KR101477886B1 - Bacillus spp., identified from lugworm and microbial cleaning agent. - Google Patents

Abstract

The present invention is based on the finding that 31 colonies selected according to the color and shape of a colony emerging from a MA medium, which are endogenous to Perinereis aibuhitensis , After culturing, 11 strains contained in the genus Bacillus were isolated based on the 16S rRNA nucleotide sequence. Analysis of the diversity of 11 isolates contained in Bacills genus (genus) Bacills algicola, Bacills anthrasis , Bacills aquimaris, Bacills barbaricus, Bacills hwajinpoensis, Bacills nanhiensis, Bacills vietnamensis including both spp Bacillus seven types (species). The lug ( Perinereis aibuhitensis ) and two species of Bacillus strains isolated from the worms are highly resistant to salinity and protein, carbohydrate, and lipid degradation, and can be used to purify the tidal environment contaminated with organic matter.

Description

(Bacillus spp., Identified from lugworm and microbial cleaning agent.) Using a heterologous Bacillus strain identified from a nematode.

The present invention relates to two kinds of Bacillus (Bacillus) and the new strain of the organic matter using the same cleaning agent contained in the Bacillus with a salinity tolerance that endogenous the lug (Perinereis aibuhitensis) known to be a very good resolution in the coastal organisms flats.

The intertidal zone is continuously exposed to environmental pollution due to its proximity from human settlements and is the most sensitive area in the coastal ecosystem. Therefore, understanding of the degradation of degradable materials by organisms in the intertidal zone is very important from an ecological point of view.

Because of its ability to adapt to stressful environmental conditions, researchers are encouraged to use these worms as an environment watcher, as well as a potentially important ecological species for monitoring programs .

Many benthic organisms that are fed with filtration are commonly used to remove and purify organics from water in homes and ecosystems, and several species of lupins , including Perinereis sp., Are known to be used uncontrolled in shrimp farm environments. In the study of the effects of large invertebrates on bacterial communities in the intertidal zone, oysters, clams, and mollusks are known to be the main repository of Vibrio spp.

(Gili and Coma, 1998; Orejas et al., 2000; Prieur et al., 1990) have been extensively studied in relation to the nutritional function of airborne germs, including suspension worms, Sabella spallanzanii has been reported to have the ability to accumulate and concentrate bacteria very effectively from the surrounding environment (Stabili et al., 2006b). In particular, intestinal microbes in animals are composed of hundreds of different bacteria that can enhance digestion and absorption of nutrients or increase body resistance to infectious diseases (Tannock, 1998; Walker and Duffy, 1998).

In general, data obtained from cultured bacteria do not represent whole bacteria present in a specific environment, so that interpretation of microbial diversity is very limited. In order to overcome this, recently, genomic DNA was isolated directly from the environment to construct a metagenomic library After 16S rRNA sequencing, microbial diversity has been actively studied.

Studies of bacteria by these methods not only provide meaningful information on the role of heterotrophic bacteria in biogeochemical cycling as well as potential metabolic activity, but also in that many species belonging to Bacillus can be cultured, A study on Korea will also provide meaningful value.

Based on this, the present invention focused on analysis of Bacillus bacterium diversity in coastal tidal flat earthworms. 31 colonies selected according to the color and shape of colonies grown on MA medium were separately selected, Bacillus strains were identified based on rRNA base sequences.

31 colonies were screened, purified, and identified on the basis of 16S rRNA sequence. As a result, 11 strains contained in Bacillus were isolated, and Bacillus Results in Bacillus know coke (Bacills algicola) Analysis of the diversity of 11 isolates contained the (genus), Bacillus anthraquinone system (Bacills anthrasis), Bacillus Aquitania Maris (Bacills aquimaris), Bacillus Barbary kusu (Bacills barbaricus), Bacillus Hwajinpo N-Sys (Bacills hwajinpoensis), Bacillus nanhi N-Sys (Bacills nanhiensis), Bacillus Viet Nam N-Sys (Bacills of Bacillus spp vietnamensis) including all seven kinds (species). The lug (Perinereis aibuhitensis ).

Korean Patent Publication No. 10-08689010 relates to a microorganism preparation comprising a Bacillus spp. Strain having an antagonistic ability against fungi, and more particularly, to a microbial agent containing an antagonistic bacillus spp. A Bacillus amyloliquefaciens A-2 strain, a Bacillus amyloliquefaciens A-7 strain, a Bacillus amyloliquefaciens A-7 strain, a Bacillus amyloliquefaciens A-7 strain, a Bacillus amyloliquefaciens A- And their formulation techniques are disclosed. Korean Patent Publication No. 10-10863670 relates to a new strain Bacillus ariabatai LS9 having plant growth promoting activity and its use and more specifically to a Bacillus ariabatai LS9 strain having a plant growth promoting activity or a culture of a strain And a method for promoting plant growth using the strain are disclosed. Korean Patent Registration No. 10-08507430 discloses a Bacillus subtilis strain having antimicrobial activity against Staphylococcus aureus and a prodrug using the same. However, the above-mentioned strains are species derived from land plants or animals, , But it is not identified on the basis of marine animals with salinity tolerance.

Bacillus species are known to occupy microbial populations of marine organisms in the marine environment (Kennedy et al., 1998; Hovda et al., 2007) and are commercially available as gram-positive, (Ziaei-Nejad et al., 2006; Gatesoupe, 1999), which is the most commonly used probiotics. In particular, B. subtilis is known to be used as food for many organisms, as well as the ability to produce proteases and other enzymes that can contribute to the host's natural digestion activity (Ziaei-Nejad et al., 2006; Verschuere et al., 2000). B. subtilis is known to prevent colonization of host organisms by potential pathogenic microorganisms through production of antimicrobial agents or competition for nutrition and space (Vaseeharan and Ramasamy, 2003). B. licheniformis has been shown to produce antiviral agents (Arena et al., 2006) and B. pumilus (Sugita et al., 1998), commonly found in marine fish, is associated with digestive activities such as extracellular proteases, amylases, and cellulases (Ghosh et al., 2002). In addition, B. subtilis has been shown to help trout growth and immune resistance when administered to trout, such as B. licheniformis or B. pumilus (Raida et al., 2003; Bagheri et al., 2008). Several species of Bacillus have been proposed as antibiotic substitutes in shrimp farming (Banerjee et al., 2007).

The present invention is to identify novel Bacillus strains from Bacillus (Bacillus) strain, which is known closely associated with the digestion and biological immune nutrient Microbe that endogenous to the lug (Perinereis aibuhitensis) known to be very excellent organic resolution in coastal tidal And to provide an organic cleaning agent which can be useful for purifying the environment contaminated with organic substances by verifying the protein, carbohydrate and lipid decomposition ability of the new strains.

In the present invention, a lignite ( Perinereis aibuhitensis ) were analyzed based on the culture method. Bacillus sp. Bacillus sp. Bacillus sp .

31 colonies were selected and sorted according to the color and shape of colonies grown on MA medium. Then, 11 strains contained in Bacillus were isolated from 16S rRNA sequences. Results Bacillus know coke (Bacills algicola) Analysis of the diversity of 11 isolates contained in Bacills genus (genus), Bacillus anthraquinone system (Bacills anthrasis), Bacillus Aquitania Maris (Bacills aquimaris), Bacillus Barbary kusu (Bacills barbaricus), Bacillus Hwajinpo N-Sys (Bacills hwajinpoensis), Bacillus nanhi N-Sys (Bacills nanhiensis), Bacillus Viet Nam N-Sys (Bacills of Bacillus spp vietnamensis) including all seven kinds (species). The lug (Perinereis aibuhitensis ).

The systematic analysis of the 16S rRNA sequences of the 11 Bacillus strains revealed in the present invention was broadly classified into five groups. Among them, Bacillus strain Bacillus anthracis (hereinafter referred to as "CBW4") and Bacillus virginaceus strain (Hereinafter referred to as "EBW4").

Both the CBW4 and EBW4 showed DNAse activity and protease activity by degrading casein. In addition, CBW4 had enzymatic activity to degrade strach, but EBW4 showed no starch degradation, and both CBW4 and EBW4 showed lipase activity. In particular, the strains CBW2 and CBW4 are Bacillus vietnamensis 15-1 ^T and < RTI ID = 0.0 &^gt; Bacillus anthracis ATCC 14578 ^T and , And 90.7% and 94.2%, respectively. EBW4 showed homology with Bacillus hwajinpoensis SW-72 ^T And 96.8% homology, indicating a new species.

In the present invention, strains CBW2 and CBW4 isolated from the worms of the present invention have excellent protein, carbohydrate and lipid-resolving ability and are resistant to salt, so they may be useful for purifying the tidal-environment contaminated with organic matter.

Figure 1 shows a photograph of a snakehead.
Fig. 2 is a schematic diagram showing the identification process of a Bacillus strain derived from a nematode.
Fig. 3 shows the nucleotide sequence of 16S rDNA gene of Bacillus strain CBW4 isolated and isolated from the worms.
Fig. 4 shows the nucleotide sequence of 16S rDNA gene of Bacillus strain EBW4 isolated and isolated from the nematode.
Fig. 5 is a schematic diagram showing the systematic relationship of the Bacillus strain derived from the identified wormwood.
Figure 6 shows the patented microorganism deposit of the genus Bacillus (Accession No .: KACC91722P).
Figure 7 shows the patented microorganism deposit of the genus Bacillus (Accession No .: KACC91749P).
Fig. 8 is a graph showing the distribution of Bacillus sp. Is a schematic view showing a method for producing a microorganism preparation from a strain.
Figure 9 shows an example of a microbial preparation prepared using a Bacillus sp.

The present invention relates to a novel heterologous strain having a salt resistance which is cultured from a strain of the genus Bacillus sp. Of KACC91722P and KACC91749P identified from a nematode, and having at least one organic compound resolution selected from protein, carbohydrate, lipid and surfactant The present invention provides a microbial purification agent for purification of tidal flats using a Bacillus strain.

The present invention also provides a microorganism preparation for purification of tidal flats containing the culture of the strain as an active ingredient and a method for decomposing any one or more organic substances selected from protein, carbohydrate, lipid and surfactant immobilized on the tidal flat by using the strain .

The new strains CBW2 and CBW4 of the Bacillus subtilis strain CBW2 and CBW4 of the present invention were isolated and identified for the first time by the present inventors and based on the culture method, microorganisms endemic to the lagoon ( Perinereis aibuhitensis ) The first isolates were identified.

The 16S rDNA sequences of the isolated strains were analyzed by molecular genetic analysis. As a result, the systematic analysis of the 16S rRNA sequences of eleven Bacillus strains revealed that they were divided into five groups, The medium strains CBW2 and CBW4 are Bacillus vietnamensis 15-1 ^T and < RTI ID = 0.0 &^gt; Bacillus anthracis ATCC 14578 ^T and , And 90.7% and 94.2%, respectively. EBW4 showed homology with Bacillus hwajinpoensis SW-72 ^T 96.8% homology, indicating that it was a new species.

Therefore, the present inventors named the newly isolated strain of the present invention, which has the 96.8% homology with Bacillus hwajinpoensis SW-72T, as "Bacillus sp. EBW4", and as of October 26, 2012 And deposited with the National Center for Agronomy and Genetic Resources (KACC) of the National Academy of Agricultural Science and received the deposit number KACC91749P.

The inventors of the present invention named the new strain of the present invention as "Bacillus sp. CBW4" and deposited it on the National Institute of Agricultural Science and Technology (KACC) on June 8, 2012 and deposited the deposit number KACC91722P .

Hereinafter, the method for identifying a new strain in the barnacles and the organic matter decomposition ability of the strain will be described with reference to examples.

1 Identification and isolation of strains from the marshes

1.1 Strain isolation and culture conditions

In the present invention, the lupine ( Perinereis aibuhitensis ) were harvested from the paddy field in Suncheon Bay. FIG. 1 is a photograph of a cockroach, and FIG. 2 is a schematic diagram showing the identification process of a Bacillus strain derived from a cockroach.

In order to isolate the microorganisms present in the barn, the live worms were placed in a conical tube containing 50 mL of BM buffer (Mikesell et al., 1993), washed three times in the vortex, and again briefly in 70% And washed. The washed ridge was cut with a sterilized razor blade and then cut into several very short segments. The ridge was vortexed in a conical tube containing 50 mL of BM buffer, and the endogenous microorganism of the rug was allowed to escape into the buffer.

The pellet was collected by centrifugation at 12,000 rpm, and 2 mL of BM buffer was added thereto. The microbial cells were inoculated into 100 μl of the solid Bacto marine agar 2216 (MA) medium. MA medium was used to increase the microbial diversity that can be analyzed by the culture method using both the 1:10 and 1: 2 diluted medium and the undiluted medium at the same time. LB or LB agar medium was used as the primary culture medium, and detailed culture conditions were detailed in the growth test according to the environmental condition, because all strains isolated were able to grow in LB .

1.2. Total genomic DNA isolation and purification

Each strain was inoculated in 50 mL of LB medium, cultured at 30 ° C. and 180 rpm for 24 hours, and harvested by centrifugation (6,300 ㅧ g, 10 min, 4 ° C). The recovered cultured cells were resuspended in 5 mL of TES buffer (0.1 M Tris-HCl (pH 7.0), 0.01 M EDTA, 1 M NaCl), 200 μl of lysozyme (10 mg / mL) Lt; / RTI > 100 μl of 10% SDS was added and mixed slowly to prevent physical DNA cleavage. Then, 50 μl proteinase K (20 mg / ml) and 5 μl RNase were added and reacted at 37 ° C for 30 minutes. Add 5 ml of TES buffer, add 10 ml of phenol, and mix well.

The supernatant was transferred to a new tube and treated twice with phenol / chloroform / isoamyl alcohol and once with chlorofrom. The supernatant was transferred to a new tube, and 1/10 volume of 3M sodium acetate (PH.54) and 2-3 volumes of 100% ethanol were added and left at room temperature for 30 minutes.

After the centrifugation, the supernatant was discarded, the salts were washed with 70% ethanol, and centrifuged again. The supernatant was discarded, ethanol was removed from the air, and the precipitate was dissolved in sterilized water to be used in the next experiment. After electrophoresis on 0.8% agarose gel, the DNA band was identified and quantified using a UV-spectrophotometer (F 800, Beckman Coulter, Fullerton, USA).

For purification of total DNA, crude DNA was dissolved in 10 ml of cesium chloride solution, ethidium bromide was added, dialyzed against 102,200um membrane filter for 30 minutes, and the DNA solution was recovered and concentrated. Then, a sample for amplification or cloning of a specific gene Were used.

1.3. 16S rRNA gene sequence and phylogenetic analysis

Bacterial 16S rDNA sequencing was performed by culturing the isolates in liquid medium at 30 ° C for 2 days to 3 days. The chromosomal DNA extracted from the chromosomal DNA was used as a template for PCR reaction and the primers were Eub27F: 5 '-AGA GTT TGA TCC A set of TGG CTC AG- 3 'and Eub 1522R: 5' -AAG GAG GTG ATC CAR CCG CA- 3 'was used.

The PCR reaction was performed using GeneAmp PCR System 9700 (Applied Biosystems, CT). The reaction conditions were 95 ° C for 5 minutes, 1 minute at 95 ° C, 1 minute at 55 ° C, 1 minute at 72 ° C 30 times and finally at 72 ° C for 1 minute and then stored at 4 ° C. The amplified DNA was recovered by gel extraction kit (SolGent) after electrophoresis on 1.0% agarose gel.

The recovered DNA is then ligation in pGEM-T easy vector (Promega, Madison, Wisconsin) E. coli JM109. The plasmid DNA of the transformed clones was isolated and DNA sequencing was performed bidirectionally using an ABI 377 automated sequencer (Applied Biosystem, Foster, USA) using a promoter primer or SP6 promoter primer.

The determined nucleotide sequence was analyzed using a database of Ribosomal Database Project-II (http://rdp.cme.msu.edu) GenBankhttp: //ncbi.nlm.nih.gov). The systematic relationship of the microorganisms determined by the nucleotide sequence was shown to be related to the 16S rNA nucleotide sequence of the type strain using EZtaxon.

1.4 Culture-Dependent Endosperm Segregation Results

Bacterial isolates were obtained by inoculating 0.1 mL of BM buffer containing the extract of Rubus coreus into MA medium prepared by adjusting the concentration of three kinds of nutrients by dilution method. Thirty colonies were screened and cultured according to the color and shape of the colonies grown on MA medium. Then, based on the 16S rRNA sequence, 11 strains were identified as Bacillus genus, 1 strain Micrococcus , 1 strain of Pantoea , 7 strains of Shewanella and 10 strains of Vibrio (Table 1).

Identification of lugid-derived bacteria based on 16S rRNA sequence Strain Closest Relatives Similarity (%) Diff / Total nt CBW1 Shewanella marisflavi SW-117 ^T 98.4 23/1441 CBW2 Bacillus vietnamensis 15-1 ^T 90.7 127/1362 CBW3 Bacillus barbaricus V2-BIII-A2 ^T 96.8 46/1419 CBW4 Bacillus anthracis ATCC 14578 ^T 94.2 76/1305 CBW5 Bacillus nanhaiensis JSM 082006 ^T 99.2 11/1429 CBW6 Shewanella marisflavi SW-117 ^T 98.8 18/1441 CBW7 Bacillus nanhaiensis JSM 082006 ^T 99.6 5/1401 CBW8 Vibrio azureus LC2-005 ^T 99.2 10/1298 CBW9 Bacillus algicola KMM 3737 ^T 98.5 22/1443 CBW10 Vibrio azureus LC2-005 ^T 98.8 15/1298 CBW11 Micrococcus yunnanensis YIM 65004 ^T 99.0 13/1393 CBW12 Vibrio azureus LC2-005 ^T 99.2 11/1298 CBW13 Vibrio azureus LC2-005 ^T 99.2 10/1298 CBW14 Bacillus barbaricus V2-BIII-A2 ^T 99.0 14/1419 CBW15 Bacillus vietnamensis 15-1 ^T 98.0 27/1311 CBW16 Shewanella marisflavi SW-117 ^T 98.6 20/1439 CBW17 Shewanella marisflavi SW-117 ^T 97.5 36/1435 CBW18 Pantoea septica LMG 5345 ^T 99.6 5/1340 CBW19 Shewanella marisflavi SW-117 ^T 99.7 4/1416 CBW20 Bacillus aquimaris TF-12 ^T 99.4 8/1403 CBW21 Bacillus aquimaris TF-12 ^T 99.2 11/1401 EBW1 Vibrio azureus LC2-005 ^T 98.5 19/1297 EBW2 Vibrio azureus LC2-005 ^T 99.2 10/1298 EBW3 Vibrio azureus LC2-005 ^T 99.4 8/1295 EBW4 Bacillus hwajinpoensis SW-72 ^T 96.8 47/1446 EBW5 Vibrio azureus LC2-005 ^T 99.2 11/1298 EBW6 Shewanella aquimarina SW-120 ^T 98.0 30/1438 EBW7 Shewanella aquimarina SW-120 ^T 98.0 29/1440 EBW8 Vibrio azureus LC2-005 ^T 99.2 11/1298 EBW9 Vibrio azureus LC2-005 ^T 99.2 10/1298

1.5 Origin of lug Bacillus  Analysis of strain diversity and phylogenetic relationships

Bacillus in the present invention know coke (Bacillus algicola), Bacillus anthraquinone-shi (Bacillus anthrasis), Aquitania Maris Bacillus (Bacillus aquimaris), Bacillus Barbary kusu (Bacillus barbaricus), N-Sys Hwajinpo Bacillus (Bacillus hwajinpoensis , Bacillus The various species of Bacillus such as nanhiensis), Viet Nam N-Sys Bacillus (Bacillus vietnamensis) showed that live within the lug.

Based on the 16S rRNA sequences of 11 Bacillus strains identified in the present invention, the strain relationships were divided into five groups. Among them, strains CBW2 and CBW4 were identified as Bacillus vietnamensis 15-1 ^T Bacillus anthracis ATCC 14578 ^T and (90.7%) and 94.2% (94.2%), respectively. Fig. 3 shows the nucleotide sequence of the 16S rDNA gene of Bacillus strain CBW4 isolated and isolated from the nematode, and Fig. 4 shows the nucleotide sequence of the 16S rDNA gene of Bacillus strain EBW4 isolated and isolated from the nematode. Fig. 5 shows the results of the identification of Bacillus sp. Is a schematic diagram showing the systematic relationship of the strain. The invention of microbes endogenous to the worms has been found for the first time in the present invention, and an in-depth analysis of the functions of these microbes needs to be performed.

In the present invention, Perinereis information on a variety of Bacillus appeared as a dominant species of bacteria separated from aibuhitensis) was investigated in detail the characteristics of the two Bacillus strains CBW4 EBW4 and a superior resolution, the organic substance selected as the noticed that first reported in the present invention. FIG. 6 shows the patented microbial susceptibility of the genus Bacillus (Accession No .: KACC91722P), and FIG. 7 shows the patented microbial susceptibility of the genus Bacillus (Accession No .: KACC91749P).

2. Growth Characteristics of Newborn Environmental Conditions Identified in Rug

end. Strain Growth Test by Environmental Condition

In the present invention, the growth of the strain was examined by varying three environmental conditions such as temperature, pH, and salinity. The strains were inoculated into LB medium and incubated at 4 ℃, 15 ℃, 25 ℃, 30 ℃, 40 ℃, 45 ℃, 50 ℃ and 55 ℃, respectively. The growth was monitored by inoculating the LB liquid medium with LB liquid medium at 40 ° C or less and then culturing in a water bath at 40 ° C or less.

The optimal salinity range of the bacterial growth was adjusted to 1%, 3%, 5%, 7%, 10%, 11%, 12%, 13%, 14% and 15% The optimal pH range of the bacterial growth was adjusted by adjusting the pH of the LB medium to pH 1 from pH 3 to pH 11 using NaOH and HCl. The growth of the strain was confirmed by incubation for 5 days at the optimum growth temperature of each strain after the strain inoculation.

I. Selection of lobsters Bacillus  Test results on the ability of the strain to use the medium

The growth performance of two Bacillus strains selected in the present invention was tested using MA (marine agar), NA (nutrient agar), TSA (tryptic soy agar), R ₂ A and LB (Luria-Bertani) EBW4 was not able to grow in LB, TSA, or NA medium, unlike Bacillus strain, which was derived from land or freshwater. Both strains were grown in MA medium containing a large amount of salinity, (Table 2).

Growth ability test in various media Strain MA LB TSA NA R ₂ A CBW4
+ +
+
-
+
EBW4 + -
-
+
+

3. New physiological and biochemical characteristics of the newborn

end. Morphological, physiological and biochemical characterization of bacteria

For the morphological and physiological characteristics of bacteria, strains were inoculated on MA (marine agar) solid medium and cultured at 25 ℃. First, the shape, color, and size of single colonies formed by 5-7 days of culture were observed. The shape and size of the strain were observed using optical microscope and scanning electron microscope. During the electron microscopic observation, the cultured bacteria were fixed with 4% glutaraldehyde for 2 hours and then washed with 50 mM phosphate buffer (pH 7.2). The cells were fixed in 2% osmium tetroxide solution for 1 hour and dehydrated with 40, 50, 60, 70, 80, 90, 100% ethanol and lyophilized. After that, gold coating was performed using an ion sputter and observed with an electron microscope.

The motility of the strain was confirmed by semisolid medium containing 0.4% agar. Oxidase - related oxidase was analyzed by colony color change using Oxidase Reagent Kit (BioM ㅹ rieux). Catelase was determined to be bubbles by adding 3% and hydrogen peroxide to the cells.

The physiology of other isolated strains ?? Biochemical characteristics were determined by using various API kits (BioM ㅹ rieux), and the characteristics of the strains were investigated using API 20E, APL 20NE, API 50CHB, and API ZYM kit simultaneously. At this time, the strain was cultured in MA solid medium at 25 ° C for 2 days to 3 days. The grown cells were suspended in a sterilized 3% sea salt solution and were performed according to the manufacturer's instructions. Nitrate reduction reaction, esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, naphtol-AS-Bi-phosphohydrolase, glucose acidification, gelatin liquefaction, arginine dihydrolase, urease, β-glucosidase, β -galactosidase, alkaline phosphatase, lipase (C14), etc., and indole production. We also investigated the assimilation of carbon sources such as mannitol, gluconate, malate, citrate, glucose, arabinose, mammose, N-acetyl-glucosamine, maltose, caprate, adipate and phenyl-acetate.

I. Selection of lobsters Bacillus  Analysis of physiological biochemical characteristics of the strain

Biochemical and biochemical characteristics of Bacillus strains were analyzed using API 20NE kit. Both strains CBW4 and EBW4 had β-glucosidase and protease enzyme activity and could utilize maltose (Table 3).

Characterization of Bacillus strains using API 20NE kit Test Reactions / Enzymes CBW4 EBW4 NO ₃ reduction of nitrates to nitrites +/- +/- TRP İndole production - - GLU 산화 - - ADH arginine dihydrolase - - URE urease - - ESC hydrolysis (β-glucosidase) + + GEL hydrolysis (protease) + + PNPG β-galactosidase - w + GLU glucose assimilation W + w + ARA arabinose assimilation W + w + MNE mannose assimilation - w + MAN mannitol assimilation W + + NAG N-acetyl-glucosamine assimilation W + w + MAL maltose assimilation + + GNT gluconate assimilation W + W + CAP caprate assimilation - - ADI adipate assimilation W + w + MLT malate assimilation w + w + CIT citrate assimilation W + w + PAC phenyl-acetate assimilation W + w +

As a result of the test using API 20E kit, CBW4 showed high activity of arginine dihydrolase, lysine decarboxylase and gelatinase, while EBW4 had β-glucosidase and gelatinase activity (Table 4).

Characterization of Bacillus strains using API 20E kit Test Reactions / Enzymes CBW4 EBW4 OPNG Beta-galactosidase - + ADH Arginine dihydrolase + - LDC Lysine decarboxylase + - ODC Ornithine decarboxylase - - CIT Citrate utilization - - H ₂ S H2S production - - URE Urease - - TDA Tryptophane deaminase - - IND Indole production - - VP Acetone production - - GEL gelatinase + + GLU Glucose F / O - - MAN Mannitol F / O - - INO Inositol F / O - - SOR Sorbitol F / O - - RHA Rhamnose F / O - - SAC Sucrose F / O - - MEL Melibiose F / O - - AMY Amygdaline F / O - - ARA Arabinose F / O - -

As a result of the test using the API 50CHB / E kit, both CBW4 and EBW4 showed the ability to utilize glycerol, ribose, glucose, fructose, esculin, cellobiose, maltose, trehalose and starch. In addition, CBW4 was able to utilize arbutin, salicin, and lactose, while EBW4 was not available, but mannose, mannitol, α-methyl-D-glucoside, N-acethyl-glucosamine, melibiose, sucrose, raffinose, and D-turanose (Table 5).

Characterization of Bacillus strains using API 50CHB / E kit No. Substrate CBW4 EBW4 0 CONTROL - - One GLYcerol + + 2 ERYthritol - - 3 DARAbinose - - 4 LArbinose - - 5 RIBose + + 6 DXYLose - - 7 LXYLose - - 8 ADOnitol - - 9 beta -Methyl-D-Xyloside - - 10 GALzctose - + 11 GLUcose + + 12 FRUctose + + 13 MaNnosE - + 14 SorBosE - - 15 RHAmnose - - 16 DULCitol - - 17 INOsitol - - 18 MANnitol - + 19 SORbitol - - 20 alpha -Methyl-D-Mannoside w + - 21 alpha -Methyl-D-Glucoside w + + 22 N-Acethyl-Glucosamine w + + 23 AMYgdalin w + w + 24 ARButin + w + 25 Esculin + + 26 SALicin + w + 27 CELlobiose + + 28 MALtose + + 29 LACtose + - 30 MELibiose - + 31 Sucrose - + 32 TREhalose + + 33 INUlin - - 34 MeLeZitose - w + 35 RAFfinose - + 36 Starch + + 37 GLYcoGen w + + 38 XyLiTol - - 39 GENtiobiose - - 40 DTURanose - + 41 DLYXose - - 42 DTAGatose - - 43 DFUCose - - 44 LFUCose - - 45 DArabitoL - - 46 LArabitol - - 47 GlucoNaTe - w + 48 2-keto-Gluconate - W + 49 5-keto-Gluconate - -

As a result of enzymatic activity test using API ZYM kit, both strains CBW4 and EBW4 showed alkaline phosphatase, leucine arylamidase and α-chymotrypsin activity. In addition, CBW4 has very high acid phosphatase activity and β-glucosidase activity. EBW4 had an esterase activity that was not detected in the CBW4 strain (Table 6).

Characterization of Bacillus strains using API ZYM kit NO. Enzyme Assayed For CBW4 EBW4-1 One Control 2 Alkaline phosphatase 4+ 4+ 3 Esterase (C4) One- 3+ 4 Esterase Lipase (C8) One- 2- 5 Lipase (C14) 0- 0- 6 Leucine arylamidase 4+ 3+ 7 Valine arylamidase 2- 2- 8 Crystine arylamidase One- One- 9 Trypsin 2- 0- 10 alpha-chymotrypsin 3+ 3+ 11 Acid phospatase 5+ 0- 12 Naphtol-AS-B1-phosphohydrolase One- 0- 13 alpha -galactosidase 0- 0- 14 β-galactosidase 0- 2- 15 β-glucuronidase 0- 0- 16 α-glucosidase 2- 2- 17 β-glucosidase 4+ 0- 18 N-acetyl-β-glucosaminidase 0- 0- 19 α-mannosedase 0- 0- 20 alpha-fucosidase 0- 0-

4. Application of Newborn Tidal Flats Identified in a Rug

end. Polymerase chain reaction activity test

In order to examine various Bacillus macromolecule resolving abilities, a novel heterologous Bacillus strain CBW4 (accession number: KACC91722P) and EBW4 (SEQ ID NO: 2) isolated in the present invention were added to LB solid medium containing cellulose, starch, casein and surfactant (Tween 80) Accession No .: KACC91749P) were inoculated at the same ratio, and then enzyme, cellulase, amylase, protease, and lipase activity related to decomposition of these polymers were examined.

The cellulolytic activity of the strain was determined after 30 days of incubation at 30 ° C for 3 days, followed by 30 minutes of staining with congo red, followed by discoloration with 1M NaCl for 5 minutes, staining with iodine for 30 minutes and discoloration with 1M NaCl for 5 minutes As shown in Fig. The casein and Tween 80 were cultured for 2 days and 3 days, respectively, and their resolution was confirmed by a transparent ring observed with naked eyes without performing any staining process. Alkaline protease activity was determined in 100 mM Tris-HCl buffer (pH 10.0) using Hammerstan casein (Merck) at a concentration of 0.5% (Joo et al., 2002). One Unit of Enzyme Activity is defined as the amount of enzyme that releases 1 g of tyrosine per minute at 50 캜 under standard assay conditions.

I. Selection of lobsters Bacillus  Results of Polymerase Enzyme Activity Test of Strain

Both the CBW4 and EBW4 strains showed DNAse activity and protease activity by degrading casein. CBW4 had an enzymatic activity to degrade strach, but EBW4 did not have starch resolution. In addition, both CBW4 and EBW4 were shown to degrade most of the tween, indicating lipolytic enzyme activity.

Therefore, in the present invention, two Bacillus strains isolated from the nematode can be used to purify the tidal environment contaminated with organic matter, because of their excellent protein, carbohydrate and lipid decomposition (Table 7).

Polymerase Enzyme Activity Test Strain Starch  One% DNAse CM / cellulose Casein  One% Tween 20 40 60 80 CBW4 +
+
-
+
+
+
+
+
EBW4 -
+ - +
- + + +

All. Manufacture of new tidal flat purification agent

FIG. 8 is a schematic view showing a method for producing a microorganism preparation from a Bacillus strain derived from a nematode, and FIG. 9 shows an example of a microorganism preparation prepared using a Bacillus strain derived from a nematode .

A new strain Bacillus sp. CBW4 (accession number: KACC91722P) and EBW4 (accession number: KACC91749P) identified in a rat culture were inoculated into a liquid culture at a ratio of 1: 1 and cultured at 25-30 ° C for 1-2 days The microorganism was prepared as a powdery microorganism through a step of aseptically drying a part of the product obtained in the culturing step, a remaining part of the product as a liquid microorganism, and aseptically pulverizing the product obtained after the drying step.

In the case of a liquid preparation, food coloring may be added as an excipient for color control, vitamin C may be added as an excipient for improving shelf life, and Tween 80 may be added as an excipient for activating enzyme ability.

In the case of a solid powder, rice bran, degreasing steel, molasses and the like are additionally added as an excipient for fermentation, and silicate or the like can be added as an excipient for improving shelf stability.

Bacterial studies by culture methods provide meaningful information on the role of heterotrophic bacteria in biochemical circulation as well as potential metabolic activity, as well as the ability to cultivate numerous species belonging to Bacillus , Bacillus strains CBW4 and EBW4 have excellent protein, carbohydrate, and lipid degradation ability and are resistant to salinity, so they can be useful for purifying tidal environment contaminated with organic matter.

Agricultural Biotechnology Research Institute KACC91749P 20121026 Agricultural Biotechnology Research Institute KACC91722P 20120608

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Bacillus spp., Identified from lugworm and microbial cleaning          agent <130> P201212064 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 1378 <212> RNA <213> Bacillus sp. EBW4 <400> 1 tcgagcgaat ggattgagag cttgctctta tgaagttagc ggcggacggg tgagtaacac 60 gtgggtaacc tgcccataag actgggataa ctccgggaaa ccggggctaa taccggataa 120 tattttgaac tgcatggttc gaaattgaaa ggcggcttcg gctgtcactt atggatggac 180 ccgcgtcgca ttagctagtt ggtgaggtaa cggctcacca aggcaacgat gcgtagccga 240 cctgagaggg tgatcggcca cactgggact gagacacggc ccagactcct acgggaggca 300 gcagtaggga atcttccgca atggacgaaa gtctgacgga gcaacgccgc gtgagtgatg 360 aaggctttcg ggtcgtaaaa ctctgttgtt agggaagaac aagtgctagt tgaataagct 420 ggcaccttga cggtacctaa ccagaaagcc acggctaact acgtgccagc agccgcggta 480 atacgtaggt ggcaagcgtt atccggaatt attgggcgta aagcgcgcgc aggtggtttc 540 ttaagtctga tgtgaaagcc cacggctcaa ccgtggaggg tcattggaaa ctgggagact 600 tgagtgcaga agaggaaagt ggaattccat gtgtagcggt gaaatgcgta gagatatgga 660 ggaacaccag tggcgaaggc gactttctgg tctgtaactg acactgaggc gcgaaagcgt 720 ggggagcaaa caggattaga taccctggta gtccacgccg taaacgatga gtgctaagtg 780 ttagagggtt tccgcccttt agtgctgaag ttaacgcatt aagcactccg cctggggag 840 acggccgcaa ggctgaaact caaaggaatt gacgggggcc cgcacaagcg gtggagcatg 900 tggtttaatt cgatgcaacg cgaagaacct tacctactct tgacatccag agaattcgct 960 agagatagct tagtgccttc gggagctctg agacaggtgc tgcatggctg tcgtcagctc 1020 gtgttgtgaa atgttgggtt aagtcccgca acgagcgcaa cccttatcct tacttgccag 1080 cgggtcatgc cgggaacttt agggagactg ccggtgataa accggaggaa ggtggggacg 1140 acgtcaagtc atcatggccc ttacgagtag ggctacacac gtgctacaat ggcgagtaca 1200 gagggttgcg aagccgcgag gtggagctaa tctcagaaag ctcgtcgtag tccggattgg 1260 agtctgcaac tcgactccat gaagtcggaa tcgctagtaa tcgtggatca gaatgccacg 1320 gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca ccatgggagt gggctgca 1378 <210> 2 <211> 1374 <212> RNA <213> Bacillus sp. CBW4 <400> 2 tcgagcgaat gatgaggagc ttgctcctct gatttagcgg cggacgggtg agtaacacgt 60 gggtaatctg cctgtaagac ggggataact ccgggaaacc ggggctaata ccggataaca 120 agagaagaag catttcttct ttttgaaagt cggtttcggc tgacacttac agatgagccc 180 gcggcgcatt agctagttgg tgaggtaacg gctcaccaag gcgacgatgc gtagccgacc 240 tgagagggtg atcggccaca ctgggactga gacacggccc agactcctac gggaggcagc 300 agtagggaat cttcggcaat gggcgaaagc ctgaccgagc aacgccgcgt gagcgatgaa 360 ggccttcggg tcgtaaagct ctgttgttag agaagaacaa gtacgagagt aactgctcgt 420 accttgacgg tacctaacca gaaagccacg gctaactacg tgccagcagc cgcggtaata 480 cgtaggtggc aagcgttatc cggaattatt gggcgtaaag cgcgcgcagg cggtctctta 540 agtctgatgt gaaagcccac ggctcaaccg tggagggtca ttggaaactg ggagacttga 600 gtgcaggaga gaaaagtgga attccacgtg tagcggtgaa atgcgtagag atgtggagga 660 acaccagtgg cgaaggcggc tttttggcct gtaactgacg ctgaggcgcg aaagcgtggg 720 gagcaaacag gattagatac cctggtagtc cacgccgtaa acgatgagtg ctaggtgttg 780 gggggttcca ccctcagtgc tgaagttaac acattaagca ctccgcctgg ggagtacgac 840 cgcaaggttg aaactcaaag gaattgacgg gggcccgcac aagcagtgga gcatgtggtt 900 taattcgaag caacgcgaag aacccttacc aggtcttgac atcctctgac cactctagag 960 atagagcttt ccccttcggg ggacagagtg acaggtggtg catggttgtc gtcagctcgt 1020 gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttgacctta gttgccagca 1080 ttcagttggg cactctaagg tgactgccgg tgacaaaccg gaggaaggtg gggatgacgt 1140 caaatcatca tgccccttat gacctgggct acacacgtgc tacaatggat ggtacaaagg 1200 gttgcgaagc cgcgaggcca agccaatccc aaaaagccat tctcagttcg gattgtaggc 1260 tgcaactcgc ctacatgaag ccggaattgc tagtaatcgc ggatcagcat gccgcggtga 1320 atacgttccc gggccttgta cacaccgccc gtcacaccac gagagtttgt aaca 1374

Claims (7)

( Bacillus sp.) CBW4 (accession number: KACC91722P) and Bacillus sp. EBW4 (accession number KACC91749P), which have salinity tolerance identified from the nematode, Wherein the culture contains an alkaline phosphatase, an acid phosphatase, an arginine dihydrolase, a lysine decarboxylase, and a culture medium. , Beta-glucosidase, or estradiol. &Lt; Desc / Clms Page number 24 &gt; delete delete delete delete delete delete

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