• The Plant Pathology Journal
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• v.26 no.3
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• pp.238-244
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• 2010

Spatial and temporal distribution of Bacillus licheniformis N1 was investigated over time on the leaves, petioles and crowns of the strawberry plants. Bacterial population on the strawberry plants was quantified over time by selective plating. Bacterial population of N1 containing a plasmid pWH43G carrying green fluorescent protein (GFP) declined relatively faster on the plant surface as compared to the Strain N1 itself. However, this result was found to be enough to utilize the strain to visualize bacterial colonization on the plant surface. When B. licheniformis N1 was treated together with Silwet L-77 at 0.03%, the bacterial population on plant surface persisted for up to 7 days. B. licheniformis N1 (pWH43G) containing Silwet L-77 was applied on the strawberry plants and the GFP expressing bacteria were visualized by confocal laser scanning microscopy. Bacterial persistence was also investigated in a growth chamber and in a plastic house after N1 bioformulation treatment on the strawberry plant. The Strain N1 colonized three different tissues well and persisted over 3 to 5 days on the strawberry plants. They formed bacterial aggregates on plant surfaces for at least 3 days, resulting in a biofilm to resist fluctuating plant surface environment. However, the bacterial persistence dramatically declined after 7 days in all tested tissues in a plastic house. This study suggest that B. licheniformis N1 colonizes the strawberry plant surface and persists for a long time in a controlled growth chamber, while it can not persist over 7 days on the plant surface in a plastic house.

• Journal of Life Science
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• v.11 no.2
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• pp.173-180
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• 2001

This study was conducted to estimate the cultural characteristics, the production of antibiotic, and the selection of optimal media for mass culture of Bacillus licheniformis N1 isolate which was previously reported as an antagonistic bacterium to Botrytis cinerea. We investigated initial pH, temperatures and shaking speed for good cultural conditions and antibiotics production by N1 isolate. According to the results, the optimal conditions of initial pH, temperatures, and shaking speed were determined to be pH 5.0~5.5, 30~35$^{\circ}C$ and 250 rpm, respectively. Also, the optimal conditions for the antagonism by N1 isolate highly appeared in the initial pH as 5.0, and the mycelial growth inhibition was high when the substances used such as glucose or corn starch as carbon sources, and biji(soybean curd residue) flour as a nitrogen source. Furthermore, inhibitory area was significantly expanded, when 3% or 5% of corn starch was added into 5% of Biji flour as nitrogen source, were respectivley selected for mass culture of N1 isolate. Among them, 5% Biji flour medium showed higher cell density more than 10 times that in NB medium after 48 hour incubation. Therefore, the optimal medium was determined as 5% biji flour added 3~5% of corn starch for high density of cells.

• The Plant Pathology Journal
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• v.26 no.2
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• pp.170-177
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• 2010

Bacillus licheniformis N1, previously developed as a biofungicide formulation N1E to control gray mold disease of plants, was investigated to study the bacterial traits that may be involved in its biological control activity. Two N1E based formulations, bacterial cell based formulation PN1E and culture supernatant based formulation SN1E, were evaluated for disease control activity against gray mold disease of tomato and strawberry plants. Neither PN1E nor SN1E was as effective as the original formulation N1E. Fractionation of antifungal compounds from the bacterial culture supernatant of B. licheniformis N1 indicated that two different cyclic lipopeptides were responsible for the antimicrobial activity of the N1 strain. These two purified compounds were identified as iturin A and surfactin by HPLC and LCMS. The purified lipopeptides were evaluated for plant disease control activity against seven plant diseases. Crude extracts and purified compounds applied at 500 ${\mu}g/ml$ concentration controlled tomato gray mold, tomato late blight and pepper anthracnose effectively with over 70% disease control value. While iturin showed broad spectrum activity against all tested plant diseases, the control activity by surfactin was limited to tomato gray mold, tomato late blight, and pepper anthracnose. Although antifungal compounds from B. licheniformis N1 exhibited disease control activity, our results suggested that bacterial cells present in the N1E formulation also contribute to the disease control activity together with the antifungal compounds.

• The Plant Pathology Journal
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• v.25 no.4
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• pp.344-351
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• 2009

A biocontrol bacterium Bacillus licheniformis N1 grown in nutrient broth showed no chitinolytic activity, while its genome contains a gene which encodes a chitinase. The gene for chitinase from B. licheniformis N1 was amplified by PCR and the deduced amino acid sequence analysis revealed that the chitinase exhibited over 95% identity with chitinases from other B. licheniformis strains. Escherichia coli cells carrying the recombinant plasmid displayed chitinase activity as revealed by the formation of a clear zone on chitin containing media, indicating that the gene could be expressed in E. coli cells. Chitinase gene expression in B. licheniformis N1 was not detected by RT-PCR analysis. The protein was over-expressed in E. coli BL21 (DE3) as a glutathione S-transferase fusion protein. The protein could also be produced in B. subtilis 168 strain carrying the chitinase gene of N1 strain. The crude protein extract from E. coli BL21 carrying GST fusion protein or culture supernatant of B. subtilis carrying the chitinase gene exhibited enzyme activity by hydrolyzing chitin analogs, 4-methylumbelliferyl-$\beta$-D-N,N'-diacetylchitobioside and 4-methylumbelliferyl-$\beta$-D-N,N',N"-triacetylchitotrioside. These results indicated that even though the chitinase gene is not expressed in the N1 strain, the coding region is functional and encodes an active chitinase enzyme. Furthermore, B. subtilis 168 transformants expressing the chitinase gene exhibited antifungal activity against Fulvia fulva by suppressing spore germination. Our results suggest that the proper engineering of the expression of the indigenous chitinase gene, which will lead to its expression in the biocontrol strain B. licheniformis N1, may further enhance its biocontrol activity.

• Microbiology and Biotechnology Letters
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• v.44 no.4
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• pp.470-478
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• 2016

A bacterium producing a large amount of chitinolytic enzyme was isolated from the intestinal tract of earthworm. The isolate was identified as Bacillus licheniformis by 16S ribosomal RNA analysis and designated as B. licheniformis GA9. The enzyme was purified by 40-60% ammonium sulfate precipitation, diethyl-aminoethyl groups exchange chromatography, and gel filtration chromatography. The molecular weight was estimated to be 52.1 kDa and the N-terminal amino acid sequence was D-S-G-K-N-G-K-I-I-R-Y-YP-I-R. The optimum activity of the purified chitinolytic enzyme was shown at pH 5.0 and $40^{\circ}C$, and the enzyme was stable in the ranges of $20-50^{\circ}C$ and pH 5.0-6.0. Enzyme activity was increased by $Co^{2+}$, while it was inhibited by $Cu^{2+}$ and $Fe^{2+}$. But it was recovered by chelating metals with ethylenediaminetetraacetic acid. The $K_m$ and $V_{max}$ values of the purified enzyme were 4.02 mg/ml and 0.52 mg/min, respectively. The chitinolytic enzyme characterized in this study has potential applications in areas such as biotechnology, biomedicine, agriculture, and nutrition.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.7
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• pp.946-950
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• 2014

Bacillus licheniformis was grown in minimal nutrient medium containing 1% (w/v) of distillers dried grain with soluble (DDGS), palm kernel meal (PKM), wheat bran (WB) or copra meal (CM), and the enzyme activity of endoglucanase, ${\beta}$-glucosidase, xylanase and reducing sugars was measured to investigate a possibility of using cost-effective agricultural residues in producing cellulolytic and hemicellulolytic enzymes. The CM gave the highest endoglucanase activity of 0.68 units/mL among added substrates at 48 h. CM yielded the highest titres of 0.58 units/ml of ${\beta}$-glucosidase, compared to 0.33, 0.23, and 0.16 units/mL by PKM, WB, and DDGS, respectively, at 72 h. Xylanase production was the highest (0.34 units/mL) when CM was added. The supernatant from fermentation of CM had the highest reducing sugars than other additional substrates at all intervals (0.10, 0.12, 0.10, and 0.11 mg/mL respectively). It is concluded that Bacillus licheniformis is capable of producing multiple cellulo- and hemicellololytic enzymes for bioethanol production using cost-effective agricultural residues, especially CM, as a sole nutrient source.

• Journal of Microbiology and Biotechnology
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• v.17 no.3
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• pp.438-444
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• 2007

Bacillus licheniformis N1 is a biological control agent to control gray mold diseases caused by Botrytis cinerea. Various formulations of B. licheniformis N1 were generated and evaluated for the activity to control strawberry gray mold. The wettable powder type formulation N1E was selected in pot experiments with remarkable disease control activity on both strawberry leaves and flowers. The N1E formulation contained 400 g of com starch, 50 ml of olive oil, and 50 g of sucrose per a liter of bacterial fermentation culture. Optimum dilution of N1E to appropriately control the strawberry gray mold appeared to be 100-fold dilution in plastic house artificial infection experiments. The significant reduction of symptom development in the senescent leaves was apparent by the treatment of N1E at 100-fold dilution when N1E was applied before Bo. cinerea inoculation, but not after the inoculation. Both artificial infection experiments in a plastic house and natural infection experiments in the farm plastic house under production conditions revealed that the disease severity of gray mold on strawberry leaves and flowers was significantly reduced by N1E treatment. The disease control value of N1E on strawberry leaves was 81% under production conditions, as compared with the 61.5% conferred by a chemical fungicide, iprodione. This study suggests that our previously generated formulation of B. licheniformis N1 will be effective to control strawberry gray mold by its preventive activity.

• KSBB Journal
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• v.6 no.1
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• pp.105-109
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• 1991

In separating alkaline protease from the bacteria (Bacillus licheniformis) fermentation broth using reverse micelle, effects of various factors;ionic strength, pH and surfactant concentration, on separation efficiency were studied. KCl controls the ionic strength. The lower KCl concentration was in the feed solution, the more protein and activity was recovered. The higher KCl concentration was in the stripping solution, the more protein and activity was recovered. Using sodium-di-2-ethylhexyl sulfosuccinate(Aerosol-OT or AOT) as a surfactant, the higher AOT concentration in the solvent, the more activity and protein were recovered. 0.1N NaOH and IN HCl were used to adjust pH. Maximum recovery of protein mass and activity were obtained at feed solution of pH 5.3. Maximum activity was recovered at stripping solution of pH 7.5

• Journal of Microbiology and Biotechnology
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• v.12 no.5
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• pp.773-779
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• 2002

A gene (hap) encoding aminopeptidase from the chromosomal DNA of Bacillus licheniformis was cloned. The gene is 1,347 bp long and encodes a 449 amino acid preproprotein with a major mature region of 401 amino acids (calculated molecular mass 43,241 Da). N-Terminal sequence of the purified protein revealed a potential presence of N-terminal propeptide. The deduced primary amino acid sequence and the mass analysis of the purified protein suggested that a C-terminal peptide YSSVAQ was also cleaved off by a possible endogeneous protease. Tho amino acid sequence displayed 58% identity with that of the aminopeptidase from alkaliphilic Bacillus halodurans. This bacterial enzyme was overexpressed in recombinant Escherichia coli and Bacillus subtilis cells. Clones containing the intact hap gene, including its own promoter and signal sequence, gave rise to the synthesis of extracellular and thrmostable enzyme by B. subtilis transformants. The secreted protein exhibited the same biochemical properties and the similar apparent molecular mass as the B. lichenzyormis original enzyme.

• Microbiology and Biotechnology Letters
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• v.26 no.5
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• pp.420-426
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• 1998

An extracellular glutamyl aminopeptidase (EC 3.4.11.7) producing bacterium was isolated from soil and identified as Bacillus licheniformis based on its morphological and physiological characteristics. The aminopeptidase was purified to homogeneity by ammonium sulfate precipitation, Phenyl Sepharose, Resource Q, and Superose 12 column chromatographies. The specific activity of the purified aminopeptidase was 9.2 unit/mg for glutamyl p-nitroanilide with 17.6 purification folds. The purified aminopeptidase had an estimated molecular mass of 64 kDa consists of two different subunits (42 kDa and 22 kDa), and its isoeletric point was 5.2 measured by isoelectric focusing. The optimum pH and temperature of the aminopeptidase were 8.0 and 55$^{\circ}C$, respectively. The aminopeptidase was inhibited by EDTA and 1,10-phenanthroline, suggesting it be a metalloenzyme. Comparing with other aminopeptidase, the enzyme showed relatively high activity against peptide having glutamic acid as N-terminal.