Purification, molecular properties and specificity of a thermoactive and thermostable proteinase from Pyrococcus abyssi, strain st 549, hyperthermophilic archaea from deep‐sea hydrothermal ecosystem

Abstract

A protease was isolated and purified from the supernatant of a culture of hyperthermophilic archaebacteria : Pyrococcus abyssi strain st 549. Purification consisted of three chromatographic steps. The enzyme purification yield was 4% and the purification factor 890. This protease is a seryl-protease hydrolyzing proteins and peptides with a preference for cleavage at the aromatic and hydrophobic residues in P1 and PP1 positions. Its activity is optimal at 95³C and at pH 9. The electrophoretic mobility of the protease observed by zymogram suggests that it can adopt several oligomer forms. Three of them predominate displaying apparent molecular masses of 150, 105 and 60 kDa. Interdependence of the observed bands was revealed by changing the denaturation conditions of the samples (temperature, SDS concentration) before electrophoresis.z 1998 Federation of European Biochemical Societies.

Milk-clotting activity of enzyme extracts from sunflower and albizia seeds and specific hydrolysis of bovine κ-casein

“…Enzyme activities of plant extracts were detected by zymography, adapted from the method of Dib, Chobert, Dalgalarrondo, Barbier, and Haertle´(1998). A quantity of 3 mg of each of plant crude extract or protein extract or chymosin was added to 1 mL of 0.125 M Tris-HCl buffer, pH 6.8, containing 5% (w/v) SDS, 1% (w/v) sucrose, and 0.05% (w/v) bromophenol blue.…”

Desulfurococcus kamchatkensis sp. nov., a novel hyperthermophilic protein-degrading archaeon isolated from a Kamchatka hot spring

“…A number of proteases produced by hyperthermophilic archaea of the genera Desulfurococcus (Cowan et al, 1987), Sulfolobus (Fusek et al, 1990;, Pyrococcus (Eggen et al, 1990;Dib et al, 1998), Thermococcus (Klingeberg et al, 1995), Pyrobaculum (Völkl et al, 1994) and others have been described during past decades. Using the zymogram method (Tsiroulnikov et al, 2004), añ 120 kDa thermostable proteinase, active at 85 u C and pH 6.6 and 9.0 (data not shown), was found in the cell envelope fraction of strain 1221n T , grown on a-keratin.…”

The Order Thermococcales and the Family Thermococcaceae

“…27.3a). Thermococcales genomes encode numerous proteases, and several extracellular proteases have been characterized (Dib et al 1998;Kannan et al 2001;Klingeberg et al 1995;Morikawa et al 1994;Ward et al 2002b). The peptides enter the cell through an uncharacterized transporter system (Albers et al 2004) and are most likely converted to their discrete constituent amino acids intracellularly by a number of cytosolic proteases (e.g., Bauer et al 1996aBauer et al , 1997Ghosh et al 1998;Halio et al 1996;…”