• The Plant Pathology Journal
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• 제25권1호
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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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• 제38권1호
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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.

• 한국환경과학회지
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• 제29권3호
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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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• 제44권5호
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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.

• 한국미생물·생명공학회지
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• 제34권1호
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• pp.7-14
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• 2006

가금류의 feather은 전 세계적으로 발생되지 않는 지역이 없을 만큼 광범위하게 발생되어지는 폐기물로써, 현재까지 많은 연구자들에 의하여 그 이용성에 대한 연구가 활발하게 이루지고 있다. 그러나 disulfide 결합, 수소결합 및 이온결합에 의하여 매우 강한 결합력을 가지는 feather 구성 단백질의 화학적인 성질 때문에 분해하여 사용하기에 많은 어려움이 있다. 가금류 폐기물을 이용한 사료의 제조과정에서 가열 및 가압 등의 과정을 거치면서 아미노산의 손실이 발생하고 낮은 소화율 등이 문제로 야기되어지고 있다. 이러한 문제를 해결하기 위한 노력으로 미생물이 생산하는 keratinolytic protease를 이용하고자 산업적으로 이용 가능성을 가지는 균주를 분리하였다. 토양 시료로부터 Bacillus sp. SMMJ-2, FL-3, NO-4 및 RM-12 4종의 keratinolytic protease 생성균을 분리하였다. 이 균주들은 5.0% skim milk agar plate에서 높은 protease 활성을 나타내었으며, 2.0% whole chicken feather agar plate에서도 많은 점질성의 물질을 생산하였다. Keratinolytic protease 생산을 위하여 배지4($0.7%\;K_{2}HPO_{4},\;0.2%\;KH_{2}PO_{4},\;0.1%$ fructose, 1.2% soybean meal, $0.01%\;Na_{2}CO_3$, pH 7.0)에서 Bacillus sp. SMMJ-2에 의한 keratinolytic protease의 생산을 조사한 결과, 배양 3일에 가장 높은 keratinolytic protease 활성을 나타내었다. 그러나, Basal medium(0.05% NaCl, $0.03%\;Na_{2}HPO_{4},\;0.04%\;NaH_{2}PO_{4},\;0.5%$ whole chicken feather, pH 7.0)에 유일한 탄소 및 질소원으로 whole chicken feather를 첨가하고 배양한 결과, $30^{\circ}C$에서 일주일 이상 배양하였을 때 feather은 모두 분해되었으나 깃대는 분리하지 못하였고 배지 4에 질소원으로 1.2% whole chicken feather를 첨가하였을 때 40시간 이내에 깃대를 포함한 feather를 모두 분해하였다. Bacillus sp. SMMJ-2에 의한 keratinolytic protease의 생산은 접종 후 9시간이 지나면서 생산되기 시작하여 24시간 동안 배양하였을 때 최대 활성의 86%를 나타내는 것으로 조사되었다. Bacillus sp. SMMJ-2에 의하여 생산되어지는 keratinolytic protease 활성은 $30^{\circ}C$, 180 rpm으로 3일간 배양했을 때 106 units/ml/min 이였으며, protease 활성은 540 units/ml/min을 나타내었다. 온도와 pH에 대한 효소의 안정성은 $50\%$ acetone을 이용하여 분리한 효소로 조사한 결과, $30{\sim}50^{\circ}C$까지는 80% 이상의 잔존효소활성을 나타내었고, $60^{\circ}C$ 이상에는 20분간 열처리로 효소가 거의 실활 되었다. 효소의 pH 안정성은 pH $6.0{\sim}12.0$에서는 비교적 안정한 것으로 조사되었다.

• Asian-Australasian Journal of Animal Sciences
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• 제14권12호
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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.

• 농업생명과학연구
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• 제46권1호
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• pp.163-176
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• 2012

인천 연안 갯벌에서 분리한 호알카리성 Bacillus clausii I-52로부터 세포외 알카리성 단백질 분해효소(BCAP)의 발현 및 생산성을 증가시키기 위하여 BCAP promoter, ribosome 결합 서열, 신호서열, 전구체 서열 및 활성형 BCAP 유전자를 cloning한 재조합 plasmid pHPS9-fuBCAP을 penicillin-protoplast 법으로 B. clausii I-52의 염색체 DNA에 integration 하였고, 도입된 plasmid pHPS9-fuBCAP 유전자는 PCR에 의해 확인하였다. 가장 높은 단백질 분해효소 상대 활성을 보이는 선별된 transformant C5를 생산 최적화 배지(대두박 2%, 밀가루 1%, 구연산나트륨 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%, 물엿 2.5%, 탄산나트륨 0.6%)에서 액침 배양법(배양온도, $37^{\circ}C$; 배양 시간, 48 h; 교반 속도, 650 rpm; 통기 속도, 1 vvm)으로 배양하여 단백질 분해효소를 발현 및 분비시켰을 때, BCAP 발현 양(134,670 U/ml)은 wild-type(83,960 U/ml)에 비하여 약 1.6 배 증가하였으며, 비활성도(91,611.5 U/mg 단백질)는 wild-type(71,760 U/mg 단백질)에 비하여 약 1.3 배 증가하였다. 또한, B. clausii I-52 염색체 DNA에 integration된 pHPS9-fuBCAP plasmid는 단백질 발현과 함께 8일간의 계대배양 동안에 안정하게 유지되고 있음을 확인하였다.

• 한국식품영양과학회지
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• 제46권9호
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• pp.1114-1121
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• 2017

전통장류로부터 식품 유해요소를 생성하지 않는 Bacillus 균주를 분리하여 세포외효소 활성(amylase, protease, cellulase, xylanase)을 측정한 후, 단백질 분해 활성이 우수한 14개 균주와 비교균주 1균주를 선발하였다. 선발된 균주에 대해 16S rRNA 유전자를 이용한 균주 동정을 실시한 결과, B. amyloliquefaciens 10종, B. methylotrophicus 1종, B. velezensis 1종, B. subtilis 3종이 분리 동정되었다. 그중 B. subtilis JBG17019, B. amyloliquefaciens JBD17076, B. amyloliquefaciens JBD17109 균주에서 식중독미생물에 대한 증식 억제능이 확인되었다. Glutamic acid 대사와 관련한 발효특성을 확인하기 위하여 선발된 Bacillus 균주에 대해 glutamate, glutamine 및 ${\gamma}$-PGA 생성능을 측정하였다. 발효특성과 ${\gamma}$-PGA 생성능에 대한 다변량 요인분석을 주성분(PCA) 추출법으로 분석한 결과, PC1(효소 활성(amylase, cellulase, xylanase), PC2(${\gamma}$-PGA 생성능) 및 PC3(protease, glutamate 및 glutamine)의 3가지 주성분이 분류되었다. 주성분(PC)의 추출에 따라 B. amyloliquefaciens JBD17076 및 B. subtilis JBG17019 균주는 우수한 효소 활성 및 ${\gamma}$-PGA 생성을 하는 것으로 평가되었다.

• 미생물학회지
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• 제38권1호
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• pp.50-53
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• 2002

백강균(Beauveria bassiana DGUM 34001)은 $24^{\circ}C$의 온도와pH 7.0의 초기 pH에서 최적의 균사체 생육을나타내었다. 사용한 복합배지 중 mushroom complex배지(MCM)에서 가장 우수한 균사생육을 나타내었다. Czapek-Dox 한천배지를 최소배지로 각각 탄소원, 질소원 및 인산원의 영향을 조사한 결과, glucose, soytone 및 sodium phosphate ($NaH_2$$PO_4$)에서 가장 우수한 균사 생육을 보여주었다. MCM액체배지에서 균사 배양 후 세포외 효소 활성을 측정한 결과,$\alpha$-amylase, lipase, chitinase, CMCase및 pretense의 비효소활성은 각각 297.0, 0.058, 0.33, 0.21 및 22.8 units/mg protein이었다. Casein과 soluble chitosan을 첨가할 경우 세포외 분비 pretense와 chitinase의 효소 활성 이 증가되었다.

• The Plant Pathology Journal
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• 제28권4호
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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.