Peptide synthesis catalyzed by a haloalkaliphilic protease from the archaeon Natrialba magadii.

RUIZ DM; IANNUCCI N; CASCONE O; DE CASTRO RE

Carlos Paz (Cordoba)

Congreso; XLIV Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2008

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Microbial proteases are widely used in biotechnology and industry. They catalyze the hydrolysis/synthesis of peptide bonds. Most proteases used in biotechnology inactivate under the harsh conditions required for synthesis reactions such as the presence of organic solvents. Haloarchaeal proteases offer an advantage over their mesohalic counterparts as they function optimally under high salt, meaning low water activity, a feature in common with aqueous-organic solvent mixtures. We have isolated and characterized a solvent-tolerant protease from the haloalkaliphilic archaeon Natrialba magadii (Nep). In this investigation, the potential application of Nep as biocatalyst in peptide synthesis reactions was examined. Nep catalyzed the synthesis of Ac-Phe-Gly-Phe-NH2 from Ac-Phe-OEt ester and Gly-Phe-NH2 amide substrates in the presence of 30% (v/v) DMSO, 1.5 M or 0.5 M NaCl at room temperature. The purification and identification of the peptide product was assessed by RP-HPLC and ESI-MS analysis, respectively. In the presence of 1.5 M NaCl the tripeptide yield was 54% after 1 h and reached the maximum after 24 h (67%). Nep preincubated with PMSF catalyzed the synthesis of the tripeptide and prevented hydrolysis of the product at longer reaction times. The native and recombinant enzymes were similarly effective as biocatalysts in peptide synthesis. Supported by ANPCyT, UNMDP, and CONICET.