BACKGROUND: Catechol-O-methyltransferase (COMT, EC 2.1.1.6) has been implicated in several human diseases including Parkinson's disease and the most appropriated therapy depends on the efficacy of the COMT inhibitors applied. Therefore, more effective drugs for COMT inhibition are important, and the development of such inhibitors requires research with recombinant COMT. RESULTS: The time-course production of biologically active hexa-histidine-tagged COMT in methanol-induced Pichia pastoris cultures was evaluated and immobilized-metal affinity chromatography was applied as the main capture step for the purification of the target enzyme that proved to be extremely efficient and selective. The best strategy allowed recovering soluble COMT at 300 m mol L-1 imidazol in a highly purified fraction with a purification fold of 81 and a bioactivity recovery of 57.35%. Using this strategy, a concentration of 3.68 mg L-1 of shake-flask culture of highly purified recombinant COMT was obtained. Finally, the MALDI-TOF/TOF analysis of the purified recombinant SCOMT demonstrated that it is correctly processed since no modifications in the primary sequence were observed. CONCLUSION: A new strategy was developed and implemented for the biosynthesis and purification of biologically active soluble COMT in a highly purified fraction with the ability to be inhibited by commercial Parkinson's disease inhibitors: 3,5-dinitrocatechol and entacapone. (C) 2016 Society of Chemical Industry