein sequences of qualified quality from the generated trifunctional hydrolase protein sequences.

[0083] For example, the USM Lysozyme Predictor is used to screen out qualified lysozyme protein sequences from the generated lysozyme protein sequences. The USM Chitinase Predictor is used to screen out qualified chitinase protein sequences from the generated chitinase protein sequences. The USM PETase Predictor is used to screen out qualified PETase protein sequences from the generated PETase protein sequences.

[0084] 3. Synthesis of target trifunctional hydrolase

[0085] 1) Plasmid construction

[0086] Amplify the gene for the target protein (trifunctional hydrolase with amino acid sequence as shown in SEQ ID NO:1) by homologous recombination. Insert the target gene into the corresponding pJL1 expression vector by restriction digestion and ligation using Hieff Clone™ One Step Cloning Kit. Sequence the expression vector to confirm the correct insertion of the target gene. Transform the expression vector with the target gene into DH5α competent cells. Select a single clone for vector amplification. Extract the plasmid using a kit similar to the PureStrand HQ 96 Plasmid DNA Purification Kit (Thermo Cat# K210096).

[0087] 2) Cell-free protein synthesis

[0088] The plasmid extracted in step 1) was added to the EP tube containing the cell-free expression reaction system. The reaction was maintained at 30°C for 3 hours in the EP tube by an orbital shaker at an appropriate stirring speed.

[0089] 3) Protein purification

[0090] Pretreatment: The precipitate in the solution obtained in step 2) was removed by centrifugation, and the cell-free protein synthesis (CFPS) supernatant was collected for protein purification.

[0091] Protein purification: The target protein is extracted from the CFPS supernatant by affinity chromatography or a combination of multiple chromatography methods. After each purification step, the crude sample is buffer exchanged and then subjected to the next purification step. The final purified product is exchanged into the storage buffer.

[0092] Protein quality assessment: SDS-PAGE and UV spectroscopy (OD 280 ) and other methods to assess the purity and content of crude samples after each purification step and the final protein product.

[0093] Among them, the results of western blots detection of the expressed trifunctional hydrolases can be found in Figure 2 . Figure 2 The first column on the far left is the marker band, and the immunoblot band in the yellow frame is the trifunctional hydrolase protein expression band (the amino acid sequence is shown in SEQ ID NO: 1).

[0094] 4. Detection of trifunctional hydrolase capacity and thermal stability

[0095] 4.1 Lysozyme activity detection:

[0096] The lysozyme activity of each enzyme to be tested (each trifunctional hydrolase obtained in step 3 of this example) was tested using a lysozyme activity detection kit (Thermos Fisher EnzChekTM Lysozyme Assy Kit) in a reaction system with different pH values ​​and an environmental condition of 37°C.

[0097] 4.2 PETase activity detection:

[0098] Mix 10 μL of enzyme stock solution (each trifunctional hydrolase obtained in step 3 of this example) with 80 μL of activity detection buffer (50 mM p-nitrophenylbutyrate (pNPB, 50 mM Tris-HCl, pH 8.0), incubate at 37°C for 30-90 min, and detect the absorbance signal of the product p-nitrophenol (pNP) at 405 nm (reference: https: / / doi.org / 10.1016 / j.jhazmat.2023.131574).

[0099] 4.3 Chitinase activity detection:

[0100] Mix 20 μL of enzyme stock solution (each trifunctional hydrolase obtained in step 3 of this example) with 80 μL of activity detection buffer (125 μM chitotriose substrate Mu-(GlcMAc)3, 200 mM sodium phosphate, pH 8.0), incubate at the test temperature (such as 37°C, 85°C, etc.) for 2-6 hours, and detect the fluorescence signal of the product tetramethylumbelliferone at 372 nm as the excitation wavelength and 445 nm as the emission wavelength (reference: DOI: 10.1080 / 14756366.2021.1931862).

[0101] 4.4 Test results

[0102] The test results are shown in Table 1. The trifunctional hydrolase of this example simultaneously exhibited lysozyme activity, chitinase activity and PETase activity at 37°C.

[0103] Table 1:

[0104]

[0105] In Table 1, RSD is Relative Standard Deviation, which reflects the degree of deviation between multiple experimental results.

[0106] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0107] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present application. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present application.

Claims

1. A polypeptide, characterized in that The amino acid sequence of the polypeptide is shown in SEQ ID NO:

1.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the polypeptide of claim 1.

3. An expression vector, characterized in that: The expression vector carries the nucleic acid molecule of claim 2.

4. A recombinant cell, characterized in that The recombinant cell carries the nucleic acid molecule of claim 2 or the expression vector of claim 3, or The recombinant cell expresses the polypeptide of claim 1.

5. A method for preparing a polypeptide, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO: 1; The method comprises: The recombinant cell according to claim 4 is cultured to obtain the polypeptide.

6. A reagent or a kit, characterized in that: Comprising the polypeptide of claim 1.

7. A composition, characterized in that Comprising the polypeptide of claim 1.

8. Use of the polypeptide according to claim 1 in preparing a product, wherein the product is used for at least one of the following: Lyse bacteria, hydrolyze chitin and / or PET, Disposal of waste, Antifungal, Food preservation.

9. The use according to claim 8, characterized in that The product is selected from food preservatives, antimicrobial agents, waste treatment agents, and medicines.

10. The use according to claim 8, characterized in that The product described is a fungus inhibitor.

11. The use according to claim 8, characterized in that The product is a trifunctional hydrolase of lysozyme, chitinase and PETase.

12. A method for treating waste, characterized in that: include: The waste is contacted with the polypeptide according to claim 1, the polypeptide prepared by the method according to claim 5, the reagent or kit according to claim 6, or the composition according to claim 7, wherein the waste contains bacteria, chitin and / or PET.

13. A method for hydrolyzing PET, characterized in that: include: The polypeptide according to claim 1, the polypeptide prepared by the method according to claim 5, the reagent or kit according to claim 6, or the composition according to claim 7 is used to contact a sample, wherein the sample contains PET.

14. A method for hydrolyzing bacteria and / or chitin for non-disease diagnosis and treatment purposes, characterized in that: include: The polypeptide according to claim 1, the polypeptide prepared by the method according to claim 5, the reagent or kit according to claim 6, or the composition according to claim 7 is used to contact a sample, wherein the sample contains bacteria and / or chitin.

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