• The Plant Pathology Journal
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• v.25 no.1
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• pp.62-69
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• 2009

In this study, we characterized the bacterial strain KJ2C12 in relation with its biocontrol activity against Phytophthora capsici on pepper, and identified this strain using morphological, physiological, biochemical, fatty acid methyl ester, and 16S rRNA gene sequence analyses. Strain KJ2C12 significantly (P=0.05) reduced both final disease severity and areas under the disease progress curves of 5-week-old pepper plants inoculated with P. capsici compared to buffer-treated controls. As for the production of antibiotics, biofilms, biosurfactant, extracellular enzyme, HCN, and swarming activity, strain KJ2C12 produced an extracellular enzyme with protease activity, but no other productions or swarming activity. However, Escherichia coli produced weak biofilm only. Strain KJ2C12 could colonize pepper roots more effectively in a gnotobiotic system using sterile quartz sand compared to E. coli over 4 weeks after treatments. However, no bacterial populations were detected in 10 mM $MgSO_4$ buffer-treated controls. Strain KJ2C12 produced significantly higher microbial activity than the $MgSO_4$-treated control or E. coli over 4 weeks after treatments. Bacterial strain KJ2C12 was identified as Bacillus luciferensis based on morphological, physiological, and biochemical characteristics as well as FAME and 16S rRNA gene sequence analyses. In addition, these results suggested that B. luciferensis strain KJ2C12 could reduce Phytophthora blight of pepper by protecting infection courts through enhanced effective root colonization with protease production and an increase of soil microbial activity.

• Journal of Microbiology
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• v.38 no.1
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• pp.1-7
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• 2000

Eight strains of Aeromonas hydrophila isolated from diseased trout in Korea were characterized and compared with an American type strain by various methods including biochemical and physiological tests, PCR, randomly amplified polymorphic DNA (RAPD), plasmid profiling, and gel electrophoresis of total, membrane, and extracellular proteins. Virulence factors such as surface array proteins, cytotoxin, hemolysin, haemagglutinin, and protease were also investigated. The Korean strains showed heterogeneity in Iysine decarboxylase production, utilization of various carbon sources, and production of acetoin. Five strains had the same profiles of total and membrane proteins. Six strains haemagglutinated with trout red blood cells (RBCs) which was inhibited by fucose, galactose, and mannose, except for No. 1 where haemagglutination was inhibited by only galactose and mannose, but not by fucose. Four isolates haemagglutinated with human RBCs which was inhibited by fucose and mannose yet not by galactose. The type strain haemagglutinated only with trout RBCs which was inhibited by fucose, galactose, and mannose. Every isolate secreted protease, hemolysin, cytotoxin, and siderophore, but no enterotoxin. Results showed that the Korean isolates, except for No.7, had very different biochemical and molecular characteristics from those of the American type strain.

• Journal of Environmental Science International
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• v.29 no.3
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• pp.249-255
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• 2020

Particulate matter with an aerodynamic diameter of less than 2.5 μM (PM_2.5) is one of the major environmental pollutants. Di(2-ethylhexyl) phthalate (DEHP), an endocrine disrupting chemical in PM_2.5, has been utilized for the manufacturing of polyvinyl chloride to increase the flexibility of final products. In the present study, we investigated the ecotoxicological effect of DEHP on the viability of skin keratinocytes (HaCaT). DEHP induced apoptotic cell death mediated by phosphorylation of extracellular signal-regulated kinase through the production of intracellular Reactive Oxygen Species (ROS). Interestingly, we found that DEHP induces the phosphorylation of the nuclear factor-kappa B responsible for the expression of cleaved caspase-3 as an executional cell death protease in HaCaT cells. On the basis of these results, we suggest that DEHP in PM_2.5 induces the apoptotic death of human keratinocytes via ROS-mediated signaling events.

• Journal of Microbiology
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• v.44 no.5
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• pp.537-547
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• 2006

This study was designed to determine whether or not Vibrio vulnificus metalloprotease VvpE can promote iron uptake via the proteolytic cleavage of human hemoglobin. We found that V. vulnificus utilized hemoglobin as an iron source more efficiently via the vulnibactin-mediated iron-uptake system than via the HupA-mediated iron-uptake system and, of the proteases produced by V. vulnificus, VvpE was found to be the only protease capable of destroying hemoglobin. However, VvpE expression, on both the transcriptional and protein levels, was suppressed in iron-limited media. However, vvpE transcription, but not extracellular VvpE production, was reactivated by the addition of hemoglobin or inorganic iron into iron-limited media. Moreover, vvpE transcription began only in the late growth phase when V. vulnificus had already consumed most of the iron for growth. In addition, neither vvpE mutation nor in trans vvpE complementation affected the ability of V. vulnificus to acquire iron or to grow in iron-limited media or in cirrhotic ascites containing hemoglobin. Hemoglobin added into iron-limited media was not destroyed, but gradually formed an insoluble aggregate during culture; this aggregation of hemoglobin occurred regardless of vvpE mutation or complementation. These results indicate that VvpE is not required for efficient iron uptake from hemoglobin. On the contrary, hemoglobin or iron is required for efficient vvpE transcription. In addition, a discrepancy exists between vvpE transcription and extracellular VvpE production in iron-limited media containing inorganic iron or hemoglobin, which suggests that additional unknown posttranscriptional events may be involved in the extracellular production of VvpE.

• Microbiology and Biotechnology Letters
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• v.34 no.1
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• pp.7-14
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• 2006

Feather, generated in large quantities as a byproduct of commercial poultry processing, is almost pure keratin, which is not easily degradable by common professes. Four strains, SMMJ-2, FL-3, NO-4 and RM-12 were isolated from soil for production of extracellular keratinolytic protease. They were identified as Bacillus sp. based on their morphological and physiological characteristics. They shown high protease activity on 5.0% skim milk agar medium and produced a substrate like mucoid on keratin agar medium. Bacillus sp. SMMJ-2 had a faster production time for producing keratinolytic protease than other strains. This strain did not completely degrade whole chicken feather for five days in basal medium but completely degraded whole chicken feather when supplied with nitrogen source for 40hours in keratinolytic producing medium ($0.7%\;K_{2}HPO_{4},\;0.2%\;KH_{2}PO_{4},\;0.1%$ fructose, 1.2% whole chicken feather, $0.01%\;Na_{2}CO_3$, pH 7.0). When supplied with chicken feather as nitrogen source, keratinolytic protease activity was 89 units/ml/min. When soybean meal was used as nitrogen source, the keratinolytic protease production reached a maximum of 106 units/ml/min after 48 hours under $30^{\circ}C$, 180 agitation. To isolate the keratinolytic protease, the culture filtrate was precipitated with $(NH_4)_{2}SO_4$ and acetone. The recovery rate of keratinolytic protease was about 96% after treatment with 50% acetone. The enzyme was stable in the range of $30{\sim}50^{\circ}C$ and pH $6.0{\sim}12.0$.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.12
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• pp.1769-1774
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• 2001

Bacillus licheniformis strains L-25 and PWD-1 are two thermophilic feather-degrading bacteria. Despite isolated from different environmental conditions, they were both capable of breaking down chicken feathers and growing in a medium in which feather was the only source of carbon and nitrogen. A 1.46-kb keratinase gene (ker B) was isolated from strain L-25 by a polymerase chain reaction (PCR) using L-25 genomic DNA as templates. Sequencing results reveal that ker B shares great sequence identity with a previously published keratinase gene of B. licheniformis PWD-1 (ker A). Only two amino acids differences were found in the deduced amino acid sequence between the keratinases from L-25 and PWD-1. However several nucleotide changes were found upstream of the putative promoter region. Protease inhibition studies indicated that neutral protease activity accounted for approximate 25 to 30% of total extracellular proteolytic activity produced by strain L-25 in the feather medium. In contrast, no measurable neutral protease activity was produced by strain PWD-1 in the feather medium. When glucose (1%), a common catabolic repressor, was added into the feather medium, L-25 was still able to grow and produce keratinase. Strain PWD-1 produced no neutral protease activity and its growth was severely inhibited in the feather medium containing glucose. L-25 produced an enhanced level of keratinase in the feather medium in comparison with PWD-1.

• Journal of agriculture & life science
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• v.46 no.1
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• pp.163-176
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• 2012

TTo increase productivity of a strong extracellular alkaline protease (BCAP), stable strains of Bacillus clausii I-52 carrying another copy of BCAP gene in the chromosome were developed. Integrative vector, pHPS9-fuBCAP carrying BCAP promoter, ribosome binding site, signal sequence and active protease gene was constructed and transferred into B. clausii I-52, and integration of the constructed plasmid into chromosome was identified by PCR. An investigation was carried out on BCAP production by B. clausii I-52 and transformant C5 showing the highest relative activity of alkaline protease using submerged fermentation. Maximum enzyme activity was produced when cells were grown under the submerged fermentation conditions at $37^{\circ}C$ for 48 h with an aeration rate of 1 vvm and agitation rate of 650 rpm in a optimized medium (soybean meal 2%, wheat flour 1%, sodium citrate 0.5%, $K_2HPO_4$ 0.4%, $Na_2HPO_4$ 0.1%, NaCl 0.4%, $MgSO_47H_2O$ 0.01%, $FeSO_47H_2O$ 0.05%, liquid maltose 2.5%, $Na_2CO_3$ 0.6%). A protease yield of approximately 134,670U/ml was achieved using an optimized media, which show an increase of approximately 1.6-fold compared to that of non-transformant (83,960 U/ml). When the stability of transformant C5 was examined, the integrated plasmid pHPS9-fuBCAP was detected in the transformant after cultivation for 8 days, suggesting that it maintained stably in the chromosomal DNA of transformant C5.

• Journal of the Korean Society of Food Science and Nutrition
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• v.46 no.9
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• pp.1114-1121
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• 2017

Bacillus strains not producing harmful components were isolated from Korean traditional soybean products. Extracellular enzyme activities (amylase, protease, cellulase, and xylanase) of isolated Bacillus strains were measured, and Bacillus strains with high protease activity were selected. The selected 15 strains were identified as Bacillus amyloliquefaciens (10), Bacillus methylotrophicus (1), Bacillus velezensis (1), and Bacillus subtilis (3). Among them, B. subtilis JBG17019, B. amyloliquefaciens JBD17076, and B. amyloliquefaciens JBD17109 showed antimicrobial activities against food-borne microorganisms. The production abilities of glutamate, glutamine, and poly-${\gamma}$-glutamic acid (${\gamma}$-PGA) of the selected Bacillus strains were measured to analyze fermentation characteristics related to glutamic acid metabolism. The factor for multivariate was analyzed by the principal components analysis (PCA) method between fermentation characteristics and ${\gamma}$-PGA production. The three principal components were classified according to the PCA method: PC1 [enzyme activity (amylase, cellulase, and xylanase)], PC2 (${\gamma}$-PGA), and PC3 (protease, glutamate, and glutamine). As a result, B. amyloliquefaciens JBD17076 and B. subtilis JBG17019 strains were evaluated as having excellent enzyme activity and ${\gamma}$-PGA production.

• Korean Journal of Microbiology
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• v.38 no.1
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• pp.50-53
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• 2002

The optimum temperature and pH for mycelial growth of B. bassiana DGUM 34001 were $24^{\circ}C$ and pH 7.0, respectively. Among the complex media used, mushroom complex medium (MCM) was the most favorable for mycelial growth. When Czapek-Dox medium was used as a minimal medium, glucose was an excellent source for carbon and energy. Soytone and sodium phosphate were favorable constituent for culture medium as a source of organic nitrogen and phosphorus, respectively. When the fungus was grown in MCM broth, the specific activity of extracellular enzyme of ${\alpha}$-amylase, lipase, chitinase, CMCase and pretease were 297.0, 0.058, 0.33, 0.21 and 22.8 units/mg protein, respectively. When various sources of organic nitrogen and chitin were supplemented to determine the production of enzymes, casein and soluble chitosan enhanced the production of extracellular protease and chitinase.

• The Plant Pathology Journal
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• v.28 no.4
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• pp.439-445
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• 2012

The chitinase producing Lysobacter enzymogenes C-3 has previously been shown to suppress plant pathogens in vitro and in the field, but little is known of the regulation of chitinase production, or its role in antimicrobial activity and biocontrol. In this study, we isolated and characterized chitinase-defective mutants by screening the transposon mutants of L. enzymogenes C-3. These mutations disrupted genes involved in diverse functions: glucose-galactose transpoter (gluP), disulfide bond formation protein B (dsbB), Clp protease (clp), and polyamine synthase (speD). The chitinase production of the SpeD mutant was restored by the addition of exogenous spermidine or spermine to the bacterial cultures. The speD and clp mutants lost in vitro antifungal activities against plant fungal pathogens. However, the gluP and dsbB mutants showed similar antifungal activities to that of the wild-type. The growth of the mutants in nutrient rich conditions containing chitin was similar with that of the wild-type. However, growth of the speD and gluP mutants was defective in chitin minimal medium, but was observed no growth retardation in the clp and dsbB mutant on chitin minimal medium. In this study, we identified the four genes might be involved and play different role in the production of extracellular chitinase and antifungal activity in L. enzymogenes C-3.