• Biotechnology and Bioprocess Engineering:BBE
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• v.6 no.3
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• pp.167-172
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• 2001

Staphylokinase (SAK) was produced in B. subtilis using two different promoter systems, i.e. the P43 and sacB promoters. To maximize SAK expression in B. subtilis, fermentation control strategies for each promoter were examined. SAK, under P43, a vegetative promoter transcribed mainly by $\sigma$(sup)B containing RNA polymerase, was overexpressed at low dissolved oxygen (D.O.) levels, suggesting that the sigB operon is somewhat affected by the energy charge of the cells. The expression of SAK at the 10% D.O. level was three times higher than that at the 50% D.O. level. In the case of sacB, a sucrose-inducible promoter, sucrose feeding was used to control the induction period and induction strength. Since sucrose is hydrolyzed by two sucrose hydrolyzing enzymes in the cell and culture broth, the control strategy was based on replenishing the loss of sucrose in the culture. With continuous feeding of sucrose, WB700 (pSAKBQ), which contains the SAK gene under sacB promoter, yielded ca. 35% more SAK than the batch culture. These results present efficient promoter-dependent control strategies in B. subtilis host system for foreign protein expression.

• Journal of Microbiology and Biotechnology
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• v.23 no.3
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• pp.390-396
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• 2013

Biosurfactants have versatile properties and potential industrial applications. A new producer, B. subtilis TU2, was isolated from the underground oil-extraction wastewater of Shengli Oilfield, China. Preliminary flask culture showed that the titer of biosurfactant obtained from the broth of TU2 was ~1.5 g/l at 48 h (718 mg/l after purification), with a reduced surface tension of 32.5 mN/m. The critical micelle concentration was measured as 50 mg/l and the surface tension maintained stability in solution with 50 g/l NaCl and 16 g/l $CaCl_2$ after 5 days of incubation at $70^{\circ}C$. FT-IR spectra exhibited the structure information of both glycolipid and lipopeptide. MALDI-TOF-MS analyses confirmed that the biosurfactant produced by B. subtilis TU2 was a blend of glycolipid and lipopeptide, including rhamnolipid, surfactin, and fengycin. The blended biosurfactant showed 86% of oil-washing efficiency and fine emulsification activity on crude oil, suggesting its potential application in enhanced oil recovery.

• Journal of Life Science
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• v.9 no.3
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• pp.314-327
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• 1999

This study was investigated to isolate identify the total bacteria and fungi from the indoor air of work-place of the shoes, paint, stainless steel, and plastic industries. The number of bacterial colonies on the nutrient agar plates were calculated by the open petridish method for 30 minutes in indoor air of work-places at the autumn and winter. The isolated bacteria were identified by Gram stain and biochemical test using API Staph and API 20E kits. The isolated fungal colonies were identified by gross appearance of the giant colonies and microscopic examination of their spore and hyphal characteristics on the slide culture method. The minimum inhibitory concentration (MIC) of several antibiotics against isolated bacteria was determined by the microdilution method with Mueller-Hinton broth. The 70-400 colonies in autumn and 54-236 colonies in winter were isolated from the indoor air of work-places of several industry. The isolation rates of Gram positive cocci, Gram positive bacilli, Gram negative bacilli, and Gram negative cocci were 46.3%, 19.8%, 17.3%, and 16.1%, respectively. In Gram positive cocci, the most strains were identified as Aerococcus spp, Micrococcus spp, and Staphylococcus spp. In Gram positive and negative bacilli, and Gram negative cocci were identified as Bacillus spp, Pseudomonas spp, and Neisseria spp, respectively. The frequently isolated fungi were Aspergillus spp, Penicillium spp and Rhizopus spp, respectively. The frequently isolated Aerococcus spp, Micrococcus spp, and Staphylococus spp were highly resistance against ampicillin, erythromycin, methicillin, and tetracycline. These results arouse our attention to microbiologic pollution in the indoor air of work-places of industries.

• Food Science and Biotechnology
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• v.17 no.4
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• pp.850-856
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• 2008

White, red, and extruded ginsengs were studied against 8 strains of food-borne pathogens and/or food spoilage microorganisms. The ginseng powders were extracted with different extractants and screened for antimicrobial activity using the disc diffusion and broth dilution techniques. The results showed that the yield of extraction was higher with increase of aqueous solution content and temperature. Preliminary screening revealed that the red ginseng extracts were most active, that has been found to be highly effective against all tested microbe except Listeria monocytogenes. Moreover, Bacillus subtilis has shown highly susceptible, which the diameters of inhibition zone values of 28 extracts were between 7 and 14 mm. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) recorded for the different crude ginseng extracts against microorganism using ranged from 6.25 to 100 mg/mL, indicated that the methanol extract of ginseng were more effective than ethanol and water extracts. The 60% methanol extract of red ginseng had the greatest effects against B. subtilis with MIC and MBC values at 6.25 mg/mL.

• Food Science and Biotechnology
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• v.15 no.4
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• pp.511-515
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• 2006

Antibiotic resistance of foodborne pathogens adapted to garlic (Allium sativum Linn.) was determined in order to understand the relationship between antibiotic resistance and garlic. The Gram (-) strains of Escherichia coli and Salmonella typhimurium and the Gram (+) strains of Bacillus cereus and Staphylococcus aureus were subcultured consecutively in a garlic broth, and the surviving colonies on the agar were selected as the adapted strains. Minimal inhibitory concentrations (MIC) for 15 antibiotics on the adapted strains were determined on Muller-Hinton Infusion agar. Adaptation to 1.3%(v/v) garlic juice increased MIC for vancomycin, aminoglycoside, and erythromycin on B. cereus, and for ampicillin and erythromycin on E. coli O157:H7. MIC of aminoglycosides, chloramphenicol, and vancomycin on the adapted S. aureus increased. The adapted S. typhimurium was more resistant to penicillin and vancomycin than the non-treated strain. The adapted S. typhimurium and S. aureus lost their antibiotic resistance in non-garlic stress conditions. However, the adapted B. cereus was still resistant to erythromycin and vancomycin, and the adapted E. coli was also resistant to erythromycin. Antibacterial garlic might increase the antibiotic resistance of E. coli, B. cereus, S. aureus, and S. typhimurium and this resistance can continue even without the stress of garlic. Therefore, garlic as a food seasoning could influence the resistance of such pathogens to these antibiotics temporarily or permanently.

• Journal of the Korean Society of Food Science and Nutrition
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• v.29 no.5
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• pp.888-892
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• 2000

Antimicrobial activity of Caesalpina sappan L. extract (CS extract) against 6 kinds of food spoilage and pathogenic organisms was studied. The growth of Listeria monocytogenes Brie 1, Escherichis coli ATCC 11775, Staphylococcus aureus ATCC 11775, and Pseudomonas fluorescens ATCC 11775 was inhibited about 4 to 5 $log_{10}$ cycle in Tryptic soy Broth(TSB) containing 1% CS extract. Bacillus subtilis KCTC 102 and Vibrio parahaemolyticus ACTT 17802 did not show apparent growth in the same medium. Effect of CS extract on preservation of ground meat was also investigated. The range of pH change was 5.0~5.2 in CS extract added ground meat, 5.2~6.0 in CS extract not added ground meat (control) during storage at 4$^{\circ}C$ for 30 days. Number of total bacteria after 15 days storage was $10^{6}$/g in CS extract added ground meat, 10$^3$/g in control. Redness of ground meats was improved significantly by addition of 1% CS extract during storage at 4$^{\circ}C$ for 30 days. The sensory quality of 1% CS extract added hamburger patty was similar to that of the control in taste, flavor, and overall acceptability.

• Bulletin of the Korean Chemical Society
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• v.1 no.1
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• pp.10-17
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• 1980

Penicillin amidohydrolase was partially purified from the fermented broth of Bacillus megaterium, and was immobilized on nylon fiber. The surface area of nylon fiber was increased by roughening it with fine sand and activated by acid treatment. The free amino groups on the nylon fiber exposed by such treatment were then utilized to immobilize the penicillin amidase. Enzymatic properties of penicillin amidohydrolase immobilized on the nylon fiber by covalent bonding and cross linking with glutaraldehyde were studied and compared with those of soluble enzyme. The optimal pH and temperature profile of immobilized enzyme showed only slightly broader peaks, and the values of kinetic constants, $K_m$, $K_{ia}$, and $K_{ip}$, of the immobilized enzyme are only slightly greater than those of the soluble enzyme. These results suggest that the mass transfer effect on the reaction rate for the penicillin amidase immobilized on nylon fiber is not so significant as the enzyme immobilized on some other support material like bentonite. The experimental results of batch reaction agreed well with the results of computer simulation for both the immobilized and soluble enzyme systems, confirming the validity of the rate equation derived which was based on the combined double inhibition by two reaction products.

• Research in Plant Disease
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• v.8 no.3
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• pp.184-188
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• 2002

An antagonistic bacteria, Bacillus licheniformis Nl strain which effectively inhibited mycelial growth of gray mold rot pathogen, Botrytis cinerea was isolated from the rhizosphere of perilla crop. Powder soy formulation by B. lichentfomis Nl strain as a biocontrol agent was developed far the first time and estimated its control effect on perilla leaves in this study. First of all, far the mass production of antifungal metabolites of B. lichentfomis Nl strain in flask liquid culture, the most effective carbon and nitrogen source were selected as glucose and tryp-tone, respectively, For the formulation, vegetative biomass of B. licheniformis Nl strain from 5-day-old liquid culture in nutrient broth added glucose and tryptone was mixed with soy flour, rice flour glucose, FeSo$_4$~7$H_2O$, and MnCl$_2$. 4$H_2O$, and dried and pulverized. In plastic house test, powder soy formulation effectually controlled gray mold rot as the control value of 93.1 %, was more effective than chemical fungicide, benomyl showing the control value of 86.1%. Thus, development of powder soy formulation of B. lichentfomis Nl will aid large-scale application of biological control in field trials.

• The Plant Pathology Journal
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• v.25 no.4
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• pp.344-351
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• 2009

A biocontrol bacterium Bacillus licheniformis N1 grown in nutrient broth showed no chitinolytic activity, while its genome contains a gene which encodes a chitinase. The gene for chitinase from B. licheniformis N1 was amplified by PCR and the deduced amino acid sequence analysis revealed that the chitinase exhibited over 95% identity with chitinases from other B. licheniformis strains. Escherichia coli cells carrying the recombinant plasmid displayed chitinase activity as revealed by the formation of a clear zone on chitin containing media, indicating that the gene could be expressed in E. coli cells. Chitinase gene expression in B. licheniformis N1 was not detected by RT-PCR analysis. The protein was over-expressed in E. coli BL21 (DE3) as a glutathione S-transferase fusion protein. The protein could also be produced in B. subtilis 168 strain carrying the chitinase gene of N1 strain. The crude protein extract from E. coli BL21 carrying GST fusion protein or culture supernatant of B. subtilis carrying the chitinase gene exhibited enzyme activity by hydrolyzing chitin analogs, 4-methylumbelliferyl-$\beta$-D-N,N'-diacetylchitobioside and 4-methylumbelliferyl-$\beta$-D-N,N',N"-triacetylchitotrioside. These results indicated that even though the chitinase gene is not expressed in the N1 strain, the coding region is functional and encodes an active chitinase enzyme. Furthermore, B. subtilis 168 transformants expressing the chitinase gene exhibited antifungal activity against Fulvia fulva by suppressing spore germination. Our results suggest that the proper engineering of the expression of the indigenous chitinase gene, which will lead to its expression in the biocontrol strain B. licheniformis N1, may further enhance its biocontrol activity.

• Proceedings of the KWS Conference
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• 2002.10a
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• pp.260-265
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• 2002

Microbiologically influenced Corrosion (MIC) is one of the most deleterious effects of metal microbe interactions. When a fresh metal surface comes in contact with a non-sterile fluid, biofilm formation is ensued. This might result in the initiation of corrosion. The sites and materials where MIC is implicated are versatile. Industries such as shipping, power generation, chemical etc are reported to be affected. The rapid and unexpected failure of AISI type 304 stainless steel was investigated in the laboratory by simulation studies for a period of 4 months. Slime and water samples from the failure site were screened for corrosion causing bacteria. Both aerobic and anaerobic nora were enumerated and identified using PCR techniques. Pseudomonas sp. and Bacillus sp. were the most common aerobic bacteria isolated from the water and slime samples, whilst sulfate reducing bacteria (SRB) were the major anaerobic bacteria. The aerobic bacteria were used for the corrosion experiments in the laboratory. Coupon exposure studies were conducted using a very dilute (0.1%V/V) nutrient broth medium. The coupons after retrieval were observed under a Scanning Electron Microscope (SEM) for the presence of MIC pits. Compared to sterile controls, metal coupons exposed to Pseudomonas sp and Bacillus sp. showed the initiation of severe pitting corrosion. However, amongst these two strains, Psudomonas sp. caused pits in a very short span of 14 days. Towards the end of the experiment, severe pitting was observed in both the cases. The detailed observation of pits showed they vary both in number and shapes. Whilst the coupons exposed to Bacillus sp. showed widely spread scales like pits, those exposed to Pseudomonas sp. showed smaller and circular pits, which had grown in number and size by the end of the experiment. From these results it is inferred that the rapid and unexpected failure of 304 SS might be due to MIC. Pseudonwnas sp. could be considered as the major responsible bacteria that could initiate pits in the metallic structures. As the appearance of pits was different in both the tested strains, it was thought that the mechanisms of pit formation are different. Experiments on these lines are being continued.