Document details

Biosynthesis of human membrane-bound Catechol-O-methyltransferase

Author(s): Soares, Rui Filipe Lopes

Date: 2012

Persistent ID: http://hdl.handle.net/10400.6/1120

Origin: uBibliorum

Subject(s): Doença de Parkinson; COMT membranar humana; Desenho Plackett-Burman; COMT (Catechol-O-methyltransferase); Proteína Catechol-O-metiltransferase


Description

Catechol-O-methyltransferase (COMT, EC 2.1.1.6) is an S-adenosyl-L-methionine-dependent methyltransferase enzyme that catalyzes the methylation of catechol substrates (catecholamines, catecholestrogens). Physiologically, it is responsible for the elimination of biologically active or toxic catechols, making it a protein with great clinical relevance as therapeutic target in serious disorders, like schizophrenia and Parkinson´s disease. To fulfill pharmaceuticals requirements, new strategies of optimization and large-scale production of COMT enzyme are crucial. Statistical optimization approaches have demonstrated their enormous value in laboratory and industrial scale, namely in biotechnological production processes, in which an incremental enhancement can be a perpetual improvement. In this work, we aimed the optimization of recombinant human membrane-bound COMT (hMBCOMT) enzymatic activity yields following a statistical optimization as a solving approach. Plackett-Burman design was used as a first optimization step to identify which factors have a significant effect in hMBCOMT productivity and activity levels, and Response Surface Methodology (RSM), by a Central Composite Design (CCD), to optimize the process. We applied Brevibacillus choshinensis cells for the biosynthesis of hMBCOMT and a semi-defined medium for cell growth. This medium was subjected to a first screening using the Plackett–Burman design to evaluate the influence of the culture parameters (chemicals and physicals) in hMBCOMT enzymatic activity levels. Enzymatic activity were measured in a high performance liquid chromatography (HPLC) coupled to a coulochemical detector. Among the eleven variables tested, polypeptone, ammonium sulfate, glucose and temperature were selected owing to their significant effect on human MBCOMT enzymatic activity. The biological human MBCOMT activity obtained with the semi-defined medium in Plackett-Burman design were very promising, while were higher than the obtained with 2SYNm medium, a traditional growth medium for Brevibacillus cells of this work. Typically, we obtained values of 93nmol/h for hMBCOMT total enzymatic activity and 30 nmol/h/mg of specific activity with protein in its native form, without the use of any kind of detergents on protein solubilization step. Based on the results of Plackett–Burman design, a CCD was adopted to define optimal components concentration and temperature in order to maximize our response. The CCD model presented a multiple correlation coefficient value of 0.635 and a significant lack of fit, showing the lack aptness of the model to the process optimization and the failure to attain the optimal concentration of each variable.

Document Type Master thesis
Language English
Contributor(s) Soares, Rui Filipe Lopes
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