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Applied and Environmental Microbiology, June 2001, p. 2436-2444, Vol. 67, No. 6
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.6.2436-2444.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Purification and Characterization of a Psychrophilic, Calcium-Induced, Growth-Phase-Dependent Metalloprotease from the Fish Pathogen Flavobacterium psychrophilum

P. Secades, B. Alvarez, and J. A. Guijarro*

Área de Microbiología, Departamento de Biología Funcional, Facultad de Medicina, Instituto Universitario de Biotecnología de Asturias, Universidad de Oviedo, 33006 Oviedo, Spain

Received 27 November 2000/Accepted 22 March 2001

Flavobacterium psychrophilum is a fish pathogen that commonly affects salmonids. This bacterium produced an extracellular protease with an estimated molecular mass of 55 kDa. This enzyme, designated Fpp1 (F. psychrophilum protease 1), was purified to electrophoretic homogeneity from the culture supernatant by using ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic chromatography, and size exclusion chromatography. On the basis of its biochemical characteristics, Fpp1 can be included in the group of metalloproteases that have an optimum pH for activity of 6.5 and are inhibited by 1,10-phenanthroline, EDTA, or EGTA but not by phenylmethylsulfonyl fluoride. Fpp1 activity was dependent on calcium ions not only for its activity but also for its thermal stability. In addition to calcium, strontium and barium can activate the protein. The enzyme showed typical psychrophilic behavior; it had an activation energy of 5.58 kcal/mol and was more active at temperatures between 25 and 40°C, and its activity decreased rapidly at 45°C. Fpp1 cleaved gelatin, laminin, fibronectin, fibrinogen, collagen type IV, and, to a lesser extent, collagen types I and II. Fpp1 also degraded actin and myosin, basic elements of the fish muscular system. The presence of this enzyme in culture media was specifically dependent on the calcium concentration. Fpp1 production started early in the exponential growth phase and reached a maximum during this period. Addition of calcium during the stationary phase did not induce Fpp1 production at all. Besides calcium and the growth phase, temperature also seems to play a role in production of Fpp1. In this study we found that production of Fpp1 depends on factors such as calcium concentration, growth phase of the culture, and temperature. The combination of these parameters corresponds to the combination in the natural host during outbreaks of disease caused by F. psychrophilum. Consequently, we suggest that environmental host factors govern Fpp1 production.

* Corresponding author. Mailing address: Área de Microbiologia, Departamento de Biología Funcional, Facultad de Medicina, Universidad de Oviedo, 33006 Oviedo, Spain. Phone: 34985104218. Fax: 34985103148. E-mail: JAGA{at}sauron.quimica.uniovi.es.

Applied and Environmental Microbiology, June 2001, p. 2436-2444, Vol. 67, No. 6
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.6.2436-2444.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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