An extremely thermostable extracellular proteinase from a strain of the archaebacterium Desulfurococcus growing at 88° C

Abstract

An organism growing at 88 degrees C that closely resembles Desulfurococcus mucosus produced a single extracellular proteinase. We have purified this enzyme and carried out a preliminary characterization. The proteinase, which is a serine-type enzyme, had a molecular mass of 52,000 Da by SDS/polyacrylamide-gel electrophoresis, but only 10,000-13,000 Da by gel-permeation chromatography. Molecular mass values from sucrose-gradient centrifugation were of the same order as those from SDS/polyacrylamide-gel electrophoresis. It had an isoelectric point of 8.7, and was inhibited by di-isopropyl phosphorofluoridate, phenylmethanesulphonyl fluoride and chymostatin. Substrate-specificity studies suggested a possible preference for hydrophobic residues on the C-terminal side of the splitting point. The thermostability of this enzyme is probably greater than any other reported proteinase (t1/2 at 95 degrees C, 70-90 min; t1/2 at 105 degrees C, 8-9 min). Ca2+ chelation does not appear to be implicated in stabilization of the protein structure. The stability of the Desulfurococcus proteinase was not greatly affected by the presence of reducing reagents (e.g. dithiothreitol), some chaotropic agents (e.g. NaSCN) and some detergents, but activity was lost rapidly at 95 degrees C in the presence of the oxidizing agent NaBO3. Proteolytic activity was readily detected at temperatures up to and including 125 degrees C, although denaturation was very rapid above 115 degrees C. A number of Figures supporting some of the findings reported in this paper have been deposited in supplement SUP 50137 (14 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1987) 241, 5.

Overexpression and characterization of a prolyl endopeptidase from the hyperthermophilic archaeon Pyrococcus furiosus

“…PEPase ability to hydrolyze azocasein was determined by the method of Cowan et al (7). For these experiments, 100 l (11 g) of partially purified enzyme was added to 900 l of azocasein solution (7).…”

“…For these experiments, 100 l (11 g) of partially purified enzyme was added to 900 l of azocasein solution (7). Samples were incubated at 85°C for 30 min, and the absorbance was determined at 410 nm.…”

“…Samples were incubated at 85°C for 30 min, and the absorbance was determined at 410 nm. A unit of activity is defined as described previously (7).…”

Purification and Characterization of an Intracellular Chymotrypsin-Like Serine Protease fromThermoplasma volcanium

“…Several thermophilic and hyperthermophilic archaea produce significant levels of intra-and extracellular proteolytic enzymes with high intrinsic molecular stability. Most of these enzymes, which are recognized as the serine type, have been detected in the genera Pyrococcus (Pyrolysin and PfpI), 5,6) Desulforococcus (Archaelysin), 7) Sulfolobus, [8][9][10] and Thermococcus. [11][12][13] Genome sequence data revealed even more expansive proteolytic genotypes of these organisms than can be inferred from biochemical analyses.…”

Purification and Some Properties of a Thermostable Metal Proteinase Produced by Thermomicrobium sp. KN-22 Strain.

“…Therefore, enzymes produced by thermophiles have been developed as reagents for clinical analyses. With respect to proteinases produced by extreme thermophiles, only a few enzymes, namely, caldolysin,1} aqualysin,2) and archaelysin, 3) have been reported. We have been searching for novel thermostable proteinases, and wehave already reported a thermostable acid proteinase4) produced by a moderate thermophile.…”