• Journal of Applied Biological Chemistry
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• 제50권4호
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• pp.202-210
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• 2007

A total of 45 bacterial strains were isolated from the traditional Korean soybean-fermented food, Chungkookjang. Among these strains, seven strains were selected and identified based on morphological, physiological, and biochemical characteristics, as well as phylogenetic analysis using 16S rDNA sequences. All strains were Gram-positive, aerobic, motile, oxidase-positive, rod-shaped, and endospore-forming bacteria, and produced extracellular enzymes such as amylase, cellulase, lipase, protease, and xylanase. The isolates were grown in the presence of 0-11% (w/v) NaCl. Growth was optimal at pH 6-9 and at temperatures of $30-45^{\circ}C$. According to VITEK automicrobic system tests and supplementary tests, the isolates were similar to several species of the genus Bacillus. The phylogenetic analysis of seven bacterial strains based on comparisons of 16S rDNA sequences, revealed that the strains were closely related to Bacillus species. The identification of strains that produced surfactin was also carried out, based on PCR screening of the sfp gene. Among the seven isolated strains, six yielded a surfactin-positive result with PCR.

• Mycobiology
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• 제35권1호
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• pp.21-24
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• 2007

To evaluate which dye is effective in a plate assay for detecting extracellular cellulase activity produced by fungi, four chromogenic dyes including remazol brilliant blue, phenol red, congo red, and tryphan blue, were compared using chromagepic media. For the comparison, 19 fungal species belonging to three phyla, ascomycota, basidiomycota, and zygomycota were inoculated onto yeast nitrogen-based media containing different carbon substrates such as cellulose (carboxylmethyl and avicel types) and cellobiose labeled with each of the four dyes. Overall, the formation of clear zone on agar media resulting from the degradation of the substrates by the enzymes secreted from the test fungi was most apparent with media containing congo red. The detection frequency of cellulase activity was also most high on congo red-supplemented media. The results of this study showed that congo red is better dye than other three dyes in, a plate assay for, fungal enzyme detection.

• BMB Reports
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• 제35권2호
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• pp.236-238
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• 2002

Based on the zymography analysis, Bacillus sp. DJ-4 (screened from Doen-Jang, a Korean traditional fermented food) secretes seven extracellular fibrinolytic enzymes (EFEs; 68, 64, 55, 45, 33, 27, and 13 kDa) in culture broth. These seven EFEs were analyzed by newly applied SDS-fibrin zymography combined with gradient polyacrylamide (SDS-FZGP). This improved gel system was used with a 5-20% acrylamide gradient in a fibrin zymogram gel for the separation of proteins with molecular masses from below 10kDa to over 100kDa on one gel plate. Using this system, high molecular weight bands (HMWBs) were clearly and sharply resolved. We also examined the relationship between an acrylamide concentration and the enzymatic activity of EFE using densitometric analysis.

• 대한화장품학회지
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• 제28권1호
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• pp.31-41
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• 2002

The production of matrix matalloproteinases(MMPs) by the UV irradiated skin fibroblast and the degradation of extracellular matrix(ECM) by these enzymes is known as one of the main reasons of photoaging. Recently, Fisher group showed that the MMP expression is mainly regulated by the mitogen-activated protein(MAP) kinas family, such as extracellular signal-regulated kinase(ERK), c-Jun amino-terminal kinase(JNK) and p38, each of which forms a signaling pathway. In this work we first examined the effect of nitric oxide (NO) on the production of MMP-1 and MMP-2 by the human dermal fibroblasts (HDFs). NO is a multifunctional messenger molecule generated from L-arginine and involved in many kinds of signaling pathway. We found that the treatment of HDF with NO donor, sodium nitroprusside (SNP) enhanced the expression of MMPs with or without UV irradiation and the treatment with nitric oxide synthase (NOS) inhibitors resulted in the significant decrease of MMPs production. From these results, we concluded that the production of MMPs by the UV irradiated HDF is regulated through the signaling pathway involving NO and cyclic GMP.

• Journal of Applied Biological Chemistry
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• 제65권2호
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• pp.93-99
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• 2022

Lysosome organelle extract (LOE) was derived from egg whites. The lysosome is an intracellular organelle that contains several hydrolysis enzymes. Previous studies have reported that LOE performs important functions, such as melanin de-colorization and anti-melanin production in B16F10 melanoma cells. However, its principal molecular and cellular mechanisms have not been elucidated till date. In non-cytotoxic conditions, LOE significantly inhibited α-MSH induced melanin synthesis of murine B16F10 cells. The anti-melanogenic activity of LOE was mediated by suppressing the mRNA expression of tyrosinase enzyme, tyrosinase related protein-1/2 (TRP-1/2), and microphthalmia-associated transcription factor (MITF) genes. By performing western blot analysis, we found that LOE significantly attenuated melanogenesis. In this case, LOE helped in increasing extracellular receptor kinase (ERK) phosphorylation in α-MSH induced B16F10 cells. Furthermore, MITF is found to be a key regulatory transcription factor in melanin synthesis; it was down-regulated by LOE through ERK phosphorylation. In this experiment, PD98059 (MEK inhibitor) was used to check whether LOE directly regulated the activity of ERK. Although LOE exerted inhibitory effect on melanin synthesis, we could not observe this effect in PD98059-treated α-MSH induced B16F10. These results strongly indicate that LOE is related to ERK activation and MITF degradation in anti-skin pigmentation. Hence, LOE should be utilized as a whitening agent of skin in the near future.

• 미생물학회지
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• 제33권1호
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• pp.15-21
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• 1997

남조류 분해세균으로 알려진 Moraxella sp. CK-1의 세포외 자가용균효소의 특성을 규명하였다. Moraxella sp. CK-1은 대수증기식 초기부터 세포외부로 자가용균효소를 분비하며, 이들 효소는 60-$70^{\circ}C$, pH 9.0의 최적 활성 조건을 나타내었다. 이들 효소는 $Na^{+}$, $K^{+}$, $Li^{+}$등의 이온에 의해 활성이 촉진되고, $Ba^{2+}$, $Mg^{2+}$, $Ca^{2+}$ 및 $Mn^{2+}$ 등의 이온에 의해 활성이 억제되며, 특히, $Fe^{2+}$와 $Cu^{2+}$ 이온 존재하에서는 효소의 활성이 거의 나타나지 않았다. Micrococcusluteus 세포를 기질로 이용하여 renaturing, SDS-PAGE를 수행한 결과 Moraxella sp. CK-1은 분자량이 각각 30,32,38 그리고 41 kDa인 4가지 효소를 분비하며, 이중 32와 41 kDa의 효소는 생장 초기부터 사멸기까지 지속적으로 배지에 분포하는 반면, 38 kDa의 효소는 대수증식기 중기 이전, 30 kDa의 효소는 대수증식기 중기 이후에 배지에 분포하였다. M. luteus의 SDS-insoluble peptidoglycan에 효소추출물을 처리하였을 때 반응액의 유리 아미노기 농도가 증가하는 것은 이들 효소가 N-acetylmuramyl-L-ananine amidase 또는 endopeptidase라는 사실을 시사한다.

• 한국식품영양과학회지
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• 제39권1호
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• pp.47-53
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• 2010

제주 전통된장으로부터 세포외효소(protease, fibrinolytic enzyme, amylase, cellulase, lipase) 분비능이 우수한 세균을 분리한 후 16S rRNA 유전자 분석과 생리적 특성을 분석하여 균주를 확인하고자 하였다. Protease 분비능은 JR14, JR19, JR25, JR32, JR38, JR47과 JR64가 표준균주인 Bacillus subtilis KCCM12027보다 활성이 높았다. Amylase 분비능은 JR6, JR25, JR38, JR56, JR81에서 나타난 반면 표준균주인 KCCM12027에서는 나타나지 않았다. Cellulase 분비능은 JR6, JR14, JR48과 JR65가 다른 분리균주 또는 표준균주보다 높았으며, lipase 분비능은 JR14와 JR48이 높았다. 혈전용해 활성은 positive control인 plasmin의 용해 영역에 비하여 JR19가 192%로 가장 높았고, hemolysis 활성도 높았다. 혈전용해능이 있는 균주인 JR19, JR32, JR47, JR64의 배양액을 zymography한 결과, 25～75 kDa 사이에서 4～5개의 밴드가 확인되었다. 된장으로부터 분리한 균주들의 16s rRNA 유전자 염기서열을 분석한 결과 모두 Bacillus species와 99%의 상동성을 나타내었으며, 혈전용해능이 가장 우수한 JR19는 B. stratosphericus $41KF2a^T$와 거의 일치하였다.

• International Journal of Industrial Entomology and Biomaterials
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• 제22권2호
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• pp.83-93
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• 2011

Biochemical and enzymatic characterization for extracellular protease isolated from Cordyceps militaris cultivated on rice bran medium was investigated. C militaris produced proteolytic enzymes from 10 days after inoculation, maximum enzyme production was found at 25 days. The optimum temperature and pH of proteases production was at $25^{\circ}C$ and pH 7.0, respectively. The protease activity was observed in the four peaks (Pro-I, Pro-II, Pro-III, and Pro-IV) separated through Sephadex G-100 column chromatography. The separated protease was optimally active at $25^{\circ}C$. Optimum pH of the protease was between 7 and 8. Enzyme was also stable over at $30-80^{\circ}C$. The enzyme was highly stable in a pH range of 4-9. Protease activity was found to be slightly decreased by the addition of $Mg^{2+}$, $Mn^{2+}$, $Zn^{2+}$, $Fe^{2+}$ and $Cu^{2+}$, whereas inhibited by the addition of $Ca^{2+}$ and $Co^{2+}$ Protease activity was inhibited by protease inhibitor PMSF. On the other hand, the partially purified protease was investigated on proteolytic protease activity by zymogram gel electrophoresis using three substances (casein, gelatin and fibrin). Four active bands (F-I, FII, F-III, and F-IV) of fibrin degradation were revealed on fibrin zymogram gels. Both of F-II and FIII showed caseinolytic, fibrinolytic and gelatinolytic activities in three gels. Thermostability, pH stability, and pH-thermostability of the enzyme determined the residual fibrinolytic activity also displayed on fibrin zymogram gel. The only one enzyme (F-II) displayed over a broad range of temperature at $30-90^{\circ}C$. The FII displayed fibrinolytic activity in the pH range 3-5, but was inactivated in the range of pH 6-11. The F-I and F-III showed enzyme activity in the pH range of 6-11. In the pH-thermostability, the F-II only kept fibrinolytic activity after heating at $100^{\circ}C$ for 10, 20 and 30 min at pH 3 and pH 7, respectively. On the other hand, the F-II was retained activity until heating for 10 min under pH 11 condition. By using fibrin zymogram gel electrophoresis, extracellular fibrinolytic enzyme F-II from C. militaris showed unusual thermostable under acid and neutral conditions.

• 한국미생물·생명공학회지
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• 제49권3호
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• pp.403-412
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• 2021

본 연구에서는 토양 및 근권에 존재하는 Bacillus 속의 식물 생장 촉진 활성, 식물 병원성 곰팡이의 생장 억제활성, 미네랄 가용화능 및 세포 외 효소활성을 확인해 보고자 하였다. 식물 병원성 곰팡이에 대한 항진균 활성에서 DDP257은 10종의 병원성 곰팡이에서 항진균 활성이 모두 나타났다. 식물 생장 촉진 인자인 indole-3-acetic acid 생성능에서는 ANG20이 70.97 ㎍/ml로 가장 높게 나타났다. 추가적으로 1-aminocyclopropane-1-carboxylate deaminase 생성능 조사에서는 총 10종에서 생성능을 확인하였고, 질소 고정능과 siderophore 생성능 조사에서는 대부분의 분리균주에서 활성을 확인하였다. 이후 분리된 균주에 대하여 phosphate, calcite, zinc과 같은 미네랄 가용화능을 확인하였으며 세포외 효소활성에서도 대부분의 효소에서 활성이 나타났다. 특히 alkaline phosphatase, esterase (C4), acid phosphatase, naphtol-AS-BI-phosphohydrolase에서 선별된 균주 모두 유사한 활성을 보였다. 이는 Bacillus 속이 다양한 유기물과 항생물질 및 세포 외 효소를 분비함으로써 이러한 결과가 나타난 것으로 판단된다. 따라서, 본 연구 결과를 통해 토양의 환경 개선에 기여하는 균주를 활용하여 미생물 제제로써의 가능성을 제시한다.

• Journal of Microbiology and Biotechnology
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• 제18권4호
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• pp.695-703
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• 2008

A locally isolated thermophile, Geobacillus sp. SAB-40, producing thermostable extracellular amylase constitutively and an induced intracellular ${\beta}$-galactosidase was characterized and identified based on 16S rRNA sequencing. A phylogenetic analysis then revealed its closeness to Geobacillus stearothermophilus. To evaluate the effect of the culture conditions on the coproduction of both enzymes by G stearothermophilus SAB-40, a Plackett-Burman fractional factorial design was applied to determine the impact of twenty variables. Among the tested variables, $CaCI_2$, the incubation time, $MgSO_4{\cdot}7H_2O$, and tryptone were found to be the most significant for encouraging amylase production. Lactose was found to promote ${\beta}$-galactosidase production, whereas starch had a significantly negative effect on lactase production. Based on a statistical analysis, a preoptimized medium attained the maximum production of amylase and ${\beta}$-galactosidase at 23.29 U/ml/ min and 12,958 U/mg biomass, respectively, which was 3-and 2-fold higher than the yield of amylase and lactase obtained with the basal medium, respectively.