Identification of extracellular protease producing halophiles from La Pampa saltern ponds.

PAGGI, R. A.; OKAMOTO, D. N.; OLIVEIRA, L. C.; NERCESSIAN, D.; DI MEGLIO, L.G.; JULIANO, M. ; JULIANO, L.; DE CASTRO, R.

Mar del Plata

Congreso; VIII congreso de microbiologia general; 2012

Sociedad argentina de microbiologia

Halophiles are metabolically adapted to cope with elevated salt concentrations (> 2 M NaCl) and to use the scarce nutrients present in hypersaline environments. These adaptations make halophiles a valuable resource of molecules, including enzymes, with potential applicability in Biotchnology. The aim of this work was to screen for indigenous extracellular protease producing microorganisms. Haloarcula japonica, Haloarcula argentinensis, Halobacterium sp., halobacterium salinarum and Salicola sp. were previously isolated from the solar ponds (NaCl > 30%, pH 7-8) Guatraché, Colorada Grande and Salitral Negro from La Pampa, Argentina and identified by means of 16S rRNA analysis. Two different asays were performed to detect estracellular protease activity: a) growth on solid halophilic medium containing 0.8 % casein or hemoglobin as substrate; b) degradation of fluorescence resonance energy transfer peptide where Abz is the fluorescence donor and Q-EDDnp is the fluorescence acceptor. X consists of an equimolar mixture of all amino acids. In the sub-libraries Abz-GXXZXXQ-EDDnp the Z position is occupied with a specific amino acid wich is absent in the other X positions. Clear halos indicative of protease activity were detected around the streaks of Haloarcula and Halobacterium genera on agar plates containing both substrates. These archaea were the only ones that gave positive results in the assays performed with cell-free media and sub-FRETPL (Z = X, F,  and V) at pH 8 and 3 M NaCl. To characterize the proteolytic activity present in Halobacterium medium, the effect of pH on the endopeptidase activity was determined analyzing the hydrolitic rates on FRETPL-X at pH values from 4.0 to 12.0. Maximum activity was obtained in a pH range from 6.0 to 11.0 for H. salinarum and pH 7.0 to 10.0 for Halobacterium sp. When different FRETPL-Z were used at pH 8 and 3 M NaCl, the hydrolitic activities present in the media of both archaea showed similar amino acid preferences in P1 position (Z= V, L, I, F, S, E).To assign the activities detected to a specific class of proteases, the effect of class-specific protease inhibitors on hydrolisis of FRETPL-X was asayed at pH 4.0 to 12.0. The hydrolitic activity was completely abolished in the prescence of the classical serine protease inhibitor PMSF at pH between 6-8 while addition of the metallo-protease inhibitor ortho-phenanthroline produced 50% decrease on the hydrolitic activity in both microorganisms. On the other hand, the activity was not affected by inhibitors of cysteine (E-64) and metallo-protease (EDTA) inhibitors. Altogheter, these results point to FRETPL hydrolisis through tha action of two different enzymes, a serine protease with an optimal pH around 8 and a metallo-protease with optimum activity at pH 10.0. Grantes by MinCyt-CAPES, CONICET, UNMdP, FAPESP.