%0 Journal Article %T Effect of divalent metal ions on the activity and stability of ¦Â-galactosidase isolated from Kluyveromyces lactis %A PAULO ROBERTO ADALBERTO %A ANTONIO CARLOS MASSABNI %A ELEONORA CANO CARMONA %A ANT£¿NIO JOS¨¦ GOULART %J Revista de Ci¨ºncias Farmac¨ºuticas B¨¢sica e Aplicada %D 2010 %I Universidade Estadual Paulista %X In this study, it was demonstrated that ¦Â-galactosidase can be deactivated and reactivated with EDTA and divalent metal ions. The enzyme was deactivated after 20 minutes in EDTA solution. Maximal deactivation at the lowest EDTA concentration (10-3 mol.L-1) occurred in the presence of Tris-HCl buffer (pH 7.0). The enzyme recovered 50% of its initial activity after 10 minutes at Mg2+concentrations higher than 0.1 mmol.L-1. Experimental concentrations of 0.1 mmol.L-1 Mn2+ and 1.0 mmol.L-1 Co2+ were sufficient to reactivate the enzyme to around 300% of the control activity for the Mn2+ ion and nearly 100% for the Co2+ ion. The enzyme gradually lost its activity when the Co2+ concentration was 10-2 mol.L-1. Ni2+ and Zn2+ were unable to restore the catalytic activity. Km app and Vmax app were 1.95 ¡À 0.05 mmol.L-1 and 5.40 ¡À 0.86x10-2 mmol.min-1.mg-1, with o-NPG as substrate. Optimal temperature and pH were 34oC and 7.5. The half-life (t1/2) at 30¡ãC was 17.5 min for the holoenzyme and 11.0 min for the apoenzyme. With respect to pH variation, the apoenzyme proved to be more sensitive than the holoenzyme. Keywords: ¦Â-galactosidase. Divalent metallic ions. Enzyme activity. Stability. RESUMO Efeito de ¨ªons met¨¢licos divalentes na atividade e estabilidade da ¦Â-galactosidase isolada de Kluyveromyces lactis Este estudo demonstra como a ¦Â-galactosidase pode ser desativada e reativada usando EDTA e ¨ªons met¨¢licos divalentes. A enzima foi desativada ap¨®s 20 minutos na presen a de EDTA. Desativa o m¨¢xima para a menor concentra o de EDTA (10-3 mol.L-1) ocorreu na presen a do tamp o Tris-HCl. A enzima recuperou 50% de sua atividade inicial ap¨®s 10 minutos na presen a de Mg2+ em concentra es superiores a 0,1mmol.L-1. Concentra es de 10-4 e 10-3mol.L-1 de Mn2+ e Co2+ foram suficientes para reativar a enzima em 300% comparado ao controle de ¨ªons Mn2+ e aproximadamente 100% para ¨ªons Co2+. A enzima perdeu gradualmente a sua atividade quando a concentra o foi de 10-2 mol.L-1. Ni2+ e Zn2+ foram incapazes de restabelecer a atividade catal¨ªtica. Km app e Vmax app foram 1,95 ¡À 0,05 mmol.L-1 e 5,40 ¡À 0,86 x 10-2 mmol.min-1.mg-1. A temperatura e pH ¨®timos foram 34oC e 7,5. A meia vida da holoenzima foi de 17,5 min a 30oC e para a apoenzima foi de 11,0 min a 30oC. Quanto ¨¤ varia o de pH, a apoenzima provou ser mais sens¨ªvel que a holoenzima. Palavras-chave: ¦Â-galactosidase. ¨ªons met¨¢licos divalentes. Atividade enzim¨¢tica. Estabilidade. %K ¦Â-galactosidase %K divalent metallic ions %K enzyme activity %K stability %U http://serv-bib.fcfar.unesp.br/seer/index.php/Cien_Farm/article/view/1037