Faculty of Food Technology and Biotechnology, University of Zagreb
Abstract
In the present study glucose oxidase (GOD) has been isolated from a culture filtrate of Penicillium notatum. The enzyme was purified by ammonium sulphate precipitation, diethylaminoethyl (DEAE) cellulose ion-exchange chromatography and Sephadex gel filtration. This protocol gave 16.47-fold purification and 25 % recovery of the enzyme. The optimum pH and temperature for the activity were 5.4 and 45 °C, respectively. The Km and vmax values for the enzyme were 10.5 mM and 456 U/mg, respectively. A detailed kinetic study of thermal inactivation was carried out. Both enthalpy of activation (ΔH*) and entropy of activation (ΔS*) decreased at higher temperatures. Moreover, free energy of denaturation (ΔG*) increased at higher temperature, making the enzyme thermally stable. A possible explanation for the thermal inactivation of GOD at higher temperatures is also discussed.U radu je izolirana glukoza-oksidaza iz filtrata kulture Penicillium notatum. Enzim je zatim pročišćen taloženjem amonijevim sulfatom, ionsko-izmjenjivačkom kromatografijom na dietilaminoetil (DEAE) celulozi i filtracijom na Sephadex gelu. Dobiven je prinos od 25 % enzima, pročišćenog 16,47 puta. Utvrđene su ove vrijednosti: optimalna pH-vrijednost za aktivnost enzima od 5,4, optimalna temperatura od 45 C, Km=10,5 mM i vmax=456 U/mg. Provedena je detaljna kinetička studija temperaturne inaktivacije enzima. Povisivanjem temperature smanjila se entalpija (ΔH*) i entropija aktivacije enzima (ΔS*), a povećala slobodna energija (ΔG*) potrebna za denaturaciju proteina, kao i termostabilnost enzima. U radu se također raspravlja o mogućim uzrocima inaktivacije glukoza-oksidaza pri višim temperaturama
