• Journal of Life Science
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• v.16 no.4
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• pp.605-611
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• 2006

To investigate the possible use of probiont in fish farming industry, a bacterium with inhibitory effect against Vibrio anguillarum was isolated from gastrointestinal tract of the marine fish of yellow tail. It was identified to be Bacillus amyloliquefaciens H41 based on biochemical and physiological characterization. The optimal growth conditions of the isolated strain were 1% peptone, 1.5% yeast extract, 1% sucrose, 0.5% NaCl, 0.05% $MgSO_4{\cdot}7H_20$, pH 7.0-8.0, and 20 hr of incubation between $28-35^{\circ}C$ under aeration. The culture supernatant of the isolated strain showed inhibition activity against V. anguillarum. Inhibition activity was cleared by forming a clear zone by a paper-disk method. The maximal production of growth inhibition factor was induced by cultivation under 1% peptone, 1.5% yeast extract, 1% sucrose, 1% NaCl, 0.05% $MgSO_4{\cdot}7H_20$, pH 7.5 and at $35^{\circ}C$. The highest growth inhibition factor production was observed after 16-24 hr cultivation under aeration. The culture supernatant of the isolated strain showed inhibition activity whereas no inhibition activity was shown from the standard B. amyloliquefaciens KCTC 1724 strain. The growth inhibition affected only against V. anguillarum among other pathogenic Vibrios tested here.

• Journal of Microbiology and Biotechnology
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• v.21 no.11
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• pp.1166-1173
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• 2011

Meju is a traditional Korean fermented soy product used as a key element for soy sauce and doenjang. Bacilli with antimicrobial activity were isolated from meju prepared by traditional methods at Sunchang county, Jeollabukdo, Korea. Six isolates were identified as Bacillus amyloliquefaciens by recA gene sequencing and RAPD-PCR. One isolate, B. amyloliquefaciens MJ5-41, showed the strongest fibrinolytic activity. A 27 kDa active fibrinolytic enzyme, AprE5-41, was purified from the culture supernatant of MJ5-41 grown on LB by chromatographic methods. The optimum pH and temperature for purified AprE5-41 were 7.0 and $45^{\circ}C$, respectively. AprE5-41 quickly degraded $A{\alpha}$ and $B{\beta}$ chains but not the ${\gamma}$-chain of fibrinogen. AprE5-41 exhibited the highest specificity for N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide, a known substrate for ${\alpha}$-chymotrypsin, cathepsin G, and subtilisin BPN'. The structural gene, aprE5-41, was cloned by PCR and successfully expressed in B. subtilis.

• Microbiology and Biotechnology Letters
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• v.41 no.2
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• pp.249-252
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• 2013

The Bacillus KU801 strain, due to its potential in the field of probiotics for animal use, was isolated from chicken feces. Strain KU801 was identified as Bacillus amyloliquefaciens KU801 based on the results of 16S rRNA sequencing. Vegetative and spore cells of B. amyloliquefaciens KU801 were resistant to artificial gastric juice and artificial bile acid. B. amyloliquefaciens KU801 was found to inhibit the production of nitric oxide (NO) and increase the production of Interleukin-1 alpha (IL-1${\alpha}$). DNA damage induced by N-methyl-Ntion of ninitroso-guanidine (MNNG) was significantly inhibited, in a dose dependent manner, by preincubating MNNG together with B. amyloliquefaciens KU801. These results demonstrate the potential use of B. amyloliquefaciens KU801 as a feed additive.

• Journal of Microbiology
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• v.41 no.1
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• pp.58-62
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• 2003

An extracellular pretense of Bacillus amyloliquefaciens S94 was purified to apparent homogeneity. The enzyme activity was strongly inhibited by general inhibitor for serine protease, PMSF, suggesting that the enzyme is a serine pretense. The purified enzyme activity was inhibited by leucine peptidase inhibitor, bestatin, suggesting that the enzyme is a leucine endopeptidase. The maximum proteolytic activity against different protein substrates occurred at pH 10, 45$^{\circ}C$ (protein substrate) and pH 8, 45$^{\circ}C$ (synthetic substrate). The purified enzyme was specific in that it readily hydrolyBed substrates with Leu or Lys residues at P$_1$ site. The pretense had characteristics of a cold-adapted protein, which was more active for the hydrolysis of synthetic substrate in the range of 15$^{\circ}C$ to 45$^{\circ}C$, specially at low temperature.

• Journal of Life Science
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• v.18 no.6
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• pp.789-795
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• 2008

To study the possible use of probiotics in fish farming, we evaluated antagonism of antibacterial strain Bacillus amyloliquefaciens H41 against the fish pathogenic bacterium Vibrio anguillarum NCMB1. The purification of growth inhibition factor produced by B. amyloliquefaciens H41 was achieved by obtaining supernatant of this bacterium. The growth inhibition factor was purified to homogeneity by 70% ammonium sulfate precipitation, DEAE-sephadex A-50 ion exchange chromatography, sephadex G-200 gel filtration column chromatography, and sephadex G-50 gel filtration column chromatography with 40.8 fold of purification and 2.9% yield. The molecular weight of the purified growth inhibition factor was 48 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH and temperature for the growth inhibition factor were pH 7.5 and $30^{\circ}C$, respectively. The activity of growth inhibition factor was enhanced slightly by some metal ions, such as $Mg^{+2}$, $Mn^{+2}$, but was inhibited by the addition of $Co^{+2}$, $Hg^{+2}$, $Zn^{+2}$ and $Ag^{+2}$. NaCl stability of the growth inhibition factor was observed with 50% residual activity at 3% NaCl concentration. Toxicity test showed that the purified B. amyloliquefaciens H41 growth inhibition factor did not affect the live of Japanese flounder (Paralichthys olivaceus) and the effectiveness was 78% of residual lethality compared to commercial antibacterial agents.

• Journal of Microbiology
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• v.41 no.4
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• pp.345-348
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• 2003

The bio-mediated production of calcite crystals by calcinogenic bacteria has great applicable value for the restoration of deteriorated calcareous monuments, because of its high purity and coherency. An investigation of the conditions for calcite production by an alkalophilic Bacillus amyloliquefaciens CMB01 strain was made. Optimal calcite precipitation occurred when the bacterium was cultured at pH 8.0 and 30$^{\circ}C$, and in B4 medium that consisted of 0.4% yeast extract, 0.5% glucose, and 1.5% calcium acetate. Calcium ion of the bacterially induced calcite was analyzed by an inductively coupled plasma (ICP) spectrophotometer. Optical and scanning electron microscopy (SEM) of the calcite revealed a typical rombohedral polycrystalline structure.

• Microbiology and Biotechnology Letters
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• v.41 no.1
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• pp.33-43
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• 2013

A bacterium producing a fibrinolytic enzyme was isolated from Cheonggukjang. The bacterium was identified as a strain of Bacillus amyloliquefaciens by 16S rDNA analysis and designated as B. amyloliquefaciens HC188. The optimum culture medium appeared to be one containing 0.5% (w/v) maltose and 0.5% (w/v) soytone. Bacterial growth in the optimal medium at $37^{\circ}C$ reached the stationary phase after 27 h of incubation and the fibrinolytic enzyme showed optimum activity at 24 h. The enzyme was purified by 20-80% ammonium sulfate precipitation, CM Sepharose fast flow ion exchange chromatography, and Sephacryl S-200HR column chromatography. Its specific activity was 38359.3 units/mg protein and the yield was 5.5% of the total activity of the crude extracts. The molecular weight was 24.7 kDa and the amino acids of the N-terminal sequence were AQSVPYGVSQIKAPA. The fibrinolytic enzyme activity had an optimum temperature of $40^{\circ}C$ and an optimum pH of 8.0, and the enzyme was stable in the ranges $20-40^{\circ}C$ and pH 6.0-8.0. Enzyme activity was increased by $Ca^{2+}$ and $Co^{2+}$ but inhibited by $Cu^{2+}$, EDTA, and PMSF. It is suggested that the purified enzyme is a metallo-serine protease.

• Journal of fish pathology
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• v.31 no.1
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• pp.41-48
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• 2018

The utility of Bacillus spp. organisms for reduction of dissolved hydrogen sulfide ($H_2S$) in white leg shrimp (Litopenaeus vannamei) culture was tested with different combinations of Bacillus spp. microorganisms: combination A (B. subtilis + B. licheniformis); combination B (B. licheniformis + B. amyloliquefaciens); combination C (B. subtilis + B. licheniformis + B. amyloliquefaciens). Of these 3 combinations, C was effective in few hours after addition whereas B needed longer time to be effective. The $H_2S-reducing$ effect of combination C was dependent on the amount of microorganisms added to $H_2S-containing$ test solution. Exposure of white leg shrimp to $H_2S$ at 8 mg/L for 7 days led to survival of 80% and 1 mg/L for 14 days it was 82.5%. The survival rate was 97.5% when combination C was simultaneously added to shrimp tanks during $H_2S$ exposure at 1 mg/L for 14 days. It was demonstrated that combination C microorganisms (B. subtilis + B. licheniformis + B. amyloliquefaciens) can reduce dissolved $H_2S$ concentrations, and this effect can be utilized to protect white leg shrimp from $H_2S$ toxicity.

• BMB Reports
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• v.38 no.1
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• pp.41-48
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• 2005

In this study we developed the transposon-mediated shuttle vector 'Hanpvid', which composed of HaNPV (Heliothis armigera nuclear polyhedrosis virus) genomic DNA and a transposon cassette from Bacmid of Bac-to-Bac system. Hanpvid replicates in E. coli in the same way as Bacmid and retains infective function in cotton bollworm cells (Hz-AM1). Using Hanpvid we constructed a recombinant virus, which could infect Hz-AM1 cells and generate recombinant HaNPV (rHa-Bar) containing the barnase gene, a ribonuclease gene from Bacillus amyloliquefaciens. Since the expression vector carrying barnase gene cannot replicate in the absence of barstar, a specific inhibitor of barnase, we constructed a new cotton bollworm cell line (AM1-NB) using the marker rescue method. In AM1-NB barstar was integrated into the cellular chromosome to sustain the replication of rHa-Bar. To screen out recombinant HaNPV for potential use as biopesticide, Hz-AM1 and AM1-NB cell lines were infected with rHa-Bar, respectively. The results obtained indicate that Viral progenies in AM1-NB were 23 and 160 times greater than those in Hz-AM1 48 h and 72 h after infection, respectively. With additional insertion of the polyhedron gene from AcNPV (Autographa californica nuclear polyhedrosis virus) into the Hanpvid genome, rHa-Bar regained the polyhedron phenotype and its pest-killing rate greatly improved. Toxic analysis showed that the lethal dosages ($LD_{50}$) and the lethal time(s) ($LT_{50}$) of rHa-Bar were reduced by 20% and 30%, respectively, compared to wt-HaNPV in the third instar larvae of cotton bollworm. This study shows that in AM1-NB barnase can be effectively produced and used as pest-killing agent for the biological control of cotton pests.

• The Korean Journal of Food And Nutrition
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• v.33 no.4
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• pp.381-389
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• 2020

The purpose of this study was to evaluate the appearance, physicochemical, physical, and fermentation properties of the fermented soybean produced by manufacturing with inoculation the different types of microbial strains. The strains were inoculated by the NSI (natural strains inoculation), and the SSI (selective strain inoculation) were treatments. The appearance showed differences in color, viscous substance, and hardness depending on strains inoculation and fermentation duration. The pH, and total acidity were 6.40~7.26%, and 0.10~0.39% respectively with differences depending on the samples. The moisture content as the fermentation duration increased, the NSI (56.03~57.66%) decreased and the SSI (56.71~58.63%) increased. The physical characteristics of the hardness increased as the fermentation duration increased for the NSI and the SSI decreased. The color values for the L, a, and b values were 47.64~58.56, 7.15~9.08, and 12.41~17.30, respectively. The α-amylase and protease activities of the SSI were the highest among all treatments. The total viable cell counts of the fermented soybean products by strains were 5.02 to 9.77 log CFU/g, and SSI (fermentation, 48 hours) was the highest. The amino-type nitrogen contents of all samples were 301.62~746.97 mg% and the SSI showed the highest content. The amino acid had the highest glutamic acid content.