阅读说明：本技术 一种重组几丁质脱乙酰酶及其制备方法和应用 (Recombinant chitin deacetylase and preparation method and application thereof ) 是由 丁海涛 陈波 俞勇 刘克振 于 2019-11-27 设计创作，主要内容包括：本发明提供了一种重组几丁质脱乙酰酶及其制备方法和应用。本发明人分离获得一种新颖的几丁质脱乙酰酶,其不仅能够催化几丁质的脱乙酰化,还能够有效地抑制病原菌。本发明的几丁质脱乙酰酶对于低温的适应性好,既能够在原核表达的条件下被表达,又具有较高低温活性的特性。同时,本发明还优化了该低温几丁质脱乙酰酶的重组表达方法,从而使之在宿主细胞中得到了高效的表达。(The invention provides a recombinant chitin deacetylase and a preparation method and application thereof. The inventor separates and obtains a novel chitin deacetylase which can not only catalyze deacetylation of chitin, but also effectively inhibit pathogenic bacteria. The chitin deacetylase disclosed by the invention has good adaptability to low temperature, can be expressed under the condition of prokaryotic expression, and has the characteristic of higher low-temperature activity. Meanwhile, the invention also optimizes the recombinant expression method of the low-temperature chitin deacetylase, so that the low-temperature chitin deacetylase is efficiently expressed in host cells.)

1. An isolated polypeptide selected from the group consisting of:

(a) polypeptide with an amino acid sequence shown as SEQ ID NO. 2;

(b) a polypeptide which is formed by substituting, deleting or adding one or more amino acid residues to the polypeptide of (a) and has the function of the polypeptide of (a); or

(c) A polypeptide having a homology of 80% or more with the amino acid sequence of the polypeptide of (a) and having a function of the polypeptide of (a);

(d) a polypeptide formed by adding a tag sequence to the N-or C-terminus of the polypeptide of (a) or (b) or (C), or a signal peptide sequence to the N-terminus thereof.

2. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of:

(1) a polynucleotide encoding the polypeptide of claim 1;

(2) a polynucleotide complementary to the polynucleotide (1);

preferably, the polynucleotide encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO. 2; preferably, the nucleotide sequence of the polynucleotide is shown in SEQ ID NO. 1.

3. A vector comprising the polynucleotide of claim 2.

4. A genetically engineered host cell comprising the vector of claim 4, or having the polynucleotide of claim 2 integrated into its genome.

5. A method of making the polypeptide of claim 1, comprising: (i) culturing the host cell of claim 4; (ii) collecting a culture comprising the polypeptide of claim 1; (iii) isolating the polypeptide of claim 1 from the culture;

preferably, the culturing is carried out under the following conditions:

the concentration of inducer IPTG is 0.05-0.3 mM, preferably 0.08-0.15 mM;

the induction temperature is 16-35 ℃, and more preferably 18-30 ℃; more preferably 18 to 20 ℃;

the induction time is 15-20 hours; preferably 16 to 18 hours.

6. Use of the polypeptide of claim 1 for:

catalyzing the deacetylation of chitin, or for preparing a composition that catalyzes the deacetylation of chitin; preferably, chitosan is produced after deacetylation; or

Inhibiting microorganisms, or for preparing a composition having a function of inhibiting microorganisms; preferably, the microorganism is a microorganism whose cell wall comprises chitin.

7. A composition comprising an ingredient selected from the group consisting of: the polypeptide of claim 1; or the host cell of claim 5; and

an industrially or microbiologically acceptable carrier.

8. A method of catalyzing the deacetylation of chitin comprising: treating chitin or a chitin-containing material with the polypeptide of claim 1, the host cell of claim 5, or the composition of claim 8; preferably, chitosan is produced after deacetylation; preferably, the treatment is carried out under the following conditions:

the temperature is 0 to 35 ℃, preferably 5 to 25 ℃, more preferably 10 to 20 ℃;

a pH value of 5-10, preferably a pH value of 5.5-9, more preferably a pH value of 6.5-8.5;

NaCl 0.01-0.5M, preferably 0.03-0.3M, more preferably 0.05-0.2M;

containing Na⁺、K⁺、Mg²⁺、Zn²⁺And/or Ni²⁺；

Contains EDTA; and/or

Contains DTT.

9. A method of inhibiting a microorganism, comprising: treating a subject in need of inhibition of a microorganism with the polypeptide of claim 1, the host cell of claim 5, or the composition of claim 8; preferably, the microorganism is a microorganism whose cell wall comprises chitin; preferably, the treatment is carried out under the following conditions:

the temperature is 0 to 35 ℃, preferably 5 to 25 ℃, more preferably 10 to 20 ℃;

a pH value of 5-10, preferably a pH value of 5.5-9, more preferably a pH value of 6.5-8.5;

NaCl 0.01-0.5M, preferably 0.03-0.3M, more preferably 0.05-0.2M;

containing Na⁺、K⁺、Mg²⁺、Zn²⁺And/or Ni²⁺；

Contains EDTA; and/or

Contains DTT.

10. The method of claim 6, 7 or 9, wherein the microorganisms comprise: fungi; preferably the fungi include: verticillium (Verticillium) fungi, Fusarium (Fusarium) fungi, Aspergillus (Aspergillus) fungi, Penicillium (Penicillium) fungi;

more preferably, the Verticillium fungi include: verticillium dahlia (Verticillium dahlia); the fusarium fungi include: fusarium oxysporum cucumber specialization type (Fusarium oxysporum f.sp. cucumerinum); the Aspergillus fungi include: aspergillus niger (Aspergillus niger); the penicillium fungi include: penicillium macrocephalum (Penicillium macroclorotium).

Technical Field

The invention belongs to the fields of microbiology, molecular biology and biochemistry, and particularly relates to recombinant heterologous expression, preparation and application research of low-temperature chitin deacetylase; the low-temperature recombinant chitin deacetylase disclosed by the invention is mainly applied to the industries of medicines, foods, agriculture and the like.

Background

Chitin (Chitin) is a straight-chain polysaccharide formed by connecting β -N-acetyl-D-glucosamine monomers through β -1,4 glycosidic bonds, chitosan is a product of chitosan deacetylation, and generally, chitosan can be called as chitosan with the N-deacetylation degree of more than 55%.

In recent years, chitosan prepared from chitin has attracted increasing attention in the scientific and industrial fields because of its many unique biological activities. At present, chitosan is mainly applied to the fields of food, medicine, environmental protection and the like, and the application range of the chitosan is continuously expanded along with the deep research. In the industrial production process of chitosan, most of the methods adopt a hot alkali method, so that the method has the defects of high production cost, poor product uniformity, difficult control of reaction process and the like, and alkaline waste liquid is generated in the production process to pollute the environment. Therefore, the problems of high energy consumption and environmental pollution in the industrial preparation of chitosan are urgently needed to be solved.

Chitin Deacetylases (CDAs) are an enzyme species for preparing chitosan by catalyzing Chitin with an enzyme method, and can convert Chitin into chitosan by catalyzing β -N-acetyl-D-glucosamine deacetylation.

Therefore, there is a need in the art to find novel chitin deacetylases with low temperature and high activity, so as to improve the industrial production level and expand the application of the chitinase.

Disclosure of Invention

The invention aims to provide a recombinant chitin deacetylase and a preparation method and application thereof.

In a first aspect of the invention, there is provided an isolated polypeptide selected from the group consisting of: (a) polypeptide with an amino acid sequence shown as SEQ ID NO. 2; (b) a polypeptide having the function of the polypeptide (a) and formed by substitution, deletion or addition of one or more (e.g., 1 to 20, preferably 1 to 10; more preferably 1 to 5; more preferably 1 to 3) amino acid residues to the polypeptide (a); or (c) a polypeptide having a homology of 80% or more (preferably 85% or more; more preferably 90% or more; more preferably 95% or more, e.g., 98%, 99%) with the amino acid sequence of the polypeptide of (a) and having the function of the polypeptide of (a); (d) a polypeptide formed by adding a tag sequence to the N-or C-terminus of the polypeptide of (a) or (b) or (C), or a signal peptide sequence to the N-terminus thereof.

In a preferred embodiment, the polypeptide has high low temperature activity; preferably, the optimal reaction temperature is 15 ℃; preferably, it still has a catalytic activity of more than 65% at, for example, 5 ℃.

In another aspect of the invention, there is provided an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: (1) a polynucleotide encoding the polypeptide; (2) a polynucleotide complementary to the polynucleotide (1).

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

In another aspect of the invention, there is provided a vector comprising said polynucleotide.

In another aspect of the invention, there is provided a genetically engineered host cell comprising said vector, or having said polynucleotide integrated into its genome.

In a preferred embodiment, the integration comprises directed integration or random integration.

In another preferred embodiment, the cell is not a plant propagating cell or an animal stem cell.

In another preferred embodiment, the host cell is a prokaryotic cell, such as but not limited to E.coli.

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

Preferably, the culturing is carried out under the following conditions: the inducer IPTG (when the host cell is Escherichia coli) is used at a concentration of 0.05-0.3 mM, preferably 0.08-0.15 mM; the induction temperature is 16-35 ℃, and more preferably 18-30 ℃; more preferably 18 to 20 ℃; the induction time is 15-20 hours; preferably 16 to 18 hours.

In another aspect of the invention there is provided the use of said polypeptide for: catalyzing the deacetylation of chitin, or for preparing a composition that catalyzes the deacetylation of chitin; preferably, chitosan is produced after deacetylation.

In another aspect of the invention there is provided the use of said polypeptide for: inhibiting microorganisms, or for preparing a composition having a function of inhibiting microorganisms; preferably, the microorganism is a microorganism whose cell wall comprises chitin.

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

In a preferred embodiment, the composition is a pesticide composition for controlling plant diseases, preferably for controlling cotton verticillium wilt, cucumber fusarium wilt and the like.

In another aspect of the invention, there is provided a method of catalyzing the deacetylation of chitin comprising: treating chitin or chitin-containing material with said polypeptide, said host cell or said composition; preferably, chitosan is produced after deacetylation.

In another aspect of the present invention, there is provided a method of inhibiting a microorganism, comprising: treating a subject in need of microorganism inhibition (e.g., a locus, a substance, an animal or plant containing a microorganism or a processed product thereof) with said polypeptide, said host cell or said composition; preferably, the microorganism is a microorganism whose cell wall comprises chitin.

In a preferred embodiment, the treatment is carried out at a temperature of 0 to 35 ℃, preferably 5 to 25 ℃, more preferably 10 to 20 ℃ (e.g., 12, 14, 15, 16, 18 ℃).

In another preferred embodiment, the treatment is carried out at a pH of 5-10, preferably at a pH of 5.5-9, more preferably at a pH of 6.5-8.5 (e.g., pH7, 7.5, 8).

In another preferred embodiment, the treatment is performed under the condition 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).

In another preferred embodiment, in the presence of Na⁺、K⁺、Mg²⁺、Zn²⁺And/or Ni²⁺(the metal ion content is, for example, 1. + -. 0.8mM, preferably 1. + -. 0.5mM, more preferably 1. + -. 0.3 mM).

In another preferred embodiment, the treatment is carried out in the presence of EDTA (in an amount of, for example, 1. + -. 0.5%).

In another preferred embodiment, the treatment is carried out in the presence of DTT (in an amount of, for example, 1. + -. 0.5%).

In another preferred example, the treatment is carried out without the reaction system containing: li⁺、NH₄ ⁺、Ca²⁺、Mn²⁺、Cu²⁺、Fe²⁺、Fe³⁺SDS and/or TritonX-100.

In another preferred example, the treatment is carried out without the reaction system containing: acetone, ethanol, methanol and/or acetonitrile.

In another preferred embodiment, the microorganisms comprise: fungi; preferably the fungi include: verticillium (Verticillium) fungi, Fusarium (Fusarium) fungi, Aspergillus (Aspergillus) fungi, Penicillium (Penicillium) fungi. More preferably, the Verticillium fungi include (but are not limited to): verticillium dahlia (Verticillium dahlia); the fusarium fungi include (but are not limited to): fusarium oxysporum cucumber specialization type (Fusarium oxysporum f.sp. cucumerinum); the aspergillus fungi include (but are not limited to): aspergillus niger (Aspergillus niger); the penicillium fungi include (but are not limited to): penicillium macrocephalum (Penicillium macroclorotium).

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1, recombinant expression and condition optimization of chitin deacetylase of the present invention;

(a) variation in soluble expression levels of the enzyme at different induction temperatures; wherein Lane1 is an uninduced whole cell lysate, and Lane 2-6 sequentially has an induction temperature of 15 ℃, 20 ℃, 25 ℃, 30 ℃ and 35 ℃;

(b) variation in soluble expression levels of the enzyme at different inducer concentrations; wherein Lane1 is uninduced whole cell lysate, Lane 2-9 are IPTG concentration of 0mM, 0.01mM, 0.02mM, 0.05mM, 0.1mM, 0.15mM, 0.25mM, 0.3mM in sequence;

(c) when different amounts of recombinant bacteria are inoculated, the soluble expression amount of the enzyme is changed; wherein Lane1 is an uninduced whole cell lysate, and Lane 2-7 sequentially comprise inoculum sizes of 0.5%, 1%, 1.5%, 2%, 2.5% and 3%;

(d) variation in soluble expression levels of the enzyme at different induction times; wherein Lane1 is an uninduced whole cell lysate, and Lane 2-9 are sequentially induced for 4h, 8h, 12h, 16h, 20h, 24h, 28h and 32 h.

FIG. 2 purification of recombinant expression products of chitin deacetylases according to the invention;

(a) lane1 to Lane4 each represent: whole cells without IPTG induction or IPTG induction, supernatant induced by IPTG and purified chitin deacetylase;

(b) lane1 is chitin deacetylase which is stained with Coomassie Brilliant blue R-250 after active electrophoresis, and Lane2 is chitin deacetylase which is developed with Calcofluor White M2R after active electrophoresis.

FIG. 3, properties of chitinase of the invention;

(a) the optimal temperature research of the recombinant chitin deacetylase;

(b) temperature stability study of recombinant chitin deacetylase;

(c) the optimum pH research of the recombinant chitin deacetylase;

(d) researching the pH stability of the recombinant chitin deacetylase;

(e) the effect of sodium chloride on recombinant chitin deacetylase activity;

(f) effect of sodium chloride on the stability of recombinant chitin deacetylase.

FIG. 4 shows that the recombinant chitin deacetylase of the present invention can inhibit a plurality of plant pathogenic fungi

Detailed Description

Through large-scale screening and intensive research, the inventor separates a novel chitin deacetylase (low-temperature chitin deacetylase) from a strain from Antarctic, can catalyze deacetylation of chitin, can effectively inhibit pathogenic bacteria, and has a very good application prospect. The chitin deacetylase disclosed by the invention has good adaptability to low temperature, can be expressed at relatively high temperature of prokaryotic expression, and has relatively high low-temperature activity. Meanwhile, the invention also optimizes the recombinant expression method of the low-temperature chitin deacetylase, so that the low-temperature chitin deacetylase is efficiently expressed in host cells.

As used herein, the terms "polypeptide of the invention", "protein of the invention", "low temperature chitin deacetylase", "chitin deacetylase" and "chitinase" are used interchangeably and all refer to a protein or polypeptide having SEQ ID NO 2 or a fragment or variant or derivative thereof.

As used herein, "isolated" refers to a substance that is separated from its original environment (which, if it is a natural substance, is the natural environment). If the polynucleotide or polypeptide in its native state in a living cell is not isolated or purified, the same polynucleotide or polypeptide is isolated or purified if it is separated from other substances coexisting in its native state.

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 materials with which it is naturally associated. One skilled in the art can purify the chitin deacetylase using standard protein purification techniques. Substantially pure polypeptides are capable of producing a single major band on a non-reducing polyacrylamide gel. The purity of the chitin deacetylase can be analyzed by amino acid sequence analysis.

As used herein, the "microorganism" refers to a microorganism whose cell wall contains chitin, including bacteria such as fungi, actinomycetes, bacteria, or the like; preferably, the "microorganism" is a "pathogenic microorganism".

As used herein, the "pathogenic microorganism" refers to a microorganism that is hazardous to humans, animals, plants, or the environment.

As used herein, the term "comprising" means that the various ingredients can be used together in the mixture or composition of the invention. Thus, the terms "consisting essentially of and" consisting of are encompassed by the term "comprising.

As used herein, an "industrially acceptable carrier" or a "microbiologically acceptable carrier" is a solvent, suspending agent or excipient for delivering the chitin deacetylase of the present invention to a subject in need of treatment, which is controllable in toxicity, side effects, environmentally friendly or harmless to humans and animals. The carrier may be a liquid or a solid, and is preferably a carrier capable of retaining the activity of the chitin deacetylase of the present invention to a high degree.

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

The invention also includes fragments, derivatives and analogues of the chitin deacetylases. As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that retains substantially the same biological function or activity of a native chitin deacetylase of the invention. A polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which the mature polypeptide is fused to another compound, such as a compound that increases the half-life of the polypeptide, e.g. polyethylene glycol, or (iv) a polypeptide in which an additional amino acid sequence is fused to the sequence of the polypeptide (e.g. a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with an antigenic IgG fragment). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the teachings herein.

In the present invention, the term "said chitin deacetylase" refers to a polypeptide having the sequence SEQ ID NO. 2 of said chitin deacetylase activity. The term also includes variants of the sequence of SEQ ID NO. 2 that have the same function as the chitin deacetylase. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, still more preferably 1 to 10, most preferably 1 to 5) amino acids, and addition or deletion of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. For example, addition or deletion of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein; for another example, expression of only the catalytic domain of the protein, but not the carbohydrate-binding domain, can achieve the same catalytic function as the intact protein. The term therefore also includes active fragments and active derivatives of the chitin deacetylase. For example, the variation may occur outside of the conserved domain of SEQ ID NO. 2.

Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes to the chitin deacetylase DNA under high or low stringency conditions, and polypeptides or proteins obtained using antibodies against the chitin deacetylase. The invention also provides other polypeptides, such as fusion proteins comprising the chitin deacetylase or fragments thereof. In addition to almost full-length polypeptides, fragments of the chitin deacetylases are also encompassed by the present invention. Typically, the fragment has at least about 10 contiguous amino acids, typically at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of the chitin deacetylase sequence.

The induced variants may be obtained by various techniques, such as random mutagenesis by radiation or exposure to a mutagenizing agent, site-directed mutagenesis, or other known molecular biological techniques.

In the present invention, the "conservative variant of chitin deacetylase" refers to the case where at most 30, preferably at most 20, more preferably at most 10, and still more preferably at most 5 amino acids are replaced with amino acids having similar or similar properties as compared with the amino acid sequence of SEQ ID NO. 2 to form a polypeptide. These conservative variant polypeptides are preferably generated by amino acid substitutions according to Table 1.

TABLE 1

Initial residue(s)	Representative substitutions	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

The amino-terminus or the carboxy-terminus of the chitin deacetylases of the invention may also contain one or more polypeptide fragments as protein tags. Any suitable label may be used in the present invention. For example, the tag may be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ε, B, gE, and Ty 1.

For secretory expression of the translated protein (e.g., extracellularly), a host-compatible signal peptide may be added to the amino-terminal end of the chitin deacetylase. The signal peptide may be cleaved off during secretion of the polypeptide from the cell.

The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The sequence of the coding region encoding the mature polypeptide may be identical to the native coding sequence of chitin deacetylase or to the coding sequence shown in SEQ ID NO. 1 or a degenerate variant. As used herein, "degenerate variant" refers herein 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.

The polynucleotide encoding the mature polypeptide of SEQ ID NO. 2 comprises: a coding sequence encoding only the mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature polypeptide. The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, and may also include additional coding and/or non-coding sequences.

The present invention also relates to variants of the above polynucleotides which encode polypeptides having the same amino acid sequence as the present invention or fragments, analogs and derivatives of the polypeptides. The variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the polypeptide encoded thereby.

The present invention also relates to polynucleotides which hybridize to the sequences described above and which 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 which hybridize under stringent conditions (or stringent conditions) to the polynucleotides of the present invention. In the present invention, "stringent conditions" mean: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS, 60 ℃; or (2) adding denaturant during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 deg.C, etc.; or (3) hybridization occurs only when the identity between two sequences is at least 90% or more, preferably 95% or more. Moreover, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO. 2.

The polypeptides and polynucleotides of the invention are preferably provided in isolated form, more preferably purified to homogeneity.

The chitin deacetylase nucleotide full-length sequence or a fragment thereof can be obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. At present, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) have been obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art.

The invention also provides a vector comprising the polynucleotide of the invention, a genetically engineered host cell transformed with the vector of the invention or the chitin deacetylase coding sequence, and a method for producing the polypeptide of the invention by recombinant techniques.

The polynucleotide sequences of the present invention may be used to express or produce recombinant chitin deacetylases as described by conventional recombinant DNA techniques. Generally, the following steps are performed:

(1) transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding said chitin deacetylase, or with a recombinant expression vector containing the polynucleotide;

(2) a host cell cultured in a suitable medium;

(3) isolating and purifying the protein from the culture medium or the cells.

In the present invention, the chitin deacetylase polynucleotide sequence may be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors well known in the art. Any plasmid or vector may be used as long as it can replicate and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translation control elements.

Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequence encoding the chitin deacetylase and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells. Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: escherichia coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; a plant cell; insect cells of Drosophila S2 or Sf 9; CHO, COS, 293 cells, or Bowes melanoma cells. In a preferred embodiment of the invention, the host cell is a prokaryotic cell.

The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

As a preferred mode of the present invention, Escherichia coli is used as a host cell for expressing the chitin deacetylase. The invention heterologously expresses the low-temperature chitin deacetylase in the escherichia coli by constructing an expression system.

In a preferred embodiment of the present invention, the culture is performed under the following conditions: the inducer IPTG (when the host cell is Escherichia coli) is used at a concentration of 0.05-0.3 mM, preferably 0.08-0.15 mM; the induction temperature is 16-35 ℃, and more preferably 18-30 ℃; more preferably 18 to 20 ℃; the induction time is 15-20 hours; preferably 16 to 18 hours. Under the above-mentioned preferable conditions, a recombinant chitin deacetylase having a high expression level and a high enzymatic activity can be obtained.

The application of the chitin deacetylase comprises the following steps: catalyzing the deacetylation of chitin, or for preparing a composition that catalyzes the deacetylation of chitin. Preferably, chitosan is produced after deacetylation of said chitin.

The application of the chitin deacetylase also comprises inhibiting microorganisms or preparing a composition with the function of inhibiting the microorganisms. Preferably, the microorganism is a microorganism whose cell wall comprises chitin.

After obtaining the chitin deacetylase of the present invention, the skilled person can, according to the teaching of the present invention, conveniently apply the enzyme to exert a substrate-degrading effect, in particular catalyzing the deacetylation of chitin as a substrate. As a preferred mode of the present invention, there is also provided a method for degrading chitin, the method comprising: the substrate to be degraded, in particular chitin, is treated with the chitin deacetylase according to the invention.

In some of the examples of the present invention, the activity of chitin deacetylase was investigated by catalyzing the production of p-nitroanilide to p-nitroanilide. After confirming that the chitin deacetylase can catalyze the extraction of natural substrate chitin deacetylation, the inventor selects p-nitroacetanilide as an artificial substrate to carry out the enzymological property research of the chitin deacetylase.

According to the teachings of the present invention, one skilled in the art may also apply the enzyme to act as an inhibitor of microorganisms, such as pathogenic microorganisms. Comprising treating a subject in need of microorganism inhibition with said chitin deacetylase. The microorganism includes a fungus. In a preferred mode of the present invention, preferably the fungi include: verticillium (Verticillium) fungi, Fusarium (Fusarium) fungi, Aspergillus (Aspergillus) fungi, Penicillium (Penicillium) fungi; more preferably, the Verticillium fungi include (but are not limited to): verticillium dahlia (Verticillium dahlia); the fusarium fungi include (but are not limited to): fusarium oxysporum cucumber specialization (Fusarium oxysporum cumf.sp. cucumerinum); the aspergillus fungi include (but are not limited to): aspergillus niger (Aspergillus niger); the penicillium fungi include (but are not limited to): penicillium macrocephalum (Penicillium macroclorotium). The inventors found that the chitin deacetylase has an excellent degrading effect on cell wall chitin of such fungi. Therefore, the chitin deacetylase of the present invention, or cells producing the same, can be used in agricultural microbial control, such as but not limited to cotton verticillium wilt and cucumber fusarium wilt.

The present inventors also optimized the reaction system for the treatment with the chitin deacetylase, and as a preferred embodiment of the present invention, the treatment was carried out under the following conditions: the temperature is 0-35 ℃, preferably 5-25 ℃, more preferably 10-20 ℃, such as 12, 14, 15, 16, 18 ℃; a pH of 5-10, preferably a pH of 5.5-9, more preferably a pH of 6.5-8.5, such as pH7, 7.5, 8.

The inventor also finds that the application of a small amount of NaCl is beneficial to providing a good reaction environment for the chitin deacetylase and improving the catalytic activity of the chitin deacetylase; therefore, in a preferred embodiment of the present invention, NaCl is added to the enzyme reaction system (treatment system) in an amount of 0.01 to 0.5M, preferably 0.03 to 0.3M, and more preferably 0.05 to 0.2M; such as 0.06, 0.08, 0.1, 0.12, 0.15M.

The inventionIt has also been found that Na is contained in the reaction system⁺、K⁺、Mg²⁺、Zn²⁺And/or Ni²⁺Is advantageous for promoting the enzymatic activity of the chitin deacetylase of the present invention. In a preferred mode, the metal ion content is, for example, 1. + -. 0.8mM, preferably 1. + -. 0.5 mM; more preferably 1. + -. 0.3 mM.

The present inventors have also found that EDTA or DTT is contained in the reaction system to facilitate the enzymatic activity of the chitin deacetylase of the present invention. In a preferable mode, the use amount of the EDTA is 0.1 to 5 percent; preferably 0.5% to 2%, such as 1%; or, the DTT dosage is 0.1% -5%; preferably 0.5% to 2%, such as 1%.

In a preferred embodiment of the present invention, the treatment is carried out without containing in the reaction system: li⁺、NH₄ ⁺、Ca²⁺、Mn^{2 +}、Cu²⁺、Fe²⁺、Fe³⁺SDS and/or TritonX-100; nor does it contain: acetone, ethanol, methanol and/or acetonitrile.

The chitin deacetylase obtained by the invention has ideal enzyme activity, can be suitable for being recombined and expressed under the temperature condition of escherichia coli expression, and further can be suitable for enzyme reactions (including enzyme catalysis reaction or microorganism inhibition) under the low-temperature condition, thereby having wide industrial application potential.

According to the separated chitin deacetylase and the amino acid sequence thereof provided by the invention, the enzyme activity of the chitin deacetylase can be further improved or the applicable pH value range, temperature range, salt resistance, cold and heat stability and the like of the chitin deacetylase can be enlarged by means of protein molecule modification and the like by persons in the field, so that the chitin deacetylase has a good application prospect. Variants or derivatives produced by engineering the chitin deacetylases described herein using these techniques are also encompassed by the present invention.

The invention also provides a composition comprising an effective amount of the chitin deacetylase of the invention, and a dietetically or industrially acceptable carrier or excipient. Such vectors include (but are not limited to): water, buffer, glucose, glycerol, DMSO, or a combination thereof. One skilled in the art can determine the effective amount of the chitin deacetylase in the composition based on the actual use of the composition.

The composition may further comprise a substance for regulating the activity of the chitin deacetylase of the present invention. Any substance having a function of enhancing the enzymatic activity is usable. Preferably, the agent that increases the activity of the chitin deacetylase is selected from the group consisting of: na (Na)⁺、K⁺、Mg²⁺、Zn²⁺And/or Ni²⁺Or can be hydrolyzed to form Na upon addition to the substrate⁺、K⁺、Mg²⁺、Zn²⁺And/or Ni²⁺Such as sodium chloride. The substance that increases the chitin deacetylase activity may also be selected from: EDTA or DTT.

The chitin deacetylase of the present invention, the vector or host cell containing the enzyme, the composition containing the enzyme or host cell, may also be contained in a container or kit. Preferably, the kit further comprises instructions for use and the like, so as to be convenient for the application of the kit by the skilled person.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.