KR20140073228A - Bacillus spp., identified from lugworm and microbial cleaning agent. - Google Patents

Abstract

With respect to the present invention, microorganisms which are endogeneous in Perinereis aibuhitensis known to have excellent capacity to decompose organic matters in the coastal mud flat were grown in an MA medium, and 31 colonies were selected according to the color and shape of the colony and then purely cultured. As a result of identification based on the 16S rRNA nucleotide sequence, 11 strains included in the genus Bacillus were isolated. As a result of analysis of diversity of 11 strains included in the genus Bacills, it was investigated that a total of seven species of Bacills algicola, Bacills anthrasis, Bacills aquimaris, Bacills barbaricus, Bacills hwajinpoensis, Bacills nanhiensis, and Bacills vietnamensis are endogenous in Perinereis aibuhitensis. In the present invention, two Bacillus strains isolated and selected from Perinereis aibuhitensis have excellent capacity to decompose protein, hydrocarbon, and lipid, and have resistance to salt, and thus can be useful to purify a mud flat contaminated with organic matters.

Description

{Bacillus spp., Identified from lugworm and microbial cleaning agent}, which is a novel strain of Bacillus having a salt tolerance and an esterase activity,

The present invention relates to a Bacillus having a salinity tolerance and esterase activity that is endogenous to the lug (Perinereis aibuhitensis) known to be a very good resolution in the coastal organisms tidal (Bacillus) new strains and organic cleaning agent using the same.

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 gene sequencing, microbial diversity has been actively studied

Not only does this study provide valuable information on the role of heterotrophic bacteria in biogeochemical cycling as well as potential metabolic activity (Zaccone et al., 2002), and in particular numerous species belonging to Bacillus , It is possible to obtain meaningful value by studying cultured bacteria.

Based on this, the present invention focuses on the analysis of Bacillus bacterium diversity in coastal tidal flat earthworms, and 31 colonies selected according to the color and shape of colonies grown on MA medium are separately selected and cultured in pure culture Bacillus strains were identified based on the 16S rRNA sequence.

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- Its formulation technology is 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). Interestingly, 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).

At the same time as the present invention provide a 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 foreshore of the strain Protein, carbohydrate, and lipid decomposing ability of the organism, so as to purify the environment contaminated with the organic substance.

In this invention, the diversity of Bacillus bacteria known to be closely related to the digestion and bio-immunity of nutrients among endemic microbes ( Perinereis aibuhitensis ), which is known to have excellent organic decomposability in coastal tidal flats, was analyzed based on the culture method.

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. Analysis of the diversity of the 11 strains contained in the Bacills (genus), Bacillus know-Cola (Bacillus algicola), Bacillus anthraquinone system (Baci llus anthrasis), Aquitania Maris Bacillus (Bacillus aquimaris), Bacillus Barbary kusu (Bacillus barbaricus), Bacillus Hwajinpo N-Sys (Bacillus hwajinpoensis), Bacillus nanhi N-Sys (Bacillus nanhiensis), Bacillus Viet Nam N-Sys (Bacillus vietnamensis) including both spp Bacillus seven types (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 starch, but EBW4 showed no starch degradation, and both CBW4 and EBW4 showed lipase activity.

In the present invention, the two Bacillus strains isolated from the worms are excellent in protein, carbohydrate, and lipid-resolving ability and are resistant to saline, so they can be useful for purifying the environment of contaminated tidal flats.

Figure 1 shows a photograph of a snakehead.
2 is a schematic diagram showing the diversity of the Bacillus strain derived from the nematode .
Fig. 3 shows the nucleotide sequence of the 16S rDNA gene of Bacillus subtilis isolated and isolated from the worms.
Fig. 4 shows the results of a bacillus sp. Bacillus sp. Is a schematic diagram showing the systematic relationship of the strain.
Figure 5 shows the patented microorganism deposit of the genus Bacillus (Accession No .: KACC91749P) isolated.
FIG. 6 shows the results of a bacillus-derived Bacillus sp. Is a schematic view showing a method for producing a microorganism preparation from a strain.
Fig. 7 is a schematic view of a bacillus-derived Bacillus Examples of the microorganism preparation prepared using the strain sp.

The present invention relates to a novel strain having salinity tolerance cultured from a strain of Bacillus sp. Of KFCC91749P, which has been identified from a nematode, and which has at least one organic compound selected from proteins, carbohydrates, lipids and surfactants and has an esterase activity The present invention provides a microbial purification agent for tidal flat purification using Bacillus.

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 strain of the genus Bacillus, which is the new strain of the present invention, was first isolated and identified by the inventors of the present invention, and the microorganisms endemic to the lagoon ( Perinereis aibuhitensis ), which is known to have excellent organic decomposability in coastal tidal flats, Respectively.

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.

Bacills As a result of analysis of the diversity of the 11 strains contained in the genus, Bacillus alcicola algicola), Bacillus anthraquinone system (Baci llus anthrasis , Bacills aquimaris , Bacills barbaricus , Bacills hwajinpoensis , Bacills nanhiensis , Bacills vietnamensis , and the like. ) Of Bacillus spp. Was found to be endogenous to the ridge ( Perinereis aibuhitensis ).

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 Bacillus anthracis ATCC 14578 ^T ) and Bacillus anthracis (90.7%) and 94.2%, respectively. EBW4 showed homology to Bacillus hwajinpoensis SW-72 ^T and Bacillus spp. 96.8% homology, indicating that it was a new species.

Thus, the present inventors have found that Bacillus sp. hwajinpoensis SW-72 ^T ) and The isolated and identified novel strain of the present invention, which showed 96.8% homology, was designated " Bacillus & sp. EBW4, "which was deposited on October 26, 2012 at the National Institute of Agricultural Science and Technology (KACC) and granted the deposit number KACC91749P.

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

Perinereis aibuhitensis used as a strain isolate in the present invention was collected from the Suncheon Bay tidal flat farm. Fig. 1 is a photograph of the collected lugworm, and Fig. 2 is a schematic diagram showing a method of identifying a Bacillus strain derived from a winged worm.

In order to isolate the microorganisms present in the barn, the live barnacle was placed in a conical tube containing 50 mL of BM buffer (Mikesell et al., 1993) , washed three times in the vortex, briefly rinsed again in 70% Respectively. 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 into 50 mL of LB medium, cultured at 30 ° C. and 180 rpm for 24 hours, and harvested by centrifugation (6,300 × 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 obtained by centrifugation was transferred to a new tube and treated with the same amount of phenol / chloroform / isoamyl alcohol twice and once with chloroform. The supernatant was transferred to a new tube, and 1/10 volume of 3M sodium acetate (pH 5.4) 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 was ligated into pGEM-T easy vector (Promega, Madison, Wisconsin) and transformed into 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 the database of Ribosomal Database Project-II (http://rdp.cme.msu.edu) GenBank http://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

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

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 the MA medium. 11 colonies were identified on the basis of the 16S rRNA nucleotide sequence. As a result, 11 strains were identified as Bacillus 1 genus, 1 strain of Micrococcus , 1 strain of Pantoea , 7 strains of Shewanella genus, and 10 strains of genus Vibrio (Table 1).

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

Bacillus know coke (Bacillus algicola) In the present invention, the Bacillus anthraquinone system (Bacillus anthrasis), Bacillus Aquitania Maris (Bacillus aquimaris), Bacillus Barbary kusu (Ba cillus barbaricus), Bacillus Hwajinpo N-Sys (Bacillus hwajinpoensis), Bacillus nanhi N-Sys (Bacillus nanhiensis ) , Bacillus vietnamensis Bacillus species were found to be inhabited in the barnacles.

The 16S rRNA sequences of the isolated strains were analyzed by molecular genetic analysis. As a result, the systematic analysis of the 16S rRNA sequences of the 11 Bacillus strains revealed that there were 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 (90.7%) and 94.2% (94.2%), respectively.

FIG. 3 is a graph showing the results of the measurement of the homology with the Bacillus anthracis ATCC 14578 strain isolated in the present invention Bacillus sp. 4 shows the nucleotide sequence of the 16S rDNA gene of CBW4, and FIG. 4 shows a schematic diagram showing the systematic relationship of the Bacillus strain derived from the nematode. The inventions of microbes endogenous to the nematodes have been discovered for the first time in the present invention, and further research on the function of these microbial groups needs to be carried out in depth. In the present invention, information on various Bacillus species, which are dominant species among the bacteria isolated from the lupine ( Perinereis aibuhitensis ), was found to be reported for the first time in the present invention.

The present inventors have found that the isolated and identified Bacillus Viet Nam N-Sys (Bacillus vietnamensis) 15-1 ^T and Bacillus anthraquinone system (Bacillus anthracis ) ATCC 14578 ^T and Bacillus sp. CBW4 ", which showed 90.7% and 94.2% homology, respectively, was deposited with the National Institute of Agricultural Science and Technology (KACC), National Academy of Agricultural Science, on June 8, 2012 and deposited with the deposit number KACC91722P .

In addition, Bacillus hwajinpoensis SW-72 ^T ) and The new strain of the present invention, which showed 96.8% homology, was named " Bacillus sp. EBW4 ", deposited with the National Institute of Agricultural Science and Technology (KACC) on October 26, 2012 and assigned the deposit number KACC91749P. FIG. 5 is a graph showing the activity of Bacillus sp. EBW4 patented microorganism deposit.

In particular, Bacillus isolated can enhance the survival of cultured organisms through its water purification ability and can have a positive effect on promoting growth. In addition, Bacillus species capable of producing a large amount of lipase can be utilized for removing lipid- Therefore, the in-depth study of various Bacillus strains obtained in the present invention is of great value. In the present invention, the characteristics of two selected Bacillus strains, CBW4 and EBW4, were examined in detail.

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 investigated under different 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 to pH 1 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

The strains were inoculated on MA (marine agar) solid medium and cultured at 25 ℃ to examine morphological and physiological characteristics of bacteria. 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. In 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 physiological and biochemical characteristics of other isolates were investigated by using various API kit (BioMerieux), and 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 2-3 days, and the grown cells were suspended in sterilized 3% sea salt solution and 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 enzymatic activity and were able to 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 +

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 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 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 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 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-

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 undetectable esterase activity in the CBW4 strain (Table 6).

4. From the lobster  Sympathetic New caution  Application of tidal flats as cleaning agents

FIG. 6 is a schematic view showing a method for producing a microorganism preparation from a bug sprouting Bacillus strain, and FIG. 7 shows an example of a microbial preparation prepared using a Bacillus strain derived from a wing spider.

A new strain of Bacillus ( Bacillus sp. sp . ) EBW4 (Accession No .: KACC91749P) was inoculated into a liquid culture medium and cultured at 25-30 ° C for 1-2 days. A part of the resultant obtained in the culturing step was aseptically dried as a liquid microbicide, the remaining part was aseptically dried, The resultant was aseptically pulverized to prepare a powdery microbial agent.

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 the 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.

end. Polymerase chain reaction activity test

Bacillus is known to have a variety of polymeric decomposition properties, and after inoculation of the Bacillus strains isolated from the present invention into LB solid medium containing cellulose, starch, casein, and surfactant (Tween 80) Related enzymes, cellulase, amylase, protease, and lipase. 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%. 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 enzymatic activity to degrade starch 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 are highly resistant to protein, carbohydrate and lipid degradation, and thus can be used to purify the tidal environment contaminated with organic matter (Table 7).

Polymerase Enzyme Activity Test Strain
Starch 1%
DNAse
CM / cellulose
Casein 1%
Tween 20 40 60 80 CBW4 + + - + + + + + EBW4 - + - + - + + +

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 , Both Bacillus strains are highly resistant to salinity and protein, carbohydrate, and lipid degradation, so they can be useful for purifying the tidal environment contaminated with organic matter.

Agricultural Biotechnology Research Institute KACC91749 20121026

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Bacillus spp., Identified from lugworm and microbial cleaning          agent <130> P201212064 <160> 1 <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

Claims (8)

A novel strain Bacillus sp. Having a salt resistance selected from the Bacillus sp. Strain of KACC 91749P, which has been identified from the nematode, and has at least one organic compound selected from the group consisting of protein, carbohydrate, lipid and surfactant, Bacillus sp . ) EBW4
Accession No .: KACC91749P from Bacillus ( Bacillus a novel strain Bacillus sp . having a salt resistance selected from a strain of the genus Bacillus sp. and having at least one organic compound selected from the group consisting of protein, carbohydrate, lipid and surfactant and having an esterase activity sp . ) Microbial purification agent for purification of tidal flats containing a culture of EBW4 as an active ingredient.
A method for decomposing any one or more organic substances selected from proteins, carbohydrates, lipids and surfactants fixed on the tidal flats by spraying the tidal flats microorganism preparation of claim 2 on the tidal flats.
( Bacillus sp. ) EBW4 (accession number: KACC91749P), identified from the nematode, is cultivated at 25-30 ° C for 1-2 days and then cultured for 1-2 days.
The method for producing a microbial cleaning agent for tidal flat according to claim 4, comprising aseptically drying the resultant obtained in the culturing step.
The method of claim 5, further comprising aseptically pulverizing the resultant product obtained after the drying step.
The method for producing a microorganism purifying agent for tidal flat according to claim 4, wherein any one substance selected from an excipient for color control or an excipient for improving shelf life or an excipient for activating enzyme capacity is additionally added to the liquid product .
The method according to claim 6, wherein the pulverized microorganism preparation is additionally added with any one selected from excipients for fermentation or excipients for improving shelf life.

Images