A recombinant chitin deacetylase and its preparation method and application

Abstract

The present invention provides a recombinant chitin deacetylase, its preparation method, and application. The inventors have isolated and obtained a novel chitin deacetylase that not only catalyzes the deacetylation of chitin but also effectively inhibits pathogenic bacteria. The chitin deacetylase of the present invention has good adaptability to low temperatures, can be expressed under prokaryotic expression conditions, and exhibits relatively high low-temperature activity. Furthermore, the present invention also optimizes the recombinant expression method of the low-temperature chitin deacetylase, thereby enabling its efficient expression in host cells.

Description

Technical Field

[0001] The present invention belongs to the fields of microbiology, molecular biology and biochemistry. More specifically, the present invention relates to the recombinant heterologous expression, preparation and application research of a low-temperature chitin deacetylase. The low-temperature recombinant chitin deacetylase of the present invention is mainly used in the pharmaceutical, food, agricultural and other industries. Background Art

[0002] Chitin is a linear polysaccharide composed of β-N-acetyl-D-glucosamine monomers linked by β-1,4-glycosidic bonds. Chitosan is the product of chitin deacetylation. Generally, chitosan is defined as having a degree of N-deacetylation above 55%. The main differences between chitosan and chitin lie in their degree of polymerization, degree of acetylation, and acetylation pattern. Chitosan is more soluble than chitin. The hydrogen ions in chitosan molecules are hydrogenated, preventing them from forming hydrogen bonds. This makes chitosan much more chemically active than chitin, making it susceptible to chemical reactions such as acetylation, alkylation, methylation, esterification, and etherification.

[0003] In recent years, chitosan, a compound derived from chitin, has attracted increasing attention from both the scientific and industrial communities due to its numerous unique biological activities. Currently, chitosan is primarily used in the food, pharmaceutical, and environmental fields, and its application is expanding with increasing research. However, the industrial production of chitosan, which primarily utilizes a hot alkaline process, suffers from high production costs, poor product uniformity, and difficulty controlling the reaction process. Furthermore, the production process also produces alkaline wastewater, which pollutes the environment. Consequently, the high energy consumption and environmental pollution associated with the industrial production of chitosan remain pressing challenges.

[0004] Chitin deacetylases (CDAs) are enzymes that catalyze the enzymatic conversion of chitin to chitosan. They catalyze the deacetylation of β-N-acetyl-D-glucosamine to achieve the conversion of chitin to chitosan. Furthermore, some chitin deacetylases exhibit certain antifungal activity and can be used as novel insecticides and antifungal agents in modern agriculture. Although research has been conducted on the use of chitin deacetylases to produce chitosan, the chitin deacetylases available in the prior art vary widely in terms of physiological and biochemical properties, biological functions, and enzyme activity and substrate specificity. Furthermore, they generally exhibit low activity at low temperatures.

[0005] Therefore, there is an urgent need in the art to find a new chitin deacetylase with low temperature and high activity to improve the industrial production level of such enzymes and expand their applications. Summary of the Invention

[0006] The purpose of the present invention is to provide a recombinant chitin deacetylase and a preparation method and application thereof.

[0007] In the first aspect of the present invention, an isolated polypeptide is provided, which is selected from the following groups: (a) a polypeptide having the amino acid sequence shown in SEQ ID NO: 2; (b) a polypeptide formed by replacing, deleting or adding one or more (such as 1-20, preferably 1-10; more preferably 1-5; more preferably 1-3) amino acid residues of the polypeptide described in (a), and having the function of polypeptide (a); or (c) a polypeptide having an amino acid sequence of more than 80% (preferably more than 85%; more preferably more than 90%; more preferably more than 95%, such as 98%, 99%) homology with the polypeptide described in (a) and having the function of polypeptide (a); (d) a polypeptide formed by adding a tag sequence to the N- or C-terminus of the polypeptide described in (a) or (b) or (c), or adding a signal peptide sequence to its N-terminus.

[0008] In a preferred embodiment, the polypeptide has high low-temperature activity; preferably, its optimal reaction temperature is 15°C; preferably, it still has a catalytic activity of more than 65% at, for example, 5°C.

[0009] In another aspect of the present invention, an isolated polynucleotide is provided, which comprises a nucleotide sequence selected from the following groups: (1) a polynucleotide encoding a polypeptide as described; (2) a polynucleotide complementary to the polynucleotide (1).

[0010] In a preferred embodiment, the polynucleotide encodes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2; preferably, the nucleotide sequence of the polynucleotide is as shown in SEQ ID NO: 1.

[0011] In another aspect of the present invention, a vector is provided, which contains the polynucleotide.

[0012] In another aspect of the present invention, a genetically engineered host cell is provided, which contains the vector, or has the polynucleotide integrated into its genome.

[0013] In a preferred embodiment, the integration includes targeted integration or random integration.

[0014] In another preferred embodiment, the cells are not plant reproductive cells or animal stem cells.

[0015] In another preferred embodiment, the host cell is a prokaryotic cell, such as but not limited to Escherichia coli.

[0016] In another aspect of the present invention, a method for preparing the polypeptide is provided, comprising: (i) culturing the host cell; (ii) collecting the culture containing the polypeptide; and (iii) isolating the polypeptide from the culture.

[0017] Preferably, the culture is carried out under the following conditions: the concentration of the inducer IPTG (when the host cell is Escherichia coli) is 0.05-0.3 mM, preferably 0.08-0.15 mM; the induction temperature is 16-35°C, more preferably 18-30°C; more preferably 18-20°C; the induction time is 15-20 hours; preferably 16-18 hours.

[0018] In another aspect of the present invention, the use of the polypeptide is provided, for catalyzing the deacetylation of chitin, or for preparing a composition for catalyzing the deacetylation of chitin; preferably, chitosan is generated after deacetylation.

[0019] In another aspect of the present invention, the use of the polypeptide is provided for: inhibiting microorganisms, or for preparing a composition having the function of inhibiting microorganisms; preferably, the microorganism is a microorganism whose cell wall contains chitin.

[0020] In another aspect of the present invention, a composition is provided, comprising: the polypeptide or the host cell; and an industrially or microbiologically acceptable carrier.

[0021] In a preferred embodiment, the composition is a pesticide composition, which is used for preventing and controlling plant diseases, and is preferably used for preventing and controlling cotton Verticillium wilt and cucumber wilt.

[0022] In another aspect of the present invention, a method for catalyzing the deacetylation of chitin is provided, comprising: treating chitin or a substance containing chitin with the polypeptide, the host cell or the composition; preferably, chitosan is generated after deacetylation.

[0023] In another aspect of the present invention, a method for inhibiting microorganisms is provided, comprising: applying the polypeptide, the host cell or the composition to treat an object requiring microbial inhibition (such as a place containing microorganisms, a substance, an animal or plant or a processed product thereof); preferably, the microorganism is a microorganism whose cell wall contains chitin.

[0024] In a preferred embodiment, the treatment is carried out at a temperature of 0-35°C, preferably 5-25°C, more preferably 10-20°C (such as 12, 14, 15, 16, 18°C).

[0025] In another preferred embodiment, the treatment is carried out at pH 5-10, preferably pH 5.5-9, more preferably pH 6.5-8.5 (such as pH 7, 7.5, 8).

[0026] In another preferred embodiment, the treatment is carried out under the conditions of NaCl 0.01-0.5M, preferably 0.03-0.3M, more preferably 0.05-0.2M (such as 0.06, 0.08, 0.1, 0.12, 0.15M).

[0027] In another preferred embodiment, in the presence of Na + , K + Mg 2+ 、Zn 2+ and / or Ni 2+ The treatment is carried out under the following conditions (the metal ion content is 1±0.8 mM, preferably 1±0.5 mM; more preferably 1±0.3 mM).

[0028] In another preferred embodiment, the treatment is carried out in the presence of EDTA (e.g., in an amount of 1±0.5%).

[0029] In another preferred embodiment, the treatment is carried out in the presence of DTT (e.g., in an amount of 1±0.5%).

[0030] In another preferred embodiment, when performing the treatment, the reaction system does not contain: Li + NH4 + , Ca 2+ 、Mn 2+ 、Cu 2+ 、Fe 2+ 、Fe 3+ , SDS and / or TritonX-100.

[0031] In another preferred embodiment, when performing the treatment, the reaction system does not contain: acetone, ethanol, methanol and / or acetonitrile.

[0032] In another preferred embodiment, the microorganisms include fungi; preferably, the fungi include Verticillium, Fusarium, Aspergillus, and Penicillium. More preferably, the Verticillium includes (but is not limited to) Verticillium dahlia; the Fusarium includes (but is not limited to) Fusarium oxysporum f.sp. cucumerinum; the Aspergillus includes (but is not limited to) Aspergillus niger; and the Penicillium includes (but is not limited to) Penicillium macroclerotiorum.

[0033] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 , recombinant expression and condition optimization of chitin deacetylase of the present invention;

[0035] (a) Changes in soluble enzyme expression at different induction temperatures; Lane 1 represents uninduced whole-cell lysate, and Lanes 2 to 6 represent induction temperatures of 15°C, 20°C, 25°C, 30°C, and 35°C, respectively.

[0036] (b) Changes in soluble enzyme expression at different inducer concentrations; Lane 1 represents uninduced whole-cell lysate, and Lanes 2 to 9 represent IPTG concentrations of 0 mM, 0.01 mM, 0.02 mM, 0.05 mM, 0.1 mM, 0.15 mM, 0.25 mM, and 0.3 mM, respectively;

[0037] (c) Changes in soluble enzyme expression levels when inoculated with different amounts of recombinant bacteria; Lane 1 is the uninduced whole-cell lysate, and Lanes 2 to 7 are 0.5%, 1%, 1.5%, 2%, 2.5%, and 3% of the inoculum, respectively;

[0038] (d) Changes in the soluble expression level of the enzyme at different induction times; Lane 1 is the uninduced whole-cell lysate, and Lanes 2 to 9 are the induction times of 4h, 8h, 12h, 16h, 20h, 24h, 28h, and 32h, respectively.

[0039] Figure 2 , purification of the recombinant expression product of the chitin deacetylase of the present invention;

[0040] (a) Lane 1 to Lane 4 represent: whole cells without IPTG induction, IPTG-induced cells, IPTG-induced supernatant, and purified chitin deacetylase, respectively;

[0041] (b) Lane 1 is chitin deacetylase stained with Coomassie Brilliant Blue R-250 after electrophoresis, and Lane 2 is chitin deacetylase stained with Calcofluor White M2R after electrophoresis.

[0042] Figure 3 , the properties of the chitinase of the present invention;

[0043] (a) Study on the optimal temperature of recombinant chitin deacetylase;

[0044] (b) Study on the temperature stability of recombinant chitin deacetylase;

[0045] (c) Study on the optimal pH of recombinant chitin deacetylase;

[0046] (d) pH stability study of recombinant chitin deacetylase;

[0047] (e) Effect of sodium chloride on the activity of recombinant chitin deacetylase;

[0048] (f) Effect of sodium chloride on the stability of recombinant chitin deacetylase.

[0049] Figure 4 The recombinant chitin deacetylase of the present invention can inhibit multiple plant pathogenic fungi. DETAILED DESCRIPTION

[0050] The present inventor has isolated a novel chitin deacetylase (low-temperature chitin deacetylase) from a bacterial strain in Antarctica through large-scale screening and in-depth research. The chitin deacetylase can not only catalyze the deacetylation of chitin, but also effectively inhibit pathogenic bacteria, and has a very good application prospect. The chitin deacetylase of the present invention has good adaptability to low temperatures, can be expressed at a relatively high temperature of prokaryotic expression, and has higher low-temperature activity. Simultaneously, the present invention has also optimized the recombinant expression method of the low-temperature chitin deacetylase, thereby making it obtain efficient expression in host cells.

[0051] As used herein, the terms "polypeptide of the present invention", "protein of the present invention", "low-temperature chitin deacetylase", and "chitin deacetylase" are used interchangeably and refer to a protein or polypeptide having SEQ ID NO: 2 or a fragment thereof or a variant form or derivative thereof.

[0052] As used herein, "isolated" means a substance that has been separated from its original environment (in the case of a naturally occurring substance, the original environment is the natural environment). For example, polynucleotides and polypeptides in their natural state within living cells are not isolated and purified. However, the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances with which they are naturally present.

[0053] As used herein, "isolated polypeptide (low-temperature chitin deacetylase in the present invention)" means that the chitin deacetylase is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. Those skilled in the art can purify the chitin deacetylase using standard protein purification techniques. A substantially pure polypeptide will produce a single major band on a non-reducing polyacrylamide gel. The purity of the chitin deacetylase can be determined by amino acid sequence analysis.

[0054] As used herein, the "microorganism" refers to a microorganism whose cell wall contains chitin, and the microorganism includes fungi, such as fungi, actinomycetes or bacteria; preferably, the "microorganism" is a "pathogenic microorganism".

[0055] As used herein, the "pathogenic microorganism" refers to a microorganism that is harmful to humans, animals, plants or the environment.

[0056] In the present invention, the term "comprising" means that various components can be used together in the mixture or composition of the present invention. Therefore, the terms "mainly consisting of" and "consisting of" are included in the term "comprising".

[0057] As used herein, an "industrially acceptable carrier" or "microbiologically acceptable carrier" is a solvent, suspending agent, or excipient that is environmentally friendly or harmless to humans and animals and is used to deliver the chitin deacetylase of the present invention to an object requiring treatment, and is controllable in terms of toxicity and side effects. The carrier can be liquid or solid, and preferably is a carrier that can maintain the activity of the chitin deacetylase of the present invention to a high degree.

[0058] The polypeptides of the present invention may be recombinant polypeptides, natural polypeptides, or synthetic polypeptides, preferably recombinant polypeptides. The polypeptides of the present invention may be naturally purified products, or chemically synthesized products, or produced using recombinant technology from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plants, insects, and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptides of the present invention may be glycosylated or non-glycosylated. The polypeptides of the present invention may or may not include an initial methionine residue.

[0059] The present invention also includes fragments, derivatives and analogs of the chitin deacetylase. As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that substantially maintain the same biological function or activity as natural chitin deacetylase of the present invention. Polypeptide fragments, derivatives, or analogs of the present invention may be polypeptides having one or more conservative or non-conservative amino acid residues (preferred conservative amino acid residues) substituted, or alternatively, polypeptides having a substituent group in one or more amino acid residues, or polypeptides formed by merging a mature polypeptide with another compound (such as a compound that prolongs the polypeptide half-life, for example polyethylene glycol), or polypeptides formed by merging an additional amino acid sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify this polypeptide, or a fusion protein with an antigenic IgG fragment) to form the polypeptide sequence. According to the teachings of this paper, these fragments, derivatives, and analogs belong to the well-known scope of those skilled in the art.

[0060] In the present invention, the term "chitin deacetylase" refers to a polypeptide having the sequence of SEQ ID NO: 2 that exhibits chitin deacetylase activity. The term also encompasses variants of SEQ ID NO: 2 that exhibit the same function as the chitin deacetylase. These variants include (but are not limited to): deletion, insertion, and / or substitution of one or more (generally 1-50, preferably 1-30, more preferably 1-20, even more preferably 1-10, and most preferably 1-5) amino acids, as well as addition or deletion of one or more (generally within 20, preferably within 10, and even more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, substitution with amino acids having similar or similar properties generally does not alter protein function. For example, addition or deletion of one or more amino acids at the C-terminus and / or N-terminus generally does not alter protein function. For another example, expressing only the catalytic domain of a protein without the carbohydrate-binding domain can achieve the same catalytic function as the intact protein. Therefore, the term also includes active fragments and active derivatives of the chitin deacetylase.For example, variations may occur outside the conserved functional domains of SEQ ID NO:2.

[0061] Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the chitin deacetylase DNA under high or low stringency conditions, and polypeptides or proteins obtained using antibodies against the chitin deacetylase. The present invention also provides other polypeptides, such as fusion proteins comprising the chitin deacetylase or its fragments. In addition to nearly full-length polypeptides, the present invention also includes fragments of the chitin deacetylase. Typically, the fragment has at least about 10 consecutive amino acids of the chitin deacetylase sequence, usually at least about 30 consecutive amino acids, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0062] The invention also provides analogs of the chitin deacetylase protein or polypeptide. The difference between these analogs and the natural chitin deacetylase can be a difference in the amino acid sequence, or a difference in a modified form that does not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, or by site-directed mutagenesis or other known molecular biological techniques. Analogs also include analogs with residues (such as D-amino acids) that are different from natural L-amino acids, and analogs with non-natural or synthetic amino acids (such as β, γ-amino acids). It should be understood that polypeptides of the present invention are not limited to the representative polypeptides cited above.

[0063] In the present invention, "conservative variants of chitin deacetylase" refer to polypeptides having no more than 30, preferably no more than 20, more preferably no more than 10, and even more preferably no more than 5 amino acids replaced by amino acids with similar or similar properties, compared to the amino acid sequence of SEQ ID NO: 2. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table 1.

[0064] Table 1

[0065] Initial residue Representative replacement Preferred substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile;Leu;Met;Phe;Ala Leu

[0066] The amino terminus or carboxyl terminus of the chitin deacetylase of the present invention may also contain one or more polypeptide fragments as protein tags. Any suitable tag can be used in the present invention. For example, the tag can be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ε, B, gE and Ty1.

[0067] In order to secrete the translated protein (e.g., secrete it outside the cell), a host-compatible signal peptide can be added to the amino terminus of the chitin deacetylase. The signal peptide can be cleaved during the process of secretion of the polypeptide from the cell.

[0068] The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or artificially synthesized DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be identical to the native coding sequence of chitin deacetylase or the coding sequence shown in SEQ ID NO:1, or it may be a degenerate variant. As used herein, "degenerate variant" in the present invention refers to a nucleic acid sequence that encodes a protein having SEQ ID NO:2 but differs from the native coding sequence of chitin deacetylase or the coding sequence shown in SEQ ID NO:1.

[0069] Polynucleotides encoding the mature polypeptide of SEQ ID NO: 2 include: a coding sequence encoding only the mature polypeptide; a coding sequence for the mature polypeptide and various additional coding sequences; a coding sequence for the mature polypeptide (and optional additional coding sequences) and non-coding sequences. The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide or a polynucleotide further including additional coding and / or non-coding sequences.

[0070] The present invention also relates to variants of the aforementioned polynucleotides, which encode polypeptides or polypeptide fragments, analogs, and derivatives having the same amino acid sequence as the present invention. These polynucleotide variants may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is an alternative form of a polynucleotide, which may contain one or more nucleotide substitutions, deletions, or insertions that do not substantially alter the function of the encoded polypeptide.

[0071] The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize to the polynucleotides of the present invention under stringent conditions (or stringent conditions). In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, more preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.

[0072] The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and more preferably purified to homogeneity.

[0073] Described chitin deacetylase nucleotide full-length sequence or its fragment can be obtained with PCR amplification method, recombination method or artificial synthesis method usually.At present, can obtain the DNA sequence encoding protein of the present invention (or its fragment, or its derivative) by chemical synthesis fully.Then this DNA sequence can be introduced into various existing DNA molecules (or as vector) and cell as known in the art.

[0074] The present invention also provides a vector comprising the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector of the present invention or the chitin deacetylase coding sequence, and a method for producing the polypeptide of the present invention by recombinant technology.

[0075] By conventional recombinant DNA technology, the polynucleotide sequence of the present invention can be used to express or produce the chitin deacetylase of recombinant. Generally speaking, the following steps are included:

[0076] (1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding the chitin deacetylase, or a recombinant expression vector containing the polynucleotide;

[0077] (2) Host cells cultured in a suitable culture medium;

[0078] (3) Isolate and purify proteins from culture medium or cells.

[0079] In the present invention, the chitin deacetylase polynucleotide sequence can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. As long as they can replicate and be stable in the host, any plasmid and vector can be used. An important feature of an expression vector is that it usually contains an origin of replication, a promoter, a marker gene and a translation control element.

[0080] Methods well known to those skilled in the art can be used to make up the expression vector containing the DNA sequence dna of coding described chitin deacetylase and suitable transcription / translation control signal.These methods comprise in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology etc.Described DNA sequence dna can be effectively connected on the suitable promotor in the expression vector, to instruct mRNA synthesis.Expression vector also comprises the ribosome binding site and transcription terminator that translation initiation is used.In addition, expression vector preferably comprises one or more selective marker genes, to provide the phenotypic traits of the host cell that is used to select transformation.The carrier that comprises above-mentioned suitable DNA sequence dna and suitable promotor or control sequence can be used to transform suitable host cell, so that it can express protein.

[0081] Host cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells, such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; and animal cells such as CHO, COS, 293 cells, or Bowes melanoma cells. In a preferred embodiment of the present invention, the host cell is a prokaryotic cell.

[0082] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0083] The recombinant polypeptide in the above method can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0084] As a preferred embodiment of the present invention, Escherichia coli is used as a host cell to express the chitin deacetylase. The present invention constructs an expression system to heterologously express the low-temperature chitin deacetylase in Escherichia coli.

[0085] As a preferred embodiment of the present invention, the culture is carried out under the following conditions: the concentration of the inducer IPTG (when the host cell is Escherichia coli) is 0.05-0.3 mM, preferably 0.08-0.15 mM; the induction temperature is 16-35°C, more preferably 18-30°C, more preferably 18-20°C; and the induction time is 15-20 hours, preferably 16-18 hours. Under these preferred conditions, a recombinant chitin deacetylase with high expression level and high enzyme activity can be obtained.

[0086] The chitin deacetylase of the present invention can be used to catalyze the deacetylation of chitin or to prepare a composition for catalyzing the deacetylation of chitin. Preferably, chitosan is generated after the chitin is deacetylated.

[0087] The chitin deacetylase of the present invention can also be used to inhibit microorganisms or to prepare a composition having the function of inhibiting microorganisms. Preferably, the microorganism is a microorganism whose cell wall contains chitin.

[0088] After obtaining chitin deacetylase of the present invention, according to the present invention, those skilled in the art can conveniently use the enzyme to play the role of degrading substrates (particularly catalyzing the deacetylation of chitin as a substrate). As a preferred embodiment of the present invention, a method for degrading chitin is also provided, which comprises: treating a substrate to be degraded (particularly chitin) with the chitin deacetylase of the present invention.

[0089] In some embodiments of the present invention, the activity of chitin deacetylase was investigated by catalyzing the production of p-nitroaniline from p-nitroacetanilide. After confirming that chitin deacetylase can catalyze the deacetylation of its natural substrate chitin, the inventors selected p-nitroacetanilide as an artificial substrate to study the enzymatic properties of chitin deacetylase.

[0090] According to the present invention, those skilled in the art can also use the enzyme to inhibit microorganisms (such as pathogenic microorganisms). This includes treating an object requiring microbial inhibition with the chitin deacetylase. The microorganisms include fungi. In a preferred embodiment of the present invention, the fungi preferably include Verticillium, Fusarium, Aspergillus, and Penicillium. More preferably, the Verticillium includes (but is not limited to) Verticillium dahlia; the Fusarium includes (but is not limited to) Fusarium oxysporum f.sp. cucumerinum; the Aspergillus includes (but is not limited to) Aspergillus niger; and the Penicillium includes (but is not limited to) Penicillium macroclerotiorum. The inventors have discovered that the chitin deacetylase has an excellent degradation effect on the cell wall chitin of such fungi. Therefore, the chitin deacetylase of the present invention or cells producing the chitin deacetylase can be used in the prevention and control of agricultural microorganisms, such as, but not limited to, cotton Verticillium wilt and cucumber wilt.

[0091] The inventors have also optimized the reaction system for treatment using the chitin deacetylase. As a preferred embodiment of the present invention, the treatment is carried out under the following conditions: temperature 0-35°C, preferably 5-25°C, more preferably 10-20°C, such as 12, 14, 15, 16, 18°C; pH 5-10, preferably pH 5.5-9, more preferably pH 6.5-8.5, such as pH 7, 7.5, 8.

[0092] The inventors also found that the use of a small amount of NaCl is beneficial for providing a good reaction environment for the chitin deacetylase of the present invention and improving its catalytic activity; therefore, in a preferred embodiment of the present invention, 0.01 to 0.5 M, preferably 0.03 to 0.3 M, more preferably 0.05 to 0.2 M of NaCl is added to the enzyme reaction system (treatment system); for example, 0.06, 0.08, 0.1, 0.12, or 0.15 M.

[0093] The inventors also found that the reaction system contains Na + , K + Mg 2+ 、Zn 2+ and / or Ni 2+This is beneficial for promoting the enzymatic activity of the chitin deacetylase of the present invention. In a preferred embodiment, the metal ion content is 1±0.8 mM, preferably 1±0.5 mM, and more preferably 1±0.3 mM.

[0094] The inventors have also discovered that the inclusion of EDTA or DTT in the reaction system is beneficial for promoting the enzymatic activity of the chitin deacetylase of the present invention. Preferably, the amount of EDTA is 0.1% to 5%, preferably 0.5% to 2%, such as 1%; or the amount of DTT is 0.1% to 5%, preferably 0.5% to 2%, such as 1%.

[0095] As a preferred embodiment of the present invention, when the treatment is carried out, the reaction system does not contain: Li + NH4 + , Ca 2+ 、Mn 2 + 、Cu 2+ 、Fe 2+ 、Fe 3+ , SDS and / or Triton X-100; also does not contain: acetone, ethanol, methanol and / or acetonitrile.

[0096] The chitin deacetylase obtained by the present invention has ideal enzyme activity and can be recombinantly expressed under temperature conditions suitable for expression in Escherichia coli. It can also be applied to enzyme reactions (including enzyme-catalyzed reactions or inhibition of microorganisms) under low temperature conditions, and has broad industrial application potential.

[0097] According to the isolated chitin deacetylase and amino acid sequence thereof provided by the present invention, those skilled in the art can further improve its enzymatic activity or expand its applicable pH range, temperature range, salt tolerance and stability for cold and heat etc. by means such as protein molecule transformation, so its application prospect is good. Variants or derivatives generated after adopting these technical transformations of chitin deacetylase of the present invention are also included in the present invention.

[0098] The present invention also provides a composition comprising an effective amount of the chitin deacetylase of the present invention and a food science or industrially acceptable carrier or excipient. Such carriers include, but are not limited to, water, a buffer, glucose, glycerol, DMSO, or a combination thereof. Those skilled in the art can determine the effective amount of the chitin deacetylase in the composition based on the actual application of the composition.

[0099] The composition can also be added with a substance that regulates the activity of the chitin deacetylase of the present invention. Any substance that has the function of improving the activity of the enzyme is usable. Preferably, the substance that improves the activity of the chitin deacetylase is selected from: Na + , K + Mg2+ 、Zn 2+ and / or Ni 2+ or can be hydrolyzed to form Na after addition to the substrate + , K + Mg 2+ 、Zn 2+ and / or Ni 2+ The substance that improves the activity of the chitin deacetylase can also be selected from: EDTA or DTT.

[0100] The chitin deacetylase of the present invention, the vector or host cell containing the enzyme, or the composition containing the enzyme or host cell may also be contained in a container or a kit. Preferably, the kit also includes instructions for use, etc., to facilitate application by those skilled in the art.

[0101] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental procedures in the following examples, for which specific conditions are not specified, were generally performed under conventional conditions, such as those described in J. Sambrook et al., Molecular Cloning Laboratory Manual, 3rd edition, Science Press, 2002, or according to the conditions recommended by the manufacturer.

[0102] Example 1. Source and sequence information of low-temperature chitin deacetylase

[0103] The low-temperature chitin deacetylase described in the present invention is derived from the strain Pseudomonas sp.GWSMS-1 (CCTCCNO.M2019207), which has been deposited in the China Center for Type Culture Collection, the name of the depository unit is: China Center for Type Culture Collection, the deposit date is: March 27, 2019, and its deposit number is CCTCC NO.M2019207.

[0104] After obtaining the Pseudomonas sp. GWSMS-1 strain, the inventors conducted extensive research and testing on its various properties, its genetic characteristics, and the diverse proteins it produces, focusing on key functional genes and proteins, thereby screening and obtaining the low-temperature chitin deacetylase of the present invention. The amino acid sequence of the low-temperature chitin deacetylase is as follows (SEQ ID NO: 2):

[0105] MSADYPRDLIGYANNPPHPHWPNDARIALSFVLNYEEGGERNILHGDKESEAFLSEMVSAQPLQGQRNLCMESLYEYGSRAGVWRLLSLFQKHNIPLTIFAVAMAAQRHPDAIKAMADAGHEICSHGYRWIDYQNMSEAEEREHMHEAIRILTELTG QRPQGWYTGRTGPNTRRLVREEGGFLYDSDTYDDDLPYWDPASTAAKPHLVIPYTLDTNDMRFTQVQGFNTGDDFFQYLKDAFDVLYAEGVAGAPKMLTIGMHCRLLGRPARLASLARFIEYVQSHEQVWCARRVDIAKHWHATHPFNEQASKELSK

[0106] Example 2: Cloning, heterologous expression and optimization of chitin deacetylase

[0107] 1. Materials and Methods

[0108] (1) Culture medium

[0109] LB liquid medium: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L.

[0110] LB solid medium: Add 20 g / L agar powder to LB liquid medium.

[0111] (2) Codon optimization, whole gene synthesis and cloning of chitin deacetylase gene

[0112] The inventors first performed codon optimization based on the expression profile to improve expression efficiency and DNA fragment stability. The optimized sequence was inserted into the plasmid pET28a(+) to generate the recombinant plasmid pET28-CDA. The recombinant plasmid pET28-CDA was then transformed into Escherichia coli BL21(DE3) for heterologous expression.

[0113] (3) Optimization of expression conditions of recombinant chitin deacetylase gene

[0114] Inoculation volume: The inoculation volume was set to 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, and 3.0%. The recombinant bacteria were inoculated into fresh LB liquid medium according to the above settings and cultured at 37°C and 200 rpm. 600 When the pH value reached 0.6-0.8, IPTG was added to the culture medium at a final concentration of 0.1 mM (IPTG was not added to the control group), and then cultured at 20°C and 150 rpm for 10 hours, and then the bacteria were collected.

[0115] Induction temperature: inoculate the recombinant bacteria into fresh LB liquid medium at 1% inoculation volume, culture at 37°C and 200 rpm. 600 When the pH value reached 0.6-0.8, IPTG was added to the culture medium with a final concentration of 0.1 mM (IPTG was not added to the control group), and then the bacterial solution was placed at 15°C, 20°C, 25°C, 30°C and 35°C respectively, and cultured at 150 rpm for 10 hours before the bacteria were collected.

[0116] Inducer concentration: Inoculate the recombinant bacteria into fresh LB liquid medium at a 1% inoculation rate and culture at 37°C and 200 rpm. 600 When the pH value reached 0.6-0.8, IPTG at final concentrations of 0.01, 0.02, 0.05, 0.1, 0.15, 0.2, 0.25, and 0.3 mM was added to the culture medium (IPTG was not added to the control group), and then cultured at 20°C and 150 rpm for 10 hours, and then the bacteria were collected.

[0117] Induction time: inoculate the recombinant bacteria into fresh LB liquid medium at 1% inoculation volume, culture at 37°C, 200 rpm. 600 When the pH value reached 0.6-0.8, IPTG was added to the culture medium at a final concentration of 0.1 mM (IPTG was not added to the control group), and then cultured at 20°C and 150 rpm for 0, 4, 8, 12, 16, 20, 24, and 32 hours, and the bacteria were collected.

[0118] (4) Protein electrophoresis

[0119] The cells were collected by centrifugation at 10,000 g for 10 min, washed and suspended with physiological saline, and then ultrasonically disrupted. The supernatant was collected by centrifugation at 15,000 g for 10 min at 4°C, and 15 μL of the supernatant was subjected to SDS-PAGE electrophoresis.

[0120] 2. Results

[0121] (1) Sequence after codon optimization

[0122] The codon-optimized chitin deacetylase gene sequence is as follows (SEQ ID NO: 1):

[0123] ATGAGCGCGGATTATCCGCGTGATCTGATTGGCTACGCTAACAACCCGCCGCACCCGCACTGGCCGAACGATGCGCGTATCGCACTGAGCTTTGTTCTGAATTATGAAGAAGGTGGTGAACGTAACATTCTGCATGGTGATAAAGAATCTGAAGCATTCCTGTCTGAAATGGTTTCTGCTCAGCCGCTGCAAGGCCAGCGTAACCTGTGTATGGAATCTCTGTATGAATACGGTTCTCGTGCAGGCGTTTGGCGTCTGCTGAGCCTGTTCCAGAAACACAACATCCCGCTGACCATCTTCGCGGTTGCGATGGCGGCGCAGCGTCACCCGGATGCGATCAAAGCAATGGCGGATGCTGGCCACGAAATCTGCAGCCACGGTTACCGTTGGATCGATTACCAGAACATGAGCGAAGCGGAAGAACGTGAACACATGCACGAAGCGATCCGCATCCTGACCGAACTGACCGGCCAGCGTCCGCAGGGCTGGTACACCGGTCGTACCGGCCCGAACACCCGTCGTCTGGTTCGTGAAGAAGGCGGCTTCCTGTACGACTCTGACACCTACGATGATGATCTGCCGTACTGGGATCCGGCGAGCACCGCGGCGAAACCGCACCTGGTTATCCCGTACACCCTGGATACCAACGATATGCGTTTCACCCAGGTTCAGGGCTTCAACACCGGTGATGATTTCTTCCAGTACCTGAAAGATGCGTTCGATGTTCTGTACGCGGAAGGCGTTGCGGGCGCGCCGAAAATGCTGACCATCGGTATGCACTGCCGTCTGCTGGGTCGTCCGGCGCGTCTGGCGTCCCTGGCGCGTTTCATCGAATACGTTCAGAGCCACGAACAGGTTTGGTGCGCGCGTCGTGTGGATATCGCGAAACACTGGCACGCGACCCACCCGTTCAACGAACAGGCGTCTAAAGAACTGAGCAAATAA

[0124] (2) Optimization of expression conditions

[0125] Induction temperature ( Figure 1 a) When the induction temperature is 15°C, the soluble expression level of the recombinant chitin deacetylase is low; when the induction temperature is 20°C-35°C, the soluble expression level of the recombinant chitin deacetylase does not change significantly; therefore, the optimal induction temperature is 20°C.

[0126] Inducer concentration ( Figure 1 b) When the inducer concentration is lower than 0.1 mM, the soluble expression level of the recombinant chitin deacetylase is low; when the inducer IPTG concentration is higher than 0.1 mM, the soluble expression level of the recombinant chitin deacetylase does not change significantly; therefore, the optimal final inducer concentration is 0.1 mM.

[0127] Inoculum size ( Figure 1 c) When the inoculation amount was 0.5%-1.5%, the soluble expression amount of the recombinant chitin deacetylase gradually increased; when the inoculation amount was 1.5%-3.0%, the soluble expression amount of the recombinant chitin deacetylase did not change significantly; therefore, the optimal inoculation amount was 1.5%.

[0128] Induction time ( Figure 1 d) When the induction time is shorter than 16 hours, the soluble expression level of the recombinant chitin deacetylase is low; when the induction time is longer than 16 hours, the soluble expression level of the recombinant chitin deacetylase does not change significantly; therefore, the optimal induction time is 16 hours.

[0129] Under the above-mentioned preferred expression conditions, the expression level of the recombinant chitin deacetylase was 9.6 mg / L.

[0130] Example 3. Preparation of recombinant chitin deacetylase

[0131] 1. Materials and Methods

[0132] (1) Preparation of crude enzyme solution

[0133] The recombinant bacteria were inoculated into fresh LB liquid medium at a 1% inoculation rate and cultured at 37°C and 200 rpm. 600 When the pH reaches 0.6-0.8, IPTG is added to the culture medium to a final concentration of 0.1 mM. Culture is then continued at 20°C, 150 rpm for 16 hours, and the cells are collected by centrifugation. The centrifuged cells are thoroughly suspended in lysis buffer, disrupted by sonication, and then centrifuged at 15,000 g, 4°C for 10 minutes to remove cell debris. The resulting supernatant is the crude enzyme solution.

[0134] (2) Preparation of pure enzyme

[0135] Pure enzyme is prepared by Ni-NTA affinity chromatography gravity column. The crude enzyme solution is filtered through a 0.45 μm filter before loading. After the crude enzyme solution is loaded and combined with the filler, it is rinsed with approximately 10-15 times the bed volume of rinse buffer to remove impurities. The pure enzyme is then eluted with elution buffer. The eluted pure enzyme is then desalted by ultrafiltration and switched to enzyme storage buffer. After aliquoting, it is stored in a -80°C freezer until use.

[0136] (3) Activity determination

[0137] The p-nitroacetanilide method was used to determine the activity of chitin deacetylase. This method uses p-nitroacetanilide as a substrate. Chitin deacetylase can catalyze p-nitroacetanilide to generate p-nitroaniline. The activity of chitin deacetylase is characterized by measuring the absorbance of the product at 400 nm.

[0138] Drawing of standard curve: First weigh 0.1g of p-nitroaniline, dilute to 1L with distilled water, dilute to appropriate concentrations, measure the absorbance of the dilutions with different concentrations at 400nm, use distilled water as a control, and draw a standard curve with the p-nitroaniline concentration as the horizontal axis and the absorbance value as the vertical axis.

[0139] Chitin deacetylase activity assay: Take 3 mL of Tris-HCl (pH 8.0 0.05 M), add 1 mL (200 mg / L) of p-nitroacetanilide solution, then add recombinant chitin deacetylase, keep warm in a 50°C water bath for 15 min, then terminate the reaction in a boiling water bath for 3-5 min, add distilled water to make up to 10 mL, oscillate to mix, centrifuge at 3500 g for 10 min, take the supernatant and measure its absorbance at 400 nm. In the blank control experimental system, add 1 mL of inactivated enzyme solution of the corresponding concentration (inactivated in a boiling water bath), and the rest is the same as above, and measure the absorbance of the supernatant at 400 nm.

[0140] Under the conditions of the above chitin deacetylase reaction system, the amount of enzyme required to produce 1 μg of p-nitroaniline per hour is defined as one enzyme activity unit.

[0141] 2. Results

[0142] The SDS-PAGE electrophoresis of recombinant chitin deacetylase is shown in Figure 2 (a) Lanes 1 through 4 represent chitin deacetylase from uninduced cells, IPTG-induced whole cells, IPTG-induced supernatant, and purified chitin deacetylase, respectively. The figure shows a single band after ultrafiltration purification, indicating that the enzyme has reached electrophoretic purity after purification using a Ni-NTA Agarose gravity column. Comparison with a protein marker reveals that the chitin deacetylase is approximately 40 kDa.

[0143] Figure 2 b is the electrophoresis result of active protein of chitin deacetylase. The left picture is Coomassie Brilliant Blue R-250 staining, and the right picture is chitinase activity staining (see Trudel 1990). Calcofluor White M2R is used for color development. This color development reagent is a fluorescent whitening agent that absorbs specific ultraviolet light and reflects back blue light. The fluorescence intensity after it binds to the reaction product chitosan is higher than that after it binds to the substrate chitin. Therefore, a band brighter than the background will appear at the location of chitosan. Figure 2 , showing that the target bands of Coomassie brilliant blue and activity staining are in the same position on the electrophoresis gel, indicating that active chitin deacetylase has a deacetylation effect on chitin.

[0144] The specific activity of the purified recombinant chitin deacetylase was 150.5 U / mg, and the recovery rate of the total enzyme activity was about 53%.

[0145] Example 4. Properties of Chitin Deacetylase

[0146] 1. Materials and Methods

[0147] (1) Optimum temperature and temperature stability

[0148] Optimum temperature: The recombinant chitinase was reacted at 0℃, 5℃, 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, and 60℃ for 30 min under standard reaction conditions. The control group was chitin deacetylase solution inactivated in a boiling water bath for 5 min, and its activity was measured according to the chitin deacetylase activity assay method.

[0149] Temperature stability: The recombinant chitin deacetylase was treated at the above temperature conditions for 1 hour, and then the chitin deacetylase activity was measured at the optimal reaction temperature.

[0150] (2) Optimum pH and pH stability

[0151] Optimum pH: Recombinant chitinase was reacted in buffer solutions at pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0 for 30 min under standard reaction conditions, and the activity was determined using the chitin deacetylase activity assay.

[0152] pH stability: The recombinant chitinase was treated at the above pH conditions at 4°C for 1 hour, and then the chitin deacetylase activity was measured at the optimal reaction temperature.

[0153] (3) Effects of sodium chloride on the activity and stability of recombinant chitin deacetylase

[0154] Activity: Recombinant chitinase was reacted with sodium chloride at final concentrations of 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 4 M under standard reaction conditions for 30 min, and chitin deacetylase activity was measured.

[0155] Stability: The recombinant chitinase was treated with the above sodium chloride concentration at 4°C for 1 hour, and then the chitin deacetylase activity was measured at the optimal reaction temperature.

[0156] (4) Effects of metal ions on the activity of recombinant chitin deacetylase

[0157] Under the optimal pH buffer system, buffer solutions containing 1 mM each of NaCl, KCl, LiCl, NH4Cl, MgCl2, CaCl2, MnCl2, CuSO4, NiSO4·6H2O, ZnSO4, CoCl2, FeSO4·7H2O, and FeCl3·6H2O, and buffer solutions containing 1% each of EDTA, DTT, TritonX-100, and SDS were prepared, and their activity was measured according to the chitin deacetylase activity assay method.

[0158] (5) Effects of organic solvents on the activity of recombinant chitin deacetylase

[0159] The chitin deacetylase solution was placed in 10%, 20%, 30%, 40%, 50% of the organic solvent methanol, ethanol, acetone, acetonitrile, DMSO, respectively, and kept at 4°C for 1 hour, and then the chitin deacetylase activity was measured.

[0160] 2. Results

[0161] (1) Optimum temperature and temperature stability

[0162] Optimum temperature: Figure 3 As shown in Figure a, the optimal reaction temperature of the recombinant chitin deacetylase is 15°C. When the catalytic temperature is between 10°C and 20°C, the catalytic activity of the recombinant chitin deacetylase exceeds 80%. The enzyme still has 68.89% catalytic activity at a reaction temperature of 5°C, and the chitin deacetylase activity is undetectable at a catalytic temperature of 55°C. This indicates that the recombinant chitin deacetylase is a low-temperature enzyme.

[0163] Temperature stability: Figure 3 As shown in b, when the recombinant chitin deacetylase was treated at 15°C for 1 hour, the enzyme activity dropped to 76%, and when the recombinant enzyme was treated at 40°C for 1 hour, almost no enzyme activity was detected, which also shows that the recombinant chitin deacetylase is a low-temperature enzyme.

[0164] (2) Optimum pH and pH stability

[0165] Optimum pH: Figure 3 As shown in Figure c, the optimal catalytic pH of the recombinant chitin deacetylase is 7.0. When the pH is less than 6.0 or greater than 8.0, the catalytic activity decreases rapidly, indicating that the enzyme is a neutral enzyme.

[0166] pH stability: Figure 3 As shown in d: The recombinant chitin deacetylase has the highest stability at pH 8.0.

[0167] (3) Effects of sodium chloride on the activity and stability of recombinant chitin deacetylase

[0168] Activity: such as Figure 3 As shown in Figure e, when the sodium chloride concentration was between 0 and 0.5 M, the catalytic activity of the recombinant chitin deacetylase was higher than that without sodium chloride, indicating that the recombinant enzyme had a certain degree of sodium chloride tolerance. Furthermore, the catalytic efficiency of the recombinant enzyme was highest at a sodium chloride concentration of 0.1 M, while sodium chloride concentrations above 0.5 M showed an inhibitory effect on the recombinant enzyme. This indicates that low concentrations of NaCl help improve the catalytic activity of the recombinant enzyme.

[0169] Stability: As Figure 3 As shown in f, the relative enzyme activity of the recombinant chitin deacetylase was less than 50% after being stored at 4°C for 1 hour at a sodium chloride concentration higher than 1 M, indicating that the recombinant enzyme has a general tolerance to sodium chloride.

[0170] (4) Effects of metal ions on the activity of recombinant chitin deacetylase

[0171] As shown in Table 2, 1 mM Na + , K + Mg 2+ 、Zn 2+ and Ni 2+ The catalytic activity of the recombinant enzyme was promoted by the addition of nickel ions, while the other metal ions had varying degrees of inhibitory effects on the recombinant chitin deacetylase. 1% EDTA and 1% DTT promoted the catalytic activity of the recombinant chitin enzyme, while 1% SDS and 1% TritonX-100 completely inhibited the catalytic activity of the recombinant enzyme.

[0172] Table 2 Effects of metal ions and other compounds on the activity of recombinant chitin deacetylase

[0173]

[0174] (5) Effect of organic solvents on the stability of recombinant chitin deacetylase

[0175] As shown in Table 3, the recombinant chitin deacetylase had a relative enzyme activity of 76% after being stored in a 30% DMSO solution at 4°C for 1 hour, indicating that the recombinant enzyme has good tolerance to DMSO; the recombinant enzyme still had an activity of 80% after being stored in a 20% acetone solution at 4°C for 1 hour; the recombinant enzyme had poor tolerance to methanol, ethanol, and acetonitrile above 20%, and the relative enzyme activity was less than 50% after being stored in 10% acetonitrile at 4°C for 1 hour.

[0176] Table 3. Effects of organic solvents on the stability of recombinant chitin deacetylase

[0177]

[0178] Example 5, antifungal experiment

[0179] 1. Materials and Methods

[0180] (1) Culture medium

[0181] Same as Example 2.

[0182] (2) Preparation of recombinant chitin deacetylase

[0183] Same as Example 3.

[0184] (3) Antifungal test

[0185] Soak a 6mm diameter filter paper in the recombinant chitin deacetylase solution for 5 minutes, use a 0.5cm diameter punch to punch out a bacterial plate, and inoculate it into the center of a new culture dish with PDA culture medium; then place a soaked filter paper in the center of the bacterial plate, incubate at 20℃, and observe the inhibition after incubation. During the incubation period, add the experimental solution once every 2 hours on the filter paper.

[0186] (4) Fungal test strains

[0187] Verticillium dahlia CICC 2534 (Verticillium dahliae); Fusarium oxysporumf.sp.cucumerinum CICC 2532 (Fusarium oxysporum f.sp.cucumerinum); Aspergillus niger CICC 2039 (Aspergillus niger); Penicillium macrosclerotiorum CICC 40649 (Penicillium macrosclerotiorum).

[0188] 2. Results

[0189] like Figure 4The recombinant chitin deacetylase of the present invention exhibited a significant inhibitory effect against the plant pathogenic fungi Verticillium dahlia CICC 2534 and Fusarium oxysporum f.sp. cucumerinum CICC 2532. It also exhibited significant inhibitory effects against Aspergillus niger CICC 2039 and Penicillium macrosclerotiorum CICC 40649. The inhibition rates are shown in Table 4.

[0190] Table 4

[0191] strains Inhibition rate on day 6 Verticillium dahlia CICC 2534 56％ Fusarium oxysporum f.sp.cucumerinum CICC 2532 43％ Aspergillus niger CICC 2039 34％ Penicillium macrosclerotiorum CICC 40649 57％

[0192] It is known that Verticillium dahlia CICC 2534 and Fusarium oxysporumf.sp.cucumerinum CICC 2532 can cause cotton verticillium wilt and cucumber wilt, respectively, which indicates that the recombinant chitin deacetylase of the present invention has the ability to be applied to the biological control of the above two plant diseases.

[0193] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto. Sequence Listing <110> China Polar Research Center (China Polar Research Institute) <120> A recombinant chitin deacetylase and its preparation method and application <130> 198164 <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 945 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(945) <223> Codon-optimized chitin deacetylase gene sequence <400> 1 atgagcgcgg attatccgcg tgatctgatt ggctacgcta acaacccgcc gcacccgcac 60 tggccgaacg atgcgcgtat cgcactgagc tttgttctga attatgaaga aggtggtgaa 120 cgtaacattc tgcatggtga taaagaatct gaagcattcc tgtctgaaat ggtttctgct 180 cagccgctgc aaggccagcg taacctgtgt atggaatctc tgtatgaata cggttctcgt 240 gcaggcgttt ggcgtctgct gagcctgttc cagaaacaca acatcccgct gaccatcttc 300 gcggttgcga tggcggcgca gcgtcacccg gatgcgatca aagcaatggc ggatgctggc 360 cacgaaatct gcagccacgg ttaccgttgg atcgattacc agaacatgag cgaagcggaa 420 gaacgtgaac acatgcacga agcgatccgc atcctgaccg aactgaccgg ccagcgtccg 480 cagggctggt acaccggtcg taccggcccg aacacccgtc gtctggttcg tgaagaaggc 540 ggcttcctgt acgactctga cacctacgat gatgatctgc cgtactggga tccggcgagc 600 accgcggcga aaccgcacct ggttatcccg tacaccctgg ataccaacga tatgcgtttc 660 acccaggttc agggcttcaa caccggtgat gatttcttcc agtacctgaa agatgcgttc 720 gatgttctgt acgcggaagg cgttgcgggc gcgccgaaaa tgctgaccat cggtatgcac 780 tgccgtctgc tgggtcgtcc ggcgcgtctg gcgtccctgg cgcgtttcat cgaatacgtt 840 cagagccacg aacaggtttg gtgcgcgcgt cgtgtggata tcgcgaaaca ctggcacgcg 900 acccacccgt tcaacgaaca ggcgtctaaa gaactgagca aataa 945 <210> 2 <211> 314 <212> PRT <213> Pseudomonas sp. <400> 2 Met Ser Ala Asp Tyr Pro Arg Asp Leu Ile Gly Tyr Ala Asn Asn Pro 1 5 10 15 Pro His Pro His Trp Pro Asn Asp Ala Arg Ile Ala Leu Ser Phe Val 20 25 30 Leu Asn Tyr Glu Glu Gly Gly Glu Arg Asn Ile Leu His Gly Asp Lys 35 40 45 Glu Ser Glu Ala Phe Leu Ser Glu Met Val Ser Ala Gln Pro Leu Gln 50 55 60 Gly Gln Arg Asn Leu Cys Met Glu Ser Leu Tyr Glu Tyr Gly Ser Arg 65 70 75 80 Ala Gly Val Trp Arg Leu Leu Ser Leu Phe Gln Lys His Asn Ile Pro 85 90 95 Leu Thr Ile Phe Ala Val Ala Met Ala Ala Gln Arg His Pro Asp Ala 100 105 110 Ile Lys Ala Met Ala Asp Ala Gly His Glu Ile Cys Ser His Gly Tyr 115 120 125 Arg Trp Ile Asp Tyr Gln Asn Met Ser Glu Ala Glu Glu Arg Glu His 130 135 140 Met His Glu Ala Ile Arg Ile Leu Thr Glu Leu Thr Gly Gln Arg Pro 145 150 155 160 Gln Gly Trp Tyr Thr Gly Arg Thr Gly Pro Asn Thr Arg Arg Leu Val 165 170 175 Arg Glu Glu Gly Gly Phe Leu Tyr Asp Ser Asp Thr Tyr Asp Asp Asp 180 185 190 Leu Pro Tyr Trp Asp Pro Ala Ser Thr Ala Ala Lys Pro His Leu Val 195 200 205 Ile Pro Tyr Thr Leu Asp Thr Asn Asp Met Arg Phe Thr Gln Val Gln 210 215 220 Gly Phe Asn Thr Gly Asp Asp Phe Phe Gln Tyr Leu Lys Asp Ala Phe 225 230 235 240 Asp Val Leu Tyr Ala Glu Gly Val Ala Gly Ala Pro Lys Met Leu Thr 245 250 255 Ile Gly Met His Cys Arg Leu Leu Gly Arg Pro Ala Arg Leu Ala Ser 260 265 270 Leu Ala Arg Phe Ile Glu Tyr Val Gln Ser His Glu Gln Val Trp Cys 275 280 285 Ala Arg Arg Val Asp Ile Ala Lys His Trp His Ala Thr His Pro Phe 290 295 300 Asn Glu Gln Ala Ser Lys Glu Leu Ser Lys 305 310

Claims

1. An isolated polypeptide having low temperature activity, which is a chitin deacetylase for catalyzing the deacetylation of chitin, characterized in that: The polypeptide is selected from the group consisting of: (a) a polypeptide with an amino acid sequence as shown in SEQ ID NO: 2; (b) A polypeptide formed by adding a tag sequence to the N- or C-terminus of the polypeptide described in (a), or adding a signal peptide sequence to its N-terminus.

2. An isolated polynucleotide, characterized in that The nucleotide sequence of the polynucleotide is selected from the group consisting of: (1) a polynucleotide encoding the polypeptide according to claim 1; (2) A polynucleotide complementary to the polynucleotide (1).

3. The polynucleotide according to claim 2, wherein The nucleotide sequence of the polynucleotide is shown in SEQ ID NO:

1.

4. A carrier, characterized in that It contains the polynucleotide according to claim 2 or 3.

5. A genetically engineered host cell, characterized in that It contains the vector according to claim 4, or the polynucleotide according to claim 2 or 3 is integrated into its genome.

6. A method for preparing the polypeptide of claim 1, comprising: (i) culturing the host cell according to claim 5; (ii) collecting the culture containing the polypeptide according to claim 1; (iii) isolating the polypeptide according to claim 1 from the culture.

7. The method according to claim 6, wherein Cultivate under the following conditions: The concentration of inducer IPTG was 0.05-0.3 mM; Induction temperature 16~35℃; The induction time is 15 to 20 hours.

8. The method according to claim 7, wherein Cultivate under the following conditions: The concentration of inducer IPTG was 0.08-0.15 mM; Induction temperature 18-30°C; The induction time is 16 to 18 hours.

9. Use of the polypeptide according to claim 1, for: Catalyzing the deacetylation of chitin, or for preparing a composition for catalyzing the deacetylation of chitin; or The invention relates to inhibiting microorganisms or preparing a composition with the function of inhibiting microorganisms; the microorganisms are: Verticillium dahlia, a fungus of the genus Verticillium, Fusarium oxysporum f.sp.cucumerinum, a fungus of the genus Fusarium, Aspergillus niger, and Penicillium macroclerotiorum.

10. The use according to claim 9, characterized in that Chitosan is produced after deacetylation of chitin.

11. A composition having low temperature activity for catalyzing the deacetylation of chitin, comprising a component selected from the group consisting of: the polypeptide of claim 1; or the host cell of claim 5; and Industrially or microbiologically acceptable carriers.

12. A method for catalyzing the deacetylation of chitin, comprising: Chitin or a substance containing chitin is treated using the polypeptide according to claim 1, the host cell according to claim 5 or the composition according to claim 11.

13. The method according to claim 12, wherein: Chitosan is produced after deacetylation of chitin.

14. The method according to claim 13, wherein Processing is carried out under the following conditions: Temperature 0~35℃; pH 5-10; NaCl 0.01~0.5M; Contains Na + , K + Mg 2+ 、Zn 2+ and / or Ni 2+ ; Contains EDTA; and / or Contains DTT.

15. The method according to claim 14, wherein in, Temperature 5~25℃; pH 5.5-9; NaCl 0.03~0.3M.

16. The method according to claim 15, wherein in, Temperature 10-20℃; pH 6.5-8.5; NaCl 0.05~0.2M.

17. A method for inhibiting microorganisms by catalyzing the deacetylation of chitin, comprising: The polypeptide according to claim 1, the host cell according to claim 5 or the composition according to claim 11 is used to treat an object in need of inhibiting microorganisms; the microorganisms are: Verticillium dahlia, a fungus of the genus Verticillium, Fusarium oxysporum f.sp. cucumerinum, a fungus of the genus Fusarium, Aspergillus niger, and Penicillium macrosclerotiorum.

18. The method according to claim 17, wherein Processing is carried out under the following conditions: Temperature 0~35℃; pH 5-10; NaCl 0.01~0.5M; Contains Na + , K + Mg 2+ 、Zn 2+ and / or Ni 2+ ; Contains EDTA; and / or Contains DTT.

19. The method according to claim 18, wherein in, Temperature 5~25℃; pH 5.5-9; NaCl 0.03~0.3M.

20. The method according to claim 19, wherein in, Temperature 10-20℃; pH 6.5-8.5; NaCl 0.05~0.2M.

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