Biosynthesis scale-up of human soluble catechol-O-methyltransferase
Year: 2013
Type: dissertation
Abstract: Catechol-O-methyltransferase (COMT, EC 2.1.1.6) was first described in 1958. It is a Sadenosyl-L-methionine (SAM) dependent methyltransferase which catalyses the methylation of catechol substrates (catecholamines, catecholestrogens). This enzyme has several important functions in the body, acting as a barrier between the blood and various organs, through the elimination of toxic or biologically active catechols. COMT plays a particularly important role in the brain, where it participates in the metabolism of the neurotransmitter dopamine, and so its role in neurodegenerative diseases is extremely relevant, especially in Parkinson's disease. In recent years, several COMT inhibitors have been developed for Parkinson's disease treatment, and there is considerable interest in developing new and more effective inhibitors. To do so, large amounts of COMT in an active form are necessary, and the best way to achieve this is by up-scaling its production through biotechnological processes. In this work, we propose a fed-batch process for biosynthesis scale-up of the soluble isoform of COMT, using an Escherichia coli expression system for the production of the soluble isoform of this protein, testing various fermentation conditions and controlling various parameters during the process. After several different batch and fed-batch experiments, the selected fermentation conditions were 20% dissolved oxygen, an initial batch concentration of 20 g/L of glycerol and tryptone, and a constant feed of 1 g glycerol/L/h, achieving a final specific COMT activity of 442.34 nmol/h/mg. Cell viability was monitored by flow cytometry and glycerol concentration by an HPLC system.
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2121499494