• Proceedings of the PSK Conference
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• 2002.10a
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• pp.334.1-334.1
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• 2002

Streptomyces scabiei subsp. chosunensis M0137. nonadecanoic acid producer. showed the highest protease activity when grown in OSY medium (oatmeal 1.5%, soybean meal 2%, dried yeast 1 %) supplemented with. glycerol (1 %) and CaCO3 (0.1 %). Two forms of protease(SS-1 and SS-2) were fractionated and purified through Ultrogel AcA 54 gel filtration and DEAE-sepharose CL-6B column chromatography. Both proteases were practically stable in the pH range of 6-10. The optimal pH for the activities of both protease 88-1 and 8S-2 were 7.5 and 8.0. respectively. (omitted)

• Korean Journal of Microbiology
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• v.49 no.1
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• pp.78-82
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• 2013

A novel Chryseobacterium sp. JK1 strain isolated from soil had been reported that this isolate produced large amount of extracellular protease at mesophilic temperature in previous study. The optimal temperature and pH of extracellular protease were $40^{\circ}C$ and 7.0, respectively, showing narrow range of optimal temperature and relatively broad activity from pH 6.0 to 9.0. In addition, the protease showed greatest activity against skim milk and lowest against bovine serum albumin (BSA). The protease strongly inhibited by ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA) or phenylmethylsulfonyl fluoride (PMSF), and addition of cation $Ag^+$ or $Cu^{2+}$, and slightly inhibited by $Al^{3+}$. No significant inhibition was found with pepstatin, and addition of cation, $K^+$, $Ca^{2+}$, $Na^+$, $Fe^{2+}$ or $Mg^{2+}$. On the contrary, protease was enhanced by addition of divalent cation $Mn^{2+}$ (5 mM). Zymography analysis of concentrated culture supernatant revealed two major bands at 67 and 145 kDa. These results suggest that Chryseobacterium sp. JK1 strain produced extracellular neutral serine proteases which could apply in food industry.

• Korean Journal of Microbiology
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• v.46 no.1
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• pp.73-79
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• 2010

The Korea Polar Research Institute (KOPRI) has assembled a culture collection of cold-adapted bacterial strains from both the Arctic and Antarctic. To identify excellent protease-producers among the proteolytic bacterial collection (874 strains), 78 strains were selected in advance according to their relative activities and were subsequently re-examined for their extracellular protease activity on $0.1{\times}$ ZoBell plates supplemented with 1% skim milk at various temperatures. This rapid and direct screening method permitted the selection of a small group of 15 cold-adapted bacterial strains, belonging to either the genus Pseudoalteromonas (13 strains) or Flavobacterium (2 strains), that showed proteolytic activities at temperatures ranging between $5-15^{\circ}C$. The cold-active proteases from these strains were classified into four categories (serine protease, aspartic protease, cysteine protease, and metalloprotease) according to the extent of enzymatic inhibition by a class-specific protease inhibitor. Since highly active and/or cold-adapted proteases have the potential for industrial or commercial enzyme development, the protease-producing bacteria selected in this work will be studied as a valuable natural source of new proteases. Our results also highlight the relevance of the Antarctic for the isolation of protease-producing bacteria active at low temperatures.

• Korean Journal of Microbiology
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• v.46 no.1
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• pp.63-67
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• 2010

Proteases catalyze hydrolytic cleavage of a peptide bond between amino acids and occupy pivotal positions in application in physiological and commercial fields. During the screening for novel bacteria producing extracellular protease, two bacterial strains, WD12 and WD32, were isolated from rotten trees and they made clear zone on LB plates supplemented with 1% skim milk. The similarities of 16S rRNA gene sequence of either WD12 or WD32 to GenBank database showed the highest to Pseuoxanthomonas mexicana as 97.8 and 99.8%, respectively. Phylogenetic analysis showed that both isolated was located within the cluster comprising P. mexicana and P. japonesis. WD12 and WD32 were catalase- and oxidase-positive, Gram-negative rod strains. In case of WD12, it could assimilate malate, but could not assimilate D-mannose, which were different characteristics from P. mexicana. Both Pseuoxanthomonas sp. WD12 and WD32 optimally produced extracellular protease at $35-37^{\circ}C$, and maximal activity showed as 656 unit/ml and 267 unit/ml, respectively.

• Korean Journal of Microbiology
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• v.39 no.3
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• pp.148-154
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• 2003

Moraxella sp. CK-l is known to inhibits the growth of Anabaena cylindrica, a cyanobacterium. It has been documented that the ability of this growth inhibition of Anabaena cylindrica was attributed to extracellular autolysin from Moraxella sp. CK-l. However, it remains to be elucidated identification and characterization of autolysin have yet been elucidated. In this study, we tried to purify and identify autolysin secreted from Moraxella sp. CK-l. Cells were grown in a complex liquid medium (BGC-11) and culture supernatants were collected, followed by ammonium sulfate fractionation. Fractions were further separated with anion exchange column, Mono-Q, in FPLC system and analyzed by SDS/PAGE. The fraction containing high autolysin activity showed a single distinct protein peak in anion column and molecular mass of about 17 kDa in SDS/PAGE. Nterminal amino acid sequencing of the protein was analyzed, of which result showed the homology with some proteases, including extracellular serine protease, Dichelobacter nodosus.

• Korean Journal of Microbiology
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• v.34 no.3
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• pp.87-90
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• 1998

Various bacterial strains that secret extracellular fibrinolytic enzyme were screened from Doen-Jang, a traditional soybean fermented food in Korea. Five microbes of them were identified to be Bacillus sp. strains according to Bergey's manual of systematic bacteriology. The culture filtrates of B. amyloliquefaciens (2.46 plasmin unit/ml) and B. pantothenticus (3.82 plasmin unit/ml) showed a level of fibrinolytic activity that was about three times higher than that of plasmin 1.0 unit and Bacillus subtilis showed the highest fibrinolytic activity (4.94 plasmin unit/ml). All of the extracellular proteases showing the fibrinolytic activity are confirmed by SDS-PAGE followed by reverse fibrin zymogram activity assay and we proposed that some of the fibrinolytic enzymes from this work are novel enzymes.

• Korean Journal of Food Science and Technology
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• v.31 no.1
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• pp.219-223
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• 1999

Various bacterial strains that secret extracellular fibrinolytic enzyme were screened from kimchi, a traditional vegetable fermented food in Korea. Three microbes of them were identified to be Bacillus amyloliquefaciens, Bacillus brevis and Micrococcus luteus strains according to Bergey's Manual of Systematic Bacteriology. It was found that B. amyloliquefaciens, B. brevis and M. luteus produced 2.58, 1.48 and 2.03 plasmin unit/mL of fibrinolytic enzyme, respectively. All extracellular proteases showing the fibrinolytic activity were confirmed by SDS-PAGE and fibrin zymography assay and we propose that some of the fibrinolytic enzymes from this work are novel enzymes.

• Journal of Microbiology and Biotechnology
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• v.23 no.11
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• pp.1536-1543
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• 2013

Proteases produced by Xenorhabdus are known to play a significant role in virulence leading to insect mortality. The present study was undertaken to purify and characterize protease from Xenorhabdus indica, an endosymbiont of nematode Steinernema thermophilum, and to decipher its role in insect mortality and its efficacy to control Helicoverpa armigera. A set of 10 strains of Xenorhabdus isolated from different regions of India were screened for protease activity on the basis of zone of clearing on gelatin agar plates. One potent strain of Xenorhabdus indica was selected for the production of protease, and the highest production (1,552 U/ml) was observed at 15-18 h of incubation at $28^{\circ}C$ in soya casein digest broth. The extracellular protease was purified from culture supernatant using ammonium sulfate precipitation and ion-exchange chromatography. The enzyme was further characterized by SDS-PAGE and zymography, which confirmed the purity of the protein and its molecular mass was found to be ~52 kDa. Further MALDI-TOF/TOF analysis and effect of metal chelating agent 1,10-phenanthrolin study revealed the nature of the purified protease as a secreted alkaline metalloprotease. The bioefficacy of the purified protease was also tested against cotton bollworm (Helicoverpa armigera) and resulted in $67.9{\pm}0.64%$ mortality within one week. This purified protease has the potential to be developed as a natural insecticidal agent against a broad range of agriculturally important insects.

• BMB Reports
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• v.34 no.2
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• pp.134-138
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• 2001

By adding sodium chloride (2.5%) into a Bacillus amyloliquefaciens DJ-4 culture broth, two serine-type fibrinolytic proteases with a molecular weight of 29 (subtilisin DJ-4) and 38-kDa were stimulated on the SDS-fibrin zymogram or inhibitor gels. B. amyloliquefaciens DJ-4 showed the highest proteolytic activity (5.52 plasmin NIH unit/ml) on the fibrin plate based on the molar ratio when cells were subjected to the 2.5% NaCl. Using a fibrin plate, the secreted protease from this strain in the presence of 5% NaCl showed that about 49% of the enzyme's activity remained after incubation at $60^{\circ}C$ for 30 min, but as the salt concentration was increased (10% NaCl) the activity nearly disappeared (0.14 plasmin NIH unit/ml). However, through a fibrin zymography assay, three fibrinolytic enzymes (38, 53 and 80-kDa) from the cells in the presence of 10% NaCl were detected. Also, two salt-activated serine-type fibrinolytic professes (29 and 38kDa) showed thermostability from 65 to $70^{\circ}C$ for 30 min. Furthermore, these professes also showed stability, pH 6-11. In particular, 29-kDa (subtilisin DJ-4) was very stable in the pH range of 4-11 at $4^{\circ}C$ for 48 h.

• Preventive Nutrition and Food Science
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• v.14 no.1
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• pp.14-20
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• 2009

Matrix metalloproteinases (MMPs) are a group of zinc proteases that serve the function of breaking down extracellular matrix (ECM). The present study evaluated the role of N-acetylcysteine (NAC) on the increased deposition of ECM in hepatic and glomerular fibrosis caused by carbon tetrachloride ($CCl_4$). The activity of MMPs increased and the levels of tissue inhibitors of metalloproteinases 1 and 2 (TIMP-1 and TIMP-2) decreased in the liver and kidney of $CCl_4$-treated rats. Rats treated with $CCl_4$ and NAC showed increased activities of MMPs and decreased levels of TIMP-1 and TIMP-2 in the liver and kidney. Treatment with NAC resulted in the effective degradation of ECM due to an increase in the activities of MMPs and a decrease in the levels of TIMPs.