WO2021103295A1 - 一种重组几丁质脱乙酰酶及其制备方法和应用 - Google Patents

一种重组几丁质脱乙酰酶及其制备方法和应用 Download PDF

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WO2021103295A1
WO2021103295A1 PCT/CN2020/071121 CN2020071121W WO2021103295A1 WO 2021103295 A1 WO2021103295 A1 WO 2021103295A1 CN 2020071121 W CN2020071121 W CN 2020071121W WO 2021103295 A1 WO2021103295 A1 WO 2021103295A1
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polypeptide
chitin
chitin deacetylase
recombinant
polynucleotide
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PCT/CN2020/071121
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English (en)
French (fr)
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丁海涛
陈波
俞勇
刘克振
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中国极地研究中心(中国极地研究所)
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01041—Chitin deacetylase (3.5.1.41)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Definitions

  • the present invention belongs to the fields of microbiology, molecular biology, and biochemistry. More specifically, the present invention relates to a recombinant heterologous expression, preparation and application research of a low-temperature chitin deacetylase; the low-temperature recombinant chitin of the present invention Deacetylase is mainly used in medicine, food, agriculture and other industries.
  • Chitin is a linear polysaccharide formed by ⁇ -N-acetyl-D-glucosamine monomers connected by ⁇ -1,4 glycosidic bonds. Chitin is the product of chitin de-N-acetylation. Generally, the N-deacetylation degree above 55% can be called chitosan. The difference between chitosan and chitin is mainly in their polymerization degree, acetylation degree, acetylation mode and other characteristics. The solubility of chitosan is higher than that of chitin.
  • the hydrogen ions in the chitosan molecule are hydrogenated to make it impossible to form hydrogen bonds, causing the chemical properties of chitin to be much more active than chitin.
  • Chitosan is prone to acetylation, alkylation, methylation, and esterification. , Etherification and other chemical reactions.
  • chitosan prepared from chitin has many unique biological activities and has gradually attracted the attention of scientific and industrial circles.
  • chitosan is mainly used in food, medicine, environmental protection and other fields.
  • its application range is also expanding.
  • it In the process of producing chitosan in industry, because most of the hot alkali method is adopted, it not only has the disadvantages of high production cost, poor product uniformity, and difficult control of the reaction process, but also alkaline waste liquid pollution during the production process. surroundings. Therefore, the problems of high energy consumption and environmental pollution in the industrial preparation of chitosan are urgently to be solved.
  • Chitin deacetylases is an enzyme that catalyzes the preparation of chitosan from chitin by enzymatic method. It can achieve chitin by catalyzing the deacetylation of ⁇ -N-acetyl-D-glucosamine. The quality is converted to chitosan.
  • some chitin deacetylases also have certain antifungal activity and can be used as new insecticides and antifungal agents in modern agriculture.
  • the existing chitin deacetylase in the prior art is uneven in terms of physiological and biochemical properties and biological functions. The activity and substrate specificity are also mixed, and there is a general problem of low activity at low temperatures.
  • the purpose of the present invention is to provide a recombinant chitin deacetylase and its preparation method and application.
  • an isolated polypeptide which is selected from the following group: (a) a polypeptide having the amino acid sequence shown in SEQ ID NO: 2; (b) the polypeptide described in (a) passes through a Or more (such as 1-20, preferably 1-10; more preferably 1-5; more preferably 1-3) formed by substitution, deletion or addition of amino acid residues, and has a polypeptide (a) a functional polypeptide; or (c) and (a) the amino acid sequence of the polypeptide has 80% or more (preferably 85% or more; more preferably 90% or more; more preferably 95% or more, such as 98% , 99%) homology and a polypeptide with the function of polypeptide (a); (d) adding a tag sequence to the N or C-terminus of the polypeptide in (a) or (b) or (c), or at its N-terminus A polypeptide formed after adding a signal peptide sequence.
  • 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 5°C, for example.
  • an isolated polynucleotide which comprises a nucleotide sequence selected from the group consisting of: (1) a polynucleotide encoding the polypeptide; (2) ) A polynucleotide complementary to the polynucleotide (1).
  • 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.
  • a vector which contains the polynucleotide.
  • a genetically engineered host cell which contains the vector or the polynucleotide integrated in its genome.
  • the integration includes directed integration or random integration.
  • the cell is not a plant propagation cell or an animal stem cell.
  • the host cell is a prokaryotic cell, such as but not limited to Escherichia coli.
  • a method for preparing the polypeptide comprising: (i) culturing the host cell; (ii) collecting a culture containing the polypeptide; (iii) from the culture The said polypeptide is isolated from it.
  • the culture is carried out under the following conditions: the concentration of the inducer IPTG (when the host cell is E. 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; induction time 15-20 hours; preferably 16-18 hours.
  • the concentration of the inducer IPTG when the host cell is E. coli
  • the induction temperature is 16-35°C, more preferably 18-30 °C; more preferably 18-20 °C
  • induction time 15-20 hours preferably 16-18 hours.
  • 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, deacetylation Chitosan is produced after chemicalization.
  • the use of the polypeptide is provided for: inhibiting microorganisms, or for preparing a composition with the function of inhibiting microorganisms; preferably, the microorganisms are microorganisms whose cell walls contain chitin .
  • composition which comprises: the polypeptide or the host cell; and an industrially or microbiologically acceptable carrier.
  • the composition is a pesticide composition, which is used for the prevention and control of plant diseases, preferably for the prevention and control of cotton verticillium wilt and cucumber fusarium wilt.
  • a method for catalyzing the deacetylation of chitin comprising: applying the polypeptide, the host cell or the composition to chitin or containing chitin
  • the substance is processed; preferably, chitosan is generated after deacetylation.
  • a method for inhibiting microorganisms which includes: the polypeptide, the host cell, or the composition described in the right of use for objects that need to inhibit microorganisms (such as places containing microorganisms, substances , Animals, plants or their processed products); preferably, the microorganisms are microorganisms whose cell walls contain chitin.
  • the treatment is performed at a temperature of 0 to 35°C, preferably 5 to 25°C, more preferably 10 to 20°C (such as 12, 14, 15, 16, 18°C).
  • the treatment is performed under the conditions of pH 5-10, preferably pH 5.5-9, more preferably pH 6.5-8.5 (such as pH 7, 7.5, 8).
  • the treatment is performed 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).
  • the content of metal ions such as Na + , K + , Mg 2+ , Zn 2+ and/or Ni 2+ (metal ion content such as 1 ⁇ 0.8 mM, preferably 1 ⁇ 0.5 mM; more preferably 1 ⁇ 0.3mM) under the condition of processing.
  • the treatment is carried out under the condition of containing EDTA (for example, the amount is 1 ⁇ 0.5%).
  • the treatment is carried out under the condition of containing DTT (for example, the amount is 1 ⁇ 0.5%).
  • the reaction system does not contain: Li + , NH 4 + , Ca 2+ , Mn 2+ , Cu 2+ , Fe 2+ , Fe 3+ , SDS and/or TritonX-100.
  • the reaction system does not contain: acetone, ethanol, methanol and/or acetonitrile.
  • the microorganisms include: fungi; preferably, the fungi include: Verticillium fungi, Fusarium fungi, Aspergillus fungi, Penicillium (Penicillium) fungus. More preferably, the Verticillium fungi include (but not limited to): Verticillium dahlia; the Fusarium fungus includes (but not limited to): Fusarium oxysporum cucumber specialization type (Fusarium oxysporum f.sp. cucumerinum); the Aspergillus fungi include (but are not limited to): Aspergillus niger; the Penicillium fungi include (but are not limited to): Penicillium macrosclerotiorum.
  • Lane1 is the uninduced whole cell lysate
  • Lane2 ⁇ 6 are the induction temperatures of 15°C, 20°C, 25°C, 30°C, 35°C. ;
  • Lane1 is the uninduced whole cell lysate
  • Lane2-9 is the IPTG concentration 0mM, 0.01mM, 0.02mM, 0.05mM, 0.1mM. , 0.15mM, 0.25mM, 0.3mM;
  • Lane1 is the uninduced whole cell lysate
  • Lane2-7 is the inoculum volume 0.5%, 1%, 1.5%, 2%, 2.5%, 3%;
  • Lane1 is the uninduced whole cell lysate
  • Lane2-9 is the induction time 4h, 8h, 12h, 16h, 20h, 24h, 28h, 32h.
  • Lane1 ⁇ Lane4 respectively represent: whole cells without IPTG induction, IPTG induction, IPTG induced supernatant, purified chitin deacetylase;
  • Lane1 is a chitin deacetylase stained with Coomassie Brilliant Blue R-250 after active electrophoresis
  • Lane2 is a chitin deacetylase colored by Calcofluor White M2R after active electrophoresis.
  • the recombinant chitin deacetylase of the present invention can inhibit multiple plant pathogenic fungi.
  • chitin deacetylase low temperature chitin deacetylase
  • the chitin deacetylase of the present invention has good adaptability to low temperature, can be expressed at a relatively high temperature of prokaryotic expression, and has high low temperature activity.
  • the present invention also optimizes the recombinant expression method of the low-temperature chitin deacetylase, so that it can be efficiently expressed in the host cell.
  • polypeptide of the present invention 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 all refer to having SEQ ID NO: 2 Or a fragment or variant form or derivative of a protein or polypeptide.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • the polynucleotides and polypeptides in the natural state in living cells are not separated and purified, but the same polynucleotides or polypeptides are separated and purified from other substances that exist in the natural state. .
  • isolated polypeptide low-temperature chitin deacetylase in the present invention
  • isolated polypeptide low-temperature chitin deacetylase in the present invention
  • isolated polypeptide is substantially free of other proteins, lipids, carbohydrates or other naturally related proteins. substance.
  • Those skilled in the art can use standard protein purification techniques to purify the chitin deacetylase.
  • a substantially pure polypeptide can produce a single main band on a non-reducing polyacrylamide gel.
  • the purity of the chitin deacetylase can be analyzed by amino acid sequence.
  • microorganism refers to a microorganism whose cell wall contains chitin, and the microorganism includes bacteria, such as fungi, actinomycetes, or bacteria; preferably, the “microorganism” is a “pathogenic microorganism”.
  • pathogenic microorganism refers to microorganisms that are harmful to humans, animals, plants, or the environment.
  • the "containing” means that various ingredients 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 "containing”.
  • the carrier may be liquid or solid, and is preferably a carrier that can maintain the activity of the chitin deacetylase of the present invention to a higher degree.
  • the polypeptide of the present invention can be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, preferably a recombinant polypeptide.
  • the polypeptide of the present invention may be a natural purified product, or a chemically synthesized product, or produced from a prokaryotic or eukaryotic host (for example, bacteria, yeast, higher plants, insect and mammalian cells) using recombinant technology. Depending on the host used in the recombinant production protocol, the polypeptide of the present invention may be glycosylated or non-glycosylated.
  • the polypeptide of the present invention may also include or not include the initial methionine residue.
  • the present invention also includes fragments, derivatives and analogs of the chitin deacetylase.
  • fragment refers to polypeptides that substantially retain the same biological function or activity as the natural chitin deacetylase of the present invention.
  • polypeptide fragments, derivatives or analogues of the present invention may be (i) polypeptides with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide with a substitution group in one or more amino acid residues, or (iii) a mature polypeptide and another compound (such as a compound that extends the half-life of the polypeptide, such as Polyethylene glycol) fused to the polypeptide, or (iv) additional amino acid sequence fused to the polypeptide sequence to form a polypeptide (such as leader sequence or secretory sequence or sequence used to purify the polypeptide or proprotein sequence, or with The formation of fusion protein of antigen IgG fragment). According to the teachings herein, these fragments, derivatives and analogs belong to the scope well known to those skilled in the art.
  • the term "the chitin deacetylase” refers to a polypeptide having the sequence of SEQ ID NO: 2 with the chitin deacetylase activity.
  • the term also includes variant forms of SEQ ID NO: 2 that have the same function as the chitin deacetylase. These variant forms include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, more preferably 1-10, optimally 1-5) deletions, insertions and/or substitutions of amino acids, and addition or deletion of one or several (usually within 20, preferably within 10, more preferably at the C-terminal and/or N-terminal) Within 5) amino acids.
  • the function of the protein is usually not changed.
  • adding or deleting one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein; for example, expressing only the catalytic domain of the protein without expressing the carbohydrate binding domain can also be obtained.
  • the mutation can occur outside the conserved functional domain of SEQ ID NO: 2.
  • the variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, capable of hybridizing with the chitin deacetylase DNA under high or low stringency conditions
  • the present invention also provides other polypeptides, such as fusion proteins containing the chitin deacetylase or fragments thereof. In addition to almost full-length polypeptides, the present invention also includes fragments of the chitin deacetylase.
  • 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, Optimally at least about 100 consecutive amino acids.
  • the invention also provides analogs of the chitin deacetylase protein or polypeptide.
  • the difference between these analogues and the natural chitin deacetylase can be the difference in amino acid sequence, the difference in modification 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, site-directed mutagenesis or other known molecular biology techniques. Analogs also include analogs having residues different from natural L-amino acids (such as D-amino acids), and analogs having non-naturally occurring or synthetic amino acids (such as ⁇ , ⁇ -amino acids). It should be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.
  • the conservative variant of the chitin deacetylase refers to at most 30, preferably at most 20, and more preferably at most 10 compared with the amino acid sequence of SEQ ID NO: 2. More preferably, at most 5 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide. These conservative variant polypeptides are best produced according to Table 1 through amino acid substitutions.
  • 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
  • the amino terminal or carboxy terminal of the chitin deacetylase of the present invention can also contain one or more polypeptide fragments as protein tags.
  • Any suitable label can be used in the present invention.
  • the tag can be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ⁇ , B, gE and Ty1.
  • a signal peptide suitable for the host can be added to the amino terminal of the chitin deacetylase.
  • the signal peptide can be cut off during the 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 synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be a coding strand or a non-coding strand.
  • the coding region sequence encoding the mature polypeptide may be the same as the natural coding sequence of chitin deacetylase or the coding sequence shown in SEQ ID NO:1 or be a degenerate variant.
  • "degenerate variant" in the present invention refers to a protein that encodes SEQ ID NO: 2, but is different from the natural coding sequence of chitin deacetylase or the coding sequence shown in SEQ ID NO: 1. Different nucleic acid sequences.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: a coding sequence that only encodes the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences), and Non-coding sequence.
  • the term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or a polynucleotide that also includes additional coding and/or non-coding sequences.
  • the present invention also relates to variants of the above-mentioned polynucleotides, which encode polypeptides having the same amino acid sequence as the present invention, or polypeptide fragments, analogs and derivatives.
  • the variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide. It may be a substitution, deletion or insertion of one or more nucleotides, but it will not substantially change the function of the encoded polypeptide. .
  • the present invention also relates to polynucleotides that hybridize with the aforementioned 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 can hybridize with the polynucleotide of the present invention under stringent conditions (or stringent conditions).
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) adding during hybridization There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, and more Fortunately, hybridization occurs when more than 95%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and are more preferably purified to homogeneity.
  • the full-length sequence of the chitin deacetylase nucleotide or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis.
  • the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art.
  • the present invention also provides a vector containing the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or the chitin deacetylase coding sequence, and recombinant technology to produce the polypeptide of the present invention Methods.
  • the polynucleotide sequence of the present invention can be used to express or produce the recombinant chitin deacetylase. Generally speaking, there are the following steps:
  • the chitin deacetylase polynucleotide sequence can be inserted into a recombinant expression vector.
  • recombinant expression vector refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenovirus, retrovirus or other vectors well known in the art. Any plasmid and vector can be used as long as it can be replicated and stabilized in the host.
  • An important feature of an expression vector is that it usually contains an origin of replication, a promoter, a marker gene, and translation control elements.
  • an expression vector containing the DNA sequence encoding the chitin deacetylase and appropriate transcription/translation control signals can be used to construct an expression vector containing the DNA sequence encoding the chitin deacetylase and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology.
  • the DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells.
  • a vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express the protein.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; CHO, COS, 293 cells, or Bowes melanoma cells Animal cells, etc.
  • the host cell is a prokaryotic cell.
  • the obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture can be selected from various conventional mediums.
  • the culture is carried out under conditions suitable for the growth of the host cell. After the host cell has grown to a suitable cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cell is cultured for a period of time.
  • the recombinant polypeptide in the above method can be expressed in the cell or on the cell membrane, or secreted out of the cell. If necessary, the physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. 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 precipitation agent (salting out method), centrifugation, osmotic breakage, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • E. 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.
  • culture is performed 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; induction time 15-20 hours; preferably 16-18 hours.
  • the concentration of the inducer IPTG when the host cell is Escherichia coli
  • the concentration of the inducer IPTG 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
  • induction time 15-20 hours preferably 16-18 hours.
  • the use of the chitin deacetylase of the present invention includes: catalyzing the deacetylation of chitin, or for preparing a composition for catalyzing the deacetylation of chitin.
  • the chitin is deacetylated to produce chitosan.
  • the use of the chitin deacetylase of the present invention also includes inhibiting microorganisms, or for preparing a composition with the function of inhibiting microorganisms.
  • the microorganism is a microorganism whose cell wall contains chitin.
  • a method for degrading chitin comprising: treating the substrate to be degraded (especially chitin) with the chitin deacetylase of the present invention.
  • the activity of chitin deacetylase was investigated by catalyzing p-nitroacetanilide to p-nitroaniline. After confirming that the chitin deacetylase can catalyze the deacetylation of chitin from its natural substrate, the inventors selected p-nitroacetanilide as an artificial substrate to study the enzymatic properties of chitin deacetylase.
  • microorganisms such as pathogenic microorganisms.
  • chitin deacetylase to treat objects that need to inhibit microorganisms.
  • the microorganisms include fungi.
  • the fungi include: Verticillium fungi, Fusarium fungi, Aspergillus fungi, Penicillium fungi; more preferably The fungi of the genus Verticillium include (but are not limited to): Verticillium dahlia; the fungi of the genus Fusarium include (but are not limited to): Fusarium oxysporum (Fusarium oxysporum) .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 macrosclerotiorum.
  • 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 the cell producing the enzyme can be applied to the control of agricultural microorganisms, 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.
  • 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.
  • the present inventors also found that the application of a small amount of NaCl is beneficial to provide a good reaction environment for the chitin deacetylase of the present invention and improve its catalytic activity; therefore, in the preferred mode of the present invention, in the enzyme reaction system (treatment system ) Is added with 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.
  • the inventors also found that the inclusion of Na + , K + , Mg 2+ , Zn 2+ and/or Ni 2+ in the reaction system is beneficial to promote the enzymatic activity of the chitin deacetylase of the present invention.
  • the metal ion content is 1 ⁇ 0.8mM, preferably 1 ⁇ 0.5mM; more preferably 1 ⁇ 0.3mM.
  • the inventors also found that the inclusion of EDTA or DTT in the reaction system is beneficial to promote the enzymatic activity of the chitin deacetylase of the present invention.
  • 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%.
  • the reaction system does not contain: Li + , NH 4 + , Ca 2+ , Mn 2+ , Cu 2+ , Fe 2+ , Fe 3+ , SDS and/or TritonX-100; also does not contain: acetone, ethanol, methanol and/or acetonitrile.
  • the chitin deacetylase obtained by the present invention has ideal enzymatic activity, and can be adapted to be recombinantly expressed under the temperature conditions of E. coli expression, and further can be applied to enzyme reactions under low temperature conditions (including enzyme-catalyzed reactions or inhibiting microorganisms) , Has a wide range of industrial application potential.
  • chitin deacetylase and its amino acid sequence provided by the present invention, those skilled in the art can further improve its enzyme activity, or expand its applicable pH range, temperature range, and salt tolerance through protein molecular modification and other means. And the stability to cold and heat, etc., so its application prospects are good.
  • the variants or derivatives generated after using these technologies to transform the chitin deacetylase of the present invention are also included in the present invention.
  • the present invention also provides a composition, which contains an effective amount of the chitin deacetylase of the present invention and a food or industrially acceptable carrier or excipient.
  • a composition which contains an effective amount of the chitin deacetylase of the present invention and a food or industrially acceptable carrier or excipient.
  • Such carriers include (but are not limited to): water, 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 according to the actual use of the composition.
  • the composition can also be added with substances that regulate the activity of the chitin deacetylase of the present invention.
  • Any substance with the function of enhancing enzyme activity is available.
  • the substance that improves the activity of the chitin deacetylase is selected from: Na + , K + , Mg 2+ , Zn 2+ and/or Ni 2+ or can be hydrolyzed to form Na + after being added to the substrate , K + , Mg 2+ , Zn 2+ and/or Ni 2+ substances, such as sodium chloride.
  • the substance that improves the activity of the chitin deacetylase can also be selected from: EDTA or DTT.
  • the chitin deacetylase of the present invention, the vector or host cell containing the enzyme, and the composition containing the enzyme or host cell may also be contained in a container or kit.
  • the kit also includes instructions for use and the like to facilitate the application by those skilled in the art.
  • Example 1 Source and sequence information of low-temperature chitin deacetylase
  • the low-temperature chitin deacetylase of the present invention is derived from the strain Pseudomonas sp.GWSMS-1 (CCTCC NO.M2019207), which has been deposited in the China Type Culture Collection, the name of the depository unit: China Type Culture Collection Center , Preservation date: March 27, 2019, and its preservation number is CCTCC NO.M2019207.
  • the amino acid sequence of the low-temperature chitin deacetylase is as follows (SEQ ID NO: 2):
  • LB liquid medium yeast extract 5g/L, tryptone 10g/L, sodium chloride 10g/L.
  • LB solid medium add 20g/L agar powder to LB liquid medium.
  • the inventors first performed codon optimization according to the expression situation to improve the expression efficiency and the stability of DNA fragments.
  • the optimized sequence was inserted into the plasmid pET28a(+) to obtain the recombinant plasmid pET28-CDA, and then the recombinant plasmid pET28-CDA was transformed into E. coli BL21(DE3) for heterologous expression.
  • the inoculation amount is set to 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 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. When the OD 600 of the bacterial solution reached 0.6-0.8, IPTG with a final concentration of 0.1 mM was added to the medium (the control group did not Add IPTG), and then continue culturing for 10 hours at 20°C and 150 rpm, and then collect the bacteria.
  • Induction temperature Inoculate the recombinant bacteria in fresh LB liquid medium according to 1% of the inoculum, and cultivate at 37°C and 200 rpm. When the OD 600 of the bacterial solution reaches 0.6-0.8, add the final concentration of 0.1 mM to the medium. IPTG (IPTG was not added to the control group), and then the bacteria solution was placed at 15°C, 20°C, 25°C, 30°C, and 35°C, respectively, and cultured at 150rpm for 10 hours, and then the bacteria were collected.
  • Concentration of inducer Inoculate the recombinant bacteria in fresh LB liquid medium according to 1% of the inoculum, and cultivate at 37°C and 200 rpm. When the OD 600 of the bacterial solution reaches 0.6-0.8, add the final concentration of 0.01 to the medium. , 0.02, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3 mM IPTG (no IPTG added to the control group), and then continue culturing at 20°C and 150 rpm for 10 hours, and then collect the bacteria.
  • Induction time Inoculate the recombinant bacteria in fresh LB liquid medium according to 1% of the inoculum, and cultivate at 37°C and 200 rpm. When the OD 600 of the bacterial solution reaches 0.6-0.8, add the final concentration of 0.1 mM to the medium. IPTG (no IPTG added to the control group), and then continue to culture at 20°C and 150 rpm for 0, 4, 8, 12, 16, 20, 24, and 32 hours, and then collect the bacteria.
  • the cells were collected by centrifugation at 10,000g for 10min, washed and suspended with physiological saline, then the cells were disrupted by ultrasound, centrifuged at 15000g for 10min at 4°C to collect the supernatant, and 15 ⁇ L of the supernatant was taken for SDS-PAGE electrophoresis.
  • the codon optimized chitin deacetylase gene sequence is as follows (SEQ ID NO:1):
  • Induction temperature ( Figure 1a): When the induction temperature is 15°C, the soluble expression of recombinant chitin deacetylase is lower; when the induction temperature is 20°C-35°C, the recombinant chitin deacetylase can be expressed The amount of soluble expression does not change significantly; therefore, the optimal induction temperature is 20°C.
  • Inducer concentration ( Figure 1b): When the inducer concentration is lower than 0.1 mM, the soluble expression of recombinant chitin deacetylase is lower; when the inducer IPTG concentration is higher than 0.1 mM, the recombinant chitin deacetylase The soluble expression of acetylase does not change significantly; therefore, the optimal final concentration of inducer is 0.1mM.
  • Inoculation volume ( Figure 1c): When the inoculation volume is 0.5% -1.5%, the soluble expression of recombinant chitin deacetylase gradually increases; when the inoculation volume is 1.5% -3.0%, the recombinant chitin deacetylation The soluble expression amount of the enzyme does not change significantly; therefore, the optimal inoculum amount is 1.5%.
  • Induction time ( Figure 1d): When the induction time is shorter than 16 hours, the soluble expression of recombinant chitin deacetylase is lower; when the induction time is longer than 16 hours, the recombinant chitin deacetylase can be expressed There is no obvious change in the amount of soluble expression; therefore, the best induction time is 16 hours.
  • the expression amount of recombinant chitin deacetylase is 9.6 mg/L.
  • the recombinant bacteria were inoculated into fresh LB liquid medium at 1% inoculum, and cultured at 37°C and 200 rpm.
  • OD 600 of the bacterial solution reached 0.6-0.8
  • IPTG with a final concentration of 0.1 mM was added to the medium, and then The culture was continued for 16 hours at 20°C and 150 rpm, and then the cells were collected by centrifugation.
  • the cells after centrifugation were fully suspended in the lysis buffer, the cells were disrupted by ultrasound, and the cell debris was removed by centrifugation at 15000g at 4°C for 10 minutes, and the supernatant collected was the crude enzyme solution.
  • the pure enzyme is prepared by the Ni-NTA affinity chromatography gravity column.
  • the crude enzyme solution needs to be filtered with a 0.45 ⁇ m filter before loading. After the crude enzyme solution is loaded and the filler is combined, rinse with a rinse buffer for about 10-15 Double the bed volume to remove impurities, and then add elution buffer to elute the pure enzyme.
  • the eluted pure enzyme is desalted by ultrafiltration and switched to enzyme storage buffer solution, and stored in a -80°C refrigerator for later use.
  • the p-nitroacetanilide method is used to determine the activity of chitin deacetylase. This method uses p-nitroacetanilide as the substrate. Chitin deacetylase can catalyze p-nitroacetanilide to produce p-nitroaniline. The activity of chitin deacetylase was characterized by measuring the absorbance of the product at 400nm.
  • Standard curve drawing first weigh 0.1g of p-nitroaniline, distilled water to a constant volume of 1L, dilute to the appropriate concentration, measure the absorbance of different concentrations of diluents at 400nm, distilled water as a control, and take the concentration of p-nitroaniline as the horizontal Coordinates, the absorbance value is the ordinate to draw a standard curve.
  • Determination of chitin deacetylase activity Take 3mL of Tris-HCl with pH 8.0 0.05M, add 1mL (200mg/L) p-nitroacetanilide solution, then add recombinant chitin deacetylase, and keep it in a water bath at 50°C for 15 minutes , Then boiling water bath for 3-5min to terminate the reaction, add distilled water to make the volume up to 10mL, shake and mix, centrifuge at 3500g for 10min, take the supernatant and measure its absorbance at 400nm, add 1mL corresponding concentration of inactivation to the blank control experiment system Enzyme solution (inactivated in a boiling water bath), the rest is the same as above, and the absorbance of the supernatant at 400nm is measured.
  • the amount of enzyme required to produce 1 ⁇ g of p-nitroaniline per hour is defined as an enzyme activity unit.
  • the SDS-PAGE electrophoresis diagram of recombinant chitin deacetylase is shown in Figure 2(a).
  • Lanes 1 to 4 indicate the whole cell without IPTG induction, IPTG induction, and IPTG induction supernatant, after purification. From the figure, it can be seen that the enzyme only shows a single band after purification and ultrafiltration, indicating that the enzyme has reached electrophoretic purity after purification by the Ni-NTA Agarose packing gravity column. Compared with the protein Marker, the size of the chitin deacetylase is about 40kDa).
  • FIG. 2b shows the results of active protein electrophoresis of chitin deacetylase.
  • the left picture shows Coomassie Brilliant Blue R-250 staining
  • the right picture shows the activity staining of Chitinase (see Trudel 1990), which was developed by Calcofluor White M2R.
  • the chromogenic reagent is a fluorescent whitening agent, which can absorb specific ultraviolet light and reflect back to blue light.
  • the fluorescence intensity after combining with chitosan, the reaction product is higher than the fluorescence intensity after combining with the substrate chitin, so A band brighter than the background will appear where there is chitosan.
  • the target bands showing Coomassie Brilliant Blue and active staining are consistent in the position of the electrophoresis gel, indicating that active chitin deacetylase has a deacetylation effect on chitin.
  • the specific activity of recombinant chitin deacetylase was 150.5 U/mg, and the recovery rate of total enzyme activity was about 53%.
  • Optimum temperature Recombinant chitinase under standard reaction conditions at 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55 React at 60°C for 30 minutes.
  • the control group is a chitin deacetylase solution that has been inactivated in a boiling water bath for 5 minutes, and its activity is measured according to the chitin deacetylase activity detection method.
  • Temperature stability Treat the recombinant chitin deacetylase at the above temperature for 1 hour, and then measure its chitin deacetylase activity at the optimum reaction temperature.
  • Optimum pH Recombinant chitinase was reacted for 30 minutes in buffers of pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0 under standard reaction conditions, and tested according to chitin deacetylase activity Method to test its live.
  • pH stability Treat the recombinant chitinase at 4°C for 1 hour under the above pH conditions, and then measure its chitin deacetylase activity at the optimum reaction temperature.
  • Recombinant chitinase was added to the final concentration of 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4M sodium chloride and reacted for 30 minutes under standard reaction conditions, and then the chitin deacetylase activity was determined. active.
  • Stability Treat the recombinant chitinase at 4°C for 1 hour under the above sodium chloride concentration conditions, and then measure its chitin deacetylase activity at the optimal reaction temperature.
  • the optimal reaction temperature of recombinant chitin deacetylase is 15°C.
  • the catalytic activity of the recombinant chitin deacetylase is Over 80%, the enzyme still has 68.89% catalytic activity at a reaction temperature of 5°C, and no chitin deacetylase activity can be detected when the catalytic temperature is 55°C. It shows that the recombinant chitin deacetylase is a low-temperature enzyme.
  • the optimal catalytic pH of the recombinant chitin deacetylase is 7.0.
  • 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.
  • pH stability As shown in Figure 3d: the recombinant chitin deacetylase has the highest stability at pH 8.0.
  • 1 mM Na + , K + , Mg 2+ , Zn 2+ and Ni 2+ can promote the catalytic activity of the recombinase.
  • nickel ions have the most obvious promotion effect, and other metal ions have a significant effect on the recombination.
  • Chitin deacetylase has different degrees of inhibition; 1% EDTA and 1% DTT can promote the catalytic activity of chitin recombinase, and 1% SDS and 1% TritonX-100 can make the recombination. Enzyme catalytic activity showed complete inhibition.
  • the recombinant chitin deacetylase has a relative enzymatic activity of 76% after being stored in a 30% DMSO solution at 4°C for 1 hour, indicating that the recombinant enzyme has a good tolerance to DMSO;
  • the enzyme remains 80% active when stored in a 20% acetone solution at 4°C for 1 hour;
  • the recombinant enzyme has poor tolerance in methanol, ethanol, and acetonitrile above 20%, and it is stored in 10% acetonitrile at 4°C.
  • the relative enzyme activity in 1 hour is less than 50%.
  • Verticillium dahlia CICC 2534 Verticillium dahlia
  • Fusarium oxysporum f.sp. cucumerinum CICC 2532 Fusarium oxysporum cucumber specialization type
  • Aspergillus niger CICC 2039 Aspergillus niger
  • Penicillium macrosclerotiorum CICC 40649 Penicillium macrosclerotiorum
  • the recombinant chitin deacetylase of the present invention can have a good inhibitory effect on the plant pathogenic fungi Verticillium dahlia CICC 2534, Fusarium oxysporum f.sp. cucumerinum CICC 2532; it also has a good inhibitory effect on Aspergillus niger CICC 2039 and Penicillium macrosclerotiorum CICC 40649. Shows a significant inhibitory effect.
  • the inhibition rate statistics are shown in Table 4.
  • Verticillium dahlia CICC 2534 and Fusarium oxysporum f.sp. cucumerinum CICC 2532 can cause cotton verticillium wilt and cucumber fusarium wilt respectively, which indicates that the recombinant chitin deacetylase of the present invention can be applied to the above two plant diseases.

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Abstract

提供了一种重组几丁质脱乙酰酶及其制备方法和应用。该几丁质脱乙酰酶能够催化几丁质的脱乙酰化，还能够有效地抑制病原菌，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。还提供了优化的该几丁质脱乙酰酶的重组表达方法，使之能在宿主细胞中得到高效的表达。

Description

一种重组几丁质脱乙酰酶及其制备方法和应用 技术领域
本发明属于微生物学、分子生物学、生物化学领域，更具体地，本发明涉及一种低温几丁质脱乙酰酶的重组异源表达、制备及其应用研究；本发明的低温重组几丁质脱乙酰酶主要应用于医药、食品、农业等行业。
背景技术
几丁质(Chitin)是由β-N-乙酰-D-葡萄糖胺单体通过β-1,4糖苷键连接成的直链多糖，壳聚糖是几丁质脱N-乙酰基的产物，一般地，N-脱乙酰度在55％以上的就可称之为壳聚糖。壳聚糖与几丁质的区别主要在于它们的聚合度、乙酰化度、乙酰化模式等特征不同，壳聚糖的溶解性比几丁质的溶解性高。壳聚糖分子中的氢离子被氢化使其无法形成氢键，导致壳多糖的化学性质相对几丁质要活泼很多，壳聚糖很容易发生乙酰化、烷基化、甲基化、酯化、醚化等化学反应。
近年来，由几丁质制备的壳聚糖有许多独特的生物学活性逐渐引起了科学界和产业界的重视。目前，壳聚糖主要应用于食品、医药、环保等领域，随着研究的深入，其应用范围也在不断的扩展。工业上在生产壳聚糖的过程中，由于大部分采用热碱法，不仅具有生产成本高，产物均一性差，反应进程不易控制等缺点，而且在生产的过程中还会产生碱性废液污染环境。因此，工业上制备壳聚糖的高能耗和环境污染问题是亟待解决的。
几丁质脱乙酰酶(Chitin deacetylases，CDAs)是酶法催化几丁质制备壳聚糖的一种酶种，其通过催化β-N-乙酰-D-葡萄糖胺脱乙酰基即可实现几丁质转化为壳聚糖。此外，一些几丁质脱乙酰酶还具有一定的抗真菌活性，可作为新型杀虫剂和抗真菌剂应用于现代农业。尽管现在已有人员针对以几丁质脱乙酰酶制取壳聚糖进行研究，但是，现有技术中存在的几丁质脱乙酰酶在生理生化性质、生物学功能等方面参差不齐，酶活性、底物特异性方面也良莠不齐，并且普遍存在低温下活性低的问题。
因此，本领域亟待找到新型的，具有低温及高活性的几丁质脱乙酰酶，以提高此类酶的工业化生产水平、拓展其应用。
发明内容
本发明的目的在于提供一种重组几丁质脱乙酰酶及其制备方法和应用。
在本发明的第一方面，提供一种分离的多肽，该多肽选自下组：(a)具有SEQ ID NO:2所示氨基酸序列多肽；(b)由(a)所述的多肽经过一个或多个(如1-20个，较佳地1-10个；更佳地1-5个；更佳地1-3个)氨基酸残基的取代、缺失或添加而形成的，且具有多肽(a)功能的多肽；或(c)与(a)所述的多肽的氨基酸序列有80％以上(较佳地85％ 以上；更佳地90％以上；更佳95％以上，如98％，99％)同源性，且具有多肽(a)功能的多肽；(d)在(a)或(b)或(c)所述多肽的N或C末端添加标签序列，或在其N末端添加信号肽序列后形成的多肽。
在一个优选例中，所述的多肽具有较高的低温活性；较佳地，其最适反应温度为15℃；较佳地，其在例如5℃时仍然具有65％以上的催化活性。
在本发明的另一方面，提供一种分离的多核苷酸，它包含一核苷酸序列，该核苷酸序列选自下组：(1)编码如所述多肽的多核苷酸；(2)与多核苷酸(1)互补的多核苷酸。
在一个优选例中，该多核苷酸编码如SEQ ID NO:2所示氨基酸序列的多肽；较佳地，该多核苷酸的核苷酸序列如SEQ ID NO:1所示。
在本发明的另一方面，提供一种载体，它含有所述的多核苷酸。
在本发明的另一方面，提供一种遗传工程化的宿主细胞，它含有所述的载体，或其基因组中整合有所述的多核苷酸。
在一个优选例中，所述的整合包括定向整合或随机整合。
在另一优选例中，所述的细胞不是植物繁殖细胞或动物干细胞。
在另一优选例中，所述的宿主细胞是原核细胞，如但不限于大肠杆菌。
在本发明的另一方面，提供一种制备所述的多肽的方法，包括：(i)培养所述的宿主细胞；(ii)收集含有所述的多肽的培养物；(iii)从培养物中分离出所述的多肽。
较佳地，在以下条件下进行培养：诱导剂IPTG(当宿主细胞为大肠杆菌时)浓度0.05～0.3mM、较佳地0.08～0.15mM；诱导温度16～35℃、更佳地18～30℃；更佳地18～20℃；诱导时间15～20小时；较佳地16～18小时。
在本发明的另一方面，提供所述的多肽的用途，用于：催化几丁质的脱乙酰化，或用于制备催化几丁质的脱乙酰化的组合物；较佳地，脱乙酰化后生成壳聚糖。
在本发明的另一方面，提供所述的多肽的用途，用于：抑制微生物，或用于制备具有抑制微生物功能的组合物；较佳地，所述的微生物是细胞壁包含几丁质的微生物。
在本发明的另一方面，提供一种组合物，其包含：所述的多肽或所述的宿主细胞；以及工业学或微生物学上可接受的载体。
在一个优选例中，所述的组合物为农药组合物，用于植物病害防治，较佳地用于防治棉花黄萎病和黄瓜枯萎病等。
在本发明的另一方面，提供一种催化几丁质的脱乙酰化的方法，包括：应用所述的多肽、所述的宿主细胞或所述的组合物对几丁质或含有几丁质的物质进行处理；较佳地，脱乙酰化后生成壳聚糖。
在本发明的另一方面，提供一种抑制微生物的方法，包括：应用权所述的多肽、所述的宿主细胞或所述的组合物对需要抑制微生物的对象(如含有微生物的场所、物质、动植物或其加工产物)进行处理；较佳地，所述的微生物是细胞壁包含几丁质的 微生物。
在一个优选例中，在温度0～35℃、较佳地5～25℃、更佳地10～20℃(如12、14、15、16、18℃)条件下进行处理。
在另一优选例中，在pH5～10、较佳地pH5.5～9、更佳地pH6.5～8.5(如pH7，7.5，8)条件下进行处理。
在另一优选例中，在NaCl 0.01～0.5M、较佳地0.03～0.3M、更佳地0.05～0.2M(如0.06、0.08、0.1、0.12、0.15M)条件下进行处理。
在另一优选例中，在含有Na +、K +、Mg 2+、Zn 2+和/或Ni 2+(金属离子含量如1±0.8mM，较佳地1±0.5mM；更佳地1±0.3mM)条件下进行处理。
在另一优选例中，在含有EDTA(用量如为1±0.5％)条件下进行处理。
在另一优选例中，在含有DTT(用量如为1±0.5％)条件下进行处理。
在另一优选例中，进行所述处理时，反应体系中不含有：Li +、NH 4 +、Ca 2+、Mn 2+、Cu 2+、Fe 2+、Fe 3+、SDS和/或TritonX-100。
在另一优选例中，进行所述处理时，反应体系中不含有：丙酮、乙醇、甲醇和/或乙腈。
在另一优选例中，所述的微生物包括：真菌；较佳地所述真菌包括：轮枝菌属(Verticillium)真菌，镰刀菌属(Fusarium)真菌，曲霉属(Aspergillus)真菌，青霉属(Penicillium)真菌。更佳地，所述轮枝菌属真菌包括(但不限于)：大丽花轮枝孢(Verticillium dahlia)；所述镰刀菌属真菌包括(但不限于)：尖孢镰孢黄瓜专化型(Fusarium oxysporum f.sp.cucumerinum)；所述曲霉属真菌包括(但不限于)：黑曲霉(Aspergillus niger)；所述青霉属真菌包括(但不限于)：大核青霉(Penicillium macrosclerotiorum)。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
图1、本发明的几丁质脱乙酰酶的重组表达及条件优化；
(a)不同的诱导温度下，酶的可溶表达量的变化；其中Lane1为未诱导的全细胞裂解物，Lane2～6依次为诱导温度15℃，20℃，25℃，30℃，35℃；
(b)不同的诱导剂浓度下，酶的可溶表达量的变化；其中Lane1为未诱导的全细胞裂解物，Lane2～9依次为IPTG浓度0mM，0.01mM，0.02mM，0.05mM，0.1mM，0.15mM，0.25mM，0.3mM；
(c)接种不同量的重组菌时，酶的可溶表达量的变化；其中Lane1为未诱导的全细胞裂解物，Lane2～7依次为接种量0.5％，1％，1.5％，2％，2.5％，3％；
(d)不同的诱导时间下，酶的可溶表达量的变化；其中Lane1为未诱导的全细胞裂解物，Lane2～9依次为诱导时间4h，8h，12h，16h，20h，24h，28h，32h。
图2、本发明的几丁质脱乙酰酶的重组表达产物的纯化；
(a)Lane1～Lane4分别表示：未加IPTG诱导、IPTG诱导的全细胞、IPTG诱导的上清液、纯化后的几丁质脱乙酰酶；
(b)Lane1是活性电泳后经考马斯亮蓝R-250染色的几丁质脱乙酰酶，Lane2是活性电泳后经Calcofluor White M2R显色的几丁质脱乙酰酶。
图3、本发明的几丁质酶的性质；
(a)重组几丁质脱乙酰酶的最适温度研究；
(b)重组几丁质脱乙酰酶的温度稳定性研究；
(c)重组几丁质脱乙酰酶的最适pH研究；
(d)重组几丁质脱乙酰酶的pH稳定性研究；
(e)氯化钠对重组几丁质脱乙酰酶活性影响；
(f)氯化钠对重组几丁质脱乙酰酶的稳定性的影响。
图4、本发明的重组几丁质脱乙酰酶可以对于多株植物病原真菌的抑制作用。
具体实施方式
本发明人经过大规模的筛选和深入的研究，从南极来源的菌株中分离到了一种新颖的几丁质脱乙酰酶(低温几丁质脱乙酰酶)，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，具有非常好的应用前景。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相对较高的温度下被表达，又具有较高的低温活性。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。
如本文所用，术语“本发明的多肽”、“本发明的蛋白”、“低温几丁质脱乙酰酶”、“几丁质脱乙酰酶”可互换使用，都指具有SEQ ID NO:2或其片段或其变异形式或衍生物的蛋白或多肽。
如本文所用，“分离的”是指物质从其原始环境中分离出来(如果是天然的物质，原始环境即是天然环境)。如活体细胞内的天然状态下的多聚核苷酸和多肽是没有分离纯化的，但同样的多聚核苷酸或多肽如从天然状态中同存在的其他物质中分开，则为分离纯化的。
如本文所用，“分离的多肽(本发明中为低温几丁质脱乙酰酶)”是指所述几丁质脱乙酰酶基本上不含天然与其相关的其它蛋白、脂类、糖类或其它物质。本领域的技术人员能用标准的蛋白质纯化技术纯化所述几丁质脱乙酰酶。基本上纯的多肽在非还 原聚丙烯酰胺凝胶上能产生单一的主带。所述几丁质脱乙酰酶的纯度能用氨基酸序列分析。
如本文所用，所述“微生物”是指细胞壁含有几丁质的微生物，所述微生物包括菌，如真菌、放线菌或细菌等；较佳地，该“微生物”为“病原微生物”。
如本文所用，所述“病原微生物”是指对于人、动物、植物或环境具有危害性的微生物。
本发明中，所述“含有”表示各种成分可一起应用于本发明的混合物或组合物中。因此，术语“主要由...组成”和“由...组成”包含在术语“含有”中。
如本文所用，“工业学上可接受的载体”或“微生物学上可接受的载体”是用于将本发明的几丁质脱乙酰酶传送给需要处理的对象的，在毒性、副作用方面可控的、环境友好或对人畜无害溶剂、悬浮剂或赋形剂。所述载体可以是液体或固体，较佳的是能够较高程度保持本发明的几丁质脱乙酰酶的活性的载体。
本发明的多肽可以是重组多肽、天然多肽、合成多肽，优选重组多肽。本发明的多肽可以是天然纯化的产物，或是化学合成的产物，或使用重组技术从原核或真核宿主(例如，细菌、酵母、高等植物、昆虫和哺乳动物细胞)中产生。根据重组生产方案所用的宿主，本发明的多肽可以是糖基化的，或可以是非糖基化的。本发明的多肽还可包括或不包括起始的甲硫氨酸残基。
本发明还包括所述几丁质脱乙酰酶的片段、衍生物和类似物。如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明的天然几丁质脱乙酰酶相同的生物学功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或与抗原IgG片段的形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
在本发明中，术语“所述几丁质脱乙酰酶”指具有所述几丁质脱乙酰酶活性的SEQ ID NO:2序列的多肽。该术语还包括具有与所述几丁质脱乙酰酶相同功能的、SEQ ID NO:2序列的变异形式。这些变异形式包括(但并不限于)：一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，更佳地1-10个，最佳地1-5个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加或缺失一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性 能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。比如，在C末端和/或N末端添加或缺失一个或数个氨基酸通常也不会改变蛋白质的功能；又比如，仅表达该蛋白的催化结构域，而不表达碳水化合物结合结构域也能获得和完整蛋白同样的催化功能。因此该术语还包括所述几丁质脱乙酰酶的活性片段和活性衍生物。例如，变异可以发生在SEQ ID NO:2的保守功能域之外。
该多肽的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严谨度条件下能与所述几丁质脱乙酰酶DNA杂交的DNA所编码的蛋白、以及利用抗所述几丁质脱乙酰酶的抗体获得的多肽或蛋白。本发明还提供了其他多肽，如包含所述几丁质脱乙酰酶或其片段的融合蛋白。除了几乎全长的多肽外，本发明还包括了所述几丁质脱乙酰酶的片段。通常，该片段具有所述几丁质脱乙酰酶序列的至少约10个连续氨基酸，通常至少约30个连续氨基酸，较佳地至少约50个连续氨基酸，更佳地至少约80个连续氨基酸，最佳地至少约100个连续氨基酸。
发明还提供所述几丁质脱乙酰酶蛋白或多肽的类似物。这些类似物与天然所述几丁质脱乙酰酶的差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。这些多肽包括天然或诱导的遗传变异体。诱导变异体可以通过各种技术得到，如通过辐射或暴露于诱变剂而产生随机诱变，还可通过定点诱变法或其他已知分子生物学的技术。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的多肽并不限于上述例举的代表性的多肽。
在本发明中，“所述几丁质脱乙酰酶的保守性变异体”指与SEQ ID NO:2的氨基酸序列相比，有至多30个，较佳地至多20个，更佳地至多10个，更佳地至多5个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表1进行氨基酸替换而产生。
表1
最初的残基 代表性的取代 优选的取代
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
本发明的几丁质脱乙酰酶的氨基端或羧基端还可含有一个或多个多肽片段，作为蛋白标签。任何合适的标签都可以用于本发明。例如，所述的标签可以是FLAG，HA，HA1，c-Myc，Poly-His，Poly-Arg，Strep-TagII，AU1，EE，T7，4A6，ε，B，gE以及Ty1。
为了使翻译的蛋白分泌表达(如分泌到细胞外)，还可在所述几丁质脱乙酰酶的氨基酸氨基末端添加宿主适用的信号肽。信号肽在多肽从细胞内分泌出来的过程中可被切去。
本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。编码成熟多肽的编码区序列可以与几丁质脱乙酰酶的天然编码序列或与SEQ ID NO:1所示编码序列相同或者是简并的变异体。如本文所用，“简并的变异体”在本发明中是指编码具有SEQ ID NO:2的蛋白质，但与几丁质脱乙酰酶的天然编码序列或SEQ ID NO:1所示编码序列有差别的核酸序列。
编码SEQ ID NO:2的成熟多肽的多核苷酸包括：只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。
本发明还涉及上述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的多肽或多肽的片段、类似物和衍生物。此多核苷酸的变异体可以是天然发生的等位变异体或非天然发生的变异体。这些核苷酸变异体包括取代变异体、缺失变异体和插入变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个或多个核苷酸的取代、缺失或插入，但不会从实质上改变其编码的多肽的功能。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件(或严谨条件)下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC，0.1％SDS，60℃；或(2)杂交时加有变性剂，如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两 条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。并且，可杂交的多核苷酸编码的多肽与SEQ ID NO:2所示的成熟多肽有相同的生物学功能和活性。
本发明中的多肽和多核苷酸优选以分离的形式提供，更佳地被纯化至均质。
所述几丁质脱乙酰酶核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。
本发明也提供了包含本发明的多核苷酸的载体，以及用本发明的载体或所述几丁质脱乙酰酶编码序列经基因工程产生的宿主细胞，以及经重组技术产生本发明所述多肽的方法。
通过常规的重组DNA技术，可利用本发明的多聚核苷酸序列可用来表达或生产重组的所述几丁质脱乙酰酶。一般来说有以下步骤：
(1).用编码所述几丁质脱乙酰酶的多核苷酸(或变异体)，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2).在合适的培养基中培养的宿主细胞；
(3).从培养基或细胞中分离、纯化蛋白质。
本发明中，所述几丁质脱乙酰酶多核苷酸序列可插入到重组表达载体中。术语“重组表达载体”指本领域熟知的细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其他载体。只要能在宿主体内复制和稳定，任何质粒和载体都可以用。表达载体的一个重要特征是通常含有复制起点、启动子、标记基因和翻译控制元件。
本领域的技术人员熟知的方法能用于构建含编码所述几丁质脱乙酰酶的DNA序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。此外，表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状。包含上述的适当DNA序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母；植物细胞；果蝇S2或Sf9的昆虫细胞；CHO、COS、293细胞、或Bowes黑素瘤细胞的动物细胞等。在本发明的优选方式中，所述的宿主细胞为原核细胞。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用 的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
作为本发明的优选方式，采用大肠杆菌作为宿主细胞来表达所述几丁质脱乙酰酶。本发明通过构建表达系统，在大肠杆菌中异源表达所述的低温几丁质脱乙酰酶。
作为本发明的优选方式，在以下条件下进行培养：诱导剂IPTG(当宿主细胞为大肠杆菌时)浓度0.05～0.3mM、较佳地0.08～0.15mM；诱导温度16～35℃、更佳地18～30℃；更佳地18～20℃；诱导时间15～20小时；较佳地16～18小时。在上述优选的条件下，可以获得表达量高且酶活性高的重组几丁质脱乙酰酶。
本发明所述的几丁质脱乙酰酶的用途包括：催化几丁质的脱乙酰化，或用于制备催化几丁质的脱乙酰化的组合物。较佳地，所述的几丁质脱乙酰化后生成壳聚糖。
本发明所述的几丁质脱乙酰酶的用途还包括抑制微生物，或用于制备具有抑制微生物功能的组合物。较佳地，所述的微生物是细胞壁包含几丁质的微生物。
在获得了本发明的几丁质脱乙酰酶后，根据本发明的提示，本领域人员可以方便地应用该酶来发挥降解底物(特别是催化作为底物的几丁质的脱乙酰化)的作用。作为本发明的优选方式，还提供了一种降解几丁质的方法，该方法包含：用本发明所述的几丁质脱乙酰酶处理待降解的底物(特别是几丁质)。
在本发明的部分实施例中，通过催化对硝基乙酰苯胺生成对硝基苯胺来考察几丁质脱乙酰酶的活性。本发明人在确认几丁质脱乙酰酶可以催化其提天然底物几丁质脱乙酰化后，选用对硝基乙酰苯胺作为人工底物进行几丁质脱乙酰酶的酶学性质研究。
根据本发明的提示，本领域人员也可以应用该酶来发挥抑制微生物(如病原微生物)的作用。包括以所述的几丁质脱乙酰酶对需要抑制微生物的对象进行处理。所述的微生物包括真菌。在本发明的优选方式中，较佳地所述真菌包括：轮枝菌属(Verticillium)真菌，镰刀菌属(Fusarium)真菌，曲霉属(Aspergillus)真菌，青霉属(Penicillium)真菌；更佳地，所述轮枝菌属真菌包括(但不限于)：大丽花轮枝孢(Verticillium dahlia)；所述镰刀菌属真菌包括(但不限于)：尖孢镰孢黄瓜专化型(Fusarium oxysporum f.sp.cucumerinum)；所述曲霉属真菌包括(但不限于)：黑曲霉 (Aspergillus niger)；所述青霉属真菌包括(但不限于)：大核青霉(Penicillium macrosclerotiorum)。本发明人发现，所述的几丁质脱乙酰酶对于此类真菌的细胞壁几丁质具有优异的降解作用。因此，本发明的几丁质脱乙酰酶或产生该酶的细胞，能被应用于农业微生物防治上，例如但不限于棉花黄萎病和黄瓜枯萎病。
本发明人还优化了利用所述的几丁质脱乙酰酶进行处理的反应体系，作为本发明的优选方式，在以下条件下进行处理：温度0～35℃、较佳地5～25℃、更佳地10～20℃，如12、14、15、16、18℃；pH5～10、较佳地pH5.5～9、更佳地pH6.5～8.5，如pH7，7.5，8。
本发明人还发现，少量NaCl的应用有利于为本发明的几丁质脱乙酰酶提供良好的反应环境，提高其催化活性；因此，在本发明的优选方式中，在酶反应体系(处理体系)中加入NaCl 0.01～0.5M、较佳地0.03～0.3M、更佳地0.05～0.2M；如0.06、0.08、0.1、0.12、0.15M。
本发明人还发现，在反应体系中含有Na +、K +、Mg 2+、Zn 2+和/或Ni 2+有利于促进本发明的几丁质脱乙酰酶的酶活性。在优选的方式下，所述的金属离子含量如1±0.8mM，较佳地1±0.5mM；更佳地1±0.3mM。
本发明人还发现，在反应体系中含有EDTA或DTT有利于促进本发明的几丁质脱乙酰酶的酶活性。在优选的方式下，所述EDTA的用量为0.1％～5％；较佳地为0.5％～2％，如1％；或，所述的DTT用量为0.1％～5％；较佳地为0.5％～2％，如1％。
作为本发明的优选方式，进行所述处理时，反应体系中不含有：Li +、NH 4 +、Ca 2+、Mn 2+、Cu 2+、Fe 2+、Fe 3+、SDS和/或TritonX-100；也不含有：丙酮、乙醇、甲醇和/或乙腈。
本发明获得几丁质脱乙酰酶，酶活性理想，并且能够适于在大肠杆菌表达的温度条件下被重组表达，进一步又能够适用于低温条件下的酶反应(包括酶催化反应或抑制微生物)，有着广泛的工业应用潜力。
根据本发明所提供的分离的几丁质脱乙酰酶及其氨基酸序列，本领域人员可以通过蛋白分子改造等手段进一步提高其酶活力、或扩大其适用的PH值范围、温度范围、耐盐性及对于冷、热的稳定性等，因此其应用前景良好。采用这些技术改造本发明所述几丁质脱乙酰酶后生成的变体或衍生物也被包含在本发明中。
本发明还提供了一种组合物，它含有有效量的本发明的几丁质脱乙酰酶以及食品学上或工业上可接受的载体或赋形剂。这类载体包括(但并不限于)：水、缓冲液、葡萄糖、甘油、DMSO或其组合。本领域技术人员可根据组合物的实际用途确定组合物中所述几丁质脱乙酰酶的有效量。
所述的组合物中还可添加调节本发明的几丁质脱乙酰酶活性的物质。任何具有提高酶活性功能的物质均是可用的。较佳地，提高所述几丁质脱乙酰酶活性的物质选自： Na +、K +、Mg 2+、Zn 2+和/或Ni 2+或在添加至底物后可水解形成Na +、K +、Mg 2+、Zn 2+和/或Ni 2+的物质，如氯化钠。提高所述几丁质脱乙酰酶活性的物质还可选自：EDTA或DTT。
本发明的几丁质脱乙酰酶、含有该酶的载体或宿主细胞、含有该酶或宿主细胞的组合物，还可被包含在容器或试剂盒中。较佳地，所述的试剂盒中还包括使用说明书等，以便于本领域技术人员应用。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
实施例1、低温几丁质脱乙酰酶的来源及序列信息
本发明所述的低温几丁质脱乙酰酶来源于菌株Pseudomonas sp.GWSMS-1(CCTCC NO.M2019207)，该菌株已在中国典型培养物保藏中心保藏，保藏单位名称：中国典型培养物保藏中心，保藏日期：2019年3月27日，其保藏编号为CCTCC NO.M2019207。
获得该Pseudomonas sp.GWSMS-1菌株后，本发明人对其各方面性能、其基因特点及其产生的多种多样的蛋白进行了大量的研究和试验，对重点的功能性基因和蛋白进行着重研究，从而筛选获得本发明所述的低温几丁质脱乙酰酶。所述的低温几丁质脱乙酰酶的氨基酸序列如下(SEQ ID NO:2)：
Figure PCTCN2020071121-appb-000001
实施例2、几丁质脱乙酰酶的克隆、异源表达和优化
1、材料与方法
(1)培养基
LB液体培养基：酵母提取物5g/L、胰蛋白胨10g/L、氯化钠10g/L。
LB固体培养基：LB液体培养基中加入琼脂粉20g/L。
(2)几丁质脱乙酰酶基因的密码子优化、全基因合成及克隆
本发明人首先根据表达情况进行了密码子优化，以提高表达效率及DNA片段的稳定性。将优化后的序列插入至质粒pET28a(+)中，得到重组质粒pET28-CDA，随后 将重组质粒pET28-CDA转化至大肠杆菌BL21(DE3)中进行异源表达。
(3)重组几丁质脱乙酰酶基因的表达条件优化
接种量：接种量设置为0.5％、1.0％、1.5％、2.0％、2.5％、3.0％。按上述设置将重组菌接种于新鲜LB液体培养基中，于37℃，200rpm条件下培养，当菌液OD 600达到0.6-0.8时向培养基中加入终浓度为0.1mM的IPTG(对照组未添加IPTG)，然后于20℃，150rpm条件下继续培养10小时后收集菌体。
诱导温度：按1％接种量将重组菌接种于新鲜LB液体培养基中，于37℃，200rpm条件下培养，当菌液OD 600达到0.6-0.8时向培养基中加入终浓度为0.1mM的IPTG(对照组未添加IPTG)，随后将菌液分别置于15℃、20℃、25℃、30℃和35℃条件下，150rpm继续培养10小时后收集菌体。
诱导剂浓度：按1％接种量将重组菌接种于新鲜LB液体培养基中，于37℃，200rpm条件下培养，当菌液OD 600达到0.6-0.8时向培养基中分别加入终浓度为0.01、0.02、0.05、0.1、0.15、0.2、0.25、0.3mM的IPTG(对照组未添加IPTG)，然后于20℃，150rpm条件下继续培养10小时后收集菌体。
诱导时间：按1％接种量将重组菌接种于新鲜LB液体培养基中，于37℃，200rpm条件下培养，当菌液OD 600达到0.6-0.8时向培养基中加入终浓度为0.1mM的IPTG(对照组未添加IPTG)，然后于20℃，150rpm条件下分别继续培养0、4、8、12、16、20、24、32小时后收集菌体。
(4)蛋白电泳
10000g离心10min收集菌体，用生理盐水洗涤并悬浮菌体，然后超声破碎菌体细胞，于4℃条件下15000g离心10min收集上清，取15μL上清液进行SDS-PAGE电泳。
2、结果
(1)密码子优化后序列
密码子优化后的几丁质脱乙酰酶基因序列如下(SEQ ID NO:1)：
Figure PCTCN2020071121-appb-000002
Figure PCTCN2020071121-appb-000003
(2)表达条件优化
诱导温度(图1a)：当诱导温度为15℃时，重组几丁质脱乙酰酶的可溶表达量较低；当诱导温度为20℃-35℃时，重组几丁质脱乙酰酶的可溶表达量变化不明显；因此，最佳诱导温度为20℃。
诱导剂浓度(图1b)：当诱导剂浓度为低于0.1mM时，重组几丁质脱乙酰酶的可溶表达量较低；当诱导剂IPTG浓度高于0.1mM时，重组几丁质脱乙酰酶的可溶表达量变化不明显；因此，最佳诱导剂终浓度为0.1mM。
接种量(图1c)：当接种量为0.5％-1.5％时，重组几丁质脱乙酰酶的可溶表达量逐步增加；当接种量为1.5％-3.0％时，重组几丁质脱乙酰酶的可溶表达量变化不明显；因此，最佳接种量为1.5％。
诱导时间(图1d)：当诱导时间短于16小时的时候，重组几丁质脱乙酰酶的可溶表达量较低；当诱导时间长于16小时的时候，重组几丁质脱乙酰酶的可溶表达量变化不明显；因此，最佳诱导时间为16小时。
上述较佳的表达条件下，重组几丁质脱乙酰酶的表达量为9.6mg/L。
实施例3、重组几丁质脱乙酰酶的制备
1、材料与方法
(1)粗酶液制备
按1％接种量将重组菌接种于新鲜LB液体培养基中，于37℃，200rpm条件下培养，当菌液OD 600达到0.6-0.8时向培养基中加入终浓度为0.1mM的IPTG，然后于20℃，150rpm条件下分别继续培养16小时后离心收集菌体。离心后的菌体充分悬浮于裂解缓冲液中，通过超声破碎细胞，随后于15000g，4℃离心10min去除细胞碎片，收集得到的上清液即为粗酶液。
(2)纯酶制备
通过Ni-NTA亲和层析重力柱进行纯酶的制备，粗酶液在上样前需用0.45μm的滤器过滤，粗酶液上样和填料结合后，使用漂洗缓冲液漂洗约10-15倍的床体积以清除杂蛋白，随后加入洗脱缓冲液洗脱纯酶。洗脱下的纯酶再通过超滤脱盐并切换成储酶缓冲液，分装后储藏于-80℃冰箱中待用。
(3)活性测定
采用对硝基乙酰苯胺法来测定几丁质脱乙酰酶的活性，该方法以对硝基乙酰苯胺为作用底物，几丁质脱乙酰酶可催化对硝基乙酰苯胺生成对硝基苯胺，通过测定产物 在400nm处的吸光值来进行几丁质脱乙酰酶的活性表征。
标准曲线绘制：首先称量0.1g对硝基苯胺，蒸馏水定容1L，分别稀释至合适浓度，测定不同浓度的稀释液在400nm处的吸光值，蒸馏水作对照，以对硝基苯胺浓度为横坐标，吸光值为纵坐标绘制标准曲线。
几丁质脱乙酰酶活性测定：取pH 8.0 0.05M的Tris-HCl 3mL，加入1mL(200mg/L)的对硝基乙酰苯胺溶液，然后加入重组几丁质脱乙酰酶，50℃水浴保温15min，然后沸水浴3-5min终止反应，加入蒸馏水定容到10mL，振荡混匀，3500g离心10min，取上清液测定其在400nm处的吸光值，空白对照实验体系中添加1mL相应浓度的灭活酶液(沸水浴灭活)，其余同上，测定上清液在400nm处的吸光值。
在上述几丁质脱乙酰酶促反应体系条件下，每小时产生1μg对硝基苯胺所需的酶量，定义为一个酶活力单位。
2、结果
重组几丁质脱乙酰酶的SDS-PAGE电泳图如图2(a)，第1条到第4条泳道分别表示未加IPTG诱导、IPTG诱导的全细胞及IPTG诱导的上清液、纯化后的几丁质脱乙酰酶，从图可以看出该酶经过纯化超滤后只显示一个单一的条带，说明该酶经过Ni-NTA Agarose填料重力柱纯化后已经达到了电泳纯。与蛋白Marker比较结果显示，该几丁质脱乙酰酶的大小约为40kDa左右)。
图2b为几丁质脱乙酰酶的活性蛋白电泳结果，左图为考马斯亮蓝R-250染色，右图为几丁质酶的活性染色(参见Trudel 1990)，利用Calcofluor White M2R显色。该显色试剂是一种荧光增白剂，能吸收特定的紫外光反射回蓝光，其与反应产物壳聚糖结合后的荧光强度比其与底物几丁质结合后的荧光强度高，所以在有壳聚糖的位置会出现比背景更亮的条带。根据图2，显示考马斯亮蓝和活性染色的目的条带在电泳胶的位置上一致，说明活性几丁质脱乙酰酶对于几丁质具有脱乙酰作用。
纯化后重组几丁质脱乙酰酶的比活力为150.5U/mg，总酶活力回收率约为53％。
实施例4、几丁质脱乙酰酶性质
1、材料与方法
(1)最适温度与温度稳定性
最适温度：重组几丁质酶在标准反应条件下分别于0℃、5℃、10℃、15℃、20℃、25℃、30℃、35℃、40℃、45℃、50℃、55℃、60℃中反应30min，对照组为沸水浴灭活5min的几丁质脱乙酰酶液，按照几丁质脱乙酰酶活性检测方法测其活性。
温度稳定性：将重组几丁质脱乙酰酶在以上温度条件下处理1小时，然后在最适反应温度下测定其几丁质脱乙酰酶的活性。
(2)最适pH与pH稳定性
最适pH：重组几丁质酶在标准反应条件下分别于pH 3.0、4.0、5.0、6.0、7.0、8.0、9.0、10.0、11.0的缓冲液中反应30min，按照几丁质脱乙酰酶活性检测方法测其活。
pH稳定性：将重组几丁质酶在以上pH条件下4℃处理1小时，然后在最适反应温度下测定其几丁质脱乙酰酶的活性。
(3)氯化钠对重组几丁质脱乙酰酶活性和稳定性的影响
活性：重组几丁质酶在标准反应条件下分别加入终浓度为0.1、0.5、1、1.5、2、2.5、3、3.5、4M的氯化钠反应30min，然后测定几丁质脱乙酰酶的活性。
稳定性：将重组几丁质酶在上述氯化钠浓度条件下4℃处理1小时，然后在最适反应温度下测定其几丁质脱乙酰酶的活性。
(4)金属离子等对重组几丁质脱乙酰酶活性影响
在最适pH的缓冲体系下分别配制含有NaCl、KCl、LiCl、NH 4Cl、MgCl 2、CaCl 2、MnCl 2、CuSO 4、NiSO 4·6H 2O、ZnSO 4、CoCl 2、FeSO 4·7H 2O、FeCl 3·6H 2O各1mM的缓冲液，含有EDTA、DTT、TritonX-100、SDS各1％的缓冲液，按照几丁质脱乙酰酶活性检测方法测其活性。
(5)有机溶剂对重组几丁质脱乙酰酶活性影响
将几丁质脱乙酰酶溶液分别置于体积分数为10％、20％、30％、40％、50％的有机溶剂甲醇、乙醇、丙酮、乙腈，DMSO中4℃保温1小时，然后测定几丁质脱乙酰酶的活性。
2、结果
(1)最适温度与温度稳定性
最适温度：如图3a所示，重组几丁质脱乙酰酶的最适反应温度为15℃，当催化温度在10℃-20℃之间时，该重组几丁质脱乙酰酶的催化活性超过了80％，该酶在反应温度为5℃时仍然具有68.89％的催化活性，当催化温度为55℃时已经检测不到几丁质脱乙酰酶活性。说明该重组几丁质脱乙酰酶为低温酶。
温度稳定性：如图3b所示，当该重组几丁质脱乙酰酶在15℃处理1小时后酶活性即下降到76％，当该重组酶在40℃处理1小时后基本检测不到酶活性，也同样说明该重组几丁质脱乙酰酶是一个低温酶。
(2)最适pH与pH稳定性
最适pH：如图3c所示，该重组几丁质脱乙酰酶的最适催化pH为7.0，当pH小于6.0或大于8.0时催化活性迅速降低，说明该酶是一个中性酶。
pH稳定性：如图3d所示：该重组几丁质脱乙酰酶在pH 8.0时的稳定性最高。
(3)氯化钠对重组几丁质脱乙酰酶活性和稳定性的影响
活性：如图3e所示，当氯化钠的浓度在0-0.5M时重组几丁质脱乙酰酶的催化活性高于未加氯化钠时的水平，说明该重组酶具有一定的氯化钠耐受性，并且在氯化钠浓度为0.1M时该重组酶的催化效率最高，而氯化钠的浓度高于0.5M时表现为对该重组酶的抑制作用。可见低浓度NaCl有助于重组酶的催化活性的提高。
稳定性：如图3f所示，该重组几丁质脱乙酰酶在氯化钠浓度高于1M、4℃条件下保存1小时后相对酶活性低于50％，说明该重组酶对氯化钠的耐受性一般。
(4)金属离子等对重组几丁质脱乙酰酶的酶活影响
如表2所示，1mM的Na +、K +、Mg 2+、Zn 2+和Ni 2+对该重组酶的催化活性有促进作用，其中镍离子的促进作用最明显，其它金属离子对重组几丁质脱乙酰酶均有不同程度的抑制作用；1％的EDTA和1％的DTT对几丁质重组酶的催化活性有促进作用，1％的SDS和1％的TritonX-100对该重组酶催化活性表现出完全抑制作用。
表2、金属离子及其它化合物对重组几丁质脱乙酰酶活性的影响
Figure PCTCN2020071121-appb-000004
(5)有机溶剂对重组几丁质脱乙酰酶稳定性的影响
如表3所示，该重组几丁质脱乙酰酶在30％的DMSO溶液中4℃保存1小时后具有76％的相对酶活性，说明该重组酶对DMSO的耐受性很好；该重组酶在20％的丙酮溶液中4℃保存1小时仍然具有80％的活性；该重组酶在20％以上的甲醇、乙醇、乙腈中的耐受性较差，在10％的乙腈中4℃保存1小时相对酶活力低于50％。
表3、有机溶剂对重组几丁质脱乙酰酶稳定性的影响
Figure PCTCN2020071121-appb-000005
实施例5、抗真菌实验
1、材料与方法
(1)培养基
同实施例2。
(2)重组几丁质脱乙酰酶的制备
同实施例3。
(3)抗真菌实验
将直径为6mm的滤纸片浸泡于重组几丁质脱乙酰酶液中5min，用直径为0.5cm的打孔器打取菌盘，接种到新的具有PDA培养基的培养皿的中央位置；然后在菌盘中央处放1片浸泡好的滤纸片，在20℃培养后观察抑制情况，培养期间，每间隔2个小时在滤纸片上滴加1次实验溶液。
(4)真菌供试菌株
Verticillium dahlia CICC 2534(大丽花轮枝孢)；Fusarium oxysporum f.sp.cucumerinum CICC 2532(尖孢镰孢黄瓜专化型)；Aspergillus niger CICC 2039(黑曲霉)；Penicillium macrosclerotiorum CICC 40649(大核青霉)。
2、结果
如图4，本发明的重组几丁质脱乙酰酶可以对于植物病原真菌Verticillium dahlia CICC 2534、Fusarium oxysporum f.sp.cucumerinum CICC 2532具有良好的抑制效果；对Aspergillus niger CICC 2039和Penicillium macrosclerotiorum CICC 40649也表现出具有显著性的抑制作用。抑制率统计如表4。
表4
菌株 第6天抑制率
Verticillium dahlia CICC 2534 56％
Fusarium oxysporum f.sp.cucumerinum CICC 2532 43％
Aspergillus niger CICC 2039 34％
Penicillium macrosclerotiorum CICC 40649 57％
已知Verticillium dahlia CICC 2534和Fusarium oxysporum f.sp.cucumerinum CICC 2532分别可以引起棉花黄萎病和黄瓜枯萎病，这表明本发明所述的重组几丁脱乙酰酶有应用于上述两种植物病害的生物防治的能力。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (17)

  1. 一种分离的多肽，其特征在于，该多肽选自下组：
    (a)具有SEQ ID NO:2所示氨基酸序列多肽；
    (b)由(a)所述的多肽经过一个或多个氨基酸残基的取代、缺失或添加而形成的，且具有多肽(a)功能的多肽；或
    (c)与(a)所述的多肽的氨基酸序列有80％以上同源性，且具有多肽(a)功能的多肽；
    (d)在(a)或(b)或(c)所述多肽的N或C末端添加标签序列，或在其N末端添加信号肽序列后形成的多肽。
  2. 一种分离的多核苷酸，其特征在于，它包含一核苷酸序列，该核苷酸序列选自下组：
    (1)编码如权利要求1所述多肽的多核苷酸；
    (2)与多核苷酸(1)互补的多核苷酸。
  3. 如权利要求2所述的多核苷酸，其特征在于，该多核苷酸编码如SEQ ID NO:2所示氨基酸序列的多肽；或，该多核苷酸的核苷酸序列如SEQ ID NO:1所示。
  4. 一种载体，其特征在于，它含有权利要求2所述的多核苷酸。
  5. 一种遗传工程化的宿主细胞，其特征在于，它含有权利要求4所述的载体，或其基因组中整合有权利要求2所述的多核苷酸。
  6. 一种制备权利要求1所述的多肽的方法，包括：(i)培养权利要求4所述的宿主细胞；(ii)收集含有权利要求1所述的多肽的培养物；(iii)从培养物中分离出权利要求1所述的多肽。
  7. 如权利要求6所述的方法，其特征在于，在以下条件下进行培养：
    诱导剂IPTG浓度0.05～0.3mM、较佳地0.08～0.15mM；
    诱导温度16～35℃、更佳地18～30℃；更佳地18～20℃；
    诱导时间15～20小时；较佳地16～18小时。
  8. 如权利要求6所述的方法，其特征在于，在以下条件下进行培养：
    诱导剂IPTG浓度0.08～0.15mM；
    诱导温度18～30℃；较佳地18～20℃；
    诱导时间16～18小时。
  9. 权利要求1所述的多肽的用途，用于：
    催化几丁质的脱乙酰化，或用于制备催化几丁质的脱乙酰化的组合物；或
    抑制微生物，或用于制备具有抑制微生物功能的组合物。
  10. 如权利要求9所述的用途，其特征在于，催化几丁质的脱乙酰化后，生成壳聚糖；或，所述的微生物是细胞壁包含几丁质的微生物。
  11. 一种组合物，其包含选自下组的成分：权利要求1所述的多肽；或权利要求5所述的宿主细胞；以及工业学或微生物学上可接受的载体。
  12. 一种催化几丁质的脱乙酰化的方法，包括：应用权利要求1所述的多肽、权利要求5所述的宿主细胞或权利要求8所述的组合物对几丁质或含有几丁质的物质进行处理；较佳地，脱乙酰化后生成壳聚糖。
  13. 如权利要求12所述的方法，其特征在于，在以下条件下进行处理：
    温度0～35℃、较佳地5～25℃、更佳地10～20℃；
    pH5～10、较佳地pH5.5～9、更佳地pH6.5～8.5；
    NaCl 0.01～0.5M、较佳地0.03～0.3M、更佳地0.05～0.2M；
    含有Na +、K +、Mg 2+、Zn 2+和/或Ni 2+；
    含有EDTA；和/或
    含有DTT。
  14. 一种抑制微生物的方法，包括：应用权利要求1所述的多肽、权利要求5所述的宿主细胞或权利要求11所述的组合物对需要抑制微生物的对象进行处理；较佳地，所述的微生物是细胞壁包含几丁质的微生物。
  15. 如权利要求14所述的方法，其特征在于，在以下条件下进行处理：
    温度0～35℃、较佳地5～25℃、更佳地10～20℃；
    pH5～10、较佳地pH5.5～9、更佳地pH6.5～8.5；
    NaCl 0.01～0.5M、较佳地0.03～0.3M、更佳地0.05～0.2M；
    含有Na +、K +、Mg 2+、Zn 2+和/或Ni 2+；
    含有EDTA；和/或
    含有DTT。
  16. 如权利要求9、10、11或14所述，其特征在于，所述的微生物包括：真菌；较佳地所述真菌包括：轮枝菌属(Verticillium)真菌，镰刀菌属(Fusarium)真菌，曲霉属(Aspergillus)真菌，青霉属(Penicillium)真菌。
  17. 如权利要求16所述，其特征在于，所述轮枝菌属真菌包括：大丽花轮枝孢(Verticillium dahlia)；所述镰刀菌属真菌包括：尖孢镰孢黄瓜专化型(Fusarium oxysporum f.sp.cucumerinum)；所述曲霉属真菌包括：黑曲霉(Aspergillus niger)；所述青霉属真菌包括：大核青霉(Penicillium macrosclerotiorum)。
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