Extracellular protease of Natrialba magadii : purification and biochemical characterization

Abstract

A serine protease secreted by the haloalkaliphilic archaeon Natrialba magadii at the end of the exponential growth phase was isolated. This enzyme was purified 83 fold with a total yield of 25% by ethanol precipitation, affinity chromatography, and gel filtration. The native molecular mass of the enzyme determined by gel filtration was 45 kDa. Na. magadii extracellular protease was dependent on high salt concentrations for activity and stability, and it had an optimum temperature of 60 degrees C in the presence of 1.5M NaCl. The enzyme was stable and had a broad pH profile (6-12) with an optimum pH of 8-10 for azocasein hydrolysis. The protease was strongly inhibited by diisopropyl fluorophosphate (DFP), phenylmethyl sulfonylfluoride (PMSF), and chymostatin, indicating that it is a serine protease. It was sensitive to denaturing agents such as SDS, urea, and guanidine HCl and activated by thiol-containing reducing agents such as dithiotreitol (DTT) and 2-mercaptoethanol. This protease degraded casein and gelatin and showed substrate specificity for synthetic peptides containing Phe, Tyr, and Leu at the carboxyl terminus, showing that it has chymotrypsin-like activity. Na. magadii protease presented no cross-reactivity with polyclonal antibodies raised against the extracellular protease of Natronococcus occultus, suggesting that although these proteases share several biochemical traits, they might be antigenically unrelated.

Purification and Characterization of Two Novel Halotolerant Extracellular Proteases fromBacillus subtilisStrain FP-133

“…For example, an extracellular serine protease from the halophilic archaeon Natrialba magadii does not show any activity at a concentration less than 5.8% (w/v) NaCl. 32) Some reports suggest that the amino acid composition of a protein determines its halophilism or halotolerance. 33) In general, the halophilism of an enzyme strengthens in the predominant existence of acidic amino acid residues, an increased amount of small hydrophobic amino acid residues such as glycine, L-alanine, and L-valine, and a decreased amount of aliphatic amino acid residues, but no reports describing the characteristics of amino acid composition in halotolerant enzymes have been published.…”

Purification and biochemical characterization of the haloalkaliphilic archaeonNatronococcus occultus extracellular serine protease

“…No positive signals were detected in the culture medium of any of these strains (not shown). Moreover, Natrialba magadii, which produces an extracellular serine protease that was purified and characterized in our laboratory (GIMENEZ et al 2000) and which has an N-terminal sequence 85% homologous to halolysins 172 P1 and R4 did not show immunoreactivity with anti-EP antibody.…”

Autocatalytic Maturation of the Tat-Dependent Halophilic Subtilase Nep Produced by the Archaeon Natrialba magadii

“…The protease activity was determined using azocasein as a substrate as previously described (10). Briefly, aliquots of the cellular or extracellular fractions were incubated at 45°C in a reaction mix containing 50 mM Tris-HCl buffer (pH 8), 1.5 M NaCl, and 0.5% (wt/vol) azocasein.…”