• 미생물학회지
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• 제29권6호
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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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• 제28권12호
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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.

• 한국축산식품학회지
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• 제22권1호
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• pp.81-86
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• 2002

쇠고기 분쇄육으로부터 박테리오신 생산균주를 분리하여 형태학적, 생화학적 특성을 조사한 결과 Lactobacillus plantarum ssp. 와 유사하게 나타났으며 당발효성 실험결과 95%의 신뢰도로 L. plantarum으로 동정되어 L. plantarum KU107로 명명하였다. L. plantarum KU107이 생산하는 박테이로신은 trypsin과 pepsin의 처리에 의해 활성이 소실되었으며 pH 2와 12에서도 활력이 완전하게 소실되지 않는 pH 안정성과 열안정성을 갖는 것으로 조사되었다. 또한, 이 박테리오신은 Bacillus cereus, Listeria inoccua, Listeria monacytogenes, Staphylococcus aureus, Staphylococcus intermedius KCCM 11806, 그리고 Yersinia enterocolitica에 대해서도 항균 활성을 나타내었다. S. aureus가 접종된 쇠고기 분쇄육에 박테리오신을 처리한 경우, 대조구와는 다르게 저장 7일째까지 접종된 S. aureus 초기균수와 유의적인 차이없이 성장을 지연시킴을 알 수 있었으며 저장 14일까지 대조구에 비해 유의적으로 현저히 적은 균수를 나타내었다.