Influence of additives on the stability of Vaccinium myrtillus peroxidase in reversed micellar systems Emílio Pedro
Arlindo Joaquim* Maria Raquel
Aires-Barros *Corresponding author Keywords: compatible
solutes, antibody structure, unwanted proteolysis
Peroxidases (EC.1.11.1.7) are heme-containing enzymes which catalyse the oxidation of a large number of substrates by reduction of hydrogen peroxide. A cationic peroxidase (VMPxC1) isolated and purified from a cell suspension of Vaccinium myrtillus, a heme-containing glycoprotein with a isoelectric point close to 9 and a molecular weight of aproximately 34000 Da, has been used in this work. To optimize the effect of experimental parameters on the stability of (VMPxC1) cationic peroxidase microencapsulated in AOT-reversed micelles a 24 factorial design was used. The factors considers were the water and surfactant concentrations, molarity and buffer pH. With the results obtained from this experimental planing the response surface curves were established. The best results were obtained with a surfactant concentration of 50mM, a water concentration of 500mM, and a ionic strengh of 80mM at pH 4.6. These conditions were used to evaluate the effect of different additives, namely sugars, polyols and alcohols on the stability of the enzyme in the reversed micelles system of 50mM AOT in isooctane. An increase on the activity retention was observed in the presence of the two sugars, melezitose and trehalose, and two linear polyols, sorbitol and mannitol, wich is dependent on the type and concentration used. Melezitose (50mM) led to the highest peroxidase stabilisation, at temperature of 30ºC, increasing the half-life time about 3-fold compared to the enzyme preparation without additive. |
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