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

Abstract

A organism growing at 88.degree.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 52000 Da by SDS/polyacrylamide-gel electrophoresis, but only 10000-13000 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.degree.C, 70-90 min; t1/2 at 105.degree.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.degree.C in the presence of the oxidizing agent NaBO2. Proteolytic acitvity was readily detected at temperatures up to and including 125.degree.C, although denaturation was very rapid above 115.degree.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.