• Title, Summary, Keyword: Bacillus subtilis K-20

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Cloning of aprE86-1 Gene Encoding a 27-kDa Mature Fibrinolytic Enzyme from Bacillus amyloliquefaciens CH86-1

  • Lee, Ae-Ran;Kim, Gyoung-Min;Kwon, Gun-Hee;Lee, Kang-Wook;Park, Jae-Yong;Chun, Ji-Yeon;Cha, Jae-Ho;Song, Young-Sun;Kim, Jeong-Hwan
    • Journal of Microbiology and Biotechnology
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    • v.20 no.2
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    • pp.370-374
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    • 2010
  • A gene encoding the major secreted fibrinolytic protein of Bacillus amyloliquefaciens CH86-1 was cloned from genomic DNAs. DNA sequencing showed that the gene, aprE86-1, could direct the synthesis of a mature protein 275 amino acids in length after processing. When aprE86-1 was introduced into B. subtilis, a mature 27-kDa protein was produced as expected. The fibrinolytic activity of the B. subtilis transformant (TF) was higher than that of B. amyloliquefaciens CH86-1, showing the possibility of increasing the fibrinolytic activity of Bacillus strains through genetic engineering.

Characterization of a Novel Fibrinolytic Enzyme Produced from Bacillus subtilis BK-17 (Bacillus subtilis BK-17 유래 혈전용해 효소의 특성)

  • Hyun Bek;Lim Hak-Seob;Chung Kyung Kae;Choi Yung Hyun;Choi Byung Tae;Seo Min-Jeong;Kim Ji-Eun;Ryu Eun-Ju;Huh Man Kyu;Joo Woo Hong;Jeong Young Kee
    • Journal of Life Science
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    • v.15 no.6
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    • pp.987-993
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    • 2005
  • A bacterium, producing a fibrinolytic enzyme, was screened from a decaying rice plant. The bacterium was identified as Bacillus subtilis by morphological, biochemical, and physiological properties and named Bacillus subtilis BK-17. The fibrinolytic enzyme (BK) was purified from supernatant of Bacillus subtilis BK-17 culture broth. The molecular weight was 31 kDa as determined by SDS-PAGE. The effect of temperature, pH, and plasminogen on the activity of the bacillokinase (BK) was analysed and the activity was compared with urokinase. The optimal temperature and pH were $50^{circ}C$ and pH 7, pH 8, respectively. The BK activity was inhibited to $45\%$, $35\%$, and $23\%$ with 1mM EDTA, $Zn^{2+}$, and $Ca^{2+}$, respectively. However, $Mg^{2+}$, $Mn^{2+}$, and $Co^{2+}$ ions did not have any significant effect on the enzyme activity The BK showed the artivity in the both plates, plasminogen-free fibrin plate and plasminogen-rich fibrin plate. The result indicates that the BK can directly act the fibrin. In comparison of fibrinolytic activity with urokinase on the fibrin plate, the BK shows about 20 folds higher activity than that of the urokinase.

Dried Bacillus subtilis Culture Reduced Ammonia Gas Release in Poultry House

  • Santoso, U.;Ohtani, S.;Tanaka, K.;Sakaida, M.
    • Asian-Australasian Journal of Animal Sciences
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    • v.12 no.5
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    • pp.806-809
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    • 1999
  • The present study showed the advantages of dried Bacillus subtilis culture (DBSC) supplementation on reducing ammonia gas release in the poultry house. In Experiment 1, 65-week-old Hyline W-36 hens were raised in individual wire-floor cages in a windowless house, and divided into two groups of 180 hens each. One group was fed diets without DBSC as the control and another group was fed a diet supplemented with 2% DBSC. In Experiment 2, 2-week-old broiler chicks were divided into 3 treatment groups of 20 chicks each and maintained in individual floor cages. One group was fed the diet without DBSC and other two groups were fed the diet supplemented with 1 or 2% DBSC, respectively. In experiment 1, DBSC consistently reduced ammonia gas release in the laying house (p<0.01) and manure storage facilities (p<0.01). incubation of feces for 1, 2, 3, 4, 5, 6, 24 or 48 hours showed that DBSC consistently reduced ammonia gas release. In Experiment 2, DBSC reduced ammonia gas release in the broiler house; however, DBSC had no effect on total N, urate-N and ammonia-N contents of feces, but it improved cumulative N utilization and decreased serum urea-N concentration when chicks when chicks were fed 1% DBSC.

Studies on the Isolation of Auxotrophic Mutants of Bacillus subtilis and Escherichia coli (Adenine 요구변이주(要求變異株)의 분리(分離)에 관(關)한 연구(硏究))

  • Kim, H.S.;Lee, C.Y.;Lee, K.H.;Kim, S.S.
    • Applied Biological Chemistry
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    • v.11
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    • pp.137-141
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    • 1969
  • In order to obtain amino acids and nucleic acid derivatives from adenine auxotrophic mutants of Bacillus subtilis and Escherichia coli, vitamin, nucleic acid analogue, streptomycin as well as ultraviolet light were adopted for the production of adenine auxotrophic mutants and the results showed efficient production of desired mutants. 1. Ultraviolet ray $(2530\;{\AA}\;2080\;erg/mm^2)$ irradiation to Bacillus subtilis and E. coli at a distance of 30 cm for 80-90 sec. and for 15-20 sec. respetively induced four and eight strains of auxotrophic mutants. 2. Treatment of aminopterine$(200\;{\mu}g/ml)$ inhibited the growth of Bacillus sultilis significantly but a subsequent irradiation of ultraviolet light at the above mentioned conditions induced six times as much mutants as compared to the irradiation alone. In case of E. coli a similar tendency was observed with treatment of streptomycin$(200\;{\mu}g/ml)$ with doubled induction rate of adenine auxotrophic mutants as compared to the irradiation alone.

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Combinatorial Methylerythritol Phosphate Pathway Engineering and Process Optimization for Increased Menaquinone-7 Synthesis in Bacillus subtilis

  • Chen, Taichi;Xia, Hongzhi;Cui, Shixiu;Lv, Xueqin;Li, Xueliang;Liu, Yanfeng;Li, Jianghua;Du, Guocheng;Liu, Long
    • Journal of Microbiology and Biotechnology
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    • v.30 no.5
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    • pp.762-769
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    • 2020
  • Vitamin K2 (menaquinone) is an essential vitamin existing in the daily diet, and menaquinone-7 (MK-7) is an important form of it. In a recent work, we engineered the synthesis modules of MK-7 in Bacillus subtilis, and the strain BS20 could produce 360 mg/l MK-7 in shake flasks, while the methylerythritol phosphate (MEP) pathway, which provides the precursor isopentenyl diphosphate for MK-7 synthesis, was not engineered. In this study, we overexpressed five genes of the MEP pathway in BS20 and finally obtained a strain (BS20DFHG) with MK-7 titer of 415 mg/l in shake flasks. Next, we optimized the fermentation process parameters (initial pH, temperature and aeration) in an 8-unit parallel bioreactor system consisting of 300-ml glass vessels. Based on this, we scaled up the MK-7 production by the strain BS20DFHG in a 50-l bioreactor, and the highest MK-7 titer reached 242 mg/l. Here, we show that the engineered strain BS20DFHG may be used for the industrial production of MK-7 in the future.

Purification and Characterization of Phytase from Bacillus subtilis (Bacillus subtilis가 생산하는 Phytase의 정제 및 특성)

  • Koh Hyun-Jung;Chu In-Ho;Chung Kun-Sub
    • Microbiology and Biotechnology Letters
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    • v.34 no.1
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    • pp.40-46
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    • 2006
  • A bacterial strain producing high level of a phytase was isolated from cattle feces and identified as Bacillus subtilis, and designated as Bacillus sp. CF 5-26. The production of the phytase from Bacillus sp. CF 5-26 reached the highest level after 72 hours at $37^{\circ}C$. The optimum condition of the media for the production of phytase was 10% rice bran extract, 0.1% whey protein powder, $0.01%\;CaCl_{2},\;0.01%\;KH_{2}PO_4$. The phytase was purified 20.3 folds with ethanol precipitation, Sephadex G-100, CM Sepharose CL-6B and Sephacryl S-100-HR column chromatography. The molecular weight of the purified enzyme was estimated to be 66 kDa on SDS-polyacrylamide gel electrophoresis. The purified phytase activity was stable up pH 5.0, 7.0, 11.0 and the remaining activity was 50% when it was treated at $100^{\circ}C$ for 1 hour. The substrate specificity of phytase was most active against sodium phytate and inositol polyphosphate compound. And the phytase hydrolysed tripolyphosphate and pyrophosphate a little. The Km value for the sodium phytate was 0.64 mM and the Vmax value was $4.41\;{\mu}mol/min$.

Cloning, High-Level Expression, Purification, and Properties of a Novel Endo-${\beta}$-1,4-Mannanase from Bacillus subtilis G1 in Pichia pastoris

  • Vu, Thi Thu Hang;Quyen, Dinh Thi;Dao, Thi Tuyet;Nguyen, Sy Le Thanh
    • 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.

Overexpression of aprE2, a Fibrinolytic Enzyme Gene from Bacillus subtilis CH3-5, in Escherichia coli and the Properties of AprE2

  • Jeong, Seon-Ju;Cho, Kye Man;Lee, Chang Kwon;Kim, Gyoung Min;Shin, Jung-Hye;Kim, Jong Sang;Kim, Jeong Hwan
    • Journal of Microbiology and Biotechnology
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    • v.24 no.7
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    • pp.969-978
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    • 2014
  • The aprE2 gene with its prosequence from Bacillus subtilis CH3-5 was overexpressed in Escherichia coli BL21(DE3) by using plasmid pET26b(+). After IPTG induction, active and mature AprE2 was produced when cells were grown at $20^{\circ}C$, whereas inactive and insoluble enzyme was produced in a large amount when cells were grown at $37^{\circ}C$. The insoluble fraction was resuspended with 6 M guanidine-HCl and dialyzed against 2 M Tris-HCl (pH 7.0) or 0.5 M sodium acetate (pH 7.0) buffer. Then active AprE2 was regenerated and purified by a Ni-NTA column. Purified AprE2 from the soluble fraction had a specific activity of $1,069.4{\pm}42.4U/mg$ protein, higher than that from the renatured insoluble fraction. However, more active AprE2 was obtained by renaturation of the insoluble fraction. AprE2 was most stable at pH 7 and $40^{\circ}C$, respectively. The fibrinolytic activity of AprE2 was inhibited by PMSF, but not by EDTA and metal ions. AprE2 degraded $A{\alpha}$ and $B{\beta}$ chains of fibrinogen quickly, but not the ${\gamma}$-chain. AprE2 exhibited the highest specificity for N-succinyl-Ala-Ala-Pro-Phe-pNA. The $K_m$ and $k_{cat}/K_m$ of AprE2 was 0.56 mM and $3.10{\times}10^4S^{-1}M^{-1}$, respectively.

Antimicrobial and Antioxidant Peptide from Bacillus Strain CBS73 Isolated from Korean Food

  • Kim, Miri;Khan, Md Maruf;Yoo, Jin Cheol
    • Journal of the Chosun Natural Science
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    • v.10 no.3
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    • pp.154-161
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    • 2017
  • An antimicrobial peptides-producing Bacillus strain CBS73 was isolated from fermented food (kimchi) that produces low-molecular-weight proteins with broad-spectrum antimicrobial activity. Our goal was to explore the therapeutic potential of antimicrobial substances produced by Bacillus species. Peptide CBS73 was purified from Bacillus subtilis subsp. subtilis with identity of 99.79%. It was found to be stable at pH 4.0-10.0 and temp $20-60^{\circ}C$. A protein band around 5.2 kDa was detected in tricine-SDS-PAGE and band was confirmed by MALDI-TOF test. Peptide CBS73 showed antimicrobial activity against MDR bacteria. The minimal inhibitory concentration (MIC) of peptide CBS73 for vancomycin-resistant S. aureus (VRSA), vancomycin resistant Enterococci (VRE) and Salmonella typhimurium ranged from $10-40{\mu}g/mL$. The antioxidant activity of peptide CBS73 was measured by DPPH scavenging, reducing power activity and total phenolic content. Cell viability and NO production result showed less cytotoxic effect upto $12{\mu}g/mL$. Peptide CBS73 could be a promising antimicrobial agent for clinical application.

Enhanced pectinase and β-glucosidase enzyme production by a Bacillus subtilis strain under blue light-emitting diodes

  • Elumalai, Punniyakotti;Lim, Jeong-Muk;Oh, Byung-Teak
    • Proceedings of the Plant Resources Society of Korea Conference
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    • pp.109-109
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    • 2018
  • Bacillus subtilis B22, a chemotrophic and aerobic bacterial strain was isolated from homemade kimchi, identified by 16S rRNA gene sequencing. B22 was primarily screened by biochemical, carbon source utilization tests. B22 was used to produce pectinase and ${\beta}$-glucosidase by submerged fermentation under different light sources. B22 was incubated in pectin media and basal media (pH 7.0) under blue, green, red and white light-emitting diodes (LEDs), fluorescent white light, and in darkness at $37^{\circ}C$, orbital shaker 150 rpm for 24 hours. Fermentation under blue LEDs maximized pectinase production ($71.59{\pm}1.6U/mL$ at 24 h) and ${\beta}$-glucosidase production ($56.31{\pm}1.6U/mL$ at 24 h). Further, the production of enzyme increased to pectinase ($156{\pm}1.28U/mL$) and ${\beta}$-glucosidase ($172{\pm}1.28U/mL$) with 3% glucose as a carbon source. Activity and stability of the partially purified enzymes were higher at pH 6.0 to 8.0 and $25-55^{\circ}C$. The effect on the metal ions $Na^+$ and $K^+$ and (moderateactivity) $Mn^{2+}$ and $Ni^{2+}$ increased activity, while $Hg^{2+}$, $Cu^{2+}$, $Fe^{2+}$, and $Fe^{2+}$ inhibited activity. EDTA, phenylmethylsulfonyl fluoride and 5,5-dithiobis (2-nitrobenzoicacid) reduced activity, while tetrafluoroethylene and 1,10-phenanthroline inhibited activity. The amylase was highly tolerant of the surfactants TritonX-100, Tween-20, Tween-80 and compatible with organic solvents methanol, ethanol, isoamylalcohol, isopropanol, t-butylalcohol and the oxidizing agents hydrogen peroxide, sodium perborate and sodium hypochlorite, although potassium iodide and ammonium persulfate reduced activity. These properties suggest utility of pectinase and ${\beta}$-glucosidase produced by B. subtilis B22 under blue LED-mediated fermentation for industrial applications.

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