JP2007061038A - Chitinase derived from bacterium of genus paenibacillus and gene encoding the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means for efficiently producing a chitin oligosaccharide. <P>SOLUTION: The invention provides a new bacterial strain of the genus Paenibacillus or its variant and agrochemicals, an antibacterial agent against plant pathogens or a polysaccharide decomposing agent containing the bacterial strain; a method for producing an oligosaccharide by bringing the new bacterial strain of the genus Paenibacillus or its variant into contact with a polysaccharide or a polysaccharide-containing material (e.g. shell of snow crabs such as Echizen crab); a polypeptide composed of an amino acid sequence having a sequence identity of ≥70% to an amino acid sequence obtained by removing a secretory signal sequence from a specific amino acid sequence and having chitin decomposition activity; a polynucleotide encoding the polypeptide; etc. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention provides a novel Paenibacillus genus bacterium and mutants thereof, a novel chitinase derived therefrom, a gene thereof, a method for using them, and the like.

  Now Echizen crab is an expensive taste only in winter, but in the past it was eaten as a snack. And the crab shells that have been eaten have been used as fertilizer in the fields. The reason why crab husks have been used as fertilizers in this way is that, for a long time, farmers in Fukui Prefecture have been thought that crab husks increase the disease resistance of plants and increase their fruits. However, the reason why crab shells increase plant disease resistance has not been well understood.

  By the way, chitin oligosaccharides are known as sugars with sweetness, and many physiological functions are recognized by animal experiments and cultured cell experiments. Such physiological functions include, for example, immunostimulatory action, elicitor activity (activation of plant defense mechanism), cholesterol lowering action, blood lysozyme induction (rabbit), bifidobacteria growth promotion (intestinal function), hyaluron Examples thereof include an increase in acid amount (use in cosmetics) and a plant seed germination promoting action. Furthermore, since chitin oligosaccharides are difficult to digest, application as diet sweeteners is expected, and it is also considered useful as a substrate for measuring lysozyme and chitinase activity. Therefore, development of an efficient production method of chitin oligosaccharides exhibiting various useful properties as described above is required.

  An object of this invention is to provide the means which enables the efficient manufacture of chitin oligosaccharide.

As a result of intensive studies, the present inventors have investigated a field in which crab husks are used as fertilizer as described above, so that a new kind of bacterium, Paenibacillus fuchinensis, which degrades chitin, which is the main component of crab husks ( Paenibacillus fukuinensis) was discovered. The inventors have also found that this bacterium has antibacterial activity against phytopathogenic fungi. Paenibacillus fuchinensis can explain the tradition that Echizen crab shells become fertilizers and pesticides because it produces giraffe oligosaccharides by degradation of chitin and has antibacterial activity. In addition, the present inventors succeeded in isolating and identifying a novel chitinolytic enzyme from this bacterium, and completed the present invention. That is, the present invention is as follows:
[1] Paenibacillus genus bacteria represented by accession number FERM P-20620 or mutants thereof;
[2] A method for producing an oligosaccharide, comprising contacting a Paenibacillus bacterium represented by accession number FERM P-20620 or a mutant thereof with a polysaccharide or a polysaccharide-containing product;
[3] The method according to [2] above, wherein the polysaccharide or polysaccharide-containing material is one or more polysaccharides selected from the group consisting of chitin, chitosan, and cellulose;
[4] The method of [2] or [3] above, wherein the polysaccharide-containing material is crab shell;
[5] The method according to [4] above, wherein the crab shell is a snow crab shell;
[6] A composition comprising the genus Paenibacillus represented by accession number FERM P-20620 or a mutant thereof;
[7] The composition according to [6] above, which is an antibacterial agent, an antibacterial agent or a polysaccharide decomposing agent against plant pathogens;
[8] The following polypeptide (a) or (b) or a partial peptide thereof:
(A) an amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence having 70% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2 from which the secretory signal sequence has been removed, and chitin A polypeptide having degrading activity;
(B) an amino acid sequence represented by SEQ ID NO: 4, or an amino acid sequence having 70% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 4 from which the secretory signal sequence has been removed, and chitin A polypeptide having degrading activity;
[9] A polynucleotide encoding the polypeptide of [8] above or a partial peptide thereof;
[10] An expression vector comprising the polynucleotide of [9] above;
[11] A transformant comprising the expression vector of [10] above;
[12] An antibody against the polypeptide of [8] above or a partial peptide thereof;
[13] A hybridoma that produces the antibody of [12].

  The bacterium of the present invention or a mutant thereof can be used, for example, for the production of oligosaccharides such as chitin oligosaccharides from polysaccharides or polysaccharide-containing materials (eg, snow crab shells such as Echizen crabs), or for microbial pesticides, antibacterial agents, polysaccharides. Useful as a degrading agent. The chitinase of the present invention is useful, for example, for the production of chitin oligosaccharides from polysaccharides or polysaccharide-containing materials.

(1. Bacteria and their uses)
The present invention provides a Paenibacillus bacterium or a mutant thereof having the activity of degrading polysaccharides to oligosaccharides.

  The bacterium of the present invention or a mutant thereof can degrade, for example, chitin (N-acetylglucosamine), chitosan (glucosamine), glucan (eg, peptide glucan or non-peptide glucan) or cellulose (glucose) as a polysaccharide. The bacterium of the present invention or a mutant strain thereof can also secrete the above-mentioned polysaccharide degrading enzymes such as chitin degrading enzyme (chitinase), chitosan degrading enzyme (chitosanase), cellulose degrading enzyme (glucanase, cellulase) and the like. The bacterium of the present invention or a mutant strain thereof may further have antibacterial activity against phytopathogenic fungi. A phytopathogenic bacterium to which the bacterium of the present invention or a mutant strain thereof can exhibit antibacterial activity is not particularly limited as long as it contains the polysaccharide as a cell wall constituent component. Preferably, the bacterium of the present invention is a Paenibacillus genus bacterium represented by accession number FERM P-20620 or a mutant strain thereof.

  The Paenibacillus genus bacterium represented by the deposit number FERM P-20620 has been deposited with the National Institute of Advanced Industrial Science and Technology (AIST), 1-1-6 Higashi 1-1-1 Tsukuba, Ibaraki Prefecture (Deposit date: August 2005) 17th).

  The mutant strain of the present invention is not particularly limited as long as it is a strain derived from the bacterium of the present invention. For example, a phenotype similar to that of the bacterium of the present invention (see Example 2.1), and / or a molecular strain ( Example 2.2)). The mutant strain of the present invention can be obtained by culturing the bacterium of the present invention in the presence of a mutagenic substance or the like, or a gene capable of enhancing the usefulness of the bacterium of the present invention (eg, a polysaccharide degrading enzyme other than the above polysaccharide). The gene can be prepared by introducing a coding gene or a gene encoding a polysaccharide-degrading enzyme such as the chitinase of the present invention.

  The bacterium of the present invention or a mutant thereof can be cultured by a method known per se used for culturing Bacillus in general. For example, the bacterium of the present invention or a mutant thereof is Tryptic Soy Broth (BECTON DICKINSON, catalog containing Digest of casein 17.0 g, Papaic digest of soybean meal 3.0 g, Dextrose 2.5 g, Sodium chloride 5.0 g and Dipotassium phosphate 2.5 g. No. 211825) (pH 7.2 ± 0.2) can be cultured with shaking at 30-42 ° C. under aerobic conditions.

  The bacterium of the present invention or a mutant strain thereof exhibits a growth inhibitory action against phytopathogenic bacteria, and the chitin oligosaccharide that can be produced by the bacterium of the present invention or a mutant strain thereof exhibits various physiological functions. Therefore, the bacterium of the present invention or a mutant thereof can be useful as, for example, an agrochemical, an antibacterial agent against phytopathogenic fungi, a polysaccharide decomposing agent, or a soil improving agent.

  The present invention also provides a method for producing an oligosaccharide, which comprises contacting the bacterium of the present invention or a mutant strain thereof with a polysaccharide or a polysaccharide-containing product.

  The polysaccharide or polysaccharide-containing product is not particularly limited as long as it is a polysaccharide that can be degraded by the Paenibacillus genus bacterium of the present invention, or a material containing the polysaccharide. Examples of such polysaccharide or polysaccharide-containing product include chitin, chitosan (chitin). And one or more polysaccharides selected from the group consisting of cellulose and materials containing the same. More specifically, the polysaccharide-containing material includes, for example, crustacean shells, mushrooms (eg, garlic mushrooms, maitake mushrooms, himematsutake (also referred to as agaricus)); for example, Bull. Agr. Shizuoka Univ. (1988)), insects (eg, flies, grasshoppers), squid-derived components (clear white tissue like hard bones found in the center of the squid), molds (eg, Aspergillus, absidia) Cell wall components. Examples of crustaceans include crabs and shrimps. Examples of crabs include snow crab (eg, Echizen crab, pine crab) and king crab.

  Crab shells such as Echizen crab are rich in chitin. Chitin oligosaccharides produced by the degradation of chitin by enzymes are known not only to improve immunity of animals but also to activate adult defense mechanisms to protect plants against external enemies such as pathogens. ing. Snow crab shells such as Echizen crab contain abundant calcium and astaxanthin in addition to chitin. Astaxanthin is a natural red pigment, and it is this pigment that turns red when crab is boiled. Astaxanthin has an antioxidant action about 10 times that of β-carotene and about 500 times that of vitamin E, and is very valuable as a health food. The bacterium of the present invention is isolated from a field using Echizen crab shell as a fertilizer in Fukui Prefecture, and is considered to be excellent in the resolution of such crab shells. Therefore, in the production method of the present invention, particularly for the purpose of producing chitin oligosaccharides for health foods, crab shells that can contain various useful ingredients in addition to chitin as described above, especially snow crab such as Echizen crab etc. It is preferable to use the shell as a polysaccharide-containing material.

  Contact of the bacterium of the present invention or its mutant strain with the polysaccharide-containing product can be carried out by a method known per se. For example, the contact of the bacterium of the present invention or the mutant thereof with a polysaccharide-containing product can be performed in soil such as a field or a medium such as a fermenter. The bacterium of the present invention was isolated from a field using crab shell as fertilizer in Fukui Prefecture, and it is considered that it can degrade polysaccharide-containing materials such as crab shell in the natural environment even if conditions are not set. Therefore, the polysaccharide contained in the polysaccharide-containing material can be decomposed by any contact mode described above.

  The production method of the present invention includes, for example, the production of chitin oligosaccharides or chitosan oligosaccharides (see Examples) having various utilities as described above, and agricultural chemicals and health containing the chitin oligosaccharides and / or chitosan oligosaccharides. It is useful for the development of foods (which may further contain calcium and astaxanthin).

(2. Novel chitinase and related invention)
The present invention provides a novel chitinase derived from the bacterium of the present invention (ie, chitinase A and chitinase B) or a partial peptide thereof. The chitinase of the present invention has an amino acid sequence represented by SEQ ID NO: 2 or 4, or an amino acid sequence obtained by removing a secretory signal sequence from the amino acid sequence represented by SEQ ID NO: 2 or 4 (hereinafter referred to as “sequence” It may be a polypeptide consisting of the same or substantially the same amino acid sequence as the amino acid sequence represented by No. 2 or 4, or its secretory signal-removed amino acid sequence ”. The chitinase of the present invention or a partial peptide thereof can be useful, for example, for the production of chitin oligosaccharides and the production of the antibody of the present invention.

  The amino acid sequence from which the secretory signal sequence has been removed from the amino acid sequence represented by SEQ ID NO: 2 can be an amino acid sequence corresponding to the 38th to the last amino acid residue in the amino acid sequence represented by SEQ ID NO: 2, The amino acid sequence from which the secretory signal sequence has been removed from the amino acid sequence represented by SEQ ID NO: 4 can be an amino acid sequence corresponding to the 32nd to the last amino acid residue in the amino acid sequence represented by SEQ ID NO: 4.

  In one embodiment, the amino acid sequence represented by SEQ ID NO: 2 or 4 or the amino acid sequence substantially identical to the secretory signal-removed amino acid sequence is the amino acid sequence represented by SEQ ID NO: 2 or 4, or the secretion signal thereof It may be an amino acid sequence having a predetermined amino acid sequence identity with the removed amino acid sequence. The degree of amino acid sequence identity is, for example, about 70%, preferably about 80%, more preferably about 90%, even more preferably about 95%, most preferably about 97%, about 98% or about 99% or more. possible. Amino acid sequence identity can be determined by a method known per se. For example, amino acid sequence identity (%) can be determined using a program commonly used in the art (eg, BLAST, FASTA, etc.) by default. In another aspect, identity (%) is determined by any algorithm known in the art, such as Needleman et al. (1970) (J. Mol. Biol. 48: 444-453), Myers and Miller (CABIOS, 1988, 4: 11-17). Needleman et al.'S algorithm is incorporated into the GAP program in the GCG software package (available at www.gcg.com) and the percent identity is, for example, BLOSUM 62 matrix or PAM250 matrix, and gap weight: 16, It can be determined by using either 14, 12, 10, 8, 6 or 4 and length weight: 1, 2, 3, 4, 5 or 6. The Myers and Miller algorithms are also incorporated into the ALIGN program that is part of the GCG sequence alignment software package. When the ALIGN program is used to compare amino acid sequences, for example, PAM120 weight residue table, gap length penalty 12, and gap penalty 4 can be used.

  In another embodiment, the amino acid sequence represented by SEQ ID NO: 2 or 4 or the amino acid sequence substantially identical to the secretory signal-removed amino acid sequence is the amino acid sequence represented by SEQ ID NO: 2 or 4, or its secretion It may be an amino acid sequence in which one or more amino acids have been substituted, added, deleted and / or inserted in the signal removal amino acid sequence. The number of amino acids to be substituted, added, deleted and / or inserted is not particularly limited as long as it is 1 or more. For example, 1 to about 100, preferably 1 to about 50, more preferably 1 to about 30. Even more preferably, it may be 1 to about 20, most preferably 1 to about 10, 1 to about 5, or 1 or 2.

  The chitinase of the present invention may have chitinolytic activity. When the chitinase of the present invention is a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or 4 or an amino acid sequence substantially identical to the secretory signal-removed amino acid sequence, the degree of chitinolytic activity is SEQ ID NO: 2. Alternatively, it can be quantitatively equivalent to the amino acid sequence represented by 4 or a polypeptide consisting of the same amino acid sequence as the secretory signal-removed amino acid sequence, but within an acceptable range (for example, about 0.1 to about 5 times, Preferably about 0.5 to about 2 times).

  The chitinase of the present invention may also have a family 18 chitinase domain, a chitin adsorption domain, a fibronectin type 3 domain as its characteristic domains. Therefore, when the chitinase of the present invention is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 or 4 or the amino acid sequence substantially the same as the secretory signal-removed amino acid sequence, these domains and / or their functions Is preferably maintained.

  The family 18 chitinase domain is an amino acid region necessary for chitinase activity possessed by the carbohydrate hydrolase of family 18. Carbohydrate hydrolases are classified into several families based on the similarity of the amino acid sequences of the active domains (Henrissat, B, and Bairoch, A. (1966) Biochem J. 316, 695-696, 2004. It is classified into 91 families as of the year). A large number of chitinases have been reported so far, and they are all classified into family 18 and 19 types of families. The family 18 chitinase domain can correspond to the 45th to 367th amino acid residues in the amino acid sequence represented by SEQ ID NO: 2, and the 38th to 349th amino acid residues in the amino acid sequence represented by SEQ ID NO: 4. Can correspond to the group.

  The chitin adsorption domain is a region that allows stronger interaction between chitinase and its substrate chitin, and is not essential for enzyme activity, but is known to increase the specific activity of the enzyme. The chitin adsorption domain can correspond to the 564th to 606th amino acid residues in the amino acid sequence represented by SEQ ID NO: 2, and the 564th to 605th amino acid residues in the amino acid sequence represented by SEQ ID NO: 4 , And amino acid residues 662 to 757.

  The function of the fibronectin type 3 domain in carbohydrate degrading enzymes is not yet well understood. However, in the chitinases reported so far, all fibronectin type 3 domains exist between the chitinase domain and the chitin adsorption domain, and so far, they may function as linkers of both domains. It is considered. However, since the fibronectin type 3 domain of chitinase A produced by the bacterium of the present invention exists not in the chitinase domain and the chitin adsorption domain but in the C-terminal region, the enzyme is very interesting. The fibronectin type 3 domain may correspond to the amino acid residues 711 to 793 in the amino acid sequence represented by SEQ ID NO: 2, and the amino acid residues from the 378th to 461th positions in the amino acid sequence represented by SEQ ID NO: 4. It may correspond to the group and amino acid residues from position 471 to position 554.

  Among the chitinases of the present invention, a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence substantially identical to the secretory signal-removed amino acid sequence exhibits high activity at low temperatures, and the optimum temperature is It can be about 30 ° C. The polypeptides are also very pH stable enzymes and may have a relative activity of about 80% or more (eg, about 80-90%) at pH 3-9. Furthermore, the present peptide is an enzyme having a relatively high thermostability that is stable at a low temperature but not rapidly inactivated at a high temperature, is stable at a low temperature of 20 to 30 ° C., and gradually increases in the range of 40 to 80 ° C. Relative activity can be reduced. For example, the relative activity decrease rate from 40 ° C. to 80 ° C. is about 1% (relative activity) / 1 ° C., and even 60-80 ° C. can have a relative activity of about 35-50%.

  On the other hand, a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 4 or an amino acid sequence substantially the same as the secretory signal-removed amino acid sequence exhibits high activity at high temperatures (about 50-60 ° C.) and is optimal. The temperature can be about 60 ° C. The present polypeptide is also an enzyme having activity near neutrality, the optimum pH is around 7, and it can have a relative activity of 90% or more in the range of pH 6-8. Furthermore, this peptide can maintain a relative activity of 90% or more by heat treatment up to 20 to 50 ° C. and can be stable.

  Production of chitin oligosaccharides using the chitinase of the present invention can be carried out by a method known per se similar to the production of chitin oligosaccharides using known chitinases (for example, Seiichi Aiba, Polymer Processing, 46, 328 (1997)). See Usui T et al., Biochim Biophys Acta, 923, 302 (1987)). It is of course possible to produce chitin oligosaccharides according to the chitinase activity measurement method. As a method for measuring the activity of chitinase, for example, by using a substrate solution (potassium ferricyanide of colloidal chitin or glycol chitin (dissolving 0.5 g of potassium ferricyanide in 1 liter of 0.5 M sodium carbonate)), and by decomposing colloidal chitin or glycol chitin. There is a method of measuring the chitinase activity by quantification using the reducing sugar to be produced (see, for example, chitin, chitosan experiment manual, chitin, chitosan research group, Gihodo Publishing, p112-113, 1991). See the examples below for more details.

  The partial peptide of the present invention may be a partial peptide of a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or 4, or the same or substantially the same amino acid sequence as the secretory signal-removed amino acid sequence. The partial peptide of the present invention is at least about 8, preferably at least 10, selected from the amino acid sequence represented by SEQ ID NO: 2 or 4, or the same or substantially the same amino acid sequence as the secretory signal-removed amino acid sequence, More preferably, it may be a peptide consisting of at least 12, more preferably at least 15, most preferably 20 or more consecutive amino acids. Specifically, the partial peptide of the present invention includes, for example, the domain itself characteristic of the chitinase of the present invention or a polypeptide containing the domain.

  The present invention also provides a polynucleotide encoding the chitinase of the present invention or a partial nucleotide thereof. The polynucleotide of the present invention comprises a nucleotide sequence represented by SEQ ID NO: 1 or 3, or a nucleotide sequence obtained by removing a nucleotide sequence encoding a secretory signal sequence from the nucleotide sequence represented by SEQ ID NO: 1 or 3 (hereinafter referred to as necessary). Accordingly, the nucleotide sequence may be the same or substantially the same as “the nucleotide sequence represented by SEQ ID NO: 1 or 3 or its secretion signal-removed nucleotide sequence”). The polynucleotide of the present invention or a partial nucleotide thereof can be useful, for example, for producing the chitinase of the present invention or a partial peptide thereof.

  In one embodiment, the nucleotide sequence represented by SEQ ID NO: 1 or 3, or the nucleotide sequence represented by SEQ ID NO: 1 or 3, wherein the nucleotide sequence encoding the secretory signal sequence is removed and at least 70% of the sequence It can be a nucleotide sequence having identity. The degree of nucleotide sequence identity is, for example, about 70%, preferably about 80%, more preferably about 90%, even more preferably about 95%, most preferably about 97%, about 98% or about 99% or more. possible. Nucleotide sequence identity can be determined by methods known per se. For example, nucleotide sequence identity (%) can be determined in the same manner as amino acid sequence identity (%) described above.

  In another embodiment, the nucleotide sequence represented by SEQ ID NO: 1 or 3 or the nucleotide sequence substantially identical to the secretion signal elimination nucleotide sequence thereof is the nucleotide sequence represented by SEQ ID NO: 1 or 3, or the secretion thereof. It may be a nucleotide sequence in which one or more nucleotides have been substituted, added, deleted and / or inserted in a signal removal nucleotide sequence. The number of nucleotides to be substituted, added, deleted and / or inserted is not particularly limited as long as it is 1 or more. For example, 1 to about 300, preferably 1 to about 150, more preferably 1 to about 100. Even more preferably from 1 to about 50, most preferably from 1 to about 30, from 1 to about 20, from 1 to about 10 or from 1 to about 5.

  In still another embodiment, the nucleotide sequence represented by SEQ ID NO: 1 or 3 or a nucleotide sequence substantially identical to the secretory signal elimination nucleotide sequence is a complementary sequence of the nucleotide sequence represented by SEQ ID NO: 1 or 3 It can be a polynucleotide that can hybridize under high stringency conditions. Hybridization conditions under high stringency conditions can be set with reference to previously reported conditions (Current Protocols in Molecular Biology, John Wiley & Sons, 6.3.1-6.3.6, 1999). For example, hybridization conditions under high stringency conditions include hybridization at 6 × SSC (sodium chloride / sodium citrate) / 45 ° C., then 0.2 × SSC / 0.1% SDS / 50 to 65 ° C. More than once.

  The polynucleotide of the present invention may encode a polypeptide having chitinolytic activity. When the polynucleotide of the present invention is a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 3, or a nucleotide sequence substantially the same as the secretory signal-removed nucleotide sequence, a polypeptide encoded by the polynucleotide The degree of chitinolytic activity of can be quantitatively equivalent to the amino acid sequence represented by SEQ ID NO: 2 or 4, or a polypeptide consisting of the same amino acid sequence as the secretory signal-removed amino acid sequence, but within an acceptable range (For example, about 0.1 to about 5 times, preferably about 0.5 to about 2 times).

  The polynucleotide of the present invention may also encode a polypeptide having its characteristic domains, family 18 chitinase domain, chitin adsorption domain, fibronectin type 3 domain. Therefore, when the polynucleotide of the present invention is a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 3, or a nucleotide sequence substantially identical to the secretory signal elimination nucleotide sequence, these domains and / or It is preferred to encode a polypeptide that retains function.

  The partial nucleotide of the present invention can be a nucleotide encoding the partial peptide of the present invention. Specifically, the partial nucleotide of the present invention may be a partial nucleotide of a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 3, or the same or substantially the same nucleotide sequence as the secretory signal elimination nucleotide sequence. . Examples of the partial nucleotide of the present invention include a nucleotide encoding a domain characteristic of the chitinase of the present invention.

  The polynucleotide of the present invention can be prepared by a method known per se. For example, a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 3 or a secretion signal-removed nucleotide sequence thereof is prepared by extracting total RNA from the bacterium of the present invention or a mutant thereof, and preparing cDNA from mRNA. Cloning can be performed by PCR using appropriate primers. In addition, a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 3 or a nucleotide sequence substantially identical to the secretory signal-removed nucleotide sequence is prepared by introducing mutations into the cloned polynucleotide as described above. it can. Examples of the mutagenesis method include a synthetic oligonucleotide-directed mutagenesis method (gapped duplex) method, a method of randomly introducing point mutations (eg, treatment with nitrous acid or sulfite), a cassette mutagenesis method, and a linker scanning method. And a mismatch primer method.

  The polypeptide of the present invention can also be produced by a method known per se. For example, the polynucleotide of the present invention produced as described above is incorporated into an expression vector, the obtained recombinant vector is introduced into an appropriate host cell to obtain a transformant, the transformant is cultured, The polypeptide of the invention may be produced and then recovered. The present invention also provides such a recombinant vector and a transformant introduced with the vector.

  The expression vector is not particularly limited as long as it can express a gene encoding the polypeptide of the present invention in any host cell and produce these polypeptides. For example, a plasmid vector, a virus vector, etc. can be mentioned.

  When a bacterium, particularly E. coli, is used as a host cell, generally an expression vector can be composed of at least a promoter-operator region, a start codon, DNA encoding the polypeptide of the present invention, a stop codon, a terminator region, and a replicable unit.

  The promoter-operator region for expressing the polypeptide of the present invention in bacteria contains a promoter, an operator and a Shine-Dalgarno (SD) sequence (for example, AAGG). For example, when the host is Escherichia coli, preferred examples include those containing Trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter, tac promoter and the like. As the terminator region and the replicable unit, those known per se can be used.

  The transformant of the present invention can be prepared by introducing the above expression vector into a host cell.

  The host cell used for the preparation of the transformant is not particularly limited as long as it is compatible with the above expression vector and can be transformed. Natural cells or artificially used usually in the technical field of the present invention can be used. Various cells such as established recombinant cells (eg, bacteria such as E. coli, Bacillus, actinomycetes, eukaryotic cells such as yeast) are exemplified.

  The polypeptide of the present invention can be produced by culturing a transformant containing the expression vector prepared as described above in a nutrient medium.

  The nutrient medium preferably contains a carbon source, an inorganic nitrogen source or an organic nitrogen source necessary for the growth of the transformant. Examples of the carbon source include glucose, dextran, soluble starch, and sucrose. Examples of the inorganic or organic nitrogen source include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, large extract, and the like. Examples include soybean cake, potato extract and the like. If desired, other nutrients (for example, inorganic salts (for example, calcium chloride, sodium dihydrogen phosphate, magnesium chloride), vitamins) may be contained.

  The transformant is cultured by a method known per se. Culture conditions such as temperature, medium pH and culture time are appropriately selected so that the polypeptide of the present invention is produced in large quantities. For example, when the host is a bacterium, a liquid medium containing the above nutrient source is appropriate. Preferably, the medium has a pH of 5-8. When the host is Escherichia coli, preferred media include LB media and M9 media. In such a case, the culture can be performed, for example, at about 30 to 40 ° C. for about 5 to 30 hours with aeration and stirring. When the host is a genus Bacillus, for example, it can be performed at about 30 to 40 ° C. for about 15 to 100 hours with aeration and stirring.

  The polypeptide of the present invention can be obtained by culturing a transformant as described above and recovering, preferably isolating and purifying from the transformant.

  As an isolation and purification method, for example, a method using solubility such as salting out, solvent precipitation, dialysis, ultrafiltration, gel filtration, a method using molecular weight difference such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Methods using charges such as ion exchange chromatography and hydroxylapatite chromatography, methods using specific affinity such as affinity chromatography, methods using hydrophobic differences such as reversed-phase high-performance liquid chromatography, isoelectricity Examples thereof include a method using a difference in isoelectric point such as point electrophoresis.

In addition, a polypeptide to which a tag (for example, histidine tag, Flag tag) or the like is added is produced in the transformant, and a substance having an affinity for the tag (for example, Ni ²⁺ resin, an antibody specific for the tag) is used. Thus, the polypeptide of the present invention can be isolated and purified more easily.

  Furthermore, the polypeptide of the present invention can be synthesized in a cell-free system. In the synthesis of the polypeptide of the present invention in a cell-free system, for example, an extract from Escherichia coli, rabbit reticulocyte, wheat germ, or the like can be used. In addition, the polypeptide of the present invention can be produced by a known organic chemical method such as a solid phase synthesis method or a liquid phase synthesis method.

  Examples of the antigen used for producing the antibody of the present invention include the amino acid sequence represented by SEQ ID NO: 2 or 4, or a polypeptide comprising the same or substantially the same amino acid sequence as the secretory signal-removed amino acid sequence, Partial peptides can be used. The partial peptide is not particularly limited as long as it has immunogenicity. For example, the amino acid sequence represented by SEQ ID NO: 2 or 4, or the same or substantially the same amino acid sequence as the secretory signal-removed amino acid sequence, or the above-mentioned A peptide consisting of at least about 8, preferably at least 10, more preferably at least 12, even more preferably at least 15, most preferably 20 or more consecutive amino acids selected from the amino acid sequence encoding the domain of possible.

The antibody of the present invention may be either a polyclonal antibody or a monoclonal antibody, and can be prepared by a well-known immunological technique. This antibody may be not only a complete antibody molecule but also any fragment as long as it has an antigen binding site (CDR) for the polypeptide of the present invention, such as Fab, F (ab ′) ₂ , ScFv, minibody, etc. Is mentioned.

For example, polyclonal antibodies, the antigen (if necessary, bovine serum albumin, KLH (may be a K eyhole L impet H emocyanin) complexes crosslinked to a carrier protein, etc.), commercial adjuvants (e.g., (Complete or incomplete Freund's adjuvant) and administered subcutaneously or intraperitoneally 2 to 4 times every 2 to 3 weeks (the antibody titer of the partially collected serum is measured by a known antigen-antibody reaction) It can be obtained by collecting the whole blood about 3 to 10 days after the final immunization and purifying the antiserum. Examples of animals to which the antigen is administered include mammals such as rats, mice, rabbits, goats, guinea pigs, and hamsters.

  A monoclonal antibody can be prepared by a cell fusion method. For example, the above antigen is administered to mice subcutaneously or intraperitoneally 2-4 times together with a commercially available adjuvant, and spleen or lymph nodes are collected 3 days after the final administration, and white blood cells are collected. The leukocytes and myeloma cells (for example, NS-1, P3X63Ag8, etc.) are fused to obtain a hybridoma that produces a monoclonal antibody against the factor. Cell fusion may be PEG or voltage pulse. A hybridoma producing a desired monoclonal antibody can be selected by detecting an antibody that specifically binds to an antigen from the culture supernatant using a well-known EIA or RIA method. The hybridoma producing the monoclonal antibody can be cultured in vitro, or in vivo, such as mouse or rat, preferably mouse ascites, and the antibody can be obtained from the culture supernatant of the hybridoma and the ascites of the animal, respectively.

  The contents of all publications, including patents and patent application specifications cited in this specification, are hereby incorporated by reference herein to the same extent as if all were explicitly stated. Is.

  The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples.

Example 1: Isolation of a novel Paenibacillus genus In primary screening, soil is sampled from a field in which crab shells are periodically sown, and enriched in a medium containing colloidal chitosan as the sole carbon source. Degraded bacteria were concentrated.
Next, in the secondary screening, colonies are formed by smearing the culture solution concentrated in the primary screening on the nutrient agar medium that has become cloudy due to the inclusion of colloidal chitosan, and transparent that appears around the colonies due to the decomposition of colloidal chitosan The chitosan-degrading bacteria were purely cultured using the halo as an index. The chitosan-degrading bacterium thus obtained also formed a halo on the colloidal chitin plate.

Example 2: Analysis of novel Paenibacillus bacteria
2.1. Analysis of phenotypes The bacterial phenotypes obtained in Example 1 were analyzed. The results are shown below.
・ Cell morphology:
Neisseria gonorrhoeae: (size: 0.5-0.6 × 1.2-1.5 μm)
Motility: + (periflagellate)
Gram staining: +
Endogenous spores: +
Spore shape: ellipse Spore position: terminal Spore bulge: bulge / physiological properties:
Catalase: +
Starch hydrolysis: +
Acid production from glucose: +
・ Attitude toward oxygen: facultative anaerobic growth temperature: good growth at 30-42 ℃ ・ optimal pH: 7.2 (+: positive)

2.2. Molecular phylogenetic analysis based on the base sequence of 16S rDNA The base sequence of 16S rDNA of 1000 bp or more of this strain was determined, the DNA database (DDBJ) was accessed, and the base sequence of 16S rDNA was determined using the FASTA program. As a result, the 16S rDNA of this strain showed 90% or more homology with known Paenibacillus genus bacteria, but it was 95% or less with any known species of this genus. In addition, as a result of molecular phylogenetic analysis by NJ method after multiple alignment of base sequences of Paenibacillus genus and aerobic spores obtained from DNA database, the position of the molecular phylogenetic tree of this strain is composed of Paenibacillus genus bacteria Although in the cluster, it was located in a phylogenetic branch different from any known species.

2.3. Classification and identification results (Bergey's Manual of Systematic Bacteriology, Vol. 2, Williams & Wilkins, Baltimore (1984) and Bergey's Manual of Determinative Bacteriology (9th ed.) JT Staley, ST Williams (ed), Williams & Wilkins, Baltimore (1994)) and phylogenetic analysis revealed that this strain was a new species of Paenibacillus.
The inventors have named this new species of bacteria as Paenibacillus fukuinensis.

2.4. Polysaccharide resolution and phytopathogenic growth inhibition The polysaccharide resolution of the bacterium of the present invention was measured. As a result, the bacterium of the present invention had a resolution of chitin (N-acetylglucosamine), chitosan (glucosamine), and cellulose (glucose). In addition, in the bacterium of the present invention, a protein having such a polysaccharide resolution was secreted outside the microbial cells.

2.5. Inhibition of the growth of plant pathogens Next, an experiment for inhibiting the growth of plant pathogens by the bacteria of the present invention was conducted. After infecting strawberries with a phytopathogenic fungus (fusarium), the bacterial culture solution or medium (control) of the present invention was sprayed onto the strawberries. As a result, the strawberry sprayed with the bacterial culture solution of the present invention showed little growth of fusarium even after being allowed to stand at room temperature for several days, but the strawberry of control strawberry was propagated and fruit juice leaked out. It was. It is known that some fungi have chitin, chitosan, and cellulose as cell wall components. Since the bacterium of the present invention can decompose such cell wall components, it is considered that the growth of phytopathogenic fungi (fusarium) was inhibited.

Example 3: Isolation of a novel chitinase Paenibacillus fuchinensis was cultured in a nutrient liquid medium containing colloidal chitin. After completion of the culture, the cells and the remaining colloidal chitosan were removed by centrifugation, and the enzyme in the culture supernatant was precipitated and concentrated by adding ammonium sulfate. The obtained crude enzyme was separated by column chromatography, and a fraction having chitinase activity was collected. The results are shown in Table 1 below.

Example 4: Analysis of novel chitinase The purified chitinase fraction was confirmed by SDS-PAGE. As a result, two major bands in the vicinity of 70 kDa dalton and one in 40 kDa were detected (Fig. 1 left). ). Furthermore, when the chitinase activity of each band was examined by an activity staining method (zymogram), clear chitinase activity was detected in two bands present in the vicinity of 70 kDa dalton (FIG. 1-right).
The amino acid sequence of the N-terminal region of the three bands was analyzed using a protein sequencer. As a result, the two bands present in the vicinity of 70 kDa dalton were both amino acid sequences similar to chitinase, and the 40 kDa band was predicted to be a putative chitin adsorption domain (FIG. 1-right).
A synthetic primer for PCR was designed from the amino acid sequence of the obtained N-terminal region, and a chitinase gene corresponding to two bands present in the vicinity of 70 kDa dalton was cloned from Fuchinensis. These genes were subcloned into pQE30 (Qiagen), a vector for expressing histag fusion proteins, and transformed into E. coli. Two kinds of chitinases (Chitinase A (ChiA) and Chitinase B (ChiB)) derived from Fucuinensis expressed in Escherichia coli were purified by a nickel chelate column according to a conventional method, and their enzymatic properties were examined as follows.
(1) Determination of optimum temperature The basic assay conditions are as follows.
Enzyme amount: About 0.2 units was used as a purified enzyme. In addition, the amount of enzyme (units number) was calculated | required from the production amount per minute according to the following measuring methods except having performed reaction time for 1 minute and 11 minutes.
Enzyme reaction solution: 1 ml of 0.5% glycol chitin, 2 ml of citrate buffer, and 1 ml of enzyme solution (0.2 Units) were mixed to prepare a total solution of 4 ml.
Measurement method: 1 ml of 0.5% glycol chitin, 2 ml of 0.2M-NaHPO4-0.1M citrate buffer (pH 4.0) and 4 ml of the reaction solution containing the enzyme solution were reacted at 37 ° C. for 20 minutes while shaking. The reaction was stopped by boiling for 3 minutes and then centrifuged. 1.5 ml of the supernatant was taken into a test tube, 2 ml of the above potassium ferricyanide solution was added, and the mixture was boiled for 15 minutes. After cooling, the absorbance at 420 nm was measured, and the produced reducing sugar was quantified using N-acetylglucosamine as a standard. The enzyme unit was defined as 1 unit of the amount of enzyme that produces reduction corresponding to 1 μmol of N-acetylglucosamine per minute.
In this assay, in order to determine the optimum temperature, the assay was performed at a predetermined reaction temperature of 20 to 80 ° C. instead of 37 ° C. described in the basic assay conditions.
The results are shown in FIG. The optimum temperature of ChiA was 30 ° C, and showed a relative activity of 50% or more in the range of 20-80 ° C. On the other hand, the optimum temperature of ChiB was 60 ° C, and showed a relative activity of 50% or more in the range of 30-60 ° C. When the reaction temperature exceeded 60 ° C, the relative activity decreased rapidly to about 40%. From these results, it was clarified that ChiA is highly active at low temperatures (around 30 ° C), whereas ChiB is an enzyme exhibiting high activity at high temperatures (around 50-60 ° C).
(2) Determination of optimum pH The assay was performed according to the basic assay conditions described in (1) above.
In this assay, in order to determine the optimum pH, the enzyme was allowed to stand at a predetermined pH for 1 hour, and then the above assay was performed.
The results are shown in FIG. ChiA showed a relative activity of 80% or more over a wide range of pH3-9. Such a chitinase exhibiting high activity in a wide pH range is considered to be extremely rare. On the other hand, the optimum pH of ChiB was around 7, and showed a relative activity of 90% or more in the range of pH 6-8. From these results, it was clarified that ChiA is an enzyme with very high pH stability, and ChiB is an enzyme that shows activity near neutrality. Since ChiA can maintain activity in a very wide pH range unlike conventional chitinases, it can be used in combination with various enzymes that show activity only in a specific pH range. Very useful.
(3) Thermal stability The assay was performed according to the basic assay conditions described in (1) above.
In this assay, in order to determine thermal stability, the enzyme was allowed to stand at a predetermined temperature of 20 to 80 ° C. for 1 hour, and then the above assay was performed.
The results are shown in FIG. ChiA was stable at a low temperature of 20-30 ° C, and its relative activity decreased in the range of 40-80 ° C. On the other hand, ChiB was stable with a relative activity of 90% or more after heat treatment up to 20-50 ° C, but rapidly deactivated at 60 ° C or more. From these results, ChiA is a relatively heat-stable enzyme that is stable at low temperatures but does not rapidly inactivate at high temperatures, and ChiB is a low-heat-stable enzyme that inactivates rapidly above 60 ° C. It became clear that there was.

  The bacterium of the present invention or a mutant strain thereof makes it possible to produce oligosaccharides such as chitin oligosaccharides from, for example, polysaccharides or polysaccharide-containing materials (eg, snow crab shells such as Echizen crabs). The bacterium of the present invention or a mutant strain thereof can also be used as a microbial pesticide, an antibacterial agent, and a polysaccharide degradation agent. The chitinase of the present invention makes it possible to produce chitin oligosaccharides from, for example, polysaccharides or polysaccharide-containing materials.

It is a figure which shows the SDS-PAGE and activity staining of the chitinase fraction refine | purified from the culture supernatant of Fukuinensis. It is a figure which shows the domain of chitinase A (ChiA) and chitinase B (ChiB). It is a figure which shows the relationship between the activity and temperature of chitinase A (ChiA) and chitinase B (ChiB). It is a figure which shows the relationship between the activity of chitinase A (ChiA) and chitinase B (ChiB), and pH. It is a figure which shows the relationship between the activity of chitinase A (ChiA) and chitinase B (ChiB), and thermal stability.

Claims (13)

Paenibacillus genus bacteria represented by accession number FERM P-20620 or mutants thereof. A method for producing an oligosaccharide, comprising bringing a Paenibacillus genus bacterium represented by accession number FERM P-20620 or a mutant thereof into contact with a polysaccharide or a polysaccharide-containing product. The method according to claim 2, wherein the polysaccharide or the polysaccharide-containing material is one or more polysaccharides selected from the group consisting of chitin, chitosan, and cellulose, or a content thereof. The method according to claim 2 or 3, wherein the polysaccharide-containing material is crab shell. The method of claim 4, wherein the crab shell is a snow crab shell. A composition comprising a Paenibacillus genus bacterium represented by accession number FERM P-20620 or a mutant strain thereof. The composition according to claim 6, which is an antibacterial agent or a polysaccharide decomposing agent against pesticides, plant pathogens. The following (a) or (b) polypeptide or a partial peptide thereof:
(A) an amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence having 70% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2 from which the secretory signal sequence has been removed, and chitin A polypeptide having degrading activity;
(B) an amino acid sequence represented by SEQ ID NO: 4, or an amino acid sequence having 70% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 4 from which the secretory signal sequence has been removed, and chitin A polypeptide having degradation activity. A polynucleotide encoding the polypeptide according to claim 8 or a partial peptide thereof. An expression vector comprising the polynucleotide according to claim 9. A transformant comprising the expression vector according to claim 10. An antibody against the polypeptide according to claim 8 or a partial peptide thereof. A hybridoma producing the antibody according to claim 12.