Identification of extracellular proteases in halophiles isolated from La Pampa solar ponds

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

Copenhagen

Congreso; 14th International Symposium on Microbial Ecology; 2012

ISME

Identification of extracellular proteases in halophiles isolated from La Pampa solar ponds Oliveira, L.C.G. #;Okamoto, D.N.# ; Paggi, R.A.*;Nercessian, D*; Di Meglio, L*; Juliano, M.A.#; De Castro, R.E*; Juliano, L# *IIB, CONICET-UNMdP, Mar del Plata, Argentina. #DptoBiofísica, INFAR-UNIFESP, São Paulo, Brasil.   Halophiles are microorganisms adapted to survive in hypersaline environments.They are an excellent source of enzymes which are not only require high-salt concentrations (1–4 M range) for activity and stability, but also may be active at high temperature and pH values.These adaptationsmakehalophilic enzymesa valuable resource of molecules, including enzymeswith potential applicability in Biotechnology. Haloarcula japonica, Haloarculaargentiniensis, Halobacteriumsp., HalobacteriumsalinarumandSalicolasp. were previously isolated from the solar ponds (NaCl> 30%, pH 7-8) Guatraché, Colorada Grande andSalitral Negro fromLa Pampa(Argentina)and identifiedby means of 16S rRNA analysis. Two different assays were performed to detect extracellular proteaseactivity: a) degradation on solid halophilic mediumof casein orhemoglobin; b) detection of protease activity using a randomized library of fluorescence resonance energy transfer peptide (FRET)by cell-free supernatants. Thesesynthetic combinatorial libraries(SCL)with this general structure Abz-GXXXXXQ-EDDnp where Abz (ortho-aminobenzoic acid) is the fluorescence donorlocated at the C-terminus, and Q-EDDnp (glutamine-[N-(2,4-dinitrophenyl)-ethylenediamine]) is the fluorescence acceptor locatedat N-terminus andX consists of an equimolar mixture of all amino acids. In the sub-libraries Abz-GXXZXXQ-EDDnpthe Z position is occupied with a specific amino acid which is absent in the other X positions.On agar plates the proteolytic activity was detected withHaloarculaandHalobacterium genera. These archaea were the only ones that showed activity in the assays performedwith cell-free media and the SCL (Z = X, F, R and V)at pH 8 and 3 MNaCl. The activity in Halobacteriummedium showed better activity in a pH range from6.0 to 11.0 for H salinarum and pH 7.0 to 10.0 for Halobacteriumsp.When different Z position was used at pH 8 and 3M NaCl, the hydrolytic activities present in the media of both archaeashowedsimilar amino acid preferencesin P1position (Z = V, L, I, F, S, E).The hydrolytic activity was completely abolished in the presence of PMSF (serine proteaseinhibitor) at pH 6 to 8 whileaddition of the ortho-phenanthroline (metallo-protease inhibitor)decrease 50% onthe hydrolytic activity in both microorganisms. Altogether, these results point to two different enzymes, a serine protease with an optimal pH around 8 and a metallo-protease with optimum activity at pH 10.0. Granted by MinCyt-CAPES, CONICET, UNMdP, FAPESP.