Polypeptides and their uses, methods for preparing polypeptides, compositions or kits

Abstract

The present application discloses polypeptides and their uses, methods for preparing polypeptides, compositions or kits. The amino acid sequence of the polypeptide is shown as any one of SEQ ID NO: 1 to SEQ ID NO: 4; the method includes: culturing the recombinant cell described in the fourth aspect to obtain the polypeptide. 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; the polypeptide of the present application can be highly expressed in a protein expression system. Thus, the method of the present application can be produced on a large scale and standardized; the use of the composition or kit containing the above polypeptide plays an important role in biological control, medical applications, waste treatment and environmental protection, etc.

Description

Technical Field

[0001] This application belongs to the field of biotechnology, and specifically relates to polypeptides and their uses, methods for preparing polypeptides, compositions or kits. More specifically, it relates to a chitinase and PETase bifunctional hydrolase screened based on an artificial intelligence model and its uses. Background Art

[0002] Chitin (also known as chitosan) is the second largest natural polymer compound after cellulose, which is polymerized from N-acetyl-D-glucosamine (GlcNAc). Chitinase is an enzyme that catalyzes the hydrolysis of chitin and can break the glycosidic bond in chitin.

[0003] Polyethylene terephthalate (PET) is a common resin in life and can be divided into APET, RPET, and PETG. PETase can degrade PET plastics into its monomer, mono-2-hydroxyethyl terephthalic acid (MHET) molecules. MHET will be further degraded by MHETase in bacteria into hydroxyethyl terephthalic acid, and then decomposed into environmentally friendly terephthalic acid and ethylene glycol in water, and can be utilized by other bacteria, ultimately producing carbon dioxide and water.

[0004] Currently, no hydrolase that can simultaneously hydrolyze chitin and PET has been found. Therefore, it is very important to develop a chitin and PET bifunctional hydrolase. Summary of the Invention

[0005] This application aims to solve at least one of the technical problems existing in the prior art to some extent. For this purpose, this application provides a chitin and PET bifunctional hydrolase.

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

[0007] In the second aspect of this application, a nucleic acid molecule is proposed. According to the embodiments of this application, the nucleic acid molecule encodes the polypeptide described in the first aspect. The nucleic acid molecule of this application can encode the polypeptide of the first aspect, and this polypeptide has PETase activity, chitinase activity, and excellent thermal stability.

[0008] In the third aspect of the present application, the present application provides 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 PET enzyme activity, chitinase activity, and excellent thermal stability.

[0009] In the fourth aspect of the present application, the present application provides 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 and obtain the polypeptide of the first aspect, which has PET enzyme activity, chitinase activity, and excellent thermal stability.

[0010] In the fifth aspect of the present application, the present application provides 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 includes: culturing the recombinant cell described in the fourth aspect to obtain the polypeptide. The method of the present application can be scaled up and standardized for production, and the obtained polypeptide has PET enzyme activity, chitinase activity, and excellent thermal stability.

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

[0012] In the seventh aspect of the present application, the present application provides a reagent or kit. According to an embodiment of the present application, the reagent or kit includes the polypeptide described in the first aspect. As can be seen from the above, the polypeptide of the first aspect has excellent PET enzyme activity and chitinase activity. Therefore, the composition reagent or kit containing the above polypeptide can be applied in environmental governance, agriculture, and industry.

[0013] In the eighth aspect of the present application, the present application provides 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 above, the polypeptide of the first aspect has excellent PET enzyme activity and chitinase activity. Therefore, the composition containing the above polypeptide can be applied in the fields of environmental governance, agriculture, industry, and medicine.

[0014] In the ninth aspect of the present application, the present application provides the use of the polypeptide of the first aspect in the preparation of a product, and 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, a method for hydrolyzing chitin and / or hydrolyzing PET, or treating waste is proposed. According to an embodiment of the present application, the method includes: contacting a sample or waste containing chitin and / or PET 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.

[0016] In the eleventh aspect of the present application, a method for improving soil is proposed. According to an embodiment of the present application, the method includes: 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 following description, become apparent in part from the following description, or be learned through the practice of the present application. Description of the Drawings

[0018] The above and / or additional aspects and advantages of the present application will become apparent and be readily understood from the description of the embodiments in conjunction with the following drawings, where:

[0019] Figure 1 is the flowchart of protein design in the embodiment of the present application;

[0020] Figure 2 is the expression western blots band of the bifunctional hydrolase in the embodiment of the present application. Detailed Description of Embodiments

[0021] The embodiments of the present application will be described in detail below. The embodiments described below are exemplary and are only used to explain the present application, and should not be construed as a limitation of the present application.

[0022] It should be noted that the terms "first" and "second" are only used for descriptive purposes and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present application, unless otherwise specified, the meaning of "a plurality" is two or more.

[0023] In this document, the term "comprising" or "including" is an open expression, that is, it includes the content specified in the present application, but does not exclude other aspects of the content.

[0024] In this document, the term "pharmaceutically acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components containing the polypeptide or its derivatives and / or the mammalian being treated therewith.

[0025] In this text, 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 particular target dosage form. Except for the range where any conventional excipient is 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, their uses are also within the scope contemplated by the present application.

[0026] Except for any conventional excipient, the range where it is 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, their uses are also within the scope contemplated by the present application.

[0027] In this text, the term "treatment" refers to achieving a desired pharmacological and / or physiological effect. The effect can be prophylactic in terms of completely or partially preventing a disease or its symptoms, and / or 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 of mammals, especially humans, including: (a) preventing the occurrence of a disease or disorder in an individual who is susceptible to the disease but has not been diagnosed with it; (b) inhibiting a disease, such as blocking the progression of the disease; or (c) alleviating a disease, such as reducing the symptoms associated with the disease. "Treatment" as used herein covers any administration of a polypeptide or its derivative drug to an individual to treat, cure, alleviate, improve, reduce, or inhibit the disease of the individual, including but not limited to administering a drug containing the polypeptide described herein to an individual in need.

[0028] The present application provides a polypeptide and its uses, a method for preparing the polypeptide, a composition, or a kit, which will be described in detail below respectively.

[0029] Polypeptide

[0030] In the first aspect of the present application, the present application provides a polypeptide. According to an embodiment of the present application, the amino acid sequence of the polypeptide is shown as any one of SEQ ID NO:1 to SEQ ID NO:4. The polypeptide of the present application is designed by an artificial intelligence model to be different from the protein sequence and further screened. It has PET enzyme activity, chitinase activity, and excellent thermal stability, and can be mass-produced.

[0031] Furthermore, the polypeptide of the present application has both PET enzyme activity and chitinase activity and has application potential in multiple fields, such as:

[0032] 1) Environmental governance field:

[0033] 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 from aquatic product processing, reducing environmental pollution.

[0034] Promote material cycle: In the natural environment, the polypeptide of the present application can decompose organic substances more efficiently, accelerating the cycle of elements such as carbon and nitrogen, which is of great significance for maintaining the balance of the ecosystem.

[0035] 2) Industrial application fields:

[0036] Preparation of biodegradable materials: In the production process of biodegradable materials, the polypeptide of the present application can simultaneously process PET and other chitin-based materials, improving the degradation efficiency and processability of the materials, and contributing to the development of new environmentally friendly biodegradable materials.

[0037] Textile treatment: In the textile industry, both PET fibers and chitin fibers are used. The polypeptide of the present application can be used to treat textile waste containing these two types of fibers, realizing their recycling and reuse, reducing production costs and environmental burdens.

[0038] 3) Agricultural fields:

[0039] Biological control: Chitinase has antifungal effects and can help plants resist the invasion of pathogenic fungi. The introduction of PETase may enhance its stability and activity in plants. Thus, the polypeptide of the present application can improve the comprehensive resistance of plants to pests and diseases.

[0040] Soil improvement: In the soil, the polypeptide of the present application can decompose residual plastic films and chitinous substances, improving soil structure and fertility, and promoting crop growth.

[0041] 4) Medical fields:

[0042] Disease treatment: In the human immune response, chitinase is involved in regulating the activation and differentiation of immune cells. The fusion of PETase may endow it with new functions, such as enhancing its targeting in specific tissues or cells. Thus, the polypeptide of the present application can provide new ideas for the treatment of related diseases, such as preventing fungal infections, anti-tumor, anti-infection, and preventing and treating malaria, etc.

[0043] MSPKRIIAYFPEWKVKDEYLNYSVEDIPWKLLTHINYAFAKIVEGKLHPIEEDLFYSNMEKIKEYKKEYRDVKVLISVGGWTDSGEFSDVALNEENRKKFAKSALELVKEFNLDGVDIDWEFPVSGGLPTNKARPEDKENFTLLLKTLRDVLKEENENYLLTIAAPASYTQIHNTEPDKYHIFLDFINLMTYDFHGIWDKYTNHHSPLYGNPKDPDEKSRERANCDFAVKEYLKFGIPPEKIVLGVPFYGKGWICEDDGHNGLFAKVKGIPYGGSNPFFFIKGVIENDPNYIKFRDKYAKVPWLWNPKEKIMYSYDDEESILEKCNYVIKNNLGGIMFWEVTQDYPFKGHTLVKLIHQKFWEG (SEQ IDNO:1);

[0044] MSPKRIIAYFPEWKVKDEYLNYSVEDIPWKLLTHINYAFAKIVEGKLHPIEEDLFYSNMEKIKEYKKEYRDVKVLISVGGWTDSGEFSDVALNEENRKKFAKSALELVKEFNLDGVDIDWEFPITFDDKENFTLLLKTLRDVLKEENENYLLTIAAPASYTQIHNTEPDKYHIFLDFINLMTYDFHGIWDKYTNHHSPLYGNPKDPDEKSRERANCDFAVKEYLKFGIPPEKIVLGVPFYGKGWICEDDGHNGLFAKVKGIPYGIFDNEDHPSGSNPFFFIKGVIENDPNYIKFRDKYAKVPWLWNPKEKIMYSYDDEESILEKCNYVIKNNLGGIMFWEVTQDYPFKGHTLVKLIHQKFWEG(SEQ IDNO:2);

[0045] MSPKRIIAYFPEWKVKDEYLNYSVEDIPWKLLTHINYAFAKIVEGKLHPIEEDLFYSNMEKIKEYKKEYRDVKVLISVGGWTDSGEFSDVALNEENRKKFAKSALELVKEFNLDGVDIDWEFPCLRGPDIDKENFTLLLKTLRDVLKEENENYLLTIAAPASYTQIHNTEPDKYHIFLDFINLMTYDFHGIWDKYTNHHSPLYGNPKDPDEKSRERANCDFAVKEYLKFGIPPEKIVLGVPFYGKGWICEDDGHNGLFAKVKGIPYGIFDNEDHPSGSNPFFFIKGVIENDPNYIKFRDKYAKVPWLWNPKEKIMYSYDDEESILEKCNYVIKNNLGGIMFWEVTQDYPFKGHTLVKLIHQKFWEG (SEQID NO:3);

[0046] MSPKRIIAYFPEWKVKDEYLNYSVEDIPWKLLTHINYAFAKIVEGKLHPIEEDLFYSNMEKIKEYKKEYRDVKVLISVGGWTDSGEFSDVALNEENRKKFAKSALELVKEFNLDGVDIDWEFPCTEIDKENFTLLLKTLRDVLKEENENYLLTIAAPASYTQIHNTEPDKYHIFLDFINLMTYDFHGIWDKYTNHHSPLYGNPKDPDEKSRERANCDFAVKEYLKFGIPPEKIVLGVPFYGKGWICEDDGHNGLFAKVKGIPYGIFDNEDHPSGSNPFFFIKGVIENDPNYIKFRDKYAKVPWLWNPKEKIMYSYDDEESILEKCNYVIKNNLGGIMFWEVTQDYPFKGHTLVKLIHQKFWEG(SEQ IDNO:4).

[0047] It should be noted that in this 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 a conservative modification form 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 this 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 a conservative modification form 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 this 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 a conservative modification form 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 this application. Exemplarily, "the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 3" means that the polypeptide has the amino acid sequence of SEQ ID NO: 3, or the polypeptide has the amino acid sequence of a conservative modification form of SEQ ID NO: 3, or the polypeptide has the amino acid sequence having at least 90% identity with SEQ ID NO: 3, all of which are within the scope of protection of this application. Exemplarily, "the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 4" means that the polypeptide has the amino acid sequence of SEQ ID NO: 4, or the polypeptide has the amino acid sequence of a conservative modification form of SEQ ID NO: 4, or the polypeptide has the amino acid sequence having at least 90% identity with SEQ ID NO: 4, all of which are within the scope of protection of this application.

[0048] As used herein, the term "conservatively modified forms of an amino acid sequence" refers to amino acid modifications that do not significantly affect or alter the PET enzyme activity or chitinase activity of a polypeptide containing the amino acid sequence. Such modifications include 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 those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having basic side chains (such as lysine, arginine, histidine), amino acids having acidic side chains (such as aspartic acid, glutamic acid), amino acids having uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids having nonpolar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids having β-branched side chains (such as threonine, valine, isoleucine), and amino acids having aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine). Exemplarily, the conservative modifications are not more than 20% of the total number, preferably not more than 10% of the total number.

[0049] 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 refer to the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences 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, D.C.)). 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 the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs utilizing these algorithms are also available and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul 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.

[0050] As used herein, the term "having at least 90% sequence similarity" may refer to having sequence similarity of 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%. The sequence identity described in this application can be measured using sequence analysis software. For example, the computer program BLAST with default parameters, especially BLASTP or TBLASTN. The amino acid sequences described in this application are all shown in the N-terminal to C-terminal manner.

[0051] Nucleic acid molecules, expression vectors and recombinant cells

[0052] In a second aspect of the present application, the present application provides 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 thermal stability, especially excellent thermal stability.

[0053] According to an embodiment of the present application, the nucleic acid molecule is DNA.

[0054] It should be noted that for the nucleic acid molecules mentioned herein, those skilled in the art should understand that it actually includes either any one of the complementary double strands or both. For convenience, in this application, although only one strand is given in most cases, the other complementary strand is actually also disclosed. In addition, the nucleic acid molecule sequences in this application include DNA form or RNA form, and the disclosure of one means the disclosure of the other.

[0055] In a third aspect of the present application, the present application provides 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 connecting the above nucleic acid molecule to the expression vector, the above nucleic acid molecule can be directly or indirectly connected to the control elements on the expression vector, as long as these control elements can control the translation and expression of the above nucleic acid molecule, etc. Of course, these control elements can directly come from the expression vector itself or be exogenous, that is, not from the expression vector itself. Of course, the above nucleic acid molecule and the control elements can be operably linked.

[0056] As used herein, "operably linked" means that an exogenous gene is linked to an expression vector such that control elements within the expression vector, such as transcriptional control sequences and translational control sequences, etc., can perform their intended functions of regulating the transcription and translation of the exogenous gene. Commonly used expression vectors can be, for example, plasmids, phages, etc. After the expression vector according to some specific embodiments of the present application is introduced into a suitable recipient cell, under the mediation of a regulatory system, the expression of the aforementioned polypeptide can be effectively achieved, and thus a large amount of the polypeptide can be obtained in vitro.

[0057] In some specific embodiments of the present application, the expression vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus or a phage.

[0058] In some specific embodiments of the present application, the expression vector is a lentiviral vector.

[0059] In an alternative embodiment of the present application, the expression vector is a plasmid expression vector.

[0060] In a fourth aspect of the present application, the present application provides 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. Using this recombinant cell under suitable conditions, the aforementioned polypeptide can be effectively expressed within the recombinant cell.

[0061] 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.

[0062] 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 phage. 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, etc., insect cells such as Spodoptera frugiperda, plant cells such as Nicotiana tabacum, and mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells, etc.

[0063] According to an embodiment of the present application, the host cell is a eukaryotic cell.

[0064] 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.

[0065] It should be noted that the "suitable conditions" described in this application refer to the conditions suitable for the expression of the polypeptides described in this application. It is easy for those skilled in the art to understand that the conditions suitable for polypeptide expression include, but are not limited to, appropriate transformation or transfection methods, appropriate transformation or transfection conditions, healthy cell states, appropriate cell densities, suitable cell culture environments, and suitable cell culture times. The "suitable conditions" are not particularly limited, and those skilled in the art can optimize the optimal conditions for polypeptide expression according to the specific environment of the laboratory.

[0066] Method for preparing polypeptide

[0067] In the fifth aspect of this application, a method for preparing a polypeptide is proposed. According to the embodiments of this application, the amino acid sequence of the polypeptide is shown as any one of SEQ ID NO: 1 to SEQ ID NO: 4; the method includes: culturing the recombinant cells described in the fourth aspect to obtain the polypeptide. The method of this application can be scaled up and standardized for production, and the obtained polypeptide has PET enzyme activity, chitinase activity, and excellent thermal stability.

[0068] Use

[0069] In the sixth aspect of this application, the use of the polypeptide described in the first aspect as a bifunctional hydrolase of chitinase and PET enzyme is proposed. As can be seen from the above, the polypeptide of the first aspect has PET enzyme activity and chitinase activity, and has excellent thermal stability. Therefore, the polypeptide of this application can be used as a bifunctional hydrolase of chitinase and PET enzyme, which can degrade chitin and PET simultaneously, and can be used in environmental governance, industrial fields, and agricultural fields; it can also be used for antifungal, prevention of fungal infections, anti-tumor, anti-infection, and prevention and treatment of malaria, etc.

[0070] In this article, the "bifunctional hydrolase of chitinase and PET enzyme" refers to a bifunctional hydrolase that can degrade chitin and PET simultaneously.

[0071] Reagent or kit

[0072] In the seventh aspect of this application, a reagent or kit is proposed. According to the embodiments of this application, the reagent or kit includes the polypeptide described in the first aspect. As can be seen from the above, the polypeptide of the first aspect has excellent PET enzyme activity and chitinase activity. Therefore, the reagent or kit containing the above polypeptide can degrade chitin and PET, and can be used in environmental governance, preparation of biodegradable materials, soil improvement, etc., and can be applied in environmental governance, agriculture, industry and other fields.

[0073] Composition

[0074] In the eighth aspect of the present application, the present application provides a composition. According to an embodiment of the present application, the composition includes the polypeptide described in the first aspect. As known from the foregoing, the polypeptide of the first aspect has excellent PET enzyme activity and chitinase activity. Thus, the composition containing the above polypeptide can be applied in the fields of environmental governance, agriculture, industry, and medicine.

[0075] According to an embodiment of the present application, the composition may be a pharmaceutical composition. Further, the composition includes pharmaceutically acceptable excipients.

[0076] Use

[0077] In the ninth aspect of the present application, the present application provides a use of the polypeptide of the first aspect in the preparation of a product, and 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 known from the foregoing, the polypeptide of the first aspect has excellent PET enzyme activity and chitinase activity, and it can be applied in the fields of environmental governance, 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, antiviral, etc.

[0078] As used herein, "treating waste" means that the polypeptide of the present application or a product containing the polypeptide of the present application can achieve the purpose of treating waste by degrading an article containing chitin and / or PET.

[0079] According to an embodiment of the present application, the product includes, but is not limited to, reagents, reagent kits, and pharmaceutical compositions.

[0080] Method

[0081] In the tenth aspect of the present application, the present application provides a method for hydrolyzing chitin and / or hydrolyzing PET, or treating waste. According to an embodiment of the present application, the method includes: contacting a sample or waste containing chitin and / or PET with the polypeptide described in the first aspect, the polypeptide prepared by the method described in the fifth aspect, the reagent or reagent kit described in the seventh aspect, or the composition described in the eighth aspect. Thus, the method of the present application can hydrolyze chitin and / or hydrolyze PET, or treat waste.

[0082] In the eleventh aspect of the present application, the present application provides a method for improving soil. According to an embodiment of the present application, the method includes: 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 reagent kit described in the seventh aspect, or 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.

[0083] The solution of the present application will be explained below in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present application and should not be construed as limiting the scope of the present application. For those not specified in the embodiments regarding specific techniques or conditions, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For reagents or instruments not specified as to the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0084] Example 1:

[0085] 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 chitinase and PETase bifunctional hydrolase (hereinafter referred to as bifunctional hydrolase) to screening and optimization.

[0086] See Figure 1 , the design and screening mainly generate the bifunctional hydrolase protein sequence through the Protein Generation Model (PGM), screen out the target (qualified in quality) bifunctional hydrolase protein sequence from the generated bifunctional hydrolase protein sequences through the Quality Control Pipeline (QCP), synthesize the protein-coding DNA according to the screened target bifunctional hydrolase protein sequence, express it in the protein expression system, and detect the bifunctional hydrolase ability and thermal stability of the expressed protein through the For Wet-lab Assay.

[0087] 1. Generation of bifunctional hydrolase protein sequence

[0088] Exemplarily, the protein generation model is adopted to generate the 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, and can handle complex molecular systems.

[0089] 2. Screening of target bifunctional hydrolase protein sequence

[0090] Exemplarily, a quality control process screening is adopted to screen out the target bifunctional hydrolase protein sequences with qualified quality from the generated bifunctional hydrolase protein sequences. The quality control process screening includes the screening of the functional domain of the bifunctional hydrolase protein sequence, the screening of the sequence identity, the screening of the structure identity, and the screening using the Unified Sequence Model (USM).

[0091] Among them, the "functional domain" 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 components, biological processes, and molecular functions.

[0092] The "sequence identity" is used to indicate the amino acid sequence included in the bifunctional hydrolase protein sequence.

[0093] The "structure identity" is used to indicate the geometric structure of the bifunctional hydrolase protein sequence in three-dimensional space. For example, at least one level of category in the class, fold, superfamily, and family in the structure classification of the bifunctional hydrolase protein sequence.

[0094] The "Unified Sequence Model" is a model that combines the functions of a single sequence model and a multiple sequence alignment (MSA) model. The Unified Sequence Model is not only a general biological language model that can be applied to the screening task of bifunctional hydrolase protein sequences, but also a general multiple sequence model that can support two data forms of single sequence and multiple sequence alignment, unify the single sequence and multiple sequence modes (for example: the processing corresponding to the data of molecular formulas and the data of amino acid sequences can be compatible), and has versatility.

[0095] 3. Synthesis of the target bifunctional hydrolase

[0096] 1) Plasmid construction

[0097] The genes encoding the target proteins (bifunctional hydrolase 1 with the amino acid sequence shown in SEQ ID NO:1, bifunctional hydrolase 2 with the amino acid sequence shown in SEQ ID NO:2, bifunctional hydrolase 3 with the amino acid sequence shown in SEQ ID NO:3, bifunctional hydrolase 4 with the amino acid sequence shown in SEQ ID NO:4) were synthesized by homologous recombination amplification respectively. And using the Hieff Clone™ One Step Cloning Kit, the target genes were inserted into the corresponding pJL1 expression vectors through restriction enzyme digestion and ligation reactions. The expression vectors were sequenced to confirm the correct insertion of the target genes. The expression vectors carrying the target genes were transformed into DH5α competent cells. Single colonies were selected for vector amplification. A kit similar to the PureLink HQ 96 Plasmid DNA Purification Kit (Thermo Cat# K210096) was used to extract the plasmids.

[0098] 2) Cell-free protein synthesis

[0099] The plasmids extracted in step 1) were added to an EP tube containing a 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.

[0100] 3) Protein purification

[0101] 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.

[0102] Protein purification: The target protein was extracted from the CFPS supernatant by affinity chromatography or a combination of multiple chromatography methods. After each purification step, the crude sample was subjected to buffer exchange and then proceeded to the next purification step. The final purified product was exchanged into a storage buffer.

[0103] Protein quality assessment: Methods such as SDS-PAGE and ultraviolet spectroscopy (OD280) were used to evaluate the purity and content of the crude samples after each purification step and the final protein product.

[0104] Among them, western blots detection was performed on the expressed bifunctional hydrolase, and the western blots bands are shown in Figure 2 . Figure 2The leftmost first column is the marker band. The immunoblot bands in the yellow box are the protein expression bands of the bifunctional hydrolase. From left to right, they are the protein expression bands of bifunctional hydrolase 1 (amino acid sequence shown in SEQ ID NO:1), bifunctional hydrolase 2 (amino acid sequence shown in SEQ ID NO:2), bifunctional hydrolase 3 (amino acid sequence shown in SEQ ID NO:3), and bifunctional hydrolase 4 (amino acid sequence shown in SEQ ID NO:4).

[0105] 4. Detection of the ability and thermal stability of bifunctional hydrolase

[0106] 4.1 PET enzyme activity detection:

[0107] Mix 10 μL of the enzyme mother liquor (each bifunctional hydrolase obtained in step 3 of this example) with 80 μL of the activity assay buffer (50 mM p-nitrophenyl butyrate (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).

[0108] 4.2 Chitinase activity detection:

[0109] Mix 20 μL of the yeast solution (each bifunctional hydrolase obtained in step 3 of this example) with 80 μL of the activity assay buffer (125 μM chitotriose substrate Mu-(GlcMAc)3, 200 mM sodium phosphate, pH 8.0), incubate at the temperature to be measured (such as 37 °C, 85 °C, etc.) for 2 - 6 hours, and detect the fluorescence signal of the product 4-methylumbelliferone with an excitation wavelength of 372 nm and an emission wavelength of 445 nm (Reference: DOI: 10.1080 / 14756366.2021.1931862). The enzyme activities of three natural chitinases, OFCHTh, SMCHIB, and SMCHIC, were also tested under the same conditions for comparison. OFCHTh is from insects, and SMCHIB and SMCHIC are from Serratia. 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).

[0110] 4.3 Detection Results

[0111] The detection results are shown in Table 1. The four bifunctional hydrolases in this example simultaneously exhibited chitinase and PET enzyme activities at 37 °C, while the natural chitinases only exhibited chitinase activity; after heating at 85 °C for three hours, more than 80% of the enzyme activities of the four bifunctional hydrolases in this example were still maintained, while the natural chitinases had no remaining enzyme activity at this time.

[0112] Table 1:

[0113]

[0114] In Table 1, / represents inactivity; RSD is Relative Standard Deviation, which reflects the deviation degree among the results of multiple experiments.

[0115] In the description of this specification, the descriptions referring to terms such as "one embodiment", "some embodiments", "examples", "specific examples", or "some examples", etc. mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of this 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 can be combined in any one or more embodiments or examples in a suitable manner. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0116] Although the embodiments of this application have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limitations on this application. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of this application.

Claims

1. A chitinase and PETase bifunctional hydrolase, characterized in that: The amino acid sequence of the bifunctional hydrolase is shown in SEQ ID NO:

1.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the bifunctional hydrolase according to 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 according to claim 2 or the expression vector according to claim 3.

5. A recombinant cell, characterized in that The recombinant cell expresses the bifunctional hydrolase of claim 1.

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

7. Use of a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 as a chitinase and PETase bifunctional hydrolase.

8. A reagent or a kit, characterized in that: The invention comprises the bifunctional hydrolase according to claim 1.

9. A composition, characterized in that The invention comprises the bifunctional hydrolase according to claim 1.

10. Use of the bifunctional hydrolase according to claim 1 in preparing a product, wherein the product is used for Hydrolyzed PET.

11. Use of the bifunctional hydrolase according to claim 1 in preparing a product for hydrolyzing chitin and PET.

12. Use of the bifunctional hydrolase according to claim 1 in preparing a product, wherein the product is used to treat waste, wherein the waste contains PET or chitin and PET.

13. Use of the bifunctional hydrolase according to claim 1 in preparing a product, wherein the product is used to improve soil, wherein the soil contains PET, or contains chitin and PET.

14. A method for hydrolyzing PET, characterized in that: include: The bifunctional hydrolase according to claim 1, the bifunctional hydrolase prepared by the method according to claim 6, the reagent or kit according to claim 8, or the composition according to claim 9 is used to contact a sample, wherein the sample contains PET.

15. A method for hydrolyzing PET and chitin, characterized in that: include: The bifunctional hydrolase according to claim 1, the bifunctional hydrolase prepared by the method according to claim 6, the reagent or kit according to claim 8, or the composition according to claim 9 is used to contact a sample, wherein the sample contains PET and chitin.

16. A method for treating waste, characterized in that: include: The waste is contacted with the bifunctional hydrolase according to claim 1, the bifunctional hydrolase prepared by the method according to claim 6, the reagent or kit according to claim 8, or the composition according to claim 9, wherein the waste contains PET.

17. A method for treating waste, characterized in that: include: The waste is contacted with the bifunctional hydrolase according to claim 1, the bifunctional hydrolase prepared by the method according to claim 6, the reagent or kit according to claim 8, or the composition according to claim 9, wherein the waste contains chitin and PET.

18. A method for improving soil, characterized in that: include: The soil to be improved is contacted with the bifunctional hydrolase described in claim 1, the bifunctional hydrolase prepared by the method described in claim 6, the reagent or kit described in claim 8, or the composition described in claim 9 to degrade PET in the soil to be improved.

19. A method for improving soil, characterized in that: include: The soil to be improved is contacted with the bifunctional hydrolase described in claim 1, the bifunctional hydrolase prepared by the method described in claim 6, the reagent or kit described in claim 8, and the composition described in claim 9 to degrade PET and chitin in the soil to be improved.

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