Available online at www.sciencedirect.com J. of Supercritical Fluids 43 (2007) 283–290 Effects of compressed carbon dioxide treatment on the specificity of oxidase enzymatic complexes from mate tea leaves M.S. Primo a,b , G.C. Ceni a,b , N.S. Marcon a , O.A.C. Antunes b , D. Oliveira a , J. Vladimir Oliveira a , C. Dariva a,c,∗ a Department of Food Engineering, URI, Campus de Erechim, Av. Sete de Setembro, 1621, Erechim 99700-000, RS, Brazil b Institute of Chemistry, UFRJ, CT, Bloco A, Lab 641, Rio de Janeiro, RJ, Brazil c Institute of Research and Technology, ITP, PEP/NDTR-UNIT, Av. Murilo Dantas, 300, Farolˆ andia, Aracaju 49031-490, SE, Brazil Received 19 March 2007; received in revised form 26 June 2007; accepted 10 July 2007 Abstract This work evaluates the enzymatic activity of peroxidase (POD) and polyphenoloxidase (PPO) present in the crude extract of mate tea leaves (Ilex paraguariensis St. Hill) submitted to compressed CO 2 . The effects of temperature, exposure time, solvent reduced density, pressure, and depressurization rate on the activity of peroxidase and polyphenoloxidase were evaluated through a fractionated factorial experimental planning. Results show that temperature of 30 ◦ C, pressure of 70.5 bar, exposure time of 1 h, depressurization rate of 10 kg m -3 min -1 and carbon dioxide reduced density of 0.60 led to an enhancement of around 25% in the peroxidase activity and a polyphenoloxidase activity loss of 50%. Using this experimental condition, thermal stability at low temperature (-4 ◦ C) and the influence of successive pressurization/depressurization cycles were determined. Results suggest that it is possible to increase the specificity of the enzymatic extract towards enhancing POD or PPO activity depending on the experimental condition employed, and that the processing of enzymatic complexes with compressed CO 2 may be a promising route to increase the specificity of enzymatic extracts. © 2007 Elsevier B.V. All rights reserved. Keywords: Mate tea leaves; Enzymatic extract; Compressed carbon dioxide; Selectivity; Thermo stability 1. Introduction Brazil has a great variety of plants that can contribute as inex- haustible source of enzymes for application in several areas of great scientific and technological interest. Mate (Ilex paraguar- iensis) is an important natural product in the economic and cultural context of Brazil, with many relevant attributed prop- erties, such as anti-inflammatory, therapeutic, anti-rheumatic, stimulating, and diuretic [1–4]. Concerning the enzymes present in mate tea leaves, polyphe- nol oxidases and peroxidases are the most important. Polyphenol oxidases (PPO) are copper-containing enzymes, widespread in plants, which are synthesized early in tissue development, and stored in chloroplasts [5]. These enzymes catalyze two types of reactions involving molecular oxygen: the o-hydroxylation of monophenols to o-diphenols (cresols), cresolase activity, and ∗ Corresponding author. Fax: +55 79 3218 2115. E-mail address: claudio.dariva@pq.cnpq.br (C. Dariva). the subsequent oxidation of o-diphenols to o-quinones (cate- cholase activity) [6]. Peroxidases (POD) constitute a group of heme proteins having as main function the oxidation of sub- strates (monophenols, diphenols, etc.) at the expense of H 2 O 2 . POD are thermo stable enzymes, which can regenerate their activities after thermal treatment [7,8]. These enzymes can be employed as catalysts in many processes such as oxidation reactions, wastewater treatment, aroma production and terpene bioconversion [9]. For a long time enzymes were believed to work efficiently only in aqueous solutions. Consequently, their utilization in organic synthesis was rather scarce, once the low water solu- bility of most substrates always represented a serious obstacle [10,11]. This disadvantage, nevertheless, stimulated the search for systems based on the use of non-aqueous solvents in order to increase the solubility of hydrophobic substrates [12,13]. The use of supercritical carbon dioxide (SCCO 2 ) as a solvent in reaction medium has gained increased interest in enzymatic catalysis [14–16]. After the work of Hammond et al. [17], that described the use of supercritical fluids (SCFs) in biocatalysis, 0896-8446/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.supflu.2007.07.004