Glucose oxidase (GOX) is a glycoprotein that finds wide application in food industry and clinical analysis. The gene encoding the GOX from Penicillium variabile P16 was expressed in Pichia pastoris X 33 using the methanol inducible AOX1 promoter. Among 1 l transformants resistant toward high zeocin concentrations, six Mut(+) strains were screened in shaken flasks and the strain X33 c9, producing 0.33 U ml(-1) of heterologous GOX after 11 days of fermentation, was selected. Recombinant GOX (ca. 50 U ml(-1)) was produced in a 3-l fermenter under not optimized conditions, recovered and purified in order to characterize and to compare it with the native one. The GOX from P. pastoris had a molecular weight of 82 kDa. Comparison of carbohydrate moieties showed a slight over-glycosylation of the GOX from Pichia over the native enzyme (17 and 14%, respectively). pH behavior of the recombinant enzyme, in terms of both activity and stability, was similar to that of the native one; on the other hand, a certain difference was observed in optimal temperature for activity and in thermal stability. P. pastoris appears to be a good expression system for GOX production. (c) 2006 Elsevier Inc. All rights reserved.