Polypeptide and application thereof as well as method, composition or kit for preparing polypeptide

Abstract

The invention discloses a polypeptide and application thereof, and a method, a composition or a kit for preparing the polypeptide. The amino acid sequence of the polypeptide is shown as any one of SEQ ID NO: 1 to SEQ ID NO: 4; the method comprises the step of culturing the recombinant cell in the fourth aspect to obtain the polypeptide. The polypeptide provided by the invention has PET enzyme activity and chitinase activity, can be used as a bifunctional hydrolase with PET enzyme activity and chitinase activity, and has excellent thermal stability; the polypeptide provided by the invention can be massively expressed in a protein expression system, so that the method provided by the invention can be used for large-scale and standardized production; the composition or kit containing the polypeptide plays an important role in the aspects of biological control, medical application, waste treatment, environmental protection and the like.

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 chitinase and PETase bifunctional hydrolase obtained by screening based on an artificial intelligence model and uses thereof. Background Art

[0002] 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.

[0003] 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.

[0004] At present, no hydrolase that can hydrolyze chitin and PET at the same time has been found. Therefore, it is very important to develop a chitin and PET bifunctional hydrolase. Summary of the invention

[0005] 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 chitin and PET bifunctional hydrolase.

[0006] In the first aspect of the present application, the present application proposes a polypeptide. According to the embodiments of the present application, the amino acid sequence of the polypeptide is shown in any one of SEQ ID NO: 1 to SEQ ID NO: 4. The polypeptide of the present application has PETase activity and chitinase activity, can be used as a bifunctional hydrolase with PETase activity and chitinase activity, and has excellent thermal stability.

[0007] 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, which has PETase activity, chitinase activity, and excellent thermal stability.

[0008] 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. The expression vector of the present application carries the nucleic acid molecule described in the second aspect and can express the polypeptide of the first aspect, which has PETase activity, chitinase activity, and excellent thermal stability.

[0009] 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 of the present application can express the polypeptide of the first aspect, which has PETase activity, chitinase activity, and excellent thermal stability.

[0010] 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 any one of SEQ ID NO: 1 to SEQ ID NO: 4; the method comprises: culturing the recombinant cell described in the fourth aspect to obtain the polypeptide. The method of the present application can be produced on a large scale and in a standardized manner, and the obtained polypeptide has PETase activity, chitinase activity, and excellent thermal stability.

[0011] In the sixth aspect of the present application, the present application proposes the use of the polypeptide described in the first aspect as a chitinase and PETase bifunctional hydrolase.

[0012] 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 excellent 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.

[0013] 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 excellent PETase activity and chitinase activity. Thus, the composition containing the above polypeptide can be used in environmental management, agriculture, industry and medicine.

[0014] In the ninth aspect of the present application, the present application proposes the use of the polypeptide of the first aspect in preparing a product, wherein the product is used for at least one of the following: hydrolyzing chitin and / or hydrolyzing PET, treating waste, improving soil, antifungal, and antiviral.

[0015] In the tenth aspect of the present application, the present application proposes a method for hydrolyzing 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 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.

[0016] In the eleventh aspect of the present application, the present application proposes a method for improving soil. According to an embodiment of the present application, the method comprises: contacting the soil to be improved with 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.

[0017] 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

[0018] 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: Figure 1 A flowchart for protein design in the examples of this application; Figure 2 This is the western blots band of the expression of the bifunctional hydrolase in the examples of this application. DETAILED DESCRIPTION

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] The present application proposes a polypeptide and its use, a method for preparing the polypeptide, a composition or a kit, which will be described in detail below.

[0027] Peptides In the first aspect of the present application, the present application proposes a polypeptide. According to the embodiments of the present application, the amino acid sequence of the polypeptide is shown in any one of SEQ ID NO: 1 to SEQ ID NO: 4. The polypeptide of the present application is obtained by designing a protein sequence different from the protein sequence through an artificial intelligence model and further screening, and has PETase activity, chitinase activity, and excellent thermal stability, and can be mass-produced.

[0028] Furthermore, the polypeptide of the present application has both PETase activity and chitinase activity, and has application potential in multiple fields, such as: 1) Environmental governance: Degradation of plastic waste: PETase can degrade polyethylene terephthalate (PET) plastic, while chitinase can decompose chitin. The polypeptide of the present application can simultaneously degrade PET plastic and chitin waste, such as plastic waste in the ocean and chitin waste generated by aquatic product processing, reducing environmental pollution.

[0029] Promote material circulation: In the natural environment, the polypeptide of the present application can more efficiently decompose organic matter and accelerate the circulation of elements such as carbon and nitrogen, which is of great significance for maintaining the balance of the ecosystem.

[0030] 2) Industrial application areas: Preparation of biodegradable materials: In the production process of biodegradable materials, the polypeptides of the present application can simultaneously process PET and other chitin-based materials, improve the degradation efficiency and processability of the materials, and contribute to the development of new environmentally friendly biodegradable materials.

[0031] Textile processing: In the textile industry, both PET fibers and chitin fibers are used. The polypeptides of the present application can be used to process textile waste containing these two fibers, realize their recycling and reuse, and reduce production costs and environmental burdens.

[0032] 3) Agriculture: Biological control: Chitinase has antifungal effects and can help plants resist the invasion of pathogenic fungi. The introduction of PET enzyme may enhance its stability and activity in plants. Therefore, the polypeptide of the present application can improve the comprehensive resistance of plants to diseases and insect pests.

[0033] Soil improvement: In the soil, the polypeptide of the present application can decompose residual plastic film and chitin-like substances, improve soil structure and fertility, and promote crop growth.

[0034] 4) Medical field: Disease treatment: In the human immune response, chitinase is involved in regulating the activation and differentiation of immune cells. The fusion of PET enzyme may give it new functions, such as enhancing its targeting in specific tissues or cells. Therefore, the polypeptide of this application can provide new ideas for the treatment of related diseases, such as preventing fungal infections, anti-tumor, anti-infection and preventing malaria.

[0035] MSPKRIIAYFPEWKVKDEYLNYSVEDIPWKLLTHINYAFAKIVEGKLHPIEEDLFYSNMEKIKEYKKEYRDVKVLISVGGWTDSGEFSDVALNEENRKKFAKSALELVKEFNLDGVDIDWEFPVSGGLPTNKARPEDKENFTLLLKTLRDVLKEENENYLLTIAAPASYTQIHNTEPDKYHIFLDFINLMTYDFHGIWDKYTNHHSPLYGNPKDPDEKSRERANCDFAVKEYLKFGIPPEKIVLGVPPFYGKGWICEDDGHNGLFAKVKGIPYGGSNPFFIKGVIENDPNYIKFRDKYAKVPWLWNPKEKIMYSYDDEESILEKCNYVIKNNLGGIMFWEVTQDYPFKGHTLVKLIHQKFWEG (SEQ ID NO:1); MSPKRIIAYFPEWKVKDEYLNYSVEDIPWKLLTHINYAFAKIVEGKLHPIEEDLFYSNMEKIKEYKKEYRDVKVLISVGGWTDSGEFSDVALNEENRKKFAKSALELVKEFNLDGVDIDWEFPITFDDKENFTLLLKTLRDVLKEENENYLLTIAAPASYTQIHNTEPDKYHIFLDFINLMTYDFHGIWDKYTNHHSPLYGNPKDPDEKSRERANCDFAVKEYLKFGIPPEKIVLGVPPFYGKGWICEDDGHNGLFAKVKGIPYGIFDNEDHPSGSNPFFFIKGVIENDPNYIKFRDKYAKVPWLWNPKEKIMYSYDDEESILEKCNYVIKNNLGGIMFWEVTQDYPFKGHTLVKLIHQKFWEG(SEQ ID NO:2); MSPKRIIAYFPEWKVKDEYLNYSVEDIPWKLLTHINYAFAKIVEGKLHPIEEDLFYSNMEKIKEYKKEYRDVKVLISVGGWTDSGEFSDVALNEENRKKFAKSALELVKEFNLDGVDIDWEFPCLRGPDIDKENFTLLLKTLRDVLKEENENYLLTIAAPASYTQIHNTEPDKYHIFLDFINLMTYDFHGIWDKYTNHHSPLYGNPKDPDEKSRERANCDFAVKEYLKFGIPPEKIVLGVPFYGKGWICEDDGHNGLFAKVKGIPYGIFDNEDHPSGSNPFFFIKGVIENDPNYIKFRDKYAKVPWLWNPKEKIMYSYDDEESILEKCNYVIKNNLGGIMFWEVTQDYPFKGHTLVKLIHQKFWEG (SEQID NO:3); MSPKRIIAYFPEWKVKDEYLNYSVEDIPWKLLTHINYAFAKIVEGKLHPIEEDLFYSNMEKIKEYKKEYRDVKVLISVGGWTDSGEFSDVALNEENRKKFAKSALELVKEFNLDGVDIDWEFPCTEIDKENFTLLLKTLRDVLKEENENYLLTIAAPASYTQIHNTEPDKYHIFLDFINLMTYDFHGIWDKYTNHHSPLYGNPKDPDEKSRERANCDFAVKEYLKFGIPPEKIVLGVPFYGKGWICEDDGHNGLFAKVKGIPYGIFDNEDHPSGSNPFFFIKGVIENDPNYIKFRDKYAKVPWLWNPKEKIMYSYDDEESILEKCNYVIKNNLGGIMFWEVTQDYPFKGHTLVKLIHQKFWEG(SEQ IDNO:4)。

[0036] It should be noted that in the present application, "the amino acid sequence is as shown in SEQ ID NO: A" includes the amino acid sequence of SEQ ID NO: A, or the amino acid sequence of conservative modification of SEQ ID NO: A, or the amino acid sequence having at least 90% identity with SEQ ID NO: A, all of which are within the scope of protection of the present application. Exemplarily, "the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 1" means that the polypeptide has the amino acid sequence of SEQ ID NO: 1, or the polypeptide has the amino acid sequence of conservative modification of SEQ ID NO: 1, or the polypeptide has the amino acid sequence having at least 90% identity with SEQ ID NO: 1, all of which are within the scope of protection of the present application. Exemplarily, "the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 2" means that the polypeptide has the amino acid sequence of SEQ ID NO: 2, or the polypeptide has the amino acid sequence of conservative modification of SEQ ID NO: 2, or the polypeptide has the amino acid sequence having at least 90% identity with SEQ ID NO: 2, all of which are within the scope of protection of the present application. Exemplarily, "the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 3" means that the polypeptide has an amino acid sequence as shown in SEQ ID NO: 3, or a polypeptide has an amino acid sequence as conservatively modified in SEQ ID NO: 3, or a polypeptide has an amino acid sequence with at least 90% identity to SEQ ID NO: 3, all of which are within the scope of protection of the present application. Exemplarily, "the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 4" means that the polypeptide has an amino acid sequence as shown in SEQ ID NO: 4, or a polypeptide has an amino acid sequence as conservatively modified in SEQ ID NO: 4, or a polypeptide has an amino acid sequence with at least 90% identity to SEQ ID NO: 4, all of which are within the scope of protection of the present application.

[0037] In this article, "conservatively modified amino acid sequences" refer to amino acid modifications that do not significantly affect or change the PETase activity or chitinase activity of 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.

[0038] 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.

[0039] 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.

[0040] Nucleic acid molecules, expression vectors and recombinant cells 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 PETase activity, chitinase activity and thermal stability, especially excellent thermal stability.

[0041] According to an embodiment of the present application, the nucleic acid molecule is DNA.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] In some specific embodiments of the present application, the expression vector is a lentiviral vector.

[0047] In an optional embodiment of the present application, the expression vector is a plasmid expression vector.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] According to an embodiment of the present application, the host cell is a eukaryotic cell.

[0052] 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.

[0053] 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.

[0054] Method for preparing polypeptide 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 any one of SEQ ID NO: 1 to SEQ ID NO: 4; the method comprises: culturing the recombinant cell described in the fourth aspect to obtain the polypeptide. The method of the present application can be produced on a large scale and in a standardized manner, and the obtained polypeptide has PETase activity, chitinase activity, and excellent thermal stability.

[0055] use In the sixth aspect of the present application, the present application proposes the use of the polypeptide described in the first aspect as a chitinase and PETase bifunctional hydrolase. As can be seen from the foregoing, the polypeptide of the first aspect has PETase activity and chitinase activity, and has excellent thermal stability. Therefore, the polypeptide of the present application can be used as a bifunctional hydrolase of chitinase and PETase, which can 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, prevention of fungal infection, anti-tumor, anti-infection and prevention and treatment of malaria, etc.

[0056] Herein, "chitinase and PETase bifunctional hydrolase" refers to a bifunctional hydrolase that can degrade chitin and PET at the same time.

[0057] Reagents or kits 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 excellent PETase activity and chitinase activity. Thus, the reagent or the kit containing the above-mentioned polypeptide can degrade chitin and PET, and is used for environmental governance, preparation of biodegradable materials, soil improvement, etc., and can be applied in the fields of environmental governance, agriculture, industry, etc.

[0058] Composition 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 excellent PETase activity and chitinase activity. Thus, the composition containing the above polypeptide can be used in environmental management, agriculture, industry and medicine.

[0059] According to an embodiment of the present application, the composition may be a pharmaceutical composition, and further the composition includes a pharmaceutically acceptable excipient.

[0060] use 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: hydrolyzing chitin and / or hydrolyzing PET, treating waste, improving soil, antifungal, and antiviral. As can be seen from the foregoing, the polypeptide of the first aspect has excellent 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 hydrolyzing chitin and / or hydrolyzing PET, treating waste, improving soil, antifungal, and antiviral, etc.

[0061] 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 chitin and / or PET.

[0062] According to the embodiments of the present application, the products include but are not limited to reagents, kits, and pharmaceutical compositions.

[0063] method In the tenth aspect of the present application, the present application proposes a method for hydrolyzing chitin and / or hydrolyzing PET, or treating waste. According to an embodiment of the present application, the method comprises: contacting a sample or waste with 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, wherein the sample or waste contains chitin and / or PET. Thus, the method of the present application can hydrolyze chitin and / or hydrolyze PET, or treat waste.

[0064] In the eleventh aspect of the present application, the present application proposes a method for improving soil. According to an embodiment of the present application, the method comprises: contacting the soil to be improved with 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, and the composition described in the eighth aspect. Thus, the method of the present application degrades chitin and / or PET in the soil to be improved by the polypeptide to achieve the effect of improving the soil.

[0065] 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.

[0066] Embodiment 1: 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 chitinase and PETase bifunctional hydrolase (hereinafter referred to as bifunctional hydrolase) protein sequence to screening optimization.

[0067] See also Figure 1 The design and screening mainly involves generating bifunctional hydrolase protein sequences through a protein generation model (PGM), screening out target (quality qualified) bifunctional hydrolase protein sequences from the generated bifunctional hydrolase protein sequences through a quality control pipeline (QCP), synthesizing protein encoding DNA based on the screened target bifunctional hydrolase protein sequences, expressing them in a protein expression system, and expressing the bifunctional hydrolase ability and thermal stability of the protein through a wet-lab assay (For Wet-lab Assay).

[0068] 1. Generation of bifunctional hydrolase protein sequences For example, a protein generation model is used to generate a bifunctional hydrolase protein sequence with specific functions and structures through computational methods. The "protein generation model" is usually based on deep learning techniques such as diffusion models, language models and graph neural networks, which can handle complex molecular systems.

[0069] 2. Screening of target bifunctional hydrolase protein sequences Exemplarily, a quality control process screening is used to screen out target bifunctional hydrolase protein sequences with qualified quality from the generated bifunctional hydrolase protein sequences. The quality control process screening includes screening of the function domain, sequence identity, and structure identity of the bifunctional hydrolase protein sequences, as well as screening using a unified sequence model (USM).

[0070] Among them, the "functional region" is used to indicate the protein function of the bifunctional hydrolase protein sequence in the organism to which it belongs, for example, the protein function of the bifunctional hydrolase protein sequence in at least one description dimension of cellular elements, biological processes, and molecular functions.

[0071] "Sequence signature" is used to indicate the amino acid sequence included in the bifunctional hydrolase protein sequence.

[0072] "Structural features" are used to indicate the geometric structure of the bifunctional hydrolase protein sequence in three-dimensional space, for example, at least one level of classification of the bifunctional hydrolase protein sequence in a class, fold, superfamily, or family in structural classification.

[0073] The "unified sequence model" is compatible with the single sequence model and the multiple sequence alignment (MSA) model. The unified sequence model is not only a universal biological language model that can be applied to the screening task of bifunctional hydrolase protein sequences, but also a universal multi-sequence model that can support both single sequence and multi-sequence alignment data, unifying single sequence and multi-sequence modes (for example: the corresponding processing of molecular formula data and amino acid sequence data is compatible), and is versatile.

[0074] 3. Synthesis of target bifunctional hydrolase 1) Plasmid construction Genes for synthesizing the target proteins (bifunctional hydrolase 1 with amino acid sequence as shown in SEQ ID NO:1, bifunctional hydrolase 2 with amino acid sequence as shown in SEQ ID NO:2, bifunctional hydrolase 3 with amino acid sequence as shown in SEQ ID NO:3, and bifunctional hydrolase 4 with amino acid sequence as shown in SEQ ID NO:4) were amplified by homologous recombination. The target genes were inserted into the corresponding pJL1 expression vectors by restriction enzyme digestion and ligation using Hieff Clone™ One Step Cloning Kit. The expression vectors were sequenced to confirm the correct insertion of the target genes. The expression vectors with the target genes were transformed into DH5α competent cells. Single clones were selected for vector amplification. Plasmids were extracted using a kit similar to the Purechain HQ 96 Plasmid DNA Purification Kit (Thermo Cat# K210096).

[0075] 2) Cell-free protein synthesis 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.

[0076] 3) Protein purification 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.

[0077] 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.

[0078] Protein quality assessment: Purity and content of crude samples after each purification step as well as the final protein product were assessed using methods such as SDS-PAGE and UV spectroscopy (OD280).

[0079] The expressed bifunctional hydrolase was detected by western blots, and the western blot bands are shown in Figure 2 . Figure 2 The first column on the far left is the marker band, the immunoblot bands in the yellow frame are the protein expression bands of the bifunctional hydrolase, and from left to right they are the protein expression bands of bifunctional hydrolase 1 (amino acid sequence as shown in SEQ ID NO: 1), bifunctional hydrolase 2 (amino acid sequence as shown in SEQ ID NO: 2), bifunctional hydrolase 3 (amino acid sequence as shown in SEQ ID NO: 3), and bifunctional hydrolase 4 (amino acid sequence as shown in SEQ ID NO: 4).

[0080] 4. Detection of bifunctional hydrolase capacity and thermal stability 4.1 PETase activity detection: Mix 10 μL of enzyme stock solution (each bifunctional 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).

[0081] 4.2 Chitinase activity detection: 20 μL of enzyme stock solution (each bifunctional hydrolase obtained in step 3 of this example) was mixed with 80 μL of activity detection buffer (125 μM chitotriose substrate Mu-(GlcMAc)3, 200 mM sodium phosphate, pH 8.0), incubated at the test temperature (such as 37°C, 85°C, etc.) for 2-6 hours, and the fluorescence signal of the product tetramethylumbelliferone was detected at 372 nm as the excitation wavelength and 445 nm as the emission wavelength (reference: DOI: 10.1080 / 14756366.2021.1931862). The enzyme activities of three natural chitinases, OFCHTh, SMCHIB, and SMCHIC, were tested under the same conditions for comparison. OFCHTh comes from insects, SMCHIB and SMCHIC come from Serratia marcescens, and they are known for their high catalytic efficiency and high thermal stability (Chen, Wei, Mingbo Qu, Yong Zhou, and Qing Yang. 2018. “Structural Analysis of Group II Chitinase (ChtII) Catalysis Completes the Puzzle of Chitin Hydrolysis in Insects.” The Journal of Biological Chemistry 293 (8): 2652–60).

[0082] 4.3 Test results The test results are shown in Table 1. The four bifunctional hydrolases of this example showed both chitinase and PETase activities at 37°C, while the natural chitinase only showed chitinase activity; after heating at 85°C for three hours, the four bifunctional hydrolases of this example could still maintain more than 80% of the enzyme activity, while the natural chitinase had no residual enzyme activity.

[0083] Table 1:

[0084] In Table 1, / represents inactivity; RSD is Relative Standard Deviation, which reflects the degree of deviation between multiple experimental results.

[0085] 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.

[0086] 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 any one of SEQ ID NO: 1 to SEQ ID NO:

4.

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 any one of SEQ ID NO: 1 to SEQ ID NO: 4; The method comprises: The recombinant cell according to claim 4 is cultured to obtain the polypeptide.

6. Use of the polypeptide according to claim 1 as a chitinase and PETase bifunctional hydrolase.

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

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

9. Use of the polypeptide according to claim 1 in preparing a product, wherein the product is used for at least one of the following: Hydrolyzed chitin and / or hydrolyzed PET, Disposal of waste, Improve soil, Antifungal, Antiviral.

10. A method for hydrolyzing chitin and / or hydrolyzing PET, or treating waste, 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 7, or the composition according to claim 8 is used to contact a sample or waste, wherein the sample or waste contains chitin and / or PET.

11. A method for improving soil, characterized in that: include: The soil to be improved 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 7, or the composition according to claim 8.

Images