| Congresso Brasileiro de Microbiologia 2023 | Resumo: 224-1 | ||||
Resumo:The use of enzymes in the industrial sector demands the development of techniques and methodologies to enhance and optimize their performance. One alternative to meet this demand is the production of recombinant proteins, aiming to produce proteins with characteristics that favor their industrial-scale production. Xylanases are enzymes produced by a variety of organisms, grouped in the Glycosyl Hydrolase family, and they hydrolyze the β-1,4 linkages of the main component present in hemicellulose, xylan. This study analyzed the effects of phenolic compounds, ferulic acid and 4-hydroxybenzoic acid, on a recombinant endoxylanase, confirming previous results showing that these phenolics increase the activity of a recombinant endoxylanase from Myceliophthora heterothallica expressed in the yeast Pichia pastoris. The effects of the aforementioned phenolic compounds on the stability and enzymatic kinetics of this enzyme were analyzed. Based on the results obtained for the kinetic parameters, it can be concluded that the enzyme in question exhibits cooperative kinetic behavior, with a value of h close to 2 in all tested treatments. This cooperativity can be justified by the interaction scores of the phenolics with a cavity present on the protein surface, located opposite to its active site, so that both phenolics had little effect on the values of K0.5 but significantly increased the maximum reaction rate (Vmax), similar to type V allosteric modulators. Regarding the characterization of its optimum temperature, the endoxylanase without the phenolic compounds demonstrated an optimum temperature of 60°C, while the treatments with ferulic acid and 4-hydroxybenzoic acid showed optimum temperatures of 60°C and 50°C, respectively. In the ferulic acid treatments, it was observed that the enzyme maintained enzymatic activity above that exhibited by the control up to 90 minutes at 50°C, with an activity value equivalent to twice the control's activity at time zero. At higher temperatures, the activity was above that of the control for up to 30 minutes. The 4-hydroxybenzoic acid also had an activating effect on the enzyme, but only for 30 minutes, with 32% higher activity compared to the control. Palavras-chave: xylanase, phenolic compounds, thermal stability, allosteric enzyme Agência de fomento:FAPESP | |||||