JP2000093182A - Method for controlling fungus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To control fungi, e.g. pathogenic fungi to plants or the like, by the action of chitinase having antifungal activity against fungi containing pathogenic fungi to plants by means of using chitinase which is composed of the protein containing a specific amino acid sequence and arises from Actinomycetes. SOLUTION: The chitinase containing the amino acid sequence represented by the formula or the like is obtained by separating the chromosomal DNA of Actinomycetes, Streptomyces griseus HUT 6037, treating it with a restriction enzyme, preparing a gene library by the conventional method, screening out this by carrying out southern hybridization with the probe having the partial sequence of chitinase gene to obtain the chitinase gene, introducing into Escherichia coil or the like after combining this with a vector to transform and then cultivating the resultant transformant. Next, by using the chitinase which is composed of the protein containing the sequence of amino acid numbers 1-294 on the amino acid sequence represented by the formula and arises from Actinomycetes, fungi are controlled.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for controlling fungi using a chitinase having antibacterial activity against fungi including plant pathogens, which is produced by a DNA containing a base sequence encoding the amino acid sequence of the chitinase. The present invention relates to a fungal control method using an actinomycete-derived chitinase comprising a protein, or an actinomycete producing the chitinase, and a method for producing a chitinase having antifungal activity using a transformed microorganism.

[0002]

2. Description of the Related Art Chitinase classified in EC 3.2.1.14 (see IUB Enzyme Committee Enzyme Classification Table, Enzyme Handbook (Asakura Shoten)) is known to exist in many kinds of microorganisms, plants and animals. ing.

[0003] Plant sources include cucumber, eggplant, corn, tomato, legume, wheat germ, soybean seed, and the like.
Examples of the microorganism source include, for example, bacteria such as Serratia marcescens (Serratia marcescens) and Bacillus circulance, and yeast as Saccharomyces cerevisia (Saccharomyces cerevisia).
e), Streptomyces lividans (Strep
tomyces lividans) and Streptomyces pricatas
(Streptomyces plicatus) and the like are known (Takeshi Watanabe, Molecular Biology of Microbial Chitinase, Bioscience and Industry 50:12, 16: 1992).

[0004] Chitinase having antibacterial activity is considered to be one of defense mechanisms against plant pathogens, and has been widely studied for chitinase of plant origin. For example, chitinase derived from cowpea (Valdirene M Gomes J. Sci. Food)
Agric. 72, 86-90, 1996), cucumber leaf chitinase (C. Ji. Physiol. Molecular Plant Pathology 49).
Vol. 257, 1996), patents utilizing the antibacterial activity of beetle-derived chitinase (Japanese Patent Application Laid-Open No. 9-94089) and sugar beet-derived chitinase (Japanese Patent Application Laid-Open No. 6-507070).

On the other hand, chitinases of microbial origin having antibacterial activity are hardly known, and are slightly different from Pseudomonas chitoses.
There is a report of a chitinase derived from Pseudomonas stutzeri (Ho-Seong Lim. Jour. Microbiol. 295 (1995)). Recent reports have shown that two strains of the genus Streptomyces having chitinase activity have strong antifungal activity, but these are highly likely to be due to the antifungal substance produced by the actinomycete, It was reported that there was no clear relationship between potato strength and antifungal activity (Fran
dberg E. Schnurer J. Can. J. Microbiol. 44 Vol. 2, No. 1
21-127 1998).

[0006] As described above, these plant-derived chitinases enhance the protective function of plants against plant pathogenic bacteria, and attempts have been made to artificially incorporate them into plants. On the other hand, it is conceivable that these antibacterial chitinases are sprayed on soil or plants to obtain a controlling effect against plant pathogenic bacteria, but it is difficult to industrially produce these plant-derived chitinases, so that they are suitable for industrial production. A microbial antibacterial chitinase has been desired.

The present inventors have compared the domain structures of various chitinases. As a result, the chitinases in the natural world were classified into two groups, namely, a group that could be called family 19 of plant-derived chitinases due to differences in the conserved regions of amino acid sequences. And all others including microorganisms are family 1
It was found to be a chitinase that can be called No. 8 (Takeshi Watanabe, Chemistry and Biology, Vol. 35, No. 6, pp. 408, 1997).

[0008] However, in the process of searching for the base sequences of various chitinases derived from microorganisms, chitinase named chitinase C derived from Streptomyces griseus has the only chitinase having a domain structure common to family 19 except for plants. For the first time
(TSUYOSHI OHNO, TAKESHI WATANABE J. BACTERIOLOGY, 17
8 Vol. 17, No. 5065, 1996). However, it was not clear whether the chitinase truly exhibited antifungal activity against living fungi.

[0009]

An object of the present invention is to provide a fungal control method using an actinomycete-derived chitinase having an antibacterial activity, an actinomycete producing the chitinase, and a transformed microorganism. An object of the present invention is to provide a method for producing a chitinase having antifungal activity to be used.

[0010]

Means for Solving the Problems The present inventors named chitinase C, derived from Streptomyces griseus, against living fungi more unexpectedly than plant-derived chitinase. In addition to revealing a much stronger antifungal activity, many plant-derived chitinases exhibit antibacterial activity, whereas microbial chitinases do not show antibacterial activity due to this difference in domain structure. The present inventors have found that a chitinase having an amino acid sequence similar to that of this chitinase C is widely and ubiquitously present in actinomycetes, and furthermore that the actinomycetes themselves exhibit antifungal activity. Reached.

That is, the present invention relates to (a) the amino acid sequence of SEQ ID NO: 1
A fungal control method using an actinomycete-derived chitinase comprising a protein having a sequence represented by 94;

(B) In the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 in the sequence listing,
A fungal control method using an actinomycete-derived chitinase comprising a protein produced by a DNA having a nucleotide sequence encoding an amino acid sequence represented by:

(C) a fungal control method using an actinomycete that produces a chitinase comprising a protein having a sequence represented by amino acid numbers 1-294 in the amino acid sequence described in SEQ ID NO: 1 in the sequence listing;

(D) In the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing,
A fungal control method using an actinomycete producing a chitinase consisting of a protein produced by a DNA having a nucleotide sequence encoding an amino acid sequence represented by:

(E) The actinomycete is Streptomyces griseus (A) to (D)
A fungal control method according to any one of:

(F) Streptomyces griseus HU
The fungal control method according to any one of (a) to (d), which is T6037;

(G) base numbers 121 to 121 in the base sequence encoding the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing
DNA having a base sequence encoding the amino acid sequence represented by 1002 was introduced into a cell and transformed.
A fungal control method using a microorganism that produces chitinase,

(H) Base numbers 121 to 121 in the base sequence encoding the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing
And a method for producing a chitinase having antifungal activity using a microorganism transformed by introducing a DNA having a base sequence encoding the amino acid sequence represented by 1002 into cells.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION The chitinase used in the present invention is a protein having the amino acid sequence shown in SEQ ID NO: 1. The chitinase has a molecular weight of about 3 consisting of 294 amino acids.
It is a protein of 1400 and has strong antibacterial activity against incomplete fungi and fungi including plant pathogenic bacteria.

The DNA of the chitinase used in the present invention may be any DNA having a nucleotide sequence encoding a chitinase having the sequence represented by amino acids 1 to 294 in the amino acid sequence shown in SEQ ID NO: 1. It may be. Specific examples of such DNA include base numbers 121 to 1002 in the base sequence shown in SEQ ID NO: 2.
And a DNA having the sequence represented by

The chitinase DNA used in the present invention includes those having substitution, deletion, insertion or transposition of amino acid residues as long as the antibacterial activity of the chitinase encoded thereby is not substantially impaired. Things. The vector used for preparing the recombinant DNA for producing chitinase may be appropriately selected depending on the host microorganism into which the chitinase gene is to be introduced.

As a vector, a phage or a plasmid capable of growing in a host microorganism is suitable. Any known phage can be used as appropriate. For example, when Escherichia coli (Escherichia coli) is used as a host microorganism, for example, pBR322 and pUC119 can be used. Further, for example, Escherichia coli, Bacillus subtilis (B
acillus subtilis), as a shuttle vector capable of autonomous replication in two or more host microorganisms such as pHY300PLK, p
In addition to vectors such as HV14 and pUR32, various known shuttle vectors can be used. Preferably pU
C119 is mentioned.

The vector used in the present invention may be a vector that can be integrated into chromosomal DNA of a host cell. That is,
These DNAs may be maintained extrachromosomally as plasmids when introduced into host cells. Among these vectors, usually, vectors capable of autonomous replication in a host cell are preferable.

The DNA fragment containing the chitinase gene used for preparing the recombinant DNA may be directly ligated to a vector when the DNA contains a promoter capable of expressing the chitinase gene in a host cell. When a promoter that can be expressed in a cell is not contained, an appropriate promoter sequence may be ligated upstream of the chitinase coding region.

Alternatively, a chitinase gene may be inserted into an appropriate site of a heterologous protein expression vector including a promoter. As such a promoter, for example, when Escherichia coli is used as a host microorganism,
For example, when the trp promoter, lac promoter, and the like use Bacillus subtilis as a host, a promoter derived from the α-amylase gene of Bacillus amyloliquefaciens and the like can be mentioned.

Next, DNA and recombinant DNA containing it
The outline of the method for preparing is described. First, chromosomal DNA is collected and purified from a donor microorganism capable of producing chitinase derived from the actinomycetes of the present invention, and the chromosomal DNA is cut with an appropriate restriction enzyme or the like. As the donor microorganism, a chitinase-producing actinomycete including a transformant can be widely used. Such actinomycetes preferably include microorganisms belonging to the genus Streptomyces, of which Streptomyces griseus (Streptomyces) is an example.
griseus) HUT6037 strain.

The D digested with the restriction enzyme as described above
The NA fragment is separated by agarose gel electrophoresis and transferred to a nylon membrane or the like. On the other hand, a DNA probe is synthesized based on the nucleotide sequence estimated from the N-terminal amino acid sequence of chitinase collected from the donor microorganism.
Using this membrane and the RI-labeled DNA probe, Southern hybridization was performed. With reference to the obtained molecular weight of the positive band, cleavage of the chromosomal DNA restriction enzyme having a molecular weight corresponding to the positive band expected to contain the chitinase gene separately was performed. Obtain the fragment.

The DNA fragment and the vector are chromosomal DNA
And a vector fragment obtained by cleavage in the same manner as in
Allow the library to form. Next, a host microorganism such as Escherichia coli is transformed (transformed) with the library, and a clone containing the chitinase gene is identified and isolated by colony hybridization using the aforementioned labeled probe.

When the DNA does not contain a full-length chitinase gene, the above procedure is repeated when cutting the chromosomal DNA by controlling the degree of cleavage, using a different restriction enzyme, etc. DNA containing the full length chitinase gene is obtained.

The nucleotide sequence of the isolated DNA is determined by using an automatic sequencer or the like according to a standard method.
The chromosomal DNA of the donor microorganism used in the present invention is obtained by culturing the donor microorganism with aeration and stirring for 1 to 3 days in a liquid medium, for example,
The obtained culture can be prepared by centrifuging, collecting cells, and then lysing the cells.

As a lysis method, for example, treatment with a cell wall lysing enzyme such as lysozyme or β-glucanase, or ultrasonic treatment is used. If necessary, other enzyme agents such as protease and ribonuclease, and a surfactant such as sodium lauryl sulfate (SDS) are used in combination. In some cases, freeze-thaw treatment is used. In order to separate and purify DNA from the lysate thus obtained, phenol extraction, chloroform extraction, ethanol precipitation, ribonuclease treatment, protease treatment and the like are appropriately combined according to a standard method.

The DNA can be cut by, for example, ultrasonic treatment or restriction enzyme treatment.
After cleavage, a modifying enzyme such as phosphatase or DNA polymerase is used as necessary. A fragment of an appropriate length can be obtained from the cut DNA fragment by sucrose density gradient centrifugation or extraction from an electrophoresed gel.

A phage or a plasmid capable of growing in a host microorganism is suitable as a vector used for preparing a chromosomal DNA library. As the phage, known phages can be appropriately used. For example, when Escherichia coli is used as a host microorganism, λ phage and M13 phage can be used. As a plasmid, for example, when Escherichia coli is used as a host microorganism,
BR-322, pUC118, pUC119 and the like can be used.

Further, as a shuttle vector capable of autonomous propagation in two or more host microorganisms such as Escherichia coli and Bacillus subtilis, for example, pHY300
In addition to vectors such as PLK, pHV14 and pUR32,
Various known shuttle vectors can also be used.

The method for binding the DNA fragment and the vector fragment may be any method using a known ligase, for example, DNA purified by phenol extraction and ethanol precipitation.
The fragment and the vector fragment are mixed at an appropriate ratio, and an appropriate D
A recombinant DNA is prepared by the action of NA ligase. The host microorganism into which the chromosomal DNA library is introduced may be any host microorganism as long as the recombinant vector is stable and can autonomously replicate. For example, Escherichia coli and Bacillus sp.

A method for introducing a recombinant vector into a host microorganism is a known method, for example, when the host microorganism is Escherichia coli.
In the case of coli, the calcium method (Ledenberg, EM, Cohe
n, SM, J. Bacteriol., Vol. 119, p. 1072, 1974) and the electroporation method (Hirato Okayama, edited by Masami Matsumura, Genetic Handbook for Experimental Medicine, Yodosha 1991), and the like can be used. DNA for λ phage vector
In vitro into phage particles to infect bacteria as phage (B. Horn: Methods in Enzymolog
y, Vol. 68, p. 299, 1979).

Selection of the transformed microorganism into which the recombinant vector has been introduced can be carried out by a method using an antibiotic or by using β-1,4-N-
A method in which a transformant is inoculated on an agar plate mixed with an acetylamino sugar compound, for example, colloidal chitin or glycol chitin, and a dissolution zone due to degradation of the β-1,4-N-acetylamino sugar compound is detected by dye staining. It can be performed by such as.

As described above, Streptomyces
FIG. 1 shows a restriction enzyme fragment map of a DNA fragment containing a chitinase gene obtained from Griseus (Streptomyces griseus) HUT6037 strain. The nucleotide sequence of the coding region of the chitinase gene and its adjacent region in the DNA fragment and the amino acid sequence predicted from the nucleotide sequence are shown in SEQ ID NOs: 1 and 2.

The DNA of chitinase used in the present invention has been obtained as described above. The DNA can be synthesized based on the sequence described in the above-mentioned sequence listing. It can also be easily obtained by amplifying the chitinase gene from the chromosomal DNA of the donor by PCR (polymerase chain reaction) using oligonucleotide primers synthesized based on the nucleotide sequence.

The chitinase gene-containing vector obtained as described above can be used as it is as recombinant DNA when the promoter specific to the chitinase gene functions in a host cell into which the chitinase gene is to be introduced. it can. If the chitinase gene does not contain a promoter, or if the promoter specific to the chitinase gene does not function efficiently in the host cell, an appropriate promoter sequence may be ligated upstream of the chitinase coding region or the expression including the promoter may be used. When the chitinase gene is inserted into an appropriate site of the vector for use, a recombinant DNA can be obtained.

The present invention relates to an actinomycete producing a chitinase comprising a protein having a sequence represented by amino acid numbers 1 to 294 in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, and to an amino acid sequence described in SEQ ID NO: 2 in the sequence listing. Having a base sequence encoding the amino acid sequence represented by base numbers 121 to 1002 in the base sequence encoding the amino acid sequence of
A fungal control method using an actinomycete that produces a chitinase consisting of a protein produced by NA, or a microorganism transformed with a gene containing these DNAs introduced into cells.

These transformed microorganisms can be obtained by transforming with the above-mentioned recombinant DNA. The microorganism used as a host is not particularly limited as long as it can stably express the chitinase gene, but any microorganism that is usually used for producing a heterologous protein can be used. Specifically, Escherichia bacteria such as Escherichia coli, Bacillus bacteria such as Bacillus subtilis, actinomycetes of Streptomyces, Saccharomyces cerevisiae (Saccharomyces cerevisiae)
And the like.

When the chitinase gene is introduced into the above microorganism, introduction of a recombinant DNA using another copy number of a plasmid vector is effective in gene amplification (gene dos
ageeffect), but may be incorporated into the host chromosomal DNA. The chitinase having the antifungal activity of the present invention can be used in a microorganism which does not produce chitinase.
By introducing A, the ability to produce chitinase having antifungal activity can be imparted.

By introducing the above-mentioned DNA into a microorganism which produces chitinase having antifungal activity, the amount of chitinase production can be increased. The method of introducing a recombinant DNA into a host microorganism and the selection of a transformed microorganism into which a recombinant vector has been introduced can be performed in the same manner as described above.

The present invention also relates to a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.
The present invention also includes a method for producing a chitinase having an antifungal activity, using a chitinase-producing microorganism, which has been transformed by introducing a DNA having a nucleotide sequence encoding the amino acid sequence represented by 21 to 1002 into a cell.

That is, a microorganism which produces a chitinase having an antifungal activity transformed by the above-described method is cultured in a suitable medium, and the chitinase is collected from the culture to produce a chitinase having an antifungal activity. You can do it. Any medium can be used as long as it is usually used for culturing a normal host microorganism, and a natural medium or a synthetic medium can be used.

As the carbon source, for example, chitin, glycol chitin, β-1,4-N-acetylamino sugar compounds such as cell walls of incomplete bacteria, glucose, glycerin, ethanol and the like can be used. For production, the presence of a β-1,4-N-acetylamino sugar compound is more preferred.

As a nitrogen source, ammonium sulfate, ammonium chloride, urea, ammonia, yeast extract, polypeptone, meat extract and the like are used. As the inorganic salt, potassium phosphate, sodium phosphate, ammonium phosphate, magnesium sulfate and the like can be used. Also, vitamins, amino acids, etc. may be added as trace nutrients, or iron, cobalt, manganese, aene, etc. may be added as salts in the form of salts.

The cultivation is usually preferably performed under aerobic conditions such as aeration and agitation, shaking, etc., but can also be performed under static conditions. Typical medium compositions include, for example, 0.02% potassium chloride, 0.1% magnesium sulfate heptahydrate, 0.05%
A medium comprising iron sulfate and 0.2% colloidal chitin is inoculated with a chitinase-producing bacterium having antifungal activity, for example, Streptomyces sp., More preferably Streptomyces griseus HUT6037, and the culture temperature is 10 ℃ to 50 ℃, preferably 20 ℃ ~
The reaction is carried out at 40 ° C. and the culture pH is 3 to 9, preferably around 6 to 8.

Under the above conditions, aeration and stirring culture are usually performed for 1 to 3 days. After culture, if chitinase is present in the cells, the cells are first collected by centrifugation or the like, and then crushed to remove the enzyme from the cells. As the crushing method, the cells suspended in an appropriate buffer or the like are subjected to ultrasonic crushing, a dynomill method,
A physical method such as a freeze-thaw method, or a chemical method such as a solvent method or an enzymatic method can be used. Of course, two or more of these methods may be combined.

The extraction of chitinase can be carried out at room temperature in an aqueous suspension. However, in order to minimize the inactivation of the enzyme, the temperature should be 10 ° C. or lower, preferably 2 ° C. to 10 ° C. It is preferred to carry out in the presence of On the other hand, when the enzyme exists outside the cells (in the medium), the cells can be removed from the culture solution by centrifugation or the like and used as a crude enzyme solution or crude enzyme powder.

Further, if necessary, chitinase can be prepared from the obtained crude enzyme solution by a suitable method, for example, salting out such as ammonium sulfate fractionation, dialysis, ion chromatography, gel filtration chromatography or the like, alone or in combination thereof. Can also be purified.

Further, similarly transformed microorganisms producing chitinase having antifungal activity, preferably Escherichia coli and Bacillus subtilis, are cultured in a suitable medium, and the culture is carried out. By collecting chitinase from the product, a chitinase having antifungal activity can be produced.

The conditions for culturing the microorganism and the method for obtaining chitinase can be carried out in the same manner as the above-described culturing method and enzyme. Escherichia coli does not release chitinase extracellularly, so it is necessary to disrupt cells in order to express antifungal activity.Bacillus subtilis releases chitinase out of the cells, so the transformed microorganism Bacillus subtilis is more preferably used in the method of controlling fungi using itself.

Many of the conventionally used antifungal agents are more toxic to the human body than other antibacterial agents, and their use in foods and agricultural products has been limited. Chitinase having an antifungal activity derived from actinomycetes having higher industrial productivity than the plant obtained by the present invention, or a fungal control method using an actinomycete producing the chitinase is a safer fungal control method for food, agricultural products, and the like. Useful.

[0056]

EXAMPLES The present invention will be described below with reference to specific examples, but the present invention is not limited to these examples.

(Reference Example 1) (Streptomyces griseus HUT
Preparation of Chitinase of 6037) Culture method: Culture for production of chitinase of Streptomyces griseus HUT6037 was carried out in a Sakaguchi flask using a medium having the following composition and shaking at 30 ° C for 72 hours.
(Medium composition / liter) KCl 0.2g, K ₂ HPO
₄ 1 g, MgSO ₄ 7H ₂ O 0.5 g, FeSO ₄ 0.01
g, colloidal chitin 2 g, pH 7.0

Preparation of Crude Enzyme: The culture supernatant was recovered from the culture broth cultured under the above conditions by centrifugation (8,000 × g, 15 minutes, 4 ° C.), and finely divided into 60% saturated concentration. Ammonium sulfate in powder form was added with good dissolution, and the mixture was left overnight at 4 ° C. Centrifuge the resulting precipitate (8,00
(0 × g, 15 minutes, 4 ° C.), and the resulting pellet was dissolved in a small amount of 0.1 M sodium phosphate buffer (pH 6.0) and dialyzed against 1 mM sodium phosphate buffer (pH 6.0). This was freeze-dried to obtain a crude enzyme preparation.

Measurement of chitinase activity: The activity was measured by a modified Schales method using glycol chitin as a substrate. The specific experimental operation is as follows. Reagent substrate solution: 0.2% in 0.1 M phosphate buffer (pH 6.0)
The thing which melted glycol chitin to become. Schales reagent: 0.5 g of potassium ferricyanide 0.5 M
Dissolved in sodium carbonate.

Test method: After preincubating 1 ml of the substrate solution at 37 ° C. for 2 minutes, 0.5 ml of the enzyme solution was added and mixed to start the enzyme reaction, and 10 minutes later, 2.0 ml of Schales reagent was used.
1 was added to stop the reaction. This reaction solution was heated at 100 ° C. for 15 minutes, cooled in ice water for 3 minutes, and the absorbance at 420 nm was measured. Similarly, a blank was prepared by sequentially adding Schales reagent and substrate solution to the enzyme solution, incubating at 100 ° C. for 15 minutes, cooling in ice water for 3 minutes, and measuring the absorbance at 420 nm.

The value obtained by subtracting the absorbance of the sample from the absorbance of the blank was calculated, the amount of reducing sugar produced was determined using N-acetylglucosamine as a standard, and an enzyme producing reducing sugar corresponding to 1 μmol of N-acetylglucosamine per minute. 1 quantity
The unit was used.

Reference Example 2 (Streptomyces griseus HUT
Cloning of 6037 antibacterial chitinase gene) (Design and synthesis of hybridization probe) Stre
When ptomyces griseus HUT 6037 is cultured in a medium containing colloidal chitin as the sole carbon source, two major chitinases with slightly different isoelectric points, chitinase C1 and C2
Was detected. These two chitinases are very similar in molecular weight, pH stability, optimum pH, thermostability, substrate specificity, etc., and some of the chitinases are proteolytically lost. Therefore, in order to design a hybridization probe for cloning chitinase, proteins contained in the crude enzyme prepared from the culture supernatant of S. griseus HUT6037 were separated by SDS-polyacrylamide gel electrophoresis, and chitinase C1 and C2 were separated. The N-terminal amino acid sequence was first analyzed.

After washing with methanol, a PVDF membrane immersed in 25 mM Tris buffer containing 5% methanol for 30 minutes or more, and a polyacrylamide gel immersed in the same buffer after electrophoresis were set on Horizon Blot AE6675 (manufactured by ATTO). Then, blotting was performed at 191 mA (2.0 mA / cm) for 1 hour. After washing the blotted PVDF membrane with a sodium borate solution for 5 minutes and ultrapure water for 5 minutes,
% Methanol for 20 seconds.

Staining with a staining solution (0.1% Coomassie Brilliant Blue R250, 30% methanol, 10% acetic acid) for 1 minute,
Immediately, the solution was destained with a destaining solution (50% methanol). After decolorization until the protein band is clearly visible, use ultrapure water.
Washed twice for 5 minutes. After washing, the membrane is air-dried and chitinase
Cut out bands corresponding to C1 and C2, and use Applied Biosyste
The N-terminal amino acid sequence was analyzed using a gas-phase protein sequencer 470A of m company.

As a result, 1-GEGPGGNNGFVVSEAQFNQMFPNRNAFYTYKGLDALS-37 was obtained as the N-terminal amino acid sequence of C1. The N-terminal amino acid sequence of C2 was exactly the same as the sequence of C1, except that the eighth asparagine residue was aspartic acid. This is considered to indicate that C2 was obtained by converting asparagine in C1 to aspartic acid by deamination. Of this amino acid sequence
Using Glu-14 to Thr-29, a hybridization probe having the following nucleotide sequence is designed,
Oligonucleotide synthesis was commissioned to Greiner Japan Co., Ltd. Hybridization probe:
5'-GAGGCICAGGTCAACCAGATGTTCCCIA (A / C) CCG (I / A) A (A /
C) CGCITTCTACA-3 '

(Labeling of Oligonucleotide) This synthetic oligonucleotide was radioactively labeled by an end labeling method and used for hybridization. That is, 5 pm
ol of a solution containing a synthetic oligonucleotide
DNA polynucleotide kinase buffer (0.5M Tris-HCl
pH7.6, 1M MgCl, 50mM DTT, 1mM EDTA) 2μl, (γ-32
P] -ATP (10 mCi / μl, Daiichi Chemical Co., Ltd.) 8.4μ
l, 1 μl (10 units) of T4 DNA polynucleotide kinase was added, and the total amount was adjusted to 20 μl using sterile water, and gently mixed. This was incubated at 37 ° C. for 1 hour, and then centrifuged (6,000 rpm, 15 seconds) using a spin column to remove unreacted [γ-32
P] -ATP and impurities, STE buffer (8mM Tris-HCl pH
It was diluted to 50 μl with 8.0, 0.8 mM EDTA, 150 mM NaCl) and used for hybridization.

(Streptomyces griseus HUT6037 chromosome DN
Preparation of A) S. griseus HUT6037 was added to mannitol 10 g / l,
Polypeptone 2 g / l, meat extract 1 g / l, yeast extract 1 g / l,
In a medium (pH 7.0) consisting of 0.5 g / l of MgSO ₄ 7 H ₂ O at 30 ° C
The cells were cultured for 48 hours, and the cells were collected by centrifugation (2000 rpm, 15 minutes). 1 g of cells was suspended in 5 ml of TE buffer, and freeze-thawing was repeated several times. 33% so that the final concentration is 25%
Add sucrose and mix, then add 0.25 M EDTA 8m
l, Lysozyme solution (Tris / HCl buffer to 5mg / ml)
4 ml (dissolved in pH 8.0) was added and incubated at 37 ° C. for 60 minutes. 5 ml of 5M-NaClO _{4 and} 2 ml of 10% SDS were added, and the incubation was further continued until the cells were completely lysed.

After lysis, DNA was extracted from the lysate by phenol extraction, followed by chloroform extraction to obtain D.
NA was purified. Take the upper layer of chloroform extraction, 3 M
After adding 1/10 volume of sodium acetate ethanol and mixing, 2 times volume of ethanol was added and mixed to precipitate DNA. The resulting floc DNA is wrapped with a Pasteur pipette with a closed end, and ethanol is sufficiently removed.
Disperse in TE buffer (about 2 ml), and add RNase A solution (2
(50 mM Tris · HCl buffer solution, pH 7.5) was added so that the concentration became 00 mg / ml, and the mixture was slowly shaken overnight at room temperature to completely dissolve.

The next day, 2 mg of proteinase K was added, and the mixture was kept at 37 ° C. for 1 hour. Then, phenol extraction and ethanol precipitation were performed to recover the DNA, a small amount of TE buffer was added, and the mixture was gently shaken overnight to completely dissolve. This solution was used in subsequent experiments.

(Southern Hybridization) S. g
The chromosomal DNA of riseus HUT6037 was completely digested with several different restriction enzymes and separated by agarose gel electrophoresis. Put the agarose gel after electrophoresis in a tupper container,
A hydrolyzate (0.25 M HCl) was added, and the mixture was slowly shaken for 15 minutes. Discard the hydrolyzate and rinse with distilled water.
5 N NaOH, 1.5 M NaCl) and shaken for 15 minutes. This treatment denatures the DNA to a single strand. After rinsing with distilled water, neutralization solution (1.5 m NaCl, 0.5 M Tris buffer
(pH 8.0) for 15 minutes.

Discard the neutralizing solution and remove DN in agarose gel.
A was transferred to a nylon membrane by a capillary method.
That is, a 20 × SSC (0.3 M trisodium citrate, 3 M NaCl) solution is placed in a tapper container, a glass plate is inserted over the solution, and a filter paper (Whatman No. 3 MM) is placed over the glass plate. Both ends were immersed in a 20 × SSC solution. The gel was placed on the filter paper with care so that air bubbles did not enter, and portions other than the gel were covered with wrap. Advance on the gel
Nylon membrane (MAGNA Nylon, PORE Size 0.45μm, MSI) immersed in 2 × SSC solution for 15 minutes is layered so that air bubbles do not enter, and three filter papers and a paper towel are placed on top of it and a weight of about 500 g is placed. The DNA was transferred from the agarose gel to a nylon membrane by capillary action. After the blotting was completed, the DNA was fixed by exposing the nylon membrane to UV for 2 minutes, and then dried and stored.

The nylon membrane that had been subjected to the Southern blotting was placed in a hybridization bag, and an appropriate amount of prehybridization buffer (5 m / 100 cm ^{2 of} membrane area) was used.
After the air was evacuated, the cells were completely sealed and incubated at 37 ° C for about 1 hour. Next, remove the prehybridization buffer, add an appropriate amount of the hybridization buffer, add 1 μl of the above hybridization probe per 1 ml of the buffer, evacuate the air, completely seal and shake overnight at 37 ° C. While incubating. The nylon membrane was removed from the hybridization bag, and washed with a washing solution A (2 × SSC) at room temperature for 5 minutes.

This operation was repeated once with a different washing solution. Next, the plate was washed with a washing solution A (2 × SSC, 0.1% SDS) at 37 ° C. for 10 minutes while shaking. This operation was repeated once with a different washing solution. Finally, after rinsing with cleaning solution A,
The nylon membrane was wrapped in plastic wrap, and was overlaid with an X-ray film (manufactured by Kodak) and autoradiography was performed at -80 ° C. The compositions of the prehybridization mixture and the hybridization mixture are as follows.

Prehybridization mixture: 30% deionized formamide, 5 × SSC, 5 × Denhardt solution, 50 mM
Sodium phosphate buffer (pH 6.2), 1 mg salmon sperm DN
A, 0.1% SDS. Hybridization mixture: 30%
Deionized formamide, 5 × SSC, 5 × Denhardt solution, 50
mM sodium phosphate buffer (pH 6.2), 0.1% SDS.

(Preparation of Gene Library) As a result of Southern hybridization, a DNA of about 2 kb in chromosomal DNA cut with two restriction enzymes SalI and SphI was obtained.
The fragment was found to hybridize with this probe. Therefore, it was considered that the chitinase gene could be cloned by cloning the DNA fragment around 2 kb. Therefore, the chromosomal DNA was converted to Sal I and Sp
The DNA fragment was cut with the two restriction enzymes hI, the DNA fragments were separated by agarose gel electrophoresis, and a gel containing a DNA fragment of about 2 kb was cut out.

A DNA fragment was recovered from this gel using GENE CLEAN II (manufactured by BIO101) and the plasmid vector was recovered.
Escherichia coli E. coli JM109 was transformed by electroporation after ligation with pUC119. Colonies of the obtained transformant were subjected to colony hybridization using the above probe, and a clone having a chitinase gene was selected.

(Colony hybridization) A nylon membrane was gently overlapped on a colony of a transformant grown on an LB agar medium containing ampicillin so as to prevent air bubbles from entering, and the nylon membrane and agar were heated with a heated needle as a position marker. After piercing the hole, it was gently peeled off. The peeled nylon membrane was placed on a fresh LB agar medium containing ampicillin with the side with the bacteria facing up, and cultured at 37 ° C. for 3 hours.

The nylon membrane was removed from the medium, denatured by placing on a filter paper soaked in a denaturing solution (0.5N NaOH, 1.5M NaCl) for 5 minutes, and then placed on dry filter paper for 3 minutes. This operation was repeated once. Next, the filter was immersed in a neutralizing solution (1.5 m NaCl, 0.5 M Tris.HCl pH 8.0) for 5 minutes on a filter paper and placed on a dry filter paper for 3 minutes, and air-dried twice. The air-dried nylon membrane was treated with a washing solution (3 × SSC, 0.1% SDS) at 60 ° C. for 15 minutes, and cells remaining on the membrane were removed with a sponge. Further, the DNA was treated with a fresh washing solution for 15 minutes and irradiated with UV for several minutes to fix the DNA to the membrane. Finally, it was rinsed with 3 x SSC, air dried and stored in a desiccator until use.

Using the nylon membrane treated as described above, hybridization was carried out in the same manner as the above-mentioned Southern hybridization. The probe used was exactly the same as that used in Southern hybridization. After hybridization, autoradiography was performed to detect a colony of a transformant having DNA that hybridized with the probe. From clones of about 1,000 transformants, four clones having a chitinase gene were detected. In each of these clones, the same DNA of about 1.7 kb containing the chitinase gene was inserted into a plasmid vector, and this plasmid was named pGC01.

(Reference Example 3) Base sequence and amino acid sequence of chitinase gene The inserted DNA fragment of pGC01 containing the chitinase gene is ligated to the SalI and SphI sites of plasmid vector pUC119. XbaI, SacI, ApaI, PvuI, BamH
There are each restriction site for I. Prior to base sequence determination, various DNA fragments obtained by digestion with these restriction enzymes were subcloned into a plasmid vector pUC119. First, a large amount of the recombinant plasmid pGC01 was
Cut with two restriction enzymes I and Sph I,
The kb insert DNA fragment was separated by agarose gel electrophoresis, the gel containing the insert DNA fragment was cut out, and GENE CLE
DNA fragments were recovered from the gel by AN II (manufactured by Bio101).

This DNA fragment was ligated with SacI, PvuI, BamHI, Bs
Various DNs generated by digestion with any restriction enzyme of sHI
The A fragments were individually ligated to pUC119 and introduced into E. coli JM109 by electroporation. These DNA
Escherichia coli in which the fragment was subcloned was detected as a white colony on LB agar medium supplemented with ampicillin, X-gal, and IPTG (isopropyl-β-D-thiogalactoside).

(Preparation of Single-Stranded DNA) As a template DNA for base sequence determination, single-stranded DNA was prepared from a plasmid obtained by ligating various DNA fragments derived from the inserted DNA fragment of pGC01 with pUC119. First, single-stranded D
A high titer helper phage solution for NA preparation was prepared as follows.

Phage solution containing helper phage M13KO7
10 μl and cultured until OD600 nm = 0.8 or more.
E. coli MV 1184 culture 0.5 ml, and pre-dissolved
3.5ml of LB soft agar medium (0.7% agar) kept at 50 ℃
LB agar medium (1.5%
Agar). This is cultured at 37 ° C for about 8 hours,
A plaque was formed. 70 μg / m single plaque
lx kanamycin in 5 ml of 2xYT medium (polypeptone
16 g / l, yeast extract 10 g / l, NaCl 5 g / l, pH 7.5), incubate at 37 ° C for 16 hours, and centrifuge (8,000 × g).
, 10 min, 4 ° C), and collect the supernatant.
The phage solution was used to prepare NA.

Next, Escherichia coli JM109 having a plasmid for preparing single-stranded DNA was inoculated into 3 ml of 2 × YT medium containing 150 μg / ml of ampicillin and cultured with shaking at 33 ° C. overnight. Add 30 μl of helper phage solution (> 10
10 pfu / ml), and the mixture was allowed to stand at 37 ° C for 30 minutes.
This was inoculated into 3 ml of 2 × YT medium supplemented with 150 μg / ml ampicillin, 70 mg / ml kanamycin, and 0.01% thiamine, and cultured at 37 ° C. for 16 hours. The culture was centrifuged (8,000 × g, 5 minutes), and the supernatant was collected.

To 1 ml of the supernatant, 200 μl of a polyethylene glycol (PEG) -NaCl solution was added and mixed well.
Let stand for 15 minutes. Centrifuge (8,000 xg, 5 minutes)
The precipitated single-stranded DNA was recovered and 100 μl of TE
Dissolved in buffer. This was extracted with phenol and chloroform, and single-stranded DNA was collected by ethanol precipitation, dried in vacuo, dissolved in TE buffer, and used for nucleotide sequence determination.

(Determination of Nucleotide Sequence) Using the single-stranded DNA prepared as described above, pG containing chitinase gene was used.
The nucleotide sequence of the inserted DNA of C01 was determined. The nucleotide sequence was determined using an Auto Read Sequencing Kit (Pharmacia Biotech) for the sequencing reaction and electrophoresis with an ALF DNA sequencer (Pharmacia Biotech).

(Sequence reaction) First, A, T, G, C
Prepare each reaction tube of A mix (ddATP + dNTP), B mix (ddCTP + dNTP), G mix (ddGTP + dNTP), C mix (ddTTP + dNTP).
Was added in an amount of 2.5 μl. In another tube, single-stranded D
NA 1-2 μg, sequencing primer (Universal primer: 5'-CGACGTTGTAAAACGACGACGGCCAGT-3 '
Or Reverse primer: 5'-CAGGAAACAGCTATGAC-
3 ′) 2 μl of the annealing buffer and 2 μl of the annealing buffer were added, and sterilized water was added to make 17 μl (this is called master mix).

This is used as an annealing reaction at 60 ° C.
The mixture was heated at room temperature and left at room temperature for 10 minutes or more. Add an extension buffer to the master mix after the annealing reaction.
1 μl and 2 μl (3 units) of T7 polymerase were added and mixed, and the resulting mixture was dispensed into A, T, G, and C reaction tubes by 4.5 μl each. This was incubated at 37 ° C. for 5 minutes to perform an extension reaction, and the reaction was stopped by adding 5 μl of a stop solution. In this state, the cells were stored frozen at -20 ° C until electrophoresis by a sequencer.

For electrophoresis using a sequencer, a 6% long ranger gel containing 6 M urea was used. After washing the wells of the sequencing gel by suction and discharge with a pipetteman, immediately before heat denaturation at 95 ° C. for 3 minutes, quenched A,
Each sample in the T, G, C reaction tube was applied to each well in an amount of 4 to 6 μl, and electrophoresed at 700 V, 38 mA, and 45 ° C. The sequence was determined from the obtained waveform data. Editing and analysis of the determined sequence data is GENE
TYX Mac ver7.0 (Software Development Co., Ltd.) was used. The nucleotide sequence and the amino acid sequence thus obtained are shown in the sequence listing.

(Example 1) (Construction of Chitinase Expression System Using Escherichia coli) By expressing the chitinase gene used in the present invention in Escherichia coli, a large amount of chitinase free of other chitinase can be produced. However, little production of chitinase was observed in E. coli harboring plasmid pGC01, which was initially obtained by cloning the chitinase gene.
As a cause, it was considered that a regulatory region that would be present upstream of the coding region of the chitinase gene might inhibit the production of the chitinase protein.

Therefore, only the portion corresponding to the coding region of the chitinase gene was amplified by PCR and ligated to the expression plasmid pET12a (Novagen) to construct a high-expression system for chitinase which was not affected by the upstream region. . As a PCR primer to amplify only the coding region,
Oligonucleotides 5'-GATCATATGTATCGACGTGTCATC-3 'corresponding to both ends of the coding region and having an NdeI site at the end and BamH
Oligonucleotide 5'-GGCGGATCCACATCAGCA with I site
GCTCAG-3 'was designed and synthesized by Greiner Japan.

Using these oligonucleotides, pGC0
Using 1 as a template, a PCR reaction was performed. The PCR reaction is performed using the TaKaRa PCR Thermal Cycler as a thermal cycler.
MP (Takara Shuzo Co., Ltd.) converted to DNA polymerase with TakarA
Using la Taq, heat denaturation 98 ° C, 30 seconds, annealing 50 ° C, 3
Twenty-five cycles were performed for 0 second at 72 ° C. for 1 minute. As a result of analyzing a part of the PCR reaction solution by agarose gel electrophoresis, it was found that the expected DNA fragment having a length of about 900 base pairs was amplified.

Then, the entire amount of the reaction solution was subjected to agarose gel electrophoresis, a gel fragment containing the amplified DNA fragment was cut out, and the DNA fragment was extracted using GENECLEAN II. The extracted DNA fragment was treated with restriction enzymes NdeI and BamHI, ligated with pET12a cut with NdeI and BamHI, and introduced into E. coli BL21 by electroporation. One transformant into which pGC02 was introduced
Selection was performed on LB medium containing 00 μg / ml ampicillin.

(Production of Chitinase by Escherichia coli) pGC02
C produced by Escherichia coli BL21 strain
Are transported to the periplasmic space and accumulated there.
Therefore, it is necessary to extract the produced chitinase from the periplasmic space. E. coli BL21 strain with pGC02
The cells were pre-cultured overnight at 30 ° C. in an LB medium containing 00 μg / ml ampicillin. 10 ml of the preculture was inoculated into 1 liter of the same medium and cultured with shaking at 30 ° C. When the absorbance at 660 nm of the culture solution reached 0.6 to 1.0, IPTG was added aseptically (final concentration: 400 μM), culture was continued for about 5 hours after addition, and the cells were collected by centrifugation at 8,000 × g for 5 minutes. did.

The cells pelleted by centrifugation were added to a cooled spheroplast buffer (100 mM Tris-Hcl (pH 8.
0), 0.5 mM EDTA, 0.5 M sucrose), completely suspended, and allowed to stand on ice for 5 minutes. After standing still
Collect the cells by centrifugation at 10,000 xg for 10 minutes.
100 ml of 0 mM APMSF solution was added and suspended. This was allowed to stand on ice, and one minute later, 5 ml of a 20 mM magnesium chloride aqueous solution was added, and the mixture was centrifuged at 12,000 × g for 15 minutes to collect a supernatant. Fine powdery ammonium sulfate was added to the supernatant while being dissolved well (until the concentration reached 60% saturation), and the mixture was allowed to stand at 4 ° C. for 12 hours or more.

Thereafter, the protein precipitate generated by centrifugation at 10,000 × g for 10 minutes at 4 ° C. was recovered. This precipitate was dissolved in a small amount of 20 mM sodium phosphate buffer (pH 6.0), and the solution was dissolved in 1 mM sodium phosphate buffer (pH 6.0).
Dialysis overnight with occasional exchange of buffer. After completion of the dialysis, it was freeze-dried to obtain a crude enzyme.

(Purification of Chitinase) Hydroxyapatite sufficiently washed with deionized water was applied to a column (1.5 cm inner diameter,
(Column volume: 20 ml) and washed with 10 times the column volume of 1 mM sodium phosphate buffer (pH 6.0). The crude enzyme dissolved in a small amount of ultrapure water was applied to the column, and eluted with a 1 mM sodium phosphate buffer (pH 6.0) at a flow rate of 6 ml / hour. The eluate was collected in 10 ml portions by a fraction collector, and the UV 280 nm absorption of each fraction was measured. The chitinase activity of the UV 280 nm peak fraction was measured, and the fraction having chitinase activity was analyzed by SDS-PA
Analyzed by GE. Fractions in which chitinase was detected as a single band in SDS-PAGE were collected and freeze-dried to obtain purified chitinase. From the culture of 1 liter of E. coli BL21 strain carrying pGC02, about 10 mg of purified chitinase was finally obtained.

(Example 2) (Inhibition of growth of Trichoderma reesei by chitinase) A potato dextrose agar (PDA, Nissui Pharmaceutical Co., Ltd.) medium in which the agar concentration was adjusted to 2% was used for the antibacterial activity assay. The spores of the test bacterium were suspended in sterile water, and filtered with Kimwipe to remove mycelia from the suspension. 2.5-5 x 1 spore count
The concentration was adjusted to 03 cells / ml, and 40 μl of the mixture was absorbed on a paper disk placed in the center of a Petri dish containing a PDA medium. 30 ℃
After the spores germinated and the hypha elongated to form a colony of sufficient size (about 24 hours), a new paper disk was placed 5 mm away from the edge of the colony, and 40 μl of the chitinase solution was added. Absorbed.

After incubation at 30 ° C. for about 12 hours, inhibition of growth of mold hyphae by chitinase was observed. Some chitinases of other bacteria were used as the chitinase to be compared, and rice class I and III chitinases were used, and only a buffer was added as a control. The results are shown in Table 1.

[0100]

[Table 1]

(Example 3) (Inhibition of growth of Trichoderma harzianum by Streptomyces) Potato dextrose agar (PDA, Nissui Pharmaceutical Co., Ltd.) adjusted to an agar concentration of 2% and a nutrient agar medium containing 0.2% chitin powder. Was used for antibacterial activity assay.
Streptomyce was added to a Petri dish containing the chitin-containing PDA medium.
A paper disk having absorbed 50 μl of the culture solution obtained by the s griseus HUT6037 culture method was placed thereon, and incubated at 30 ° C. for about 48 hours to form actinomycete colonies.

At a distance of 10 mm from the end of the colony, a new paper disk was placed, and the spores of the solid culture of the test bacterium were suspended in sterile water to increase the number of spores to 2.5-5 × 10 3.
40 μl of the mixture prepared at the number of cells / ml was absorbed. A platinum loop of Streptomyces griseus HUT6037 solid culture medium (slant) was inoculated into a Petri dish containing a chitin-containing nutrient agar medium as in the case of the chitin-containing PDA medium, and incubated at 30 ° C. for about 72 hours to colonize actinomycete colonies. Formed.

A paper disk was newly placed 10 mm away from the end of the colony, and 40 μl of a solid culture spore prepared of the test bacterium was absorbed in the same manner as above.
After keeping each petri dish at 30 ° C, spores germinate and elongate mycelia to form colonies of sufficient size (about 24 hours), the degree of inhibition of growth of mold mycelia by the culture solution and cells Was observed. Bacillus circu for comparison
Lans WL-12 cells were also added as a control only with buffer. The results are shown in Table 2.

[0104]

[Table 2]

Example 4 (Inhibition of Growth of Trichoderma harzianum by Recombinant Escherichia coli BL21) A potato dextrose agar (PDA, Nissui Pharmaceutical Co., Ltd.) medium adjusted to agar concentration of 2% was used for antibacterial activity assay. Was. The spores of the solid culture of the test bacterium were suspended in sterile water, the number of spores was adjusted to 2.5-5 × 10 3 cells / ml, and 40 μl of the spores was absorbed on a paper disc placed in the center of a Petri dish containing PDA medium. . After incubating at 30 ° C, spores germinate, mycelia elongate, and colonies of sufficient size were formed (about 24 hours).
The cells were placed at a distance of mm, cultured in an LB medium containing 100 μg / ml of ampicillin, and 40 μl of a frozen and thawed cell of a recombinant E. coli BL21 strain expressing chitinase with IPTG was absorbed.

A paper disk impregnated with the lysozyme-treated BL21 strain obtained by culturing cells in the same manner on the same plate was placed at a distance of 5 mm from the colony of the test bacterium. After incubation for a period of time, growth inhibition of mold hyphae by the freeze-thawed BL21 strain and the lysozyme-treated product was observed. As a control, an untreated BL21 strain was used, and only a buffer was added as a control. The results are shown in Table 3.

[0107]

[Table 3]

[0108]

Industrial Applicability The present invention uses an actinomycete-derived chitinase having an antibacterial activity, which is superior in industrial productivity to plants, or a fungal control method using an actinomycete producing the chitinase, and a transformed microorganism. A method for producing a chitinase having antifungal activity can be provided.

[0109]

[Sequence list] SEQ ID NO: 1 Sequence length: 294 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin: Streptomyces griseus HUT6037 Met Tyr Arg Arg Val Met Ser Leu Leu Val Ala Leu Gly Ala Ile Val 16 Ala Ala Leu Ile Val Leu Pro Ala Thr Thr Ala Gln Ala Ala Thr Cys 32 Ala Thr Ala Trp Ser Ser Ser Ser Val Tyr Thr Asn Gly Gly Thr Val 48 Ser Tyr Asn Gly Arg Asn Tyr Thr Ala Lys Trp Trp Thr Gln Asn Glu 64 Arg Pro Gly Thr Ser Asp Val Trp Ala Asp Lys Gly Ala Cys Gly Thr 80 Gly Gly Glu Gly Pro Gly Gly Asn Asn Gly Phe Val Val Ser Glu Ala 96 Gln Phe Asn Gln Met Phe Pro Asn Arg Asn Ala Phe Tyr Thr Tyr Lys 112 Gly Leu Thr Asp Ala Leu Ser Ala Tyr Pro Ala Phe Ala Lys Thr Gly 128 Ser Asp Glu Val Lys Lys Arg Glu Ala Ala Ala Phe Leu Ala Asn Val 144 Ser His Glu Thr Gly Gly Leu Phe Tyr Ile Lys Glu Val Asn Glu Ala 160 Asn Tyr Pro His Tyr Cys Asp Thr Thr Gln Ser Tyr Gly Cys Pro Ala 176 Gly Gln Ala Ala Tyr Tyr Gly Arg Gly Pro Ile Gln Leu Ser Trp Asn 192 Phe Asn Tyr Lys Ala Ala Gly Asp Ala Leu Gly Ile Asn Leu Leu Ala 208 Asn Pro Tyr Leu Val Glu Gln Asp Pro Ala Val Ala Trp Lys Thr Gly 224 Leu Trp Tyr Trp Trp Asn Ser Gln Asn Gly Pro Gly Thr Met Thr Pro His 240 Asn Ala Ile Val Asn Asn Ala Gly Phe Gly Glu Thr Ile Arg Ser Ile 256 Asn Gly Ala Leu Glu Cys Asn Gly Gly Asn Pro Ala Gln Val Gln Ser 272 Arg Ile Asn Lys Phe Thr Gln Phe Thr Gln Ile Leu Gly Thr Thr Thr 288 Gly Pro Asn Leu Ser Cys 294

SEQ ID NO: 2 Sequence length: 1080 Sequence type: Nucleic acid Topology: Linear Sequence type: Chromosomal DNA Origin: Streptomyces griseus HUT6037 CGGCACCTTC TTCGAGGATG GTCTAGACCT TGACAGGTCC AGACCATCCT CGCTTGAGTA 60 TCGGCACCCC CACCGGCGGCTCGCGAGTCGTCGATCGCGTCGTCGCGTCGTCGCGTCGTCGCGT CGA CGT GTC ATG AGT CTG CTG GTC GCG CTC GGC GCG ATC GTC 168 Met Tyr Arg Arg Val Met Ser Leu Leu Val Ala Leu Gly Ala Ile Val GCG GCG CTC ATC GTT CTT CCC GCC ACC ACC GCG CAG GCG GCC ACC TGC 216 Ala Ala Leu Ile Val Leu Pro Ala Thr Thr Ala Gln Ala Ala Thr Cys GCC ACG GCC TGG AGC TCC TCC TCC GTC TAC ACG AAC GGC GGC ACG GTC 264 Ala Thr Ala Trp Ser Ser Ser Ser Val Tyr Thr Asn Gly Gly Thr Val TCG TAC AAC GGC CGT AAC TAC ACC GCC AAG TGG TGG ACC CAG AAC GAG 312 Ser Tyr Asn Gly Arg Asn Tyr Thr Ala Lys Trp Trp Thr Gln Asn Glu CGT CCC GGC ACG TCG GAC GTC TGG GCC GAC AAG GGT GCG TGT GGC ACC 360 Arg Pro Gly Thr Ser Asp Val Trp Ala Asp Lys Gly Ala Cys Gly Thr GGC GGT GAG GGC CCG GG C GGC AAC AAC GGC TTC GTC GTC AGC GAG GCC 408 Gly Gly Glu Gly Pro Gly Gly Asn Asn Gly Phe Val Val Ser Glu Ala CAG TTC AAC CAG ATG TTC CCG AAC CGG AAC GCC TTC TAC ACC TAC AAG 456 Gln Phe Asn Gln Met Phe Pro Asn Arg Asn Ala Phe Tyr Thr Tyr Lys GGC CTC ACC GAC GCC CTG AGC GCC TAC CCG GCT TTC GCC AAG ACC GGC 504 Gly Leu Thr Asp Ala Leu Ser Ala Tyr Pro Ala Phe Ala Lys Thr Gly AGC GAC GAG GTG AAG AAG CGC GAG GCC GCG GCC TTC CTC GCC AAC GTC 552 Ser Asp Glu Val Lys Lys Arg Glu Ala Ala Ala Phe Leu Ala Asn Val AGC CAC GAG ACC GGC GGA CTG TTC TAC ATC AAG GAA GTC AAC GAG GCG 600 Ser His Glu Thr Gly Gly Leu Phe Tyr Ile Lys Glu Val Asn Glu Ala AAC TAC CCG CAC TAC TGC GAC ACC ACG CAG TCG TAC GGC TGC CCG GCC 648 Asn Tyr Pro His Tyr Cys Asp Thr Thr Gln Ser Tyr Gly Cys Pro Ala GGC CAG GCC GCC TAC TAC GGC CGC GGC CCG ATC CAG CTG AGC TGG AAC 696 Gly Gln Ala Ala Tyr Tyr Gly Arg Gly Pro Ile Gln Leu Ser Trp Asn TTC AAC TAC AAG GCG GCC GGT GAC GCC CTG GGC ATC AAC CTG CTC GCC 744 Phe Asn Tyr Lys Ala Ala Gly As p Ala Leu Gly Ile Asn Leu Leu Ala AAC CCC TAC CTG GTG GAG CAG GAC CCG GCC GTG GCG TGG AAG ACC GGC 792 Asn Pro Tyr Leu Val Glu Gln Asp Pro Ala Val Ala Trp Lys Thr Gly CTC TGG TAC TGG AAC TCC CAG AAC GGC CCC GGC ACC ATG ACC CCG CAC 840 Leu Trp Tyr Trp Asn Ser Gln Asn Gly Pro Gly Thr Met Thr Pro His AAC GCC ATC GTC AAC AAC GCC GGC TTC GGC GAG ACG ATC CGC TCG ATC 888 Asn Ala Ile Val Asn Asn Ala Gly Phe Gly Glu Thr Ile Arg Ser Ile AAC GGC GCT CTG GAG TGC AAC GGC GGC AAC CCG GCG CAG GTC CAG AGC 936 Asn Gly Ala Leu Glu Cys Asn Gly Gly Asn Pro Ala Gln Val Gln Ser CGC ATC AAC AAG TTC ACG CAG TTC ACC CAG ATC CTC GGC ACC ACC ACG 984 Arg Ile Asn Lys Phe Thr Gln Phe Thr Gln Ile Leu Gly Thr Thr Thr GGT CCG AAC CTG AGC TGC TGATGTCGTA CCGCCCCTCA CCGACCCGCG 1032 Gly Pro Asn Leu Ser Cys AGGGCCGCTG AGGATCCCGA GGACCACC

[Brief description of the drawings]

FIG. 1 is a diagram showing a restriction enzyme fragment map of a DNA fragment containing a chitinase gene.

Continued on the front page F term (reference) 4B024 AA07 BA12 CA03 DA06 EA04 FA18 GA14 GA19 GA27 HA01 4B050 CC03 DD02 EE02 FF04E FF05E FF09E LL10 4H011 AA01 AA03

Claims (8)

[Claims] 1. A fungal control method using an actinomycete-derived chitinase comprising a protein having a sequence represented by amino acids 1 to 294 in the amino acid sequence of SEQ ID NO: 1 in the sequence listing. 2. A nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 in the sequence listing, wherein
A fungal control method using an actinomycete-derived chitinase comprising a protein produced by a DNA having a nucleotide sequence encoding the amino acid sequence represented by 002. 3. A method for controlling fungi using an actinomycete that produces a chitinase comprising a protein having a sequence represented by amino acids 1 to 294 in the amino acid sequence of SEQ ID NO: 1 in the sequence listing. 4. A base sequence encoding the amino acid sequence of SEQ ID NO: 2 in the sequence listing, wherein
A fungal control method using an actinomycete producing a chitinase comprising a protein produced by a DNA having a base sequence encoding the amino acid sequence represented by 002. 5. The method for controlling fungi according to claim 1, wherein the actinomycete is Streptomyces griseus. 6. The actinomycete is Streptomyces griseus HUT603.
The method for controlling fungi according to any one of claims 1 to 4, wherein the method is 7. 7. Base numbers 121 to 10 in the base sequence encoding the amino acid sequence of SEQ ID NO: 2 in the sequence listing
A fungal control method using a chitinase-producing microorganism, wherein a DNA having a base sequence encoding the amino acid sequence represented by 02 is introduced into a cell and transformed. 8. Base numbers 121 to 10 in the base sequence encoding the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing
A method for producing a chitinase having an antifungal activity, using a microorganism transformed by introducing a DNA having a base sequence encoding the amino acid sequence represented by No. 02 into a cell.