• Journal of Microbiology and Biotechnology
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• v.10 no.4
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• pp.551-553
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• 2000

A strong constitutive $P_{JH}$ promoter from Bacillus was applied to overexpress the endoxylanase gene in B. subtilis. The expression plasmid, pHJKJ4, was designed to contain the $P_{JH}$ promoter and endoxylanase promoter ($P_B$), and introduced into B. subtilis DB104. Through batch fermentation of the trasformant cell on a maltose medium, endoxylanase was produced in a growth-associated manner as the predominant protein. The total activity reached about 600 unit/ml at the end of the cultivation, which corresponded to 698 mg endoxylanase protein/l with a specific activity of 860 unit/mg protein. It was also found that the segregational plasmid instability was less than 30% and most of the endoxylanase activity was detected in the culture medium. This result suggests that the secretory production of endoxylanase can be significantly enhanced with the use of the $P_{JH}$ promoter and high-cell density culture techniques, quantitatively as well as qualitatively.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.22 no.5
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• pp.363-369
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• 1989

In this study, the biochemical characterization of strong proteolytic bacteria and their extracellular pretense isolated from fermented fish paste were experimented for the purpose of industrial large scale-production by accelerated fermentation. The results obtained were as follows: Among 4 strains isolated from fermented fish paste, B. subtilis p-4 and B. licheniformis p-5, which grow well at $40^{\circ}C$, pH 7.0 and $1\%$ of salt contents, were the best proteolytic bacteria and were shown $0.48hr^{-1}$, $0.49hr^{-1}$ of specific growth rate in TPY medium, respectively. Maximum enzyme activity of B. subtilis p-4 was 335n mole-Tyr/min.ml after 30 hrs and that of B. licheniformis p-5 was 300n mole-Tyr/min.ml after 28 hrs of shaking culture. Purified pretense produced by B. subtilis p-4 and B. licheniformis p-5 showed maximum activity at $50^{\circ}C$, pH 7.0 and molecular weight were estimated to be 18,000, 30,000 by sephadex G-100 gel filtration, respectively. These were supposed to be a kind of metal chelator sensitive neutral pretense from the result of high sensitivity against EDTA, o-phe-nanthroline and metal ions such as $Cu^{2+},\;Ni^{2+},\;Zn{2+}$.

• Journal of the Korea Organic Resources Recycling Association
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• v.19 no.4
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• pp.84-89
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• 2011

Cellulose-degrading bacteria were isolated and identified from cocopeat which has a good quality as a bulking agent in composting. Various bacteria from different sourecs of cocopeat were detected on CMC agar media, and these were found to be Burkholderi2a sp., Bacillu subtilis, Sphingomonas sp., Rhodotorula sp. & Pseudomonas sp. etc. Among these, four bacteria were further selected and analyzed for their biochemical characteristics and CMCase activities. CMCase activities of four bacteria, P. aeruginosa, P. stutzeri, B. subtilis, and P. luteola were found to be 83%, 40%, 8%, 6%, respectively, compared with that of the standard strain Cellulomonas sp.

• Journal of Life Science
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• v.12 no.1
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• pp.77-86
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• 2002

Chitin, a $\beta$-1,4 polymer of N-acetyl-D-glucosamine, is one of the most abundant organic compounds in nature. Chitinase (EC 3.2.1.14) is an enzyme that degrades chitin to chito-oligosaccharides, diacetyl rhitobiose and N-acetyl-D-glucosamine. An extracellular chitinase-producing bacterial strain was isolated from soil and named to as Bacillus subtilis JK-56. Optimum culture condition of B. subtilis JK-56 for the production of chitinase was 1% chitin, 0.5% polypepton, 0.1% KCl, 0.05% MnS $O_4$.4$H_2O$, 37$^{\circ}C$, initial pH 7.0 and 40 hour culture time. When B. subtilis JK-56 was grown in the optimum medium, one major active band and two minor active bands were detected by native-PAGE and active staining of the gel. Among them, the major band was purified from the culture supernatant by 70% ammonium sulfate precipitation and native-PAGE with BIO-RAD Model 491 Prep-Cell and named as Chi-56A. Its molecular weight was estimated to be 53kDa monomer and the isoelectric point (pI) was pH 4.3. The pH and temperature for the optimum activity of Chi-56A were pH 6.0 and $65^{\circ}C$, respectively. Chi-56A was stable up to $65^{\circ}C$ and in alkaline region. Its $K_{m}$ value for colloidal chitin was 17.33g/L. HPLC analysis of the reaction products confirmed that Chi-56A was an exo type chitinase.e.

• Microbiology and Biotechnology Letters
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• v.22 no.6
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• pp.593-598
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• 1994

A bacterium KL-57 which exhibited biosurfactant activity was isolated. This bacterium was identified as Bacillus subtilis. The biosurfactant-producing gene of B. subtilis KL-57 was cloned into R subtilis MI113 by using plasmid pTB523. The plasmid DNA from the clone was found to carry a 18 kb PstI insert. The biosurfactant-producing gene was cleaved into 4 fragments by SmaI, 3 fragments by PvulI or EcoRl, 4 fragments by PvulI and EcoRI double digestion, 5 fragments by AccI, and 2 fragments by KpnI, HindIII or BamHI. By subcloning the 18 kb Pstl insert, a 2.3 kb EcoRl fragment conferred the biosurfactant producing activity on B. subtilis cells. The 2.3 kb had one HindIII cleave site. But Two fragments, which corresponds HindIII/EcoRl termini, exhibited no biosurfactant activity.

• The Korean Journal of Mycology
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• v.24 no.4 s.79
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• pp.293-304
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• 1996

A bacterial strain, KSl, possessing strong antifungal activity was isolated from soil samples of ginseng fields and identified as Bacillus subtilis. In greenhouse test, the culture filtrate of B. subtilis KS1 showed strong protective effect against several fungal diseases of agricultural plants such as cucumber gray mold and wheat leaf rust. In addition, the crude butanol fraction of the culture filtrate exhibited antagonistic effect against several fungi including plant or human pathogens, such as Botrytis maydis, Chytridium lagenarium and Candida albicans. The antifungal compound, SW1, produced by B. subtilis KS1 was purified through consecutive chromatographic separations on a pep-RPC column and a ${\mu}$ Bondapak $C_{18}$ reverse phase column. Temperature and pH showed little effect on the stability of the compound in the ranges $-20-121^{\circ}C$ and pH 4.0-10.0, respectively. The composition and structural characteristics of SW1 were analysed by HPLC and by $^1H-,\;^1H-^1H-COSY$, NOESY, COSY-NOESY and HOHAHA NMR spectroscopy, respectively, which revealed that the compound belongs to iturin A, a typical cyclic antifungal compound produced by B. subtilis. In contrast to the previously reported iturin A compounds which have one or no $-CH_3$ side chain in the hydrophobic hydrocarbon chain of ${\beta}-amino$ acids, SW1 was shown to have a ${\beta}-amino$ acid containing 12-carbon skeleton with two $-CH_3$ side chains.

• Food Science and Preservation
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• v.15 no.2
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• pp.300-305
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• 2008

We investigated an antibacterial substance produced by Bacillus subtilis HS 25. Antibacterial activity was relatively heat-stable, with no effect caused by heating at $100^{\circ}C$ for 10 min, but a gradual decrease in activity after 15 min at $100^{\circ}C$. The antibacterial substance was more stable at pH 7.42-12 than at pH 4.5-5.0. There was strong antibacterial activity against E. coli and P. mirabilis at pH 12. The minimum inhibition concentration (MIC) of the substance was 5 mg/mL for E. coli 7.5 mg/mL for P. mirabilis and S. aureus and 15 mg/mL for S. enteritidis, K. pneunoniae, and V. parahaemolyticus. When the substance was added to cultures of E. coli, S. enteritidis, and P. mirabilis, the bacterial surfaces became irregular and deeply changed. The substance produced from B. subtilis HS 25 was not degraded by Papain but was degraded by a protease from Aspergillus orzae, pancreatin, and pepsin.

• Microbiology and Biotechnology Letters
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• v.34 no.4
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• pp.311-317
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• 2006

Using PCR amplification, we cloned a cellulase gene (ce/H) from the Bacillus subtilis AH18 which has plant growth-promoting activity and antagonistic ability against pepper blight caused by Phytophthora capsici. The 1.6 kb PCR fragment contained the full sequence of the cellulase gene and the 1,582 bp gene deduced a 508 amino acid sequence. Similarity search in protein database revealed that the cellulase of B. subtilis AH18 was more than 98% homologous in the amino acid sequence to those of several major Bacillus spp. The ce/H was expressed in E. coli under an IPTG inducible lac promoter on the vector, had apparent molecular weight of about 55 kDa upon CMC-SDS-PAGE analysis. Partially purified cellulase had not only cellulolytic activity toward carboxymethyl-cellulose (CMC) but also insoluble cellulose, such as Avicel and filter paper (Whatman No. 1). In addition, the cellulase could degrade a fungal cell wall of Phytophthora capsici. The optimum pH and temperature of the ce/H coded cellulase were determined to be pH 5.0 and $50^{\circ}C$. The enzyme activity was activated by $AgNO_3$ or $CoCl_2$. However its activity was Inhibited by $HgC1_2$. The enzyme activity was activated by hydroxy urea or sodium azide and inhibited by CDTA or EDTA. The results indicate that the cellulase gene, ce/H is an antifungal mechanism of B. subtilis AH18 against phytophthora blight disease in red-pepper.

• Microbiology and Biotechnology Letters
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• v.26 no.2
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• pp.122-129
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• 1998

It were reported that antifungal mechanism of Enterobacter cloacae is a volatile ammonia that produced by the strain in soil, and the production of ammonia is related to the bacterial urease activity. A powerful bacterium SH14 against soil-borne pathogen Fusarium solani, which cause root rot of many important crops, was selected from a ginseng pathogen suppressive soil. The strain SH14 was identified as Bacillus subtilis by cultural, biochemical, morphological method, and $API^{circledR}$ test. From several in vitro tests, the antifungal substance that is produced from B. subtilis SH14 was revealed as heat-stable and low-molecular weight antibiotic substance. In order to construct the multifunctional biocontrol agent, the urease gene of Bacillus pasteurii which can produce pathogenes-suppressive ammonia transferred into antifungal bacterium. First, a partial BamH I digestion fragment of plasmid pBU11 containing the alkalophilic B. pasteurii l1859 urease gene was inserted into the BamH I site of pEB203 and expressed in Escherichia coli JM109. The recombinant plasmid was designated as pGU366. The plasmid pGU366 containing urease gene was introduced into the B. subtilis SH14 with PEG-induced protoplast transformation (PIP) method. The urease gene was very stably expressed in the transformant of B. subtilis SH14. Also, the optimal conditions for transformation were established and the highest transformation frequency was obtained by treatment of lysozyme for 90 min, and then addition of 1.5 ${mu}g$/ml DNA and 40% PEG4000. From the in vitro antifungal test against F. solani, antifungal activity of B. subtilis SH14(pGu366) containing urease gene was much higher than that of the host strain. Genetical development of B. subtilis SH14 by transfer of urease gene can be responsible for enhanced biocontrol efficacy with its antibiotic action.

• Journal of Ginseng Research
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• v.28 no.1
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• pp.67-70
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• 2004

Bacillus subtilis B-4228 selected from ginseng field soil for prevention of rusty root was tested for the control of ginseng root rot. In petri-plate dual culture, mycelial growth of Cylindrocarpon destructans was inhibited by B-4228 and hyphal swelling of C. destructans was occurred. In pot experiment with C. destructans-contaminated soil B-4228 dipping of ginseng seedling showed significant preventive effect of root rot (p=0.01), percent healthy root 82% and 20% for treatment and control, root rot rate 6% and 50.4%, respectively.