A semi-synthetic biocatalyst was prepared by a double chemical modification of cytochrome c. Free amino groups were modified with poly(ethylene glycol) while free carboxylic groups were alkylated to form methyl esters. The double chemically modified protein, PEG-Cyt-Met, oxidized synthetic porphyrins in a ternary solvent mixture composed by methylene chloride, methanol, and phosphate buffer. The highest activity was found in the ternary systems with low water content (5%). The use of relatively hydrophobic peroxides, such as tert-butyl and cumene hydroperoxides, extended the operational life of the biocatalyst, which, in turn, resulted in an extended oxidation of the substrates tested. PEG-Cyt-Met is able to transform asphaltenes, a highly recalcitrant petroleum fraction. The huge energetic resource found as asphaltene-rich deposits is the driving force to investigate and to innovate upgrading technologies, including biotechnological strategies. © 2004 Wiley Periodicals, Inc.