KR100628416B1 - 1652-1 91163 A noble strain Bcillus atrophaeus TBM1652-1 KACC 91163P and chitinase chitosanase and antifungal compound produced from thereof - Google Patents

Abstract

The present invention produces a chitinase or chitosanase, and by its enzyme activity, Bacillus atrophaeus TBM1652-1 having low-molecular chitin or chitosan oligosaccharide-producing ability and antifungal activity As related to KACC 91163P, the chitinase or chitosanase produced by the strain of the present invention is insoluble and indigestible after digestion of chitin or chitosan of a polymer of various molecular weights to produce chitin oligosaccharides or chitosan oligosaccharides, respectively. In addition, the oligosaccharide has a high absorption rate so that various physiological functions can be expressed, and the present invention can be used as a biocontrol agent by inhibiting the growth of plant pathogens in order to decompose chitin or chitosan forming the cell wall of plant pathogens.

Bacillus atropheus TBM1652-1, chitinase, chitosanase, antifungal activity

Description

A noble strain Bcillus atrophaeus TBM1652-1 KACC 91163 P and chitinase, chitosanase and antifungal compound produced from Y. A. Bacillus atropheus TVM1652-1 BAC 91163 P and chitinase, chitosanase and antifungal substances produced by the strain.

1 is a diagram showing the decomposition pattern of colloidal chitin, glycol chitin and water-soluble chitosan by Bacillus atrophaeus TBM1652-1 KACC 91163P of the present invention,

2 is a view showing a scanning electron micrograph of the strain of the present invention,

Figure 3 is a schematic diagram based on the 16S rDNA of the strain of the present invention,

Figure 4 is a schematic diagram based on the gyrA gene of the strain of the present invention,

Figure 5 is a diagram showing the ability to produce black cattle of the brown series of the present invention,

Figure 6 is a diagram showing the molecular weight of the chitinase produced by the strain of the present invention through SDS-PAGE and Zymogram,

Figure 7 is a diagram showing a domain region having the function of the chitinase gene of the strain of the present invention,

Figure 8 is a comparison of the chitinase amino acid sequence of the strain of the present invention and the chitinase amino acid sequence of other species of the genus Bacillus,

9 is a diagram showing the effect of temperature on the chitinase activity of the strain of the present invention,

10 is a diagram showing the effect of metal ions on the chitinase activity of the strain of the present invention,

11A and 11B are diagrams of oligosaccharides produced as a reaction product by chitinase using chitin oligosaccharides (FIG. 11) a and colloidal chitin (FIG. 11B) of various molecular weights as substrates.

12 is a diagram showing the antifungal activity against plant pathogens of the strain of the present invention.

The present invention relates to Bacillus atrophaeus TBM1652-1 KACC 91163P, which produces chitinase or chitosanase, and which has low molecular weight chitin or chitosan oligosaccharide production and antifungal activity.

Chitin is a natural macromolecular substance in which N -acetylglucosamine is combined as β-1,4. It is a major shell component of shellfish such as crabs, shrimps, and insects. It is a high molecular material. Chitosan is a polymer in which the acetyl group of chitin is eliminated. It is estimated that more than 10 billion tons of chitin are produced annually in the underwater biosphere. Chitosan regulates biorhythms along with the ability to activate the body's natural healing abilities. In addition, it absorbs and excretes excess harmful cholesterol in the body, that is, it acts as a decholesterol, acts as an anti-cancer to inhibit the proliferation of cancer cells, and adsorbs chloride ions that cause blood pressure to rise and inhibits absorption in the intestines. By releasing it into the body, it has a function of inhibiting blood pressure increase and proliferating effective bacteria in the intestine and activating cells. Other effects include blood sugar control, liver function improvement, and the release of heavy metals and pollutants.

Chitin or chitosan by itself is a very high molecular material and, like cellulose, is not digested and absorbed by the human gastrointestinal tract. In other words, humans do not produce enzymes that can degrade it. Therefore, in order to effectively use chitin or chitosan as an active substance in vivo, it is necessary to produce their oligosaccharides (Jeon YJ, CH Kim, SK Kim, Preparation of chitin and chitosan oligosaccharides., The Korean Journal of Chitin Chitosan Research, 3, pp140 -155, 1998).

Chitinases that break down chitin with these effects are commonly found in a wide range of organisms, including bacteria, fungi, higher plants, insects, shellfish and some vertebrates (Bhushan B., Production and characterization of a thermostable chitinase from a new alkalophilic Baicllus sp.BG -11., Appl Microbiol . 88 , pp800-808, 2000; Flach J, PE Pilet, and P Jolles., What`s new in chitinase research ?, Experientia, 48 , pp701-716, 1992; Jeuniaux C., Chitinase.Methods Enzymol., 8 , pp644-650, 1996). Among them, chitinases produced by fungi, crustaceans, and insects modify the structures that make up chitin. In contrast, bacteria-producing chitinases break down chitin and use it as an energy or carbon source (Gooday GW., The ecology of chitin degration., Adv. microbiol Ecol., 11 , pp 387-430, 1990; Leah R, H. et al., Biochemical and molecular characterization of three barley seed proteins with antifungal properties., J. Biol. Chem ., 266 , pp 1564-1573, 1991; Roberts PW, and CP Selitrennikoff., Plant and bacterial chitinases differ in antifungal activity., J. Gen Microbiol ., 134 , pp69-76, 1988).

Chitinases can be broadly divided into two classes: endochitinase and exochitinase. Endocytase is an enzyme that cleaves the inside of the chitin polymer, and exochitinase can be further classified into β- N -acetylglucosamine and chitobiosidase. β- N -acetylglucosaminease releases monosaccharide form N -acetylglucosamine (NAG) from the chitin polymer or from outside the chitin polymer, and chitobiosidase releases the disaccharide form chitobio from chitin polymer (Blaak H, J Schnellmann et al., Characteristics of an exochitinase from Streptomyces olivaceoviridis , its correponding gene, putative protein domains and relationship to other chitinases., Eur. J. Biochem ., 214 , pp659-669, 1993).

Bacteria in her language Pseudomonas (Aeromonas) for producing such chitin dehydratase, Serratia marcescens (Serratia), Mick simple emitter (Myxobacter), a pseudo Alter Pseudomonas (Pseudoalteromonas), Vibrio (Vibrio), Streptomyces (Streptomyces) and Bacillus ( Bacillus ) genus is well known (Cody RM., Disrtivution of chitinase in Bacullus.Curr . Bicrobiol ., 19 , pp201-205, 1989; Robbins PW, K Overbye, B Benfield, and J Pero., Colning and high-level expression of chitinase-encoding gene of Streptomyces plicatus gene, 111, pp69-76, 1992;.... Ueda M, and M Arai, Purification and some properties of chitinase from Aeromonas sp No. Biochem 10S-24 Biosci Biotech,... 38 , pp 460-464, 1992).

Many studies have been conducted to control various pests affecting the yield and quality of crops, and various kinds of pesticides have been developed and used. There are two main methods for controlling plant pathogenic fungi, one of which has been used until now and is currently being used, and the biological method using antagonistic microorganisms that antagonize certain pathogenic fungi. There is a control method. The use of chemical pesticides has resulted in an innovative increase in crop yields, but has also caused several problems. In comparison, biological control using microorganisms is less harmful to the human body, does not cause damage to crops, and has a low load on the environment and high stability. And it can be applied to heating pathogens that are difficult to control with chemical pesticides, and there is little possibility of developing resistance. It is selective for harmful organisms and does not disturb ecosystems significantly, and has little adverse effect on natural and useful organisms. These advantages have led to a greater interest in the antifungal substances produced by microorganisms, and many studies have been made and are currently being used as products (Attafuah, A., and JF Bradbury., Pseudomonas antimicrobica , a new species strongly antagonistic . to plant pathogens J. Appl bacteriol, 67, pp567-573, 1989;.... Tanaka, YT, and Omura S., Agroactive compound of microbial orgin Annu Rev. Microbiol, 47, pp57-48, 1993;. Fruh , T. et al, Natural products as pesticides:...... Two example of stereo selective synthesis Pestic Sci, 46, pp307-316, 1996; Knight, SC et al, Rationale and perspectives on the development of fungicides Annu. Rev. Phytophathol ., 35 , pp 349-372, 1997). Ecological biological control methods using antagonistic strains against various plant pathogenic fungi include lysing action that degrades cell membranes of plant pathogens using fungal envelope hydrolytic enzymes, and antibiotics that directly inhibit the growth of plant pathogens by antifungal antibiotics. (antibiotic) and competitive antagonism that inhibits the growth of plant pathogens by siderophore, an iron (Fe ³⁺ ) -specific binding substance secreted by Pseudomonas sp. There is a control method used. The lytic action of phytopathogens to break down the cell wall is under active research, focusing on chitinase. Biological control is known as an effective method to control crop diseases by utilizing the enzymatic degradation mechanism of chitin in the outer membrane of plant pathogenic fungi (Paulitz TC, and JE Loper, Lack of a role for fluorescent siderphore production in the biological control of Phythium danping-off of cucumber by a strain of Pseudomonas putida.Phytopathol ., 81 , pp930-935, 1991).

Accordingly, the present inventors have been able to produce chitinase and chitosanase, and from this, the present invention has been completed by identifying the Bacillus atropheus TBM 1652-1 strain having antifungal activity.

An object of the present invention is to produce a chitinase or chitosanase, and by its enzymatic activity Bacillus atrophaeus TBM1652-1 KACC having low molecular weight of chitin or chitosan oligosaccharide and antifungal activity Relates to 91163P.

In order to achieve the above object, the present invention generates a chitinase encoded by the nucleotide sequence of SEQ ID NO: 3 or a chitosanase encoded by the nucleotide sequence of SEQ ID NO: 5 to produce a low molecule chitin or chitosan oligosaccharide production ability and antifungal activity Bacillus atrophaeus TBM1652-1 KACC 91163P is provided.

Also provided is a biological control agent comprising Bacillus atropheus TBM1652-1 KACC 91163P having antifungal activity against plant pathogens.

The plant pathogens include Botrytis cinerea, Fusarium oxysporum, Rhizoctonia solani or Sclerotinia sclerotiorum. do.

Hereinafter, the present invention will be described in detail.

The present invention provides a Bacillus atrophaeus TBM1652-1 KACC 91163P having a low molecule chitin or chitosan oligosaccharide producing ability and antifungal activity by producing a chitin or chitosan aze.

In order to isolate the strain of the present invention from a sample taken from the soil, incubated for 1-10 days, preferably 7 days at 30-40 ℃, preferably 37 ℃ in LB medium provided with a colloidal chitin as a carbon source The first strain and culture of the colloidal chitin is decomposed to form a transparent ring can be selected to redistribute the most clear and clear strain is the final selection. The order to identify a selected strain on the basis of 16S rDNA, and gyrA gene analysis can analyze the flexibility related to the other species (Janet LS et al., Phylogeny of marine Bacillus Isolates from the Gulf of Mexico., Curr. Microbiol , 41, pp 84-88, 2000; Dong, X., and JC Cote., Phylogenetic relationships between Bacillus species and related genera inferred from comparison 3 'end 16S rDNA and 5' end 16S-23S ITS nucleotide sequences.Int . J. Syst.Evol.Microbiol., 51 , pp 2087-2093, 2003).

In order to clone the gene of chitinase or chitosanase produced by the strain of the present invention, PCR may be performed by preparing a primer based on the chi gene of Bacillus subtilis known from GenBank, and the chitinase or chitosanase of The activity of the enzyme and the effect of temperature and metal ions on the enzymes can be measured by applying the DNS method.

Hereinafter, the present invention will be described in more detail with reference to the following Examples and Experimental Examples. However, the following Examples and Experimental Examples are examples of the present invention and the present invention is not limited thereto.

Example 1. Screening of Strains

In order to separate the microorganisms having high chitinase and chitosanase production ability from the alpine region of Taebaek Mountain, 1 g of soil sample was placed in 10 ml of sterile water and vigorously stirred, followed by a 6-step dilution plate method containing LB medium (polypeptone 10g / L, yeast extract 5g / L, NaCl 5g / L, agar 15g / L, 0.1% colloidal chitin, pH 7.0) and incubated for one week at 37 ° C to form a single colony and then decompose colloidal chitin in medium As a result, a strain of forming a transparent ring was first selected. The isolated strain was inoculated in liquid LB medium and cultured O / N at 37 ° C., and then streaked onto LB agar medium containing 0.1% colloidal chitin, 0.01% glycol chitin or water-soluble chitosan, and then at 37 ° C. One week and three days of incubation, the most clear and large strain showing the clear ring around the colony was finally selected as chitinase producing strain was separated and named TBM1652-1 (see Figure 1).

Example 2 Identification of Selected Strains

2-1. Morphological Characteristics of Selected Strains

Gram staining of the strain isolated from Example 1 confirmed that it was Gram-positive, and rod type (rod type) was observed by field emission scanning electron microscope (JSM-6700F, Jeol, Japan). ) Was observed to confirm that Bacillus type bacilli (see FIG. 2).

2-2. Identification through Gene Homology of Selected Strains

In addition, the strain selected in Example 1 was subjected to secondary identification through a flexible relationship with other species based on 16S rDNA and gyrA gene analysis. Chromosome DNA of TBM1652-1 strain was isolated and PCR reaction was carried out with the template. The initial denaturation step was performed 30 minutes at 95 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 1 minute starting at 1 minute at 95 ℃ and the extension step was carried out for 10 minutes at 72 ℃. As a result of the reaction, a 16S rDNA fragment of about 1.5 kb and a gyrA fragment of about 0.9 kb were obtained. The obtained PCR fragment was cloned using a pGEM T-easy vector (Promega, USA.), And nucleotide sequences were determined in both directions. The homology of the 16S rDNA sequence of SEQ ID NO: 1 and the gyrA sequence of SEQ ID NO: 2 with similar species was examined through NCBI Blast, and the genetic association with similar Bacillus species was investigated using the ClustalX program.

As a result, a Bacillus Ke, as shown in Table 1 Trojan mouse L (Bacillus atrophaeus) 16S rDNA sequences of the TBM 1652-1 Bacillus subtilis (Bacillus subtilis), Bacillus amyl quinone Lori Patience (B. amyloliquefaciens), Bacillus More than 99% homology with other species of the genus Bacillus , such as B. vallimortis ( B. vallimortis ) showed a high homology.

The gyrA sequences, on the other hand, are Bacillus subtilis , B. amyloliquefaciens , B. vallimortis , B. mojavensis , B. mojavensis , and Bacillus licheniformis . (B. licheniformis) and respectively 98%, 82%, 83%, 81%, showed a homology of 96% and 81%, on the other hand Bacillus-to Lindsay N-Sys (B. thuringensis), bacilli SIRIUS (B, cereus) Bacillus la Alliance system (B. anthracis) than was observed that the genetic relationship considerably distant. As a result of the analysis, it could be seen that distinction from species of the genus Bacillus appeared in the analysis results by gyrA sequence rather than the analysis result of 16S rDNA sequence (see FIG. 4).

Genetic homology 16S rDNA gyrase A B .subtilis 99% 82% B. vallismortis 99% 81% B. atrophaeus 99% 98% B. mojavensis 98% 96% B. amyloliquefaciens 98% 83% B. lichenifornis - 81% B. thuringiensis - - B. cereus - - B. anthracis - -

2-3. Observation of physiological changes of identified strains

Bacillus atrophaeus TBM1652-1 is Bacillus atrophaeus Physiological changes were observed by culturing in TGY medium to ensure species.

As a result, as shown in Figure 5 it can be observed that after 6 days of culture to secrete the brown pigment of the brown series. Through this, it was confirmed that Bacillus atrophaeus TBM1652-1 is Bacillus atrophaeus species. In Korea and foreign countries, strains that are mainly isolated as chitinase and chitosanase producing bacteria are Pseudomonas sp., Aeromonas sp., Acinetobacter sp. And Klebsiella. sp.), Bacillus sp. and Arthrobacter sp., etc., but Bacillus atrophaeus strains have been found for the first time (Fukumori, F. et al., Nucleotides). sequences of two cellulase genes from alkalophilic Bacillus sp.strain N-4 and their strong homology.J. Bacteiol ., 168 , pp 479-485, 1986; Conet, P., D. et al., Cloning and expression in E. coli . of Clostridium thermocellum genes coding for amino acid synthesis and cellulose hydrolysis FEMS Microbiol Leett, 16, pp137-141, 1983;..... Hall, J. and HJ Gilbert, The nucleotide sequence of a carboxymethylcellulase gene from Pseudomonas fluorescens Mol Gen Genet , 213 , pp 112-117, 1988; Wong, WKR et al. , Characterization and structure of an endoglucanase gene cen A of Cellulomonas fumi.Gen ., 44 , pp315-324, 1986).

Therefore, the present inventors deposited Bacillus atrophaeus TBM1652-1, the strain identified above, at the Agricultural Biotechnology Research Institute on May 7, 2005, and the accession number is KACC 91163P.

Experimental Example 1. Cloning of Chitinase Gene of Bacillus Atrophe TBM1652-1

In order to clone the chitinase gene of the strain identified in Example 2 based on the chi gene of Bacillus subtilis known from GenBank (P1 5′-TGT AAG CGT TTT CCC CTT GTT GTC TTC AGT GT-3 ′, PCR was performed using P2 5′-CTC TCT CTT TAT CGT TTT CTA TC-3 ′). The initial denaturation step was performed 30 times 95 ° C 30 seconds, 60 ° C 30 seconds, 72 ° C 1 minute starting 95 ° C 1 minute, and the extension step was carried out for 10 minutes at the final 72 ° C. The PCR product obtained as a result of the PCR was cloned into pGEM T-easy vector (Promega, USA) to clone the chitinase gene. In the same way, a primer was prepared based on the csn gene of Bacillus amyloliquefaciens to clone the chitosanase gene.

As a result of the above experiment, the nucleotide sequence of chitinase was as described in SEQ ID NO: 3, and the chitinase gene was composed of 1818bp nucleotides encoding 606 amino acids, and its molecular weight was 66,660 daltons. It could also be confirmed through an emogram (see FIG. 6). It was observed that there is a signal peptide (30 amino acids) involved in secretion of the protein at the N-terminal side of the amino acid, and the actual amino acid N-terminal was SSDFQVW. As a result of analyzing the functional domain, it was possible to distinguish between chitinase A region, which is responsible for decomposing chitin, and COG3979 region, where chitin is involved in protein binding. The chitinase A domain region belongs to sugar hydrolase family 18 (see FIG. 7). It is known that glutamic acid is widely used as an electron donor as a characteristic of sugar hydrolase family 18, and it can be observed that glutamic acid is also contained in chitinase produced by TBM1652-1.

The homology of the amino acid sequence with other chitinases of other Bacillus family registered in GenBank was examined in the same manner as in Example 2-2, and the chitinase of B. subtilis was tested. genes and 98%, Bacillus piece nipo miss chitin dehydratase genes and are 81%, Bacillus load lance chitin dehydratase genes and are 67%, Bacillus seokyu lance (B. circurans) of (B. halodrans) to the (B. licheniformis) It was confirmed that the homology with the chitinase gene about 85% (see Fig. 8).

In addition, the nucleotide sequence of chitosanase is the same as SEQ ID NO: 5, consisting of a total of 800bp nucleotides encoding 200 amino acids, the molecular weight was estimated to be 88,000 daltons. Chitosanase, like chitinase, was also observed to have a signal peptide (38 amino acids) involved in protein secretion.

The amino acid sequence was tested for homology with other chitosanases of the genus Bacillus family registered in GenBank in the same manner as in Example 2-2. As a result, 99% of B. amyloliquefaciens and B. amyloliquefaciens were identified. , Bacillus subtilis showed about 88% homology.

Experimental Example 2. Measurement of chitinase enzyme activity

2-1. Enzyme Activity Measurement Method

The strain of the present invention identified in Example 2 measured the activity of the chitinase enzyme. Colloidal chitin was used as a substrate and was performed by modifying the DNS method. The enzyme reaction was mixed with 0.1 ml of the culture supernatant, 0.05 ml of 1% colloidal chitin and 0.1 ml of 10 mM Tris-HCl buffer (pH 7.5), followed by reaction at 60 ° C. for 30 minutes. After the reaction, 0.75ml of the DNS reagent was added, followed by reaction for 10 minutes in boiling water. Subsequently, after removing the remaining substrate by centrifugation, the reduced equivalent weight was measured using a UV-spectrophotometer (Ultraspecta 2100C, Amersham Co.). Quantitative curves were completed using chitobio (0-0.15 mg). One unit is defined as the amount of enzyme that produces 1 micromole of chitobio per minute.

2-2. Effect of temperature on the activity of chitinase

The effects of temperature on the activity of chitinase enzymes produced by Bacillus atropheus TBM1652-1 at various temperature conditions were observed. Colloidal chitin was used as a substrate and was performed by modifying the DNS method. The optimum temperature test is a method of measuring the reduction equivalent produced by the substrate reaction at various temperatures, and the whole reaction is carried out for 30 minutes using 10mM Tris-HCl buffer (pH7.5) in 10 ~ 80 ℃ and 10 ℃ steps. After performing, enzyme activity was measured by DNS method. In addition, after performing the pre-reaction for 30 minutes at each of the various temperatures, the reaction was carried out at 60 ℃ showed the maximum activity to perform a temperature stability test.

As a result of the optimum temperature test of chitinase, as shown in FIG. 9, it was observed that at the optimum temperature, 60 ° C shows a significantly higher relative activity than other temperatures. When the relative activity at 60 ° C. was 100%, 50 ° C. was about 50%, and at 70 ° C., about 30% was shown. In addition, as a result of the temperature stability test of chitinase, it was observed that the relative activity was 90% or more at temperatures up to 10-60 ° C, 80% at 70 ° C, and 60% at 80 ° C. .

2-3. Effect of metal ions on the activity of chitinase

To observe the effect of metal ions on the activity of chitinase, Ba ²⁺ (BaCl ₂ ), Ca ²⁺ (CaCl ₂ ), Co ²⁺ (CoCl ₂ ), Cu ²⁺ (CuCl ₂ ), K ⁺ ( KCl), Fe ³⁺ (FeCl ₃ ), Mg ²⁺ (MgSO ₄ ), Mn ²⁺ (MnCl ² ), Ni ²⁺ (NiCl ² ), Cs ⁺ (CsCl), Zn ²⁺ (ZnCl ₂ ), Hg Various metal ions such as ²⁺ (HgCl ₂ ), Na ²⁺ (NaCl ₂ ), EDTA, Li ⁺ (LiCl), and the like were used in the experiment. Each metal ion was made to a stock solution to 50mM, and then enzyme activity was measured by adding 1mM, 5mM and 10mM to the enzymatic reaction, respectively.

As a result of the experiment, Fe ³⁺ , Zn ²⁺ , Cu ²⁺ , Hg ²⁺ ions lowered the activity of the enzyme to 0% at all concentrations of 1 mM, 5 mM, and 10 mM as shown in FIG. 10. Of these ions, Hg ²⁺ ions have been reported to act as potent inhibitors of most chitinase enzymes. Ni ²⁺ ions were observed to decrease the enzyme activity by 20-30% compared to the control. Co ²⁺ , Ca ²⁺ , Ba ²⁺ , Li ⁺ , Mg ²⁺ and Cs ⁺ ions were observed to increase the activity of the enzyme from 110% up to 143% at each metal ion concentration. Among them, the enzyme activity was increased to 143% at 5 mM Co ²⁺ ion. The remaining ions, such as K ⁺ , Na ⁺ , and EDTA, had little effect on enzyme activity. From these results, it was confirmed that Fe ³⁺ , Zn ²⁺ , Cu ²⁺ and Hg ²⁺ ions act as potent inhibitors of the chitinase enzyme produced by B. atrophaeus TBM1652-1 . ²⁺ , Ca ²⁺ , Ba ²⁺ ions were confirmed to act to increase the activity of the enzyme.

Experimental Example 3 Analysis of Chitinase Enzyme Products by Thin Membrane Chromatography

The chitinase produced by the strain of the present invention obtained in Example 2 was reacted with colloidal chitin and various chitin oligosaccharides (NAG1 to NAG6) as a substrate, and as a result, the product was subjected to silica gel thin film chromatography. layer chromatography (TLC). Aliquot the reaction product twice in 5, 15, 30, 60, 120 and 180 minutes in the reaction solution and drop onto a silica gel plate (Kieselgel 60; Merck Co., Berlin, Germany) and then n-butanol: It developed using the mixed developing solvent of methanol: 25% ammonia water: dH2O = 5: 4: 2: 1 (vol / vol / vol / vol). After the development, the aniline-diphenylamine reaction solution (4 mL of aniline, 4 g of diphenylamine, 200 mL of acetone, and 30 mL of 85% phosphoric acid) was sprayed onto a silica gel plate and baked at 180 ° C. for 3 minutes to observe the reaction product ( Yoon HG et al., Thermostable Chitosanase from Bacillus sp.strain CK4: Its purification, characterization, and reaction patterns.Biosci Biotechnol Biochem ., 65 , pp802-809, 2001; YJ Choi et al., Purification and characterization of chitosanase from Bacillus sp.strain KCTC 0377BP and its application for the production of chitosan oligosaccharides.Appl Environ Microbiol ., 70 , pp4522-4531, 2004).

As a result of performing the experiment, when chitotetraose (NAG4), chitopentose (NAG5) and chitohexose (NAG6) were used as substrates, chitobioses (NAG2) were used as the main reaction products, as shown in FIG. At the same time, it was confirmed that a small amount of N-acetylglucosamine (NAG) was produced. When chitotriose (NAG3) was used as a substrate, it was observed that N-acetylglucosamine (NAG) and chitobiose (NAG2) were produced as reaction products. However, it has been observed that N-acetylglucosamine (NAG1) and chitobioses (NAG2) cannot be used as reaction substrates. In addition, as shown in FIG. 11B, when a colloidal chitin was used as a substrate, a large amount of chitobiose (NAG2) and a small amount of N-acetylglucosamine (NAG) were produced. These results demonstrate that chitinase produced by B. atrophaeus TBM1652-1 produces chitobiose (NAG2) as a major reaction product.

Experimental Example 4. Antifungal Activity Test

In order to confirm whether the strain of the present invention has antifungal activity against phytopathogens, it was observed using a solid medium diffusion method and a paper disk.

39 g of potato dextrose agar was added to 1 L of sterile water, sterilized at 121 ° C. for 15 minutes using an autoclave, then cooled, poured into plates, and cooled to prepare a PDA medium. Botrytis cinerea , which causes phytopathogenic fungus gray fungus, Fusarium oxysporum , which causes vine pecking disease, root stalk and branching disease Rhizoctonia solani (Rhizoctonia solani), inoculated with bus Clermont Loti Nias Klee as Tio volume (Sclerotinia sclerotiorum), aspartate going across Scotland and this (Aspergillus niger), which causes black mold disease that causes gyunhaekbyeong week The culture was observed at 30 ° C. Botrytis cinerea , Fusarium oxysporum , Raiztonia solani by chitinase produced in B. atrophaeus TBM1652-1 strain of the present invention Rhizoctonia solani and sclerotinia sclerotiorum were observed to form a constant inhibitory ring that inhibits the growth of chitin, which constitutes the cell wall of plant pathogenic fungi.

As a result, as shown in Fig. 12, Botrytis cinerea , Fusarium oxysporum , Rhizoctonia solani , Sclerotinia sclerotinia Certain inhibitors inhibiting the growth of plant pathogenic fungi, such as sclerotiorum ), and the isolate B. atrophaeus TBM1652-1 were identified as strains that inhibit the growth of plant pathogenic fungi. Could.

As described above, the present invention produces chitinase or chitosanase, and Bacillus atrophaeus TBM1652-1 having low molecular chitin or chitosan oligosaccharide-producing ability and antifungal activity by these enzymes. As related to KACC 91163P, the chitinase or chitosanase produced by the strain of the present invention is insoluble and indigestible after digestion of chitin or chitosan of a polymer of various molecular weights, thereby producing chitin oligosaccharides or chitosan oligosaccharides, respectively. By increasing the absorption rate of the plant to express various physiological functions, and also by inhibiting the growth of plant pathogens to decompose chitin or chitosan, which forms the cell wall of plant pathogens, the present invention provides Botrytis cinerea, Fusarium oxyspo It can be used as a biocontrol agent for Fusarium oxysporum, Rhizoctonia solani or Sclerotinia sclerotiorum.

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Claims (3)

Bacillus atrophaeus produces a chitinase encoded by the nucleotide sequence of SEQ ID NO: 3 or a chitosanase encoded by the nucleotide sequence of SEQ ID NO: 5, and has a small molecule chitin or chitosan oligosaccharide-producing ability by these enzyme activities ) TBM1652-1 KACC 91163P. A biological control agent comprising the Bacillus atropheus TBM1652-1 KACC 91163P of claim 1 having antifungal activity against plant pathogens. 3. The plant pathogen of claim 2, wherein the plant pathogen is Botrytis cinerea, Fusarium oxysporum, Rhizoctonia solani or Sclerotinia sclerotium. Sclerotinia sclerotiorum).

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