Some properties of two proteinases from a luminous bacterium, Vibrio harveyi strain FLN-108.

Abstract

Two proteinases (I and II) from a marine luminous bacterium, FLN-108, were purified to homogeneity. The molecular weights ofproteinases I and II were estimated to be 49,000 and 46,000, comprising a dimer of 23,000 molecular weight subunits, respectively. These enzymes were most active at from pH 8.0 to pH 9.0 and 50°C, and stable below 45°C. These enzyme activities were inhibited by EDTAand orthophenanthrolin.Phosphoramidon inhibited the activity of proteinase II, but not that ofproteinase I. Metal ions such as Cu2+, Hg2+, and Ni2+ strongly inhibited these activities. These results indicate that the proteinases I and II are metal-chelater-sensitive, alkaline proteinases.Wehave reported the existence of proteolytic, marine luminous bacteria, and described the purification and some properties of these enzymes from the culture supernatants of Vibrio splendidus FLE-2 and Vibrio harveyi FLA-1 1 strains.1>2) Since the proteinase activity in the culture supernatant with BGPY (basal glycerol peptone yeast extract) medium,1) which is a nutritionally complete broth, was very low, it was examined whether the enzyme activity from other Vibrio harveyi strains is also lower than that with BC(basal casein) medium,2) which lacks carbon and energy sources other than the casein. We found that several strains, which were newly isolated and identified as Vibrio harveyi, could have high proteinase activity.In the BGPY medium, the proteinase activity of the culture supernatant of one of these strains, FLN-108, was 15-fold higher than that of V. harveyi FLA-ll, and this strain produced two proteinases, which had different electrophoretic mobilities.In this paper, we describe the identification of the proteolytic, marine luminous bacterium, FLN-108, which was isolated as one of one 3009 hundred and forty-one luminous strains from surface coastal water at Okinawa, Japan, and the purification and someproperties of these two proteinases from this luminous strain. MATERIALS AND METHODSIsolation and identification of the organisms.

Expression in Escherichia coli of the recombinant Vibrio anguillarum metalloprotease and its purification and characterization

“…A cysteine protease from a pathogenic Taiwanese strain of V. harveyi 820514 is a novel protease of 38 kDa that exhibited lethal toxicity to Panaeus monodon (Liu et al 1997). Alkaline protease has also been extracted from the supernatants of V. harveyi (Fukasawa et al 1988). The optimal temperature and pH for the purified metalloprotease from wild type V. anguillarum W-1 were found to be 50°C and 7.0, respectively (Chen et al 2002b).…”

Proteinaceous exotoxins of shrimp-pathogenic isolates of Vibrio penaeicida and Vibrio nigripulchritudo

“…Besides the demonstration of active infections, extracellular products (ECP) obtained by in vitro cultivation of particular Vibrio strains have been implicated as a virulence factor in a wide range of marine organisms (Fukasawa et al, 1988;Liu et al, 1996;Sainz et al, 1998;Montero and Austin 1999;Hörmansdorfer et al, 2000). In two different shrimppathogenic isolates of Vibrio harveyi Liu et al (1996), Lee et al (1999) and Montero and Austin (1999) have suggested that a cysteine protease and lipopolysaccharides respectively constituted the major toxins produced by the bacteria.…”

“…Además de la demostración de la infección activa, productos extracelulares (PEC) obtenidos de cultivos in vitro a partir de cepas particulares de Vibrio han sido implicados como factor de virulencia en un gran rango de organismos marinos (Fukasawa et al, 1988;Liu et al, 1996;Sainz et al, 1998;Montero y Austin 1999;Hörmansdorfer et al, 2000). En dos diferentes cepas de Vibrio harveyi aisladas de camarones enfermos (Liu et al, 1996;Lee et al, 1999;Montero y Austin 1999) se ha sugerido que la cistein-proteasa y los lipopolisacaridos, respectivamente, constituyen las principales exotoxinas producidas por esta bacteria.…”

Production of exotoxins by two luminous Vibrio harveyi strains known to be primary pathogens of Penaeus monodon larvae

“…Owens et al (1996) showed that there was a relationship between siderophore production and virulence for piscine infecting V. harveyi, but the same relationship was not seen for strains infecting inverte-brates. V harveyi strains are known to produce chitinases (Svitil et al 1997) and proteases (Fukasawa et al 1988, Liu et al 1996b but the relationship between these factors and virulence is not clear. It seems to be a requirement of V. harveyi strains that cause brown gill disease in adult P. monodon that the bacterium must also be infected with a bacteriophage (Ruangpan et al 1999).…”

“…Widely distributed marine toxins such as tetrodotoxin and the surugatoxins are known to have a bacterial origin (Yasumoto & Murata 1993), and both tetrodotoxin and anhydrotetrodotoxin have been shown to be produced by V. harveyi strains isolated from seawater (Simidu et al 1987). Alkaline proteases have been extracted from broth culture supernatant of 2 luminous strains of V. harveyi, originally isolated from seawater (Fukasawa et al 1988(Fukasawa et al , 1989. Similarly.…”