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

Abstract

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.

Phase variation in Flavobacterium psychrophilum: characterization of two distinct colony phenotypes

“…In the present study, a difference in ECP profiles between the smooth and rough cells was observed, as the P13 rough variant and the wild type isolate showed 5 intensely stained bands compared to no bands or weakly stained bands in the P13 smooth variant. Two of these protein bands had approximate molecular weights of 55 and 65 kDa and may be the 55 kDa Fpp1 and the 62 kDa Fpp2 metalloproteases previously found in F. psychrophilum (Secades et al 2001(Secades et al , 2003, indicating that the P13 rough cells could have a higher ability to hydrolyze a range of matrix and muscle proteins compared to the P13 smooth cells. The 55 and 65 kDa protein bands were not evident, however, in the ECP profiles of the P6 isolates, which could in part be due to the age differences between the isolates.…”

Complete genome sequence of the fish pathogen Flavobacterium psychrophilum

“…Little is known about the psychrotrophic properties of F. psychophilum enzymes: only the metalloproteases Fpp1 and Fpp2 were biochemically characterized and reported to display a psychrophilic and thermolabile behavior 13,14 . The psychrotolerant Flavobacterium frigidimaris produces seven NAD(P) + -dependent dehydrogenases 44 ; four of them were psychrophilic and thermolabile, whereas the other three were unexpectedly thermophilic and thermostable.…”

“…The two metalloproteases Fpp1 (ref. 13) and Fpp2 (ref. 14) hydrolyze a broad range of substrates, including basic elements of muscular tissues.…”

Optimization of Protease Extraction from Horse Mango (Mangifera foetidaLour) Kernels by a Response Surface Methodology

“…13) The poor recovery of the plant enzymes is further aggravated by the fact that most of them are cellularly bound. 14,15) Because of these inherent problems, researchers working on plant enzymes have incorporated various reducing agents, 16,17) activators, 18,19) and detergents 10,20) in order to improve the extraction and the stability of the enzymes, but the use of such reagents must to be optimized due to economic and technological considerations.…”