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 geothermally heated marine environment, is a peptide-fermenting, sulphur-dependent organism with an optimum growth temperature of 92 degrees C. It forms grapes of cells, which adhere to each other and to sulphur granules 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 membrane-anchored stalks, thereby enclosing a 'quasi-periplasmic space'. Two copies of a globular protease, which probably serves an exodigestive function related to the organism's energy metabolism, are attached near the middle of each stalk.


RESULTS
We have purified and characterized this protease with regard to its enzymatic properties and thermostability, and have sequenced its gene using an approach based entirely 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-inactivation to 95 degrees C in the free form and 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.
Mayr
J
J
Max-Planck-Institut für Biochemie, Martinsried, Germany.
Lupas
A
A
Kellermann
J
J
Eckerskorn
C
C
Baumeister
W
W
Peters
J
J
D000595:Amino Acid Sequence; D001105:Archaea / Q000201:enzymology*; D001425:Bacterial Outer Membrane Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013381:Subtilisins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature
D001425:Bacterial Outer Membrane Proteins; D008562:Membrane Glycoproteins; D013381:Subtilisins