The recombinant amidase from the hyperthermophylic archaeon Sulfolobus solfataricus (SSAM) a signatured amidase, was cloned, purified and characterized. The enzyme is active on a large number of aliphatic and aromatic amides over the temperature range 60-95°C and at pH values between 4.0 and 9.5, with an optimum at pH 5.0. The recombinant enzyme is a dimer of about 110kD which reversibly associates to an octamer in a pH dependent reaction. The pH dependence of the state of association was studied using gel permeation chromatography, analytical ultracentrifugation and dynamic light scattering techniques. At pH 7.0 all three techniques show the presence of two species, in about the same amount, compatible with a dimeric and an octameric form. Decreasing the pH, the dimers associated to the octameric species, increasing the pH the octameric species converted in dimers: above pH 8.0 only dimers were found, below pH 3.0 the octameric species was the only species present. Association of dimers to octamers was hampered by non polar solvents and increased by temperature. A mutant (Y41C) was obtained which did not show this behaviour. SSAM monomers were strongly bound to form dimers which could be successfully dissociated only by electrophoresis.
Oligomerization of Sulfolobus solfataricus signatured amidase is promoted by acidic pH and temperature.
CASADIO, RITA;TASCO, GIANLUCA;
2005
Abstract
The recombinant amidase from the hyperthermophylic archaeon Sulfolobus solfataricus (SSAM) a signatured amidase, was cloned, purified and characterized. The enzyme is active on a large number of aliphatic and aromatic amides over the temperature range 60-95°C and at pH values between 4.0 and 9.5, with an optimum at pH 5.0. The recombinant enzyme is a dimer of about 110kD which reversibly associates to an octamer in a pH dependent reaction. The pH dependence of the state of association was studied using gel permeation chromatography, analytical ultracentrifugation and dynamic light scattering techniques. At pH 7.0 all three techniques show the presence of two species, in about the same amount, compatible with a dimeric and an octameric form. Decreasing the pH, the dimers associated to the octameric species, increasing the pH the octameric species converted in dimers: above pH 8.0 only dimers were found, below pH 3.0 the octameric species was the only species present. Association of dimers to octamers was hampered by non polar solvents and increased by temperature. A mutant (Y41C) was obtained which did not show this behaviour. SSAM monomers were strongly bound to form dimers which could be successfully dissociated only by electrophoresis.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
