• Title/Summary/Keyword: Bacillus subtilis

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High-Level Expression and Secretion of Bacillus pumilus Lipase B26 in Bacillus subtilis Chungkookjang

  • Lee, Mi-Hwa;Song, Jae-Jun;Choi, Yoon-Ho;Hong, Seung-Pyo;Rha, Eu-Gene;Kim, Hyung-Kwoun;Lee, Seung-Goo;Poo, Har-Young;Lee, Sang-Chul;Seu, Young-Bae;Sung, Moon-Hee
    • Journal of Microbiology and Biotechnology
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    • v.13 no.6
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    • pp.892-896
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    • 2003
  • High-level expression of the lipase B26 gene from Bacillus pumilus was achieved using Bacillus subtilis Chungkookjang isolated from the Korean traditional fermented bean paste, Chungkookjang. For the secretory production of recombinant lipase B26 in a Bacillus host system, pLipB26 was constructed by ligating the lipase B26 gene into the recently designed Escherichia coli-Bacillus shuttle vector, pLipSM, and that was then transformed into B. subtilis Chungkookjang. Among the various vector, medium, and host combinations, B. subtilis Chungkookjang harboring the pLipB26 exhibited the highest lipase activity in PY medium, and B. subtilis Chungkookjang secreted two times more enzymes than B. subtilis DB 104 under the same condition. When B. subtilis Chungkookjang harboring the pLipB26 was cultured in a 5-1 jar-fermentor containing 21 of a PY medium, the maximum lipase activity (140 U/ml) and production yield (0.68 g/l) were obtained during the late exponential phase from a cell-free culture broth. Although B. subtilis Chungkookjang also secreted extracellular proteases at the late exponential phase, these results suggested the potential of B. subtilis Chungkookjang as a host for the secretory production of foreign proteins.

Cloning and Expression of A Liquefying $\alpha$-Amylase Gene from Bacillus amyloliquefaciens in Bacillus subtilis (Bacillus amyloliquefaciens 액화형 $\alpha$-amylase 유전자의 클로닝 및 Bacillus subtilis에서의 발현)

  • 김사열;송방호;이인구;서정환;홍순덕
    • Microbiology and Biotechnology Letters
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    • v.14 no.6
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    • pp.479-485
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    • 1986
  • A 5200 basepair DNA fragment containing the Bacillus amyloliquefaciens amyE gene, encoding liquefying $\alpha$-amylase (1,4-$\alpha$-1)-glucan glucanohydrolase, EC 3.2.1.1), has been inserted into BamHI site of the pUB110 and the hybrid plasmid was designated as pSKS3. The pSKS3 was transformed into the Bacillus subtilis KM2l3 as a host which is a saccharifying $\alpha$-amylase deficient mutant of Bacillus subtilis NA64, and the plasmid in the transformed cell was expressed $\alpha$-amylase production and kanamycin resistance. The $\alpha$-amylase production of the transformed cell was reduced to one fifth of that of the donor strain. The Bacillus subtilis KM2l3 tarring pSKS3 indicated that the amyE gene product is a polypeptide which has the same electrophoretic mobility with that of the Bacillus amyloliquefaciens, but different from the saccharifying $\alpha$-amylase of Bacillus subtilis NA64. It means that the amyE gene of pSKS3 originales from the Bacillus amyloliquefaciens.

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Expression of Bacillus macerans Cyclodextrin Glucanotransferase in Bacillus subtilis

  • Kim, Chang-Sup;Han, Nam-Soo;Kweon, Dae-Hyuk;Seo, Jin-Ho
    • Journal of Microbiology and Biotechnology
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    • v.9 no.2
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    • pp.230-233
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    • 1999
  • A plasmid vector was constructed for the expression and secretion of Bacillus macerans cyclodextrin glucanotransferase (CGTase) in Bacillus subtilis. The vector, pUBACGT, was composed of the ribosome-binding sequence, signal sequence, and cgt gene from B. macerans under the control of amyR2, the promoter of amyE gene coding for $\alpha$-amylase from B. subtilis var. natto. Bacillus subtilis LKS88, a mutant strain lacking genes for an amylase and two proteases, was used as a host for the transformation of the plasmid vector. The transformants were selected on kanamycin-containing Luria-Bertani plates. The starch hydrolyzing activity was observed on the starch-containing plates by the iodine method and cyclodextrin-forming activity was detected in the culture medium. A SDS-PAGE analysis showed that most of the expressed CGTase in the recombinant B. subtilis was secreted into the medium at a high expression level.

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Discrimination of Bacillus subtilis from Other Bacillus Species Using Specific Oligonucleotide Primers for the Pyruvate Carboxylase and Shikimate Dehydrogenase Genes

  • Lee, Gawon;Heo, Sojeong;Kim, Tao;Na, Hong-Eun;Park, Junghyun;Lee, Eungyo;Lee, Jong-Hoon;Jeong, Do-Won
    • Journal of Microbiology and Biotechnology
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    • v.32 no.8
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    • pp.1011-1016
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    • 2022
  • Bacillus subtilis is a useful bacterium in the food industry with applications as a starter strain for fermented food and as a probiotic. However, it is difficult to discriminate B. subtilis from other Bacillus species because of high phenotypic and genetic similarity. In this study, we employed five previously constructed multilocus sequence typing (MLST) methods for the discrimination of B. subtilis from other Bacillus species and all five MLST assays clearly distinguished B. subtilis. Additionally, the 17 housekeeping genes used in the five MLST assays also clearly distinguished B. subtilis. The pyruvate carboxylase (pyrA) and shikimate dehydrogenase (aroE) genes were selected for the discrimination of B. subtilis because of their high number of polymorphic sites and the fact that they displayed the lowest homology among the 17 housekeeping genes. Specific primer sets for the pyrA and aroE genes were designed and PCR products were specifically amplified from B. subtilis, demonstrating the high specificity of the two housekeeping genes for B. subtilis. This species-specific PCR method provides a quick, simple, powerful, and reliable alternative to conventional methods in the detection and identification of B. subtilis.

Fermentation and Quality Characteristics of Cheonggukjang Fermented with Bacillus subtilis BC-P1 (Bacillus subtilis BC-P1 균주를 이용하여 제조한 청국장의 발효 및 품질 특성분석)

  • Park, Sung-Yong;Bang, Mi-Ae;Oh, Boung-Jun;Park, Jeong-Hoon;Song, Won-Seob;Choi, Kyung-Min;Choung, Eui-Su;Boo, Hee-Ock;Cho, Seung-Sik
    • Korean Journal of Microbiology
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    • v.49 no.3
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    • pp.262-269
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    • 2013
  • The object of this study was to improve the quality of Cheonggukjang with new starter, Bacillus subtilis BC-P1. Twenty strains were isolated from the commercial cheonggukjang and 1 Bacillus strain (BC-P1) with protease activity was selected. The 16S rRNA gene sequence revealed that the BC-P1 was closely related to B. subtilis with 99% homology. The quality characteristics of chunggukjang fermented with B. subtilis BC-P1, Bacillus nato (PC) and commercial chunggukjang (NC) were investigated. The characteristics of fermentation were determined by protease, lipase, xylanase, chitinase, and fibrinolytic activities, reducing sugar, nutrient composition and amino acid contents of cheonggukjang sample. Cheonggukjang fermented with B. subtilis BC-P1 showed the strongest fibrinolytic, xylanase, and chitinase activities. Reducung sugar contents of Cheonggukjang samples were $30.16{\pm}2.11$ mg/g (NC), $28.56{\pm}1.52$ mg/g (PC), $32.39{\pm}1.87$ mg/g (BC-P1). And their total amino acid contents were 338.99 mg% (NC), 445.19 mg% (PC), 741.35 mg% (BC-P1). These results suggested that B. subtilis BC-P1 was suitable to be used as a starter to enhance the quality and effects of cheonggukjang.

Comparison of Heat Resistance of Bacillus subtilis, Geobacillus stearothermophilus, and Bacillus atrophaeus spores (Bacillus subtilis, Geobacillus stearothermophilus 및 Bacillus atrophaeus 포자의 열 저항성 비교)

  • Eun-Sun Jeong;Ju-Hee Nam;Jung-Beom Kim
    • Journal of Food Hygiene and Safety
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    • v.38 no.5
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    • pp.356-360
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    • 2023
  • We analyzed the heat resistance of non-pathogenic Bacillus atrophaeus, Bacillus subtilis, and Geobacillus stearothermophilus spores which exhibit strong heat resistance and evaluated the possibility of using them to determine direct sterilization when manufacturing retort foods. The D121-values of B. subtilis, G. stearothermophilus, and B. atrophaeus spores were 2.9±0.1 min, 4.3±0.1 min, and 3.7±0.1 min, respectively. The Z-values of B. subtilis, G. stearothermophilus, and B. atrophaeus spores were 43.0±1.4℃, 25.0±1.6℃, and 35.8±1.4℃, respectively. The D121-values of B. subtilis, G. stearothermophilus, and B. atrophaeus spores were all higher than that of Clostridium botulinum spores used to confirm retort food sterilization. Considering these results, B. subtilis, G. stearothermophilus, and B. atrophaeus spores can be used instead of the pathogenic spore-forming bacteria C. botulinum when sterilizing retort food. In addition, sterilization can be confirmed in 2 to 3 days, a shorter time than the 13 days required for existing bacterial growth experiments based on the Korean food code.

Effect of Meju Shapes and Strains on the Chemical Composition of Soybean Paste (Bacillus속과 Aspergillus oryzae로 만든 메주가 개량식 된장의 성분에 미치는 영향)

  • Seo, Jeong-Sook;Man, Eun-Mi;Lee, Taik-Soo
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.15 no.4
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    • pp.1-9
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    • 1986
  • The mashes of soybean paste were preparea using the conventional meju fermented naturally by wild microoganisms or the new types of meju fermented by pure cultures of Aspergillus oryzae, Bacillus natto and B. subtilis to elucidate changes during the aging period. The results obtained are as follows ; The soybean paste made with conventional meiu and Asp. oryzae meju showed higher content of amino nitrogen than those of B, natto and B. subtilis meju. Soybean paste made with conventional meju contained a little more content of total and reducing sugars than other soybean pastes. ph during aging period was higher than 5.0 for the Asp. oryzae paste while less than 4.5 for B. subtilis paste. Aspartic acid. threonine, serine, glutamic acid, glycine, alanine, cystine, valine, methionne, leucine and histidine for Asp. oryzae paste ; tyrosine, arginine and proline for conventional meju paste; and isoleucine and phenylalanine for B. subtilis paste were found to be peak amount 90 days after the preparation. The content of total free amino acid was high in the order of Asp. oryzae paste, conventional paste, B. natto paste and B. subtilis paste.

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Optimization of \beta-mammanase Production from Bacillus subtilis JS-1. (\beta-Mannanase를 생산하는 Bacillus subtilis JS-1의 분리 및 효소 생산성)

  • 임지수;정진우;이종수;강대경;강하근
    • Microbiology and Biotechnology Letters
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    • v.31 no.1
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    • pp.57-62
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    • 2003
  • A bacteria strain producing extracellular $\beta$-mannanase was isolated from soil and was identified as Bacillus subtilis by 16S rRNA sequence comparison and biochemical determinations. The optimum pH and temperature for the $\beta$-mannanase activity were 5.0 and 5.5$^{\circ}C$, respectively. The zymogram technique revealed a single protein band exhibiting $\beta$-mannanase activity from the culture supernatant. The molecular mass of the enzyme was estimated at approximately 130 kDa. The addition of 0.5% lactose or 0.5% locust bean gum to the LB medium caused to Increase significantly the $\beta$-mannanase productivity from Bacillus subtilis JS-1. The cells grown on LB medium supplemented with lactose produced maximal enzyme activity at the stationary phase. In contrast to this, the $\beta$-mannanase was induced at the logarithmic phase from the cells grown on LB medium supplemented with locust bean gum. The discrepancy in induction times suggests that $\beta$-mannanase was induced by different induction mechanisms depending on the carbon sources in Bacillus subtilis JS-1 .

Optimum Condition for Pigment Production and Antioxidative Activity of the Products by Bacillus subtilis DC-2 with Response Surface Methodology (반응표면 분석에 의한 Bacillus subtilis DC-2의 색소생성 및 그 생성물의 항산화성에 대한 최적조건)

  • 최웅규;지원대;정현채;최동환;정영건
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.26 no.4
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    • pp.620-624
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    • 1997
  • The conditions for color intensity and electron donating ability to $\alpha$,$\alpha$-diphenyl-$\beta$- picryl-hydrazyl (DPPH) of Bacillus subtilis DC-2 were investigated. Temperature, pH and cultivation time were chosen as three factors, and the optimal conditions of color intensity and DPPH was determined with response surface methodology. Color intensity was affected by cultivation temperature(p<0.1). DPPH was influenced by cultivation temperature(p<0.05) and pH(p<0.1). But cultivation time was affected neither color in- tensity nor DPPH. Optimal conditions of color intensity with Bacillus subtilis DC-2 were appeared at cultivation temperature of 39.$25^{\circ}C$, pH 8.83 and cultivation time of 84.41hrs. Optimal conditions of DPPH with Bacillus subtilis DC-2 were revealed at cultivation temperature of 39.19$^{\circ}C$, pH 8.84 and cultivation time of 82.21hrs.

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Changes of Taste Components and Palatability during Chunggugjang Fermentation by Bacillus subtilis DC-2 (Bacillus subtilis DC-2를 이용한 청국장 발효과정 중 맛성분 및 기호도의 변화)

  • 정영건;최웅규;손동화;지원대;임무혁;최종동
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.27 no.5
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    • pp.840-845
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    • 1998
  • This study was conducted to produce the high quality of chunggugjang. The taste compounds of chunggugjang produced with Bacillus subtilis DC-2, pigment producing bacterium, were analysed, and palatability of chunggugjang was compared to that of commercial chunggugjang. Among the volatile organic acids, the contentof acetic acid was contained more than any other volatile organic acid. The major nonvolatile organic acid was lactic acid, followed by oxalic acid and citric acid. Tartaric acid was not detected. In case of free sugars, raffinose was sharply decreased between 72 and 96 hours after fermentation. Free amino acid was increased to 20 folds at 48 hours after fermentation compared to that of stemed soybean. As a result of sensory test, it was founded that the chunggujang fermented by Bacillus subtilis DC-2 was suitable to produce for commercial purpose.

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