• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.671-674
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• 2001

Cyclodextrin glucanotransferase(CGTase) derived from recombinant Bacillus subtilis was partial purified and concentrated by ultrafiltration. The prepared CGTase were immobilized on various matrices by ionic interaction or covalent bond. CGTase covalently bound on CNBr-activated sepharose 4B were identified to be the highest immobilization activity among various immobilization methods. The optimum conditions for CGTase immobilization were determined; $30^{\circ}C$, 6Orpm, using O.2g CNBr-activated sepharose 4B in pH 6.0 phosphate buffer and 9hr immobilization.

• Microbiology and Biotechnology Letters
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• v.26 no.1
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• pp.23-33
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• 1998

The entire sequence of a 4,214,810 bp genome of the Bacillus subtilis 168 has been determined by an international project, and the completion has been announced on July 19, 1997. For the sequencing project an international consortium was established and 25 European, 7 Japanese laboratories, 2 biotechnology companies, and our laboratory participated in the project. Within this framework we determined the complete nucleotide sequence of a 53,289 bp fragment upstream of the odhA gene (181 $^{\circ}$) of the B. subtilis 168 chromosome. On the basis of the published DNA sequences of the B. subtilis sspC and odhA genes, we obtained genomic fragments by plasmid rescue and long-range PCR. The sequenced fragment contains 56 putative open reading frames (designated yojA-yolI and 9 known genes (sspC, cge cluster, orfE5, orfRMl and odhA), in which we found many interesting features. In addition, the entire nucleotide sequence of a 53,289 bp region enabled us to revise the current genetic map of this region.

• Journal of Microbiology and Biotechnology
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• v.29 no.9
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• pp.1383-1390
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• 2019

In this study, we expressed cotA laccase from Bacillus subtilis on the surface of B. subtilis spores for efficient decolorization of synthetic dyes. The cotE, cotG, and cotY genes were used as anchoring motifs for efficient spore surface display of cotA laccase. Moreover, a $His_6$ tag was inserted at the C-terminal end of cotA for the immunological detection of the expressed fusion protein. Appropriate expression of the CotE-CotA (74 kDa), CotG-CotA (76 kDa), and CotY-CotA (73 kDa) fusion proteins was confirmed by western blot. We verified the surface expression of each fusion protein on B. subtilis spore by flow cytometry. The decoloration rates of Acid Green 25 (anthraquinone dye) for the recombinant DB104 (pSDJH-EA), DB104 (pSDJH-GA), DB104 (pSDJH-YA), and the control DB104 spores were 48.75%, 16.12%, 21.10%, and 9.96%, respectively. DB104 (pSDJH-EA) showed the highest decolorization of Acid Green 25 and was subsequently tested on other synthetic dyes with different structures. The decolorization rates of the DB104 (pSDJH-EA) spore for Acid Red 18 (azo dye) and indigo carmine (indigo dye) were 18.58% and 43.20%, respectively. The optimum temperature for the decolorization of Acid Green 25 by the DB104 (pSDJH-EA) spore was found to be $50^{\circ}C$. Upon treatment with known laccase inhibitors, including EDTA, SDS, and $NaN_3$, the decolorization rate of Acid Green 25 by the DB104 (pSDJH-EA) spore decreased by 23%, 80%, and 36%, respectively.

• Applied Biological Chemistry
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• v.50 no.2
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• pp.87-94
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• 2007

A bacterium which has high enzymatic activities such as amylase, cellulase and protease was isolated from Korean traditional soybean food, doenjang. The isolated bacterium was identified to Bacillus subtilis HS25 by the test of morphological and biochemical properties according to Bergey's Manual of Systematic Bacteriology and API 50 CHL kit, and by the 16S rDNA sequence. The isolated B. subtilis HS25 had a potent antibacterial activity against food born causative or pathogenic bacteria. B. subtilis HS25 is endospore forming cell and contained flagella and abundant viscous material at the out layer of cell wall. It was rod type bacterium $(0.5{\sim}0.8{\times}3{\sim}5{\mu}m)$ having biochemical characteristics such as gram staining(+), catalase(+), oxidase(-) and hydrolysis of esculin(+). The optimal medium compositions for production of antibacterial substance in the B. subtilis HS25 were 1% of soluble starch, 0.5% of yeast extract, 0.5% of peptone and 0.05% of MgCl$_2{\cdot}6H_{2}O$. The optimum temperature and pH of the growth of the B. subtilis HS25 was 35$^{\circ}C$ and pH 7.5, respectively. The antibacterial activity was more high in neutral to a little alkaline pH (6.5-10.5) than in acidic pH. The optimal shaking speed to grow and to produce antibacterial substance of the B. subtilis HS25 was 160${\sim}$200 rpm. The optimal culture time for antibacterial activities of the bacterium were shown to be in the range of 12-36 hr.

• Microbiology and Biotechnology Letters
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• v.39 no.1
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• pp.9-19
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• 2011

The Bacillus subtilis pyrimidine biosynthetic (pyr) operon encodes all of the enzymes for the de novo biosynthesis of Uridine monophosphate (UMP) and additional cistrones encoding a uracil permease and the regulatory protein PyrR. The PyrR is a bifunctional protein with pyr mRNA-binding regulatory funtion and uracil phosphoribosyltransferase activity. To study the global regulation by the pyrR deletion, the proteome comparison between Bacillus subtilis DB104 and Bacillus subtilis DB104 ${\Delta}$pyrR in the minimal medium without pyrimidines was employed. Proteome analysis of the cytosolic proteins from both strains by 2D-gel electrophoresis showed the variations in levels of protein expression. On the silver stained 2D-gel with an isoelectric point (pI) between 4 and 10, about 1,300 spots were detected and 172 spots showed quantitative variations in which 42 high quantitatively variant proteins were identified. The results showed that production of the pyrimidine biosynthetic enzymes (PyrAA, PyrAB, PyrB, PyrC, PyrD, and PyrF) were significantly increased in B. subtilis DB104 ${\Delta}$pyrR. Besides, proteins associated carbohydrate metabolism, elongation protein synthesis, metabolism of cofactors and vitamins, motility, tRNA synthetase, catalase, ATP-binding protein, and cell division protein FtsZ were overproduced in the PyrR-deficient mutant. Based on analytic results, the PyrR might be involved a number of other metabolisms or various phenomena in the bacterial cell besides the pyrimidine biosynthesis.

• Food Science and Biotechnology
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• v.17 no.3
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• pp.527-533
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• 2008

For preparation of high quality kochujang by the traditional fermentation method, 4 types of kochujang were prepared with brick- or grain-shaped meju fermented with different strains (Aspergillus sojae, Aspergillus oryzae+Bacillus subtilis). After 100 days of fermentation at $25^{\circ}C$, the moisture, pH, salt, and ethanol content of kochujang were 40.52-43.20%, 4.71-4.82, 8.7-9.1%, and 0.75-0.94%, respectively, showing slight differences according to the strains and shapes of meju. Titratable acidities were slightly increased for up to 60 days of fermentation. The amino-type nitrogen content of kochujang prepared with brick-shaped meju (A. oryzae+B. subtilis) was the highest (164.20 mg%) among all of the kochujang types. The redness (a) value of kochujang prepared with brick-shaped meju (A. sojae) were higher (19.08) than those of other treatments (18.37-18.59). Sensory evaluation of kochujang prepared with grain-shaped meju (A. sojae) showed the highest scores for color and overall acceptability, 'at $6.43{\pm}1.87$ and $6.29{\pm}1.44$, respectively. It was estimated that high quality kochujang could be made by using meju fermented with selected strains.

• Journal of the Korean Society of Food Science and Nutrition
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• v.32 no.3
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• pp.413-417
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• 2003

To evaluate the antimicrobial activity of green tea against putrefactive microorganism in steamed bread, antibacterial activity of green tea extract against well-known strains of spoilage bacteria (Bacillus subtilis ATCC 6633, Bacillus pulmilus KCTC 3348 and Bacillus cereus IFO 12113) and mold (Aspergillus niger KCCM l1239) in bread was determined using the paper disk method. The green tea extract (GTE) showed the inhibition effects on the growth of all the strains of bacteria and mold at 1, 2, 3% levels. The activity of GTE was stable in the wide range of pH (4~9) and temperature (50~20$0^{\circ}C$). When green tea powder (GTP: 1, 3, 5%) was added to steamed bread increase of total bacterial and mold counts declined during storage at 25"C as the levels of GTP increased. By addition of 5% GTP, mold appeared 1 day late extending shelf life of steamed bread compared to control bread without GTP. Therefore, the levels of GTP added to steamed bread could be more than 5% for extended shelf life and wholesomeness of steamed bread.read.

• Journal of Veterinary Clinics
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• v.26 no.2
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• pp.117-122
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• 2009

The aim of this study is to investigate the effects of surfactin in combination with Bacillus subtilis BC1212 isolated from Korean soybean paste, on feed utilization and growth performance during 4 weeks in weaning piglets. Eighteen weaning piglets(Landrace$\times$Yorkshire$\times$Duroc; weighing $7.68{\pm}0.97\;kg$) were divided into control(n=9) and experimental groups(n=9). The treatments included a control group consisting of the basal diet with no additives(control) and an experimental group consisting of the basal diet supplemented with 1 g of surfactin C and $1.0{\times}10^9CFU$ of Bacillus subtilis BC1212/kg feed. Piglets fed Bacillus subtilis BC1212 increased in average daily weight gain and feed efficiency. In comparison with the control group, the fecal Bacillus subtilis were significantly increased and the fecal coliform bacteria were markedly reduced in the experimental group. In addition, Bacillus subtilis BC1212 had excellent acid and bile tolerance. The treatment of surfactin($50{\mu}g\;ml^{-1}$) in lipopolysaccharide(LPS)-stimulated swine peripheral blood mononuclear cells(PBMCs) for 6 h showed a significant inhibitory effect on INF-$\gamma$, TNF-$\alpha$ and NO secretion(p<0.05) in comparison with LPS treatment alone but not on IL-10 secretion, with levels of secreted IL-10 similar to those secreted by PBMCs stimulated with LPS alone. Supplementation with surfactin in combination with Bacillus subtilis BC1212 in diets improved the ecosystem of gastrointestinal tract by increasing probiotic population and enhanced the systemic immune response in weaned piglets.

• Korean Journal of Food Science and Technology
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• v.31 no.6
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• pp.1635-1641
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• 1999

Bacillus subtilis PCA20-3 was isolated from meju and was found to produce a protease. The strain produced the maximum amount of enzyme in the medium containing soytone (0.2%), soluble starch (2%), $(NH_4)_2SO_4\;(0.1%),\;CaCl_2(0.1%),\;yeast\;extract\;(0.01%),\;K_2HPO_4\;(0.1%),\;and\;KH_2PO_4\;(0.1%)$. Protease was first concentrated by ammonium sulfate (80% saturation, w/v) precipitation of culture supernatant. Then the enzyme was purified by column chromatography using CM Sephadex C-50. The collected proteins were rechromatographed using Sephadex G-100 gel filtration column. The fraction with protease active from Sephadex G-100 gel chromatography was found to be pure when examined by SDS-polyacrylamide gel electrophoresis and YMC-pak reverse phase chromatography. Specific activity, yield and purity were 76 U/mg. 2.7%, and 7.6 fold, respectively. The molecular weight of the enzyme was estimated to be 31.5 kDa by SDS-PAGE. The number of amino acids calculated from molecular weight was evaluated about 321 residues. N-terminal sequence of the enzyme was $Val^1-Pro^2-Tyr^3-Gly^4-Val^5-Ser^6-Gln^7-Gly^8-Lys^9-Ala^{10}$.

• Journal of Microbiology and Biotechnology
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• v.22 no.3
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• pp.331-338
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• 2012

A novel gene coding for an endo-${\beta}$-1,4-mannanase (manA) from Bacillus subtilis strain G1 was cloned and overexpressed in P. pastoris GS115, and the enzyme was purified and characterized. The manA gene consisted of an open reading frame of 1,092 nucleotides, encoding a 364-aa protein, with a predicted molecular mass of 41 kDa. The ${\beta}$-mannanase showed an identity of 90.2-92.9% ${\leq}95%$) with the corresponding amino acid sequences from B. subtilis strains deposited in GenBank. The purified ${\beta}$-mannanase was a monomeric protein on SDS-PAGE with a specific activity of 2,718 U/mg and identified by MALDI-TOF mass spectrometry. The recombinant ${\beta}$-mannanase had an optimum temperature of $45^{\circ}C$ and optimum pH of 6.5. The enzyme was stable at temperatures up to $50^{\circ}C$ (for 8 h) and in the pH range of 5-9. EDTA and most tested metal ions showed a slightly to an obviously inhibitory effect on enzyme activity, whereas metal ions ($Hg^{2+}$, $Pb^{2+}$, and $Co^{2+}$) substantially inhibited the recombinant ${\beta}$-mannanase. The chemical additives including detergents (Triton X-100, Tween 20, and SDS) and organic solvents (methanol, ethanol, n-butanol, and acetone) decreased the enzyme activity, and especially no enzyme activity was observed by addition of SDS at the concentrations of 0.25-1.0% (w/v) or n-butanol at the concentrations of 20-30% (v/v). These results suggested that the ${\beta}$-mannanase expressed in P. pastoris could potentially be used as an additive in the feed for monogastric animals.