KR100685519B1 - Multifunctional oligotrophic bacteria Burkholderia cepacia CR2 strain isolated from ginseng field producing antibiotic enzymes and industrial enzymes containing antibiotic activation - Google Patents

Abstract

A novel multifunctional oligotrophic bacteria, Burkholderia cepacia CR2 strain, isolated from ginseng root is provided to be able to produce antibiotic enzymes and industrial enzymes and have antibiotic activity. The Burkholderia cepacia CR2 strain produces antibiotic enzymes such as chitinase and industrial enzymes such as protease and lipase, includes 16S rDNA sequence of SEQ ID : NO. 1 having antibiotic activity, and is deposited as a deposition number of KACC 91204. To inhibit growth of pathogenic microorganism selected from the group consisting of Candida albicans, Collectrotrichum gloeosporioides and Xanthomonas, the Burkholderia cepacia CR2 strain or a culture material thereof is used.

Description

Multifunctional oligotrophic bacteria Burkholderia cepacia CR2 strain isolated from ginseng field, producing antibiotic producing antibacterial and industrial enzymes and isolated from ginseng roots with antimicrobial activity. enzymes and industrial enzymes, containing antibiotic activation}

1 is a classification diagram systematically classifying a multifunctional low-nutrition bacterium Berkholdereria Sephacia CR2 strain provided according to the present invention.

Figure 2 is an electron micrograph of a multifunctional low nutritional bacterium Berkholdereria Sephacia CR2 strain provided according to the present invention.

3 is a diagram showing the results of assaying the enzyme activity of the multifunctional low-nutrition bacteria Berkholdereria Sephacia CR2 strain provided according to the present invention.

Figure 4 is a view showing the results of assaying the phytopathogen inhibitory activity of the multifunctional low-nutrition bacteria Berkholdereria Sephacia CR2 strain provided according to the present invention.

The present invention relates to a strain of Berkholderia cepacia CR2, which is a multifunctional low-nutrition bacterium produced from a new ginseng root zone having antibacterial and industrial enzymes and having antibacterial activity. More specifically, Berkholderia Sephacia CR2 which produces various enzymes such as chitinase, protease and lipase, and inhibits the growth of pathogenic microorganisms using the strain and its culture. It relates to a strain (Accession Number KACC 91204). Unlike the conventional strains, the strain produces a variety of industrially available enzymes as multifunctional low-nutritive strains, among which chitinase can be used to control pathogenic microorganisms as antibacterial enzymes, and proteases and lipases as industrial enzymes. It can use for the used processing process. In addition, when used as a pathogen microorganism control agent using the above-mentioned Berkholderia Sephacia CR2 strain itself and its culture medium, Candida albicans , Ginseng anthrax, Colletotrichum gloeosporioides And Xanthomonas , which is a rice leaf blight bacterium, strongly inhibits growth.

With the recent expansion of agricultural imports, increasing the competitiveness of agriculture is becoming more important than ever. The continuous increase in the use of chemical fertilizers is a real problem that is not only a problem in Korea. The international community's efforts to avoid the overuse of chemical fertilizers and the resulting calamities are also intensifying. The United Nations Environment Development Council (UNCED) encourages countries to reduce their use of chemical pesticides by 25 percent, and Korea has announced that by 2010, it will reduce its consumption by 40 percent for chemical fertilizers and 50 percent for synthetic pesticides. Since it is inevitable to develop alternative pesticides to significantly reduce the amount of chemical pesticides and synthetic pesticides while maintaining agricultural productivity, biological pesticides are the only alternative to chemical fertilizers and synthetic pesticides. Thus, since the 1980s, some of the world's leading companies have invested heavily in the development of biopesticides with huge returns. In Korea, biopesticide sales account for only 0.16% of the total pesticide market, but the share of biopesticides is expected to increase rapidly.

Studies on hydrolases capable of breaking down chitin by biocontrol using microorganisms are being actively conducted. Chitin β- (1,4) N - the composition of the shell, fungal cell wall of various types of shellfish as multimers of acetyl -D- glucosamine (β- (1,4) N -acetyl- D- glucosamine). Enzymes that decompose chitin include hydrolyzing the N -acetyl-1,4-glucosamine bond of chitin to produce chitin oligosaccharides, endochitinase, and N, N' -diacetylchitobiose ( N the better exo kitty producing acetylglucosamine (N- acetylglucosamine) claim (exochitinase), etc. -, N '-diacetylchitobiose) via the Quito (chitobiase), from the non-reducing terminus of the chitin Quito via the (chitobiase) or N to break up Reported.

Most of the chitinase microorganisms isolated from the ocean and soil are actinomycetes, among which Streptomyces griseus , Streptomyces orientalis , Streptomyces erythraeus A chitinase purifying enzyme is commercially available from Sigma. Reports on the identification of chitin-degrading bacteria have been relatively poor , with Achromobacter, Aeromonas, Chromobacterium, Clostridium, Erwinia, and Serratia ( such as Serratia), Pseudomonas (Pseudomonas), and Vibrio (Vibrio) were reported. In particular, 30 strains isolated by Enterobacter liquefaciens CDC2149-57, Serratia marcescens QM B1466, and Monreal and Reese It is known as Naase producing strain. However, despite these many findings, there are only a few practically available materials due to their low activity, toxicity and resistance. Recently, as various methods for solving such problems have been sought, the separation and culturing methods of rare microorganisms have been examined, and the metagenome research for separating chitinase from non-cultured microorganisms has been actively conducted.

Lipase enzymes are mainly used in the food industry, feed industry, alternative energy industry, pharmaceutical industry, detergent industry, etc.The main producing microorganisms are various kinds such as bacteria, yeast, mold, etc. Candida and the bacterium Burkholderia were used. Lipases have been applied in various fields, and new lipase producing strains have been studied to meet such demands.

Currently, sluggish production of lipase in Korea has difficulty in process development but has difficulty in securing excellent strains. Since the action and titer of the produced enzyme are very different depending on the type of microorganism producing lipase, there is a need to find a new microorganism with lipase-producing ability and research on the production technology of the enzyme using the same is very important.

There are various microorganisms in the soil, but most of them are not separated. Most of the microorganisms that have been isolated and studied so far are rapidly growing under relatively high nutritional conditions, and many microorganisms that grow slowly under low nutritional conditions exist in the natural environment. There is active research on oligotrophic bacteria. These low-nutrition bacteria are difficult to isolate, and even if separated, even if the culture is not well or even disappears during the culture, the situation is not fully elucidated with regard to classification or physiological characteristics.

Low nutrient microorganisms are rarely used for screening bioactive substances because they are not well separated in normal nutrient medium. Therefore, the search for physiologically active substances using low-nutrition bacteria is likely to develop unique new compounds that are structurally different from the known substances, or lead substances for synthesis.

Accordingly, the present inventors selected a multifunctional low-nutrition bacterium Berkholdereria Sephacia CR2 strain isolated from ginseng root zone soil, and found that the strain has excellent production ability such as chitinase, which is chitinase antibacterial enzyme, or protease and lipase, which are industrial enzymes. It was found that the antimicrobial activity against phytopathogens is excellent, to complete the present invention.

Accordingly, it is an object of the present invention to produce an antibacterial enzyme and an industrial enzyme, and to provide a novel multifunctional low-nutrition bacterium Berkholdereria Sephacia CR2 strain isolated from the ginseng root zone having antimicrobial activity.

In order to achieve the above object, the present invention produces an antibacterial enzyme and an industrial enzyme, and provides a multifunctional low-nutrition bacterium Berkholderia Sephacia CR2 strain (Accession No. KACC 91204).

Hereinafter, the present invention will be described in detail.

The present invention is to extract a sample from the ginseng root zone soil of three years of ginseng cultivated soil of various soils of the Geumsan ginseng cultivation area to isolate microorganisms having various enzyme production and antimicrobial activity, the microorganism of the present invention is Berkholderia I was sympathized with myself. The present inventors named the microorganism Berkholderia Sephacia CR2 strain, and the strain was deposited with the Korea Agricultural Microbial Resources Center on November 30, 2005 and received the accession number (KACC 91204).

The present inventors assayed the enzyme production capacity in the selection medium to assay the enzyme activity of the Berkholderia Sephacia CR2 strain of the present invention. The Berkholderia Sephacia CR2 strain was found to have the function of simultaneously producing antimicrobial enzymes such as chitinase and industrial enzymes such as proteases and lipases (FIG. 3).

In addition, in order to confirm the antimicrobial activity spectrum of the antimicrobial material produced by the Berkholderia Sephacia CR2 strain of the present invention, a number of fungi and bacteria causing serious diseases in plants as indicative bacteria in vitro (in vitro) antimicrobial activity The assay was performed. As shown in Figure 4, the antimicrobial material of the present invention is a strong antimicrobial activity against Candida albicans , Ginseng anthracnose, Colletotrichum gloeosporioides and rice leaf blight, etc. Showed.

Hereinafter, the structure of the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited thereto.

Example 1 Isolation and Identification of Burkholderia Sephacia CR2 (KACC91204) Strains of the Invention

Samples were taken from ginseng roots of three-year-old ginseng soils of various soils in the Geumsan ginseng planting area, placed in polyethylene vinyl bags, and transported to the laboratory. Next, 10 g of bee extract, 10 g of peptone, 5 g of sodium chloride (NaCl), and 1000 ml of distilled water and nutrient broth (NB) adjusted to pH 7.0 to 7.2 from the sample. With badge 10 ^-1 times, 10 ^-2 times, 10 ^-3 , Various soil bacteria isolated using four kinds of diluted nutrient broth (DNB) media diluted in distilled water at 10 ^-4 times were incubated in DNB plate media.

To separate the pure water of a low nutrient bacteria, DNB plate body from the colonies formed on the medium by separating the pure soil bacteria, respectively, after the pre-culture broth of strain on DNB 10 ㎖ 10 ^-1 NB, 10 of the normal concentration Inoculation was carried out at ⁻² NB, 10 ⁻³ NB and 10 ⁻⁴ NB medium to determine the proliferative capacity after incubation for 1 week at 28 ° C. Among the bacteria that could not be ^grown in normal concentration of NB medium, bacteria growing in medium diluted to 10 ^{-1 to} 10 ^-4 times were purified as low trophic bacteria. Purely isolated low-trophic bacteria were stored in -80 ° C. in DNB medium diluted 10 ^{−1 to} 10 ⁻⁴ times with 20% glycerol added.

For purely isolated low-trophic bacteria Antimicrobial activity was measured. Among them, Candida albicans , the ginseng anthrax bacterium, Colletotrichum gloeosporioides , and the rice plant leaf bacterium Xanthomonas , etc. Berkholderia Sephacia CR2 strains were selected which have the ability to produce various enzymes such as proteases and lipases.

In order to classify the Berkholderia Sephacia CR2 strain of the present invention, 16S rDNA was amplified by PCR to determine the base sequence (SEQ ID NO: 1).

Primers used to amplify 16S rDNA were 27F (5'-AGAGTTTGATCCTGGCTCAG-3 '; 8-27bp of E. coli 16S rRNA portion; SEQ ID NO: 2) and 1492R (5'-AAGGAGGTGATCCAGCCGCA-3'; E.coli 16S rRNA Part 1510-1492bp; SEQ ID NO: 3).

PCR was carried out according to the following conditions. After reacting for 5 minutes at 94 ° C, repeat 1 minute for 94 ° C denaturation, 1 minute for 55 ° C annealing, 1 minute for 72 ° C extension, and finally extend for 10 minutes at 72 ° C. Was carried out. Thermal cycler, a DNA amplifier, was used for Perkin Elmer (GeneAmp PCR system 9700; Applied Biosystems). The product was electrophoresed for 30 minutes at 100 V, 25 mA in 1% agarose gel, 0.5 ㅧ TBE buffer (0.045 M Tris-borate, 0.001 M EDTA), and stained with EtBr (ethidium bromide) for 15 minutes. Confirmed below, and purified by Qiagen PCR Purification Kit (Qiagen Inc.,).

Nucleotide sequence determination of 16S rDNA includes 27F (5'-AGAGTTTGATCCTGGCTCAG-3 '; 8-27 bp of E. coli 16S rRNA; SEQ ID NO: 2), 261F (5'-AGCTAGTTGGTGGGGT-3'; E. coli 16S rRNA 246-261 bp; SEQ ID NO: 4), 530F (5'-GCCAGCMGCCGCGC-3 '; 515-530 bp of E. coli 16S rRNA portion; SEQ ID NO: 5), 780F (5'-GATTAGATACCCTGGTAG-3'; E.coli 16S rRNA Part 786-803bp; SEQ ID NO: 6), 1114F (5'-GCAACGAGCGCAACCC-3 '; 1099-1114bp of E. coli 16S rRNA part; SEQ ID NO: 7), 342R (5'-CTGCTGCSYCCCGTAG-3'; E.coli 357-342 bp of 16S rRNA portion; SEQ ID NO: 8), 517R (5'-ACCGCGGCKGCTGGC-3 '; 531-517 bp of E. coli 16S rRNA portion; SEQ ID NO: 9), 787R (5'-TACCAGGGTATCTAAT-3'; E 802-787 bp of coli 16S rRNA portion; SEQ ID NO: 10), 956R (5'-GGCGTTGTGTCSAATTAA-3 '; 973-956 bp of E. coli 16S rRNA portion; SEQ ID NO: 11), 1100R (5'-AGGGTTGCGCTCGTTG-3' Using a primer of 1115-1100bp of E. coli 16S rRNA portion; SEQ ID NO: 12), 1492R (5'-AAGGAGGTGATCCAGCCGCA-3 '; 1510-1492bp of E. coli 16S rRNA portion; SEQ ID NO: 3) After sequencing cycles using the Microseq ^™ 16S rRNA Gene Kit (Applied Biosystems), the nucleotide sequence was determined by ABI PRISM 310 Genetic Analyser (Applied Biosystems). The homology of the 16S rDNA sequences obtained through the experiment was investigated in DDBJ / EMBL GenBank DNA databases. The alignment of each nucleotide sequence was performed using the Clustal X (version 1.81) program package, and phylogenetic location of low nutrient bacteria was determined based on the neighborhood binding method.

(FIG. 1) is a diagram comparing and classifying the nucleotide sequences of 16S rDNA of the microbial strain of the present invention. As shown in Figure 1, the microbial strain of the present invention is a bacterium belonging to the same group as Burkholderia It turned out.

Burke holde Liao (Burkholderia) is a representative gram-negative of a bacillus, DNB viscous sex colony formation (FIG. 2) Optimal growth temperature is 28 ℃ low nutrient bacteria on the solid medium, such as Pseudomonas (Pseudomonas). In accordance with the above results, the present inventors have found that the microbial strain of the present invention is in Burkholderia . After confirming that the strain belongs to the strain Burkholderia cepacia CR2 ( Burkholderia cepacia CR2) was named on November 30, 2005 by the Korea Agricultural Microbiological Resources Center and received the accession number (KACC 91204) .

Example 2 Industrial Enzyme Production Capacity Assay

In order to assay the enzyme activity of the Berkholderia Sephacia CR2 strain of the present invention, the enzyme production capacity was assayed in selection medium.

Skim milk in basal medium containing 0.02% bee extract, 0.01% NaCl, 0.02% peptone, 0.01% yeast extract, and 1.2% agar for protease production assay After inoculation of low nutrient bacteria using a plate medium containing 1% milk), the enzyme production capacity was assayed with or without formation of a clear zone formed around the cells.

TBN emulsion (glyceryl tributyrate 5ml; NaCl 200mM, CaCl ₂ 10mM, 5% (w / v) 50ml gum arabic and 0.02% bee extract for assaying lipase production capacity) After inoculation of low nutrient bacteria using a plate medium containing 450 ml of a basal medium containing 0.01% NaCl, 0.02% peptone, 0.01% yeast extract, and 1.2% agar. Enzyme production capacity was assayed with or without clear zone.

To assay the ability of chitinase, basal medium containing 0.02% bee extract, 0.01% NaCl, 0.02% peptone, 0.01% yeast extract, and 1.2% agar. To this, a medium containing 1% (w / v) wet colloidal chitin was used. After inoculating low nutrient bacteria into a colloidal chitin plate medium, the enzyme production capacity was assayed with or without the formation of a clear zone formed in the cell strain.

As a result of the above experiments, it was confirmed that the Berkholderia Sephacia CR2 strain of the present invention had the function of simultaneously producing antibacterial enzymes such as chitinase and industrial enzymes such as protease and lipase. (FIG. 3)

Example 3 Antimicrobial Activity Spectrum Assay of Burkholderia Sephacia CR2 Strain

In order to confirm the antimicrobial activity spectrum of the antimicrobial substance produced by the Berkholderia Sephacia CR2 strain of the present invention, an in vitro antimicrobial assay was carried out with a number of fungi and bacteria that cause serious diseases in plants. Was performed.

Antimicrobial activity against Colletotrichum gloeosporioides , also known as anthrax, was determined by spores in PDA medium (0.4% potato starch; 2% dextrose; 0.7% LE agaro). After incubation in the center of the LE (gaga), 20ul of CR2 strain was dropped on a paper disk and incubated at 28 ° C.

The antimicrobial activity of Xanthomonas , a rice leaf blight bacterium, was investigated in YGC agar medium (1% D-glucose; 0.5% peptone; 0.5% Yeast extract; 0.3% juicy (BEF extract; 0.7% LE agarose) was mixed with Santomonas (Xanthomonas) and 20ul loading of CR2 bacteria was observed by incubating at 28 ℃.

Candida albicans is YM agar medium (Yeast extract 0.3%, Malt extract 0.3%, Peptone 0.5%, Dextrose 1%, LE agarose LE agarose) was dissolved 0.7%) and mixed with the culture in YM broth, 20ul loading of CR2 bacteria was incubated at 28 ℃ and then tested for antimicrobial activity.

As shown in (Fig. 4), the antimicrobial materials of the present invention is Candida albicans (Candida albicans), ginseng anthrax a call rekto tree Kiwoom glow Eos Poly cucumber-in death (Colletotrichum gloeosporioides) and rice huinip blight fungus Santo Pseudomonas ( Xanthomonas ) and the like showed a strong antimicrobial activity.

As described above, the Berkholderia Sephacia CR2 strain of the present invention, unlike conventional strains, produces a variety of industrially available enzymes as multifunctional low-nutrition strains, among which chitinase is used as an antibacterial enzyme to remove pathogenic microorganisms. Protease and lipase may be used for controlling, and may be used as industrial enzymes in processing processes using the same. In addition, Candida albicans , Colletotrichum gloeosporioides and Santomonas when used as a pathogen microorganism control agent using the above-mentioned Berkholderia Sephacia CR2 strain itself and its culture solution (Xanthomonas ) and the like shows excellent efficacy to strongly inhibit the growth.

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Claims (3)

A multifunctional low-nutrition bacterium Burkholderia cepacia CR2 strain (Accession No. KACC 91204) which produces antibacterial enzymes and industrial enzymes and has 16S rDNA sequence of SEQ ID NO: 1 having antibacterial activity. According to claim 1, wherein the antimicrobial enzyme is chitinase (chitinase), industrial enzymes are protease (protease) and lipase (lipase) multifunctional low-nutrition bacteria Burkholderia cepacia ( Burkholderia cepacia ) CR2 strain. Candida albicans , Colletotrichum gloeosporioides and Xanthomonas , etc., using the Burkholderia cepacia CR2 strain of claim 1 and its culture. Method of inhibiting the growth of pathogenic microorganisms selected from the group consisting of.

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