• Journal of Microbiology and Biotechnology
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• v.14 no.2
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• pp.430-434
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• 2004

Three extracellular protease-deficient Bacillus subtilis strains were transformed with the plasmid pCK98 containing the endo-$\beta$-1,4-glucanase (Eng) gene of B. subtilis BSE616. The three transformants, B. subtilis DB104 (pCK98), WB600 (pCK98) and WB700 (pCK98), produced the same high level of enzyme activity and showed similar patterns of cell growth and enzyme production. When B. subtilis DB 104 (pCK98), a two-extracellular protease deficient strain, was cultured for 22 h, almost all the secreted enzyme was found to be in the completely cleaved form by both activity staining and Western blotting studies. B. subtilis WB600 (pCK98), a six-extracellular protease-deficient strain, produced a partially cleaved form in addition to the intact form of the enzyme, although the degree of internal cleavage of the enzyme was greatly reduced. With B. subtilis WB700 (pCK98), a seven-extracellular protease-deficient strain, almost all the enzyme was produced as the intact uncleaved form. This study illustrates that a role of the V pr protease is to degrade foreign proteins produced in B. subtilis and WB700 is a suitable expression system for producing the intact form of the Eng and other foreign proteins that may lose at least part of their efficacy due to internal proteolytic cleavage.

• Food Science and Biotechnology
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• v.14 no.5
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• pp.704-708
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• 2005

Poly-y-glutamate (${\gamma}$-PGA) has great potential as a biodegradable polymer in a broad range of industrial fields such as food, cosmetics, medicine and water treatment. In order to isolate ${\gamma}$-PGA producers that are suitable for specific industrial applications, 653 Bacillus-like strains were isolated from 439 varieties of three Korean fermented foods, Cheongkukjang, Doenjang, and Kochujang, which were collected from different regions across Korea. A very high level of ${\gamma}$-PGA production was demonstrated in 4.7%, 1.8%, and 3.0% of the Bacillus-like strains isolated from Cheongkukjang, Doenjang, and Kochujang samples, respectively, which produced a viscous substance to such extent that it overflowed to the lid of the plate on the glutamate-dependent ${\gamma}$-PGA production plates. On glutamate-independent ${\gamma}$-PGA production plates, 5.1%, 5.9%, and 6.1% of Bacillus-like strains isolated from Cheongkukjang, Doenjang, and Kochujang samples, respectively, showed high production. The maximum ${\gamma}$-PGA production yields were 32.5 g/L and 5 g/L, depending on the purification methods in the glutamate-dependent media, with the higher yield resulting from a simple precipitation of ${\gamma}$-PGA by either methanol or ethanol and dialysis. The viscous substance produced by each strain showed different morphological characteristics, suggesting that isolated ${\gamma}$-PGA producers could produce various types of ${\gamma}$-PGA.

• Applied Biological Chemistry
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• v.39 no.3
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• pp.177-182
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• 1996

Effects of 14 carbohydrates supplied as carbon sources on cell growth and sporulation of, and the production of insecticidal crystal proteins by Bacillus thuringiensis strains were investigated in liquid cultures. Strains grew well in media containing any one of the 14 carbohydrates supplied, reaching maximum cell densities of $10^7{\sim}10^8\;cells/ml$ in 16.7 to 22 hours after inoculation depending on the strain. Spores first appeared in 16.7 to 24.7 hours after inoculation, and 80% sporulation was reached in 28 to 51.3 hours after inoculation depending on the strain. No change in pH of media was observed after cell multiplication. The production of total protein was highest when supplied with sucrose and was lowest with starch. More insecticidal crystal proteins were produced when supplied with glucose, lactose, maltose, or sucrose. The amount of insecticidal crystal proteins produced by the strains was proportional to that of the total protein. The relative amount of individual insecticidal crystal protein species produced by B.t. kurstaki and B.t. israelensis was not influenced by the carbohydrates supplied.

• Microbiology and Biotechnology Letters
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• v.48 no.4
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• pp.439-446
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• 2020

Two Bacillus strains, K3 and K208, both demonstrating strong fibrinolytic activities were isolated from Kimchi, a traditional Korean preparation of fermented vegetables. Isolates were subjected to various molecular biology based identification methods including RAPD-PCR and identified as B. subtilis and B. velezensis, respectively. Tryptic soy broth (TSB) was found to best maintain both the growth and the fibrinolytic activity of these strains. Culture supernatants were analyzed by SDS-PAGE and fibrin zymography, and the results indicate that a 40 and 27 kDa band seem to be responsible for the fibrinolytic activities of these two isolates and the 27 kDa band was subsequently identified as the mature form of AprE, the major fibrinolytic enzyme. Thus the aprE genes were cloned and the translated amino acid sequences demonstrated 99.3% identity with each other, and 86.5% identity with BsfA, a fibrinolytic enzyme from B. subtilis ZA400 also isolated from Kimchi, and AprE2, a fibrinolytic enzyme from B. subtilis CH3-5 isolated from Cheonggukjang, a traditional Korean fermented soy. Given this B. subtilis K3 and B. velezensis K208 may be promising starter cultures in the production of fermented foods.

• Biotechnology and Bioprocess Engineering:BBE
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• v.9 no.6
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• pp.482-489
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• 2004

Bacillus species were observed and quantified by molecular approaches, using the 16S rDNA primers/probes, in a wastewater treatment plant designed for the purpose of stimulating the growth of Bacillus species. The plant has been operating as a test plant since 1997 in the city of Ina, Japan, with excellent treatment performance. Observations by in situ hybridization, using Bacillus-specific probes, indicated that Bacillus strains were inhabited in the plant and their num­bers decreased during the treatment process. Similar results were obtained from a quantitative PCR analysis using a Bacillus-specific primer set, and the amount of DNA originating from various Bacillus species was maximally $1.91%\$ of the total DNA in the wastewater treatment tank. Clone library analysis using the Bacillus-specific primers suggested that, while the population was no­ticeably increased, the phylogenetic diversity of the increasing Bacillus species was very low.

• Journal of the Korean Society of Food Science and Nutrition
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• v.31 no.3
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• pp.389-393
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• 2002

Among the bacterial strains isolated from chungkook-jang, Bacillus subtilis CH-1, Bacills circulans K-1 and Bacillus subtitis (natto) N-1, Bacillus subtitis CH-1 showed the highest productivity of biosufactant. A-medium was selected for the basal medium in the large scale production of biosurfactant, and modified to synthetic medium which containing 2% glucose, 0.3% soy peptone, and mineral salts. The surface tension was reduced to maximal value after 96 hr after fermentation, about the 43% of initial tension. Temperature and initial pH of medium was not critical factor for the biosurfactant production. The yield of crude biosurfactant was 6 g/L under the optimum condition.

• Journal of Microbiology
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• v.40 no.4
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• pp.260-266
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• 2002

Bacterial strains were isolated from biofilms formed on glass slides submerged in seawater in Dae-Ho Dike. Eight strains showing fast attaching ability were selected and identified. Their exopolysaccharide (EPS)-producing ability and EPS properties were characterized. Based on Microlog System, 4 among the 8 strains were identified as Micrococcus luteus and the rest were Bacillus thuringiensis, Bacillus megaterium,, Staphylococcus saprophyticus and Agrobacterium vitis. A, vitis was reidentified as Sulfitobacter pontiacus based on 16S rDNA sequence data. The amount of water-soluble EPS produced by the 8 strains ranged from 0.114 to 1.329 g$.$l$\^$-1/ and the productivity was negatively correlated with the cell biomass. The molecular weight of the produced EPS ranged from 0.38 to 25.19$\times$10$\^$4/ Da. Glucose and galactose were ubiquitous sugar components. Mannose, ribose, and xylose were also major sugar components. The molecular weight and composition of the EPS showed strain-specific variation.

• Korean Journal of Environmental Agriculture
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• v.40 no.3
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• pp.194-202
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• 2021

BACKGROUND: Towards achievement of sustainable agriculture, using microbial inoculants may present promising alternatives without adverse environmental effects; however, there are challenging issues that should be addressed in terms of effectiveness and ecology. Viability and stability of the bacterial inoculants would be one of the major issues in effectiveness of microbial pesticide uses, and the changes within the indigenous microbial communities by the inoculants would be an important factor influencing soil ecology. Here we investigated the stability of the introduced bacterial strains in the soils planted with barley and its effect on the diversity shifts of the rhizosphere soil bacteria. METHODS AND RESULTS: Two different types of bacterial strains of Bacillus thuringiensis and Shewanella oneidensis MR-1 were inoculated to the soils planted with barley. To monitor the stability of the inoculated bacterial strains, genes specific to the strains (XRE and mtrA) were quantified by qPCR. In addition, bacterial community analyses were performed using v3-v4 regions of 16S rRNA gene sequences from the barley rhizosphere soils, which were analyzed using Illumina MiSeq system and Mothur. Alpha- and beta-diversity analyses indicated that the inoculated rhizosphere soils were grouped apart from the uninoculated soil, and plant growth also may have affected the soil bacterial diversity. CONCLUSION: Regardless of the survival of the introduced non-native microbes, non-indigenous bacteria may influence the soil microbial community and diversity.

• Korean Journal of Microbiology
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• v.49 no.3
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• pp.286-291
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• 2013

Soybean pastes with 8% (w/w) salinity were prepared instead of soybean paste with 14% (w/w) salinity to meet the growing demands of Korean's low sodium diet. After aging, white films had appeared on the surface of all low-salted soybean pastes [8% (w/w) salinity] unlike high-salted soybean pastes [14% (w/w) salinity]. All of eight microbes isolated from the surface film were identified as Pichia kudriavzevii. Eleven Bacillus strains with good characteristics of fermentation were isolated from traditionally fermented soybean pastes in order to preserve their unique flavors and aromas after aging, and as a result of analyzing the biochemical characteristics and 16S rRNA sequences, those were identified as B. subtilis, B. licheniformis, and B. methylotrophicus. All of the Bacillus isolates had antagonistic activities against 8 isolates of the film-forming yeasts and harbored the genes for synthesis of antimicrobial surfactants including lichenysin and/or surfactin.

• Journal of Microbiology and Biotechnology
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• v.14 no.6
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• pp.1142-1149
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• 2004

Streptococcus mutans has the capacity of inducing dental caries. Thus, to develop a novel way of preventing dental caries, a cell wall hydrolase-producing strain was isolated and its characteristics were investigated. Among 200 alkalophilic strains isolated from soil, 8 strains exhibited lytic activities against Streptococcus mutans. However, strain YU5215 with the highest cell wall hydrolase activity was selected for further study. Strain YU5215 was identified as a novel strain of Bacillus based on analyzing its 16S rDNA sequence and Bergey's Manual of Systematic Bacteriology, and thus designated as Bacillus mutanolyticus YU5215. The optimal conditions for the production of the cell wall hydrolase from Bacillus mutanolyticus YU5215 consisted of glucose ($0.8\%$), yeast extract ($1.2\%$), polypeptone ($0.5\%$), $K_{2}HPO_{4}\;(0.1\%$), $MgSO_{4}{\cdot}7H_{2}O$ ($0.02\%$), and $Na_{2}CO_{3}\;(1.0\%$) at pH 10.0. Bacillus mutanolyticus YU5215 was cultured at 30^{circ}C for 72 h to produce the cell wall hydrolase, which was then purified by acetone precipitation and CM-agarose column chromatography. The molecular weight of the lytic enzyme was determined as 22,700 Da by SDS-PAGE. When the cell wall peptidoglycan of Streptococcus mutans was digested with the lytic enzyme, no increase in the reducing sugars was observed, while the free amino acids increased, indicating that the lytic enzyme had an endopeptidase-like property. The amino terminus of the cell wall peptidoglycan digested by the lytic enzyme was determined as a glutamic acid, while the lytic site of the lytic enzyme in the Streptococcus mutans peptidoglycan was identified as the peptide linkage of L-Ala and D-Glu.