Extracellular Protease of the Haloalkaliphilic Archaeon Natrialba magadii: Partial Sequence of the Gene and Protease Activity in the Presence of Organic Solvents

GIMENEZ MI; RUIZ DM; SILVEYRA MX; PAGGI RA; DE CASTRO RE

St. Pete Beach (Florida)

Congreso; Annual Meeting Southeastern Branch of the American Society for Microbiology; 2005

American Society of Microbiology (ASM)

The haloalkaliphilic archaeon Natrilba magadii produces an extracellular protease at the end of the exponential growth denoted as NEP for Nab. magadii extracellular protease. NEP was previously purified and characterized at the biochemical level. We report the partial sequence of the gene encoding NEP and the activity and stability of this protease in the presence of organic solvents. A DNA fragment encoding NEP was isolated from a subgenomic library of Nab. magadii which contains the pre and pro peptides, the Nt of the mature protease and the promoter. The analysis of this sequence indicates that NEP is a halolysin-like protease and that it is a potential substrate for the Tat protein secretion pathway.  The activity and stability of NEP are dependent on high salt concentrations (> 1.5 M NaCl/KCl), meaning low water activity media, a feature in common with organic solvent systems. To evaluate the potential application of NEP in biocatalysis in organic media, its activity and stability were evaluated decreasing the concentration of NaCl in the presence of dimethylsulfoxide (DMSO) or dimethylformamide (DMF). The addition of DMSO (30%) to enzyme solutions containing 0.5, 0.3 and 0.15 M NaCl increased the proteolytic activity from 43 to 82%, 16 to 50% and 4 to 18%, respectively, relative to the enzyme activity measured in 1.5 M NaCl (control). Enzyme stability was highly improved by the presence of 30% DMSO in diluted solutions. The residual activity measured after incubation at 30° C for 2 h was enhanced by addition of 30% DMSO from 0 to 80%, 10 to 95% and 55 to 98% in solutions containing 0.15, 0.3 and 0.5 M NaCl, respectively. The same concentration of DMF decreased NEP activity at all the NaCl concentrations tested (1.5, 0.5, 0.3 and 0.15 M). These results contribute to the fundamental and applied aspects of enzymes from extremophiles (extremozymes).This research was supported by grants from ANPCyT, UNMDP and CONICET, Argentina.