Produção e imobilização da enzima α – acetolactato descarboxilase em agarose utilizando diferentes protocolos de ativação
Dissertation (Ms) 19/04/2016
Celina Maria Gentil de Farias Lemos
During the fermentation process of beer brewing, yeast cells use the nutrients found in medium to accelerate the metabolism and multiply. An important carbonyl compound present in the beer fermentation is the diacetyl because this leads to the final product an undesirable organoleptic characteristic that is the flavor of butter. The formation of this carbonyl compound is non-enzymatically, wherein the acetolactate is excreted from the cell during valine synthesis and then converted into diacetyl by oxidative decarboxylation. The formed diacetyl will be reassimilated by the cell and lowered essentially in acetoin. The enzyme α – acetolactate decarboxylase operates in the acetolactate formed converting it directly into acetoin and so the diacetyl is not formed. In the first stage of this study It has been evaluated the production of the enzyme α – acetolactate decarboxylase (ALDC) for seven strains of microorganisms which were isolated from a sewage treatment plant (Universidade Federal do Ceará – Campus Pici) and from a mangrove forest soil in regions of the state of Ceará – Brazil countryside. A satisfactory production of the enzyme of interest by the seven strains tested was observed, but the strains named I5 and J6 showed the best results of specific activity values, where average values were 1795.66 U / mg and 2043.36 U / mg respectively. Since the ALDC enzyme production occurs intracellularly it was necessary to rupture the cell with a lysozyme solution to facilitate the disruption of the cell membrane, enhanced by the effect of ultrasonic waves. Subsequently, has been evaluated the capacity of the immobilized ALDC enzyme in agarose-based media activated by different protocols glyoxyl-agarose, MANAE-agarose and MANAE-glutaraldehyde. To assess the enzymatic activity was used colorimetric and High Performance Liquid Chromatography (HPLC) methodologies, where the latter is known to be more precise and presented enzymatic activity values consistent with the results presented when used the first technique. In front of all the study carried out in this research can be concluded that the tested strains showed potential for biotechnological production of the ALDC enzyme and that once produced, showed good immobilization yields for the tested media.
