Intracellular proteases from the extremely thermophilic archaebacteriumSulfolobus solfataricus

Proteolytic activities from the extremely thermoacidophilic archaebacteriumSulfolobus solfataricus were detected with the aid of synthetic substrates in a cell extract fractionated by gel filtration. Two aminopeptidases (aminopeptidase I and II), three endopeptidases (proteinase I, II and III) and one carboxypeptidase could be identified. Experiments carried out with protease inhibitors led to the identification of the exopeptidases as metalloproteases. Proteinases I and II behaved as chymotrypsin-like serine proteases, and proteinase III as a cysteine protease with a trypsin-like specificity. Molecular weight values assessed with the aid of marker proteins were as follows: aminopeptidase I, >450 kDa; aminopeptidase II, 170 kDa; carboxypeptidase, 160 kDa; proteinase I, 115 kDa; proteinase II, 32 kDa; proteinase III, 27 kDa. On incubation for 15 min they retained most of their activity up to a temperature of 90°C, with the sole exception of proteinase II, which was rapidly inactivated at 60°C. Protease content was also determined in crude extracts from cells grown in a mineral medium both to the stationary and to the exponential phase, with glucose or with yeast extract as carbon sources. No dramatic change was detected depending on the growth phase; however, carboxypeptidase level was three- to four-fold higher when yeast extract was present in the medium instead of glucose; this might suggest an involvement of this enzyme in the digestion of extracellularly available peptides.