A HYPERTHERMOSTABLE PROTEASE OF THE SUBTILISIN FAMILY BOUND TO THE SURFACE-LAYER OF THE ARCHAEON STAPHYLOTHERMUS-MARINUS

Background: Staphylothermus marinus, an archaeon isolated from a geoth ermally heated marine environment, is a peptide-fermenting, sulphur-de pendent organism with an optimum growth temperature of 92 degrees C. I t forms grapes of cells, which adhere to each other and to sulphur gra nules via their surface layer. This glycoprotein layer forms a canopy which is held at a distance of about 70 nm from the cell membrane by m embrane-anchored stalks, thereby enclosing a 'quasi-periplasmic space' . Two copies of a globular protease, which probably serves an exodiges tive function related to the organism's energy metabolism, are attache d near the middle of each stalk. Results: We have purified and charact erized this protease with regard to its enzymatic properties acid ther mostability, and have sequenced its gene using an approach based entir ely on the polymerase chain reaction. The precursor form is 1345 amino acids long; between residues 64-741, it contains a domain with clear homology to subtilisins, which is interrupted by two large insertions. The enzyme has a broad substrate specificity and a pH optimum of 9.0. It is fully stable from pH 3.2 to 12.7 and is resistant to heat-inact ivation to 95 degrees C in the free form acid to 125 degrees C in the stalk-bound form. Conclusions: This protease is one of the most stable proteases known. Its high resistance towards denaturing agents makes it an interesting target for practical applications. Despite its large size, it is clearly a member of the subtilisin family and represents the only known enzyme that is a stoichiometric S-layer component.