Abstract
Catechol-O-methyltransferase (COMT, EC 2.1.1.6) plays a crucial role in dopamine metabolism which has intimately linked this enzyme to some neurodegenerative diseases, such as Parkinson's disease. In recent years, in the attempt of developing new therapeutic strategies for Parkinson's disease, there has been a growing interest in the search for effective COMT inhibitors. In order to do so, large amounts of COMT in an active form are needed, and the best way to achieve this is by up-scaling its production through biotechnological processes. In this work, a fed-batch process for the biosynthesis of the soluble isoform of COMT in Escherichia coli is proposed. This final process was selected through the evaluation of the effect of different dissolved oxygen concentrations, carbon and nitrogen source concentrations and feeding profiles on enzymatic production and cell viability, while controlling various parameters (pH, temperature, starting time of the feeding and induction phases and carbon source concentration) during the process. After several batch and fed-batch experiments, a final specific COMT activity of 442.34 nmol/h/mg with approximately 80% of viable cells at the end of the fermentation were achieved. Overall, the results described herein provide a great improvement on hSCOMT production in recombinant bacteria and provide a new and viable option for the use of a fed-batch fermentation with a constant feeding profile to the large scale production of this enzyme.