Sulfatase de fÃgado do molusco Aplysia cervina solÃvel e imobilizada em suportes sÃlidos

AUTOR(ES)

Luciana Duarte Martins da Matta

DATA DE PUBLICAÇÃO

2004

RESUMO

A heparin specific sulfatase (EC 3.1.6.1.) was identified in the liver of the mollusc Aplysia cervina, widely found in the Northeastern Brazilian coast. This enzyme was purified by ammonium sulfate and acetone precipitations and by affinity chromatography using Heparin-Sepharose CL-6B. Some physical chemical and kinetics properties of this 89.7-fold purified preparation (5.37% yield) were investigated using p-nitrophenyl sulfate (pNPS) as substrate. The optima pH and temperature were found to be 5.0 and 45oC, respectively. This enzyme retained more than 90% of its activity when incubated for 15 min at 45ÂC while lost 60% at 55ÂC. km equal to 3.71 Â 0.41 mM was found. Its activity was enhanced by MgCl2, CaCl2 and FeCl2 and inhibited by Na2S2O3, Na2SO4, KCl, C6H5Na3O7 (sodium citrate), HgCl2, Na2HPO4 and NaH2PO4. The heparin low molecular weight competed with pNPS for the active site enzyme more than the high molecular one. This enzyme was covalently immobilized on ferromagnetic polysiloxane/polyvinyl alcohol composite and Dacron yielding derivatives with specific activity and retention of 1.85 units/mg protein, 3.17 units/mg of protein, 21.23% and 36.5 %, respectively. These preparations were easily removed from the reaction mixture by a magnetic field and were reused several times without loss in their activities. They were more thermal stable than the soluble enzyme and presented the same optima pH and temperature and apparent Km. The derivative based on ferromagnetic Dacron presented higher shelf life than that on POS/PVA. MgCl2, CaCl2 e EDTA activated both immobilized sulfatase derivatives whereas Na2HPO4 and NaH2PO4 and heparin inhibited them. The action by the other ions varied according to the support

ASSUNTO(S)

dacron aplysia cervina sulfatase ciencias biologicas polisiloxano polysiloxane polyvinyl alcohol sulfatase Ãlcool polivinÃlico aplysia cervina enzima imobilizada dacron enzyme immobilization

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