INVESTIGADORES
RUIZ Diego Mario
congresos y reuniones científicas
Título:
Gene cloning and functional expression of an organic solvent – tolerant protease secreted by the haloalkaliphilic archaeon Natrialba magadii.
Autor/es:
RUIZ DM; MAUPIN-FURLOW JA; DE CASTRO RE
Lugar:
Capital Federal
Reunión:
Congreso; IV Congreso Argentino de Microbiología General (SAMIGE); 2007
Institución organizadora:
Sociadad Argentina de Microbiologia General
Resumen:
Archaea, the third domain of life, includes microorganisms which are mostly extremophiles. Haloarchaea grow optimally in 15-30% NaCl and, as an adaptation to the environment, halophilic enzymes are active and stable in low water solutions, features that are of interest for the application of halophilic enzymes as biocatalysts in aqueous-organic solvent media. The haloalkaliphilic archaeon Natrialba magadii (optimum growth in 20% NaCl and pH 12) secretes an organic solvent-tolerant protease NEP (Natrialba magadii Extracelular Protease) that has been biochemically characterized. The aim of this work was to clone and express the gene encoding NEP in the haloarchaeal host Haloferax volcanii. The complete gene for NEP was isolated from a genomic library of Nab. magadii by hybridization with a specific PCR probe. The nucleotide sequence of nep revealed an ORF of 1623 bp encoding a predicted 56.4 kDa polypeptide containing 20% acidic amino acid residues (pI 3.77) typical of haloarchaeal proteins. The translated protein, which included a putative 121-amino acid prepropeptide and 420 amino acids of mature protease, showed similarity to archaeal halolysins as well as to bacterial serine proteases of the subtilisin family. The prepropeptide contained the Tat consensus motif suggesting that NEP is a substrate of the Tat protein secretion pathway. The 5´ region contained A/T rich sequences that match the consensus of a typical haloarchaeal promoter (box A). For the heterologous expression, the coding region of nep was amplified by PCR and cloned into E. coli pET24b expression vector. Then, the sequence containing nep stop codon or the His6-tag were excised from pET-based constructs and subcloned in the shuttle vector pJAM under the haloarchaeal rRNAP2 promoter. After passage through a dam- E. coli strain, pJAM-NEP and pJAM-NEP-His6 were transformed into Haloferax volcanii DS70. Recombinant NEP (HvNEP) was successfully secreted and expressed as an active enzyme in Hfx. volcanii. Moreover, the protease activity detected in the culture medium was about 100-fold higher than that produced by Nab. magadii. The recombinant enzyme displayed similar electrophoretic mobility and antigenic behavior as native NEP. Current studies are focused on the determination of the biochemical properties and solvent tolerance of HvNEP as this will benefit basic studies as well the potential application of haloarchaeal enzymes in biotechnology. This work was supported by research grants from ANPCyT, CONICET and UNMDP.