• Korean Journal of Microbiology
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• v.29 no.6
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• pp.345-352
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• 1991

A serine proteinase of molecular weight 60 kd was purified from culture supernatant of P. aeruginosa using DEAE-Trisacryl M ion-exchange and AcA 54 gel filtration column chromatography, and the properties of serine proteinase were characterized. By means of SDS-polyacrylamide gel electrophoresis, the molecular weight of the enzyme was 55 kd. The optimal pH for the activity of purified enzyme was 7.5. The activity of the purified enzyme was completely inhibited by Di-isopropylfluorophosphate(DFP) and N-.alpha.-p-tosyl-L-lysine choloromethyl detone(TLCK) but not by other proteinase inhibitors such as E-64, pepstatin A, 1, 10-phenanthroline. The purified enzyme was capable of degrading type I and type IV collagen. Antisera obtained from hymans infected with Pseudomonas aeruginosa reacted to the purified serine proteinase in immunoblots. These results indicate that the purified enzyme is trypsin-like serine proteinase and this enzyme of P. aeruginosa may play an important role in tissue damage as a spreading factor and may be useful for serodiagnosis of Pseudomonas infections.

• Journal of Microbiology and Biotechnology
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• v.28 no.12
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• pp.2079-2094
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• 2018

Sunflower trypsin inhibitor (SFTI) is a 14-amino-acid bicyclic peptide that contains a single internal disulfide bond. We initially constructed chimeras of SFTI with N-terminal secretion signals from the Escherichia coli OmpA and Pseudomonas aeruginosa ToxA, but only detected small amounts of protease inhibition resulting from these constructs. A substantially higher degree of protease inhibition was detected from a C-terminal SFTI fusion with E. coli YebF, which radiated more than a centimeter from an individual colony of E. coli using a culture-based inhibitor assay. Inhibitory activity was further improved in YebF-SFTI fusions by the addition of a trypsin cleavage signal immediately upstream of SFTI, and resulted in production of a 14-amino-acid, disulfide-bonded SFTI free in the culture supernatant. To assess the potential of the secreted SFTI to protect the ability of a cytotoxic protein to kill tumor cells, we utilized a tumor-selective form of the Pseudomonas ToxA (OTG-PE38K) alone and expressed as a polycistronic construct with YebF-SFTI in the tumor-targeted Salmonella VNP20009. When we assessed the ability of toxin-containing culture supernatants to kill MDA-MB-468 breast cancer cells, the untreated OTG-PE38K was able to eliminate all detectable tumor cells, while pretreatment with trypsin resulted in the complete loss of anticancer cytotoxicity. However, when OTG-PE38K was co-expressed with YebF-SFTI, cytotoxicity was completely retained in the presence of trypsin. These data demonstrate SFTI chimeras are secreted in a functional form and that co-expression of protease inhibitors with therapeutic proteins by tumor-targeted bacteria has the potential to enhance the activity of therapeutic proteins by suppressing their degradation within a proteolytic environment.

• Food Science of Animal Resources
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• v.22 no.1
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• pp.81-86
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• 2002

A bacteriocin producing lactic acid bacteria was isolated from ground beef and the strain was identified as Lactobacillus plantarum ssp. by use of API carbohydrate fermentation pattern and physiological tests. The bacteriocin produced by L. plantarum KU107 exhibited a good spectrum of activity against foodborne pathogens including Bacillus cereus, Escherichia coli, Listeria ivanovii, Listeria monocytogenes, Pseudomonas aeruginosa, Pseudomonas chlororaphis, Staphylococcus aureus, Staphylococcus intermedius, Salmonella typhimurium and Yersinia enterocolitica. The bacteriocin was active over a wide pH range and stable of heat treatment, and inactivated by treatment with proteases. A bacteriocin from L. plantarum KU107 was effetive in reducing S. aureus in tryptic soy broth. On the ground beef containing S. aureus was added with the crude bacteriocin, S. aureus was inhibited during storage period at 4$\^{C}$.