| Congresso Brasileiro de Microbiologia 2023 | Resumo: 228-1 | ||||
Resumo:The application of molecular techniques in the production, analysis, and purification of enzymes has been crucial for scientific and industrial research, enabling a comprehensive and detailed study of the chemical conditions affecting enzymes and their applicability factors. In this study, a recombinant endoxylanase derived from the thermophilic fungus Myceliophthora heterothallica, expressed in the yeast Pichia pastoris, capable of hydrolyzing xylan bonds, the main component of hemicellulose, was investigated. Xylanase can be used in various industries for the processing and utilization of lignocellulosic by-products from agricultural production, resulting in the production of high-value-added chemical compounds. During the treatment or pre-treatment of the lignocellulosic biomass, the release of phenolic compounds that can negatively interfere with the enzymatic activity of xylanases and other enzymes involved in plant fiber degradation is common. Therefore, the aim of this study was to investigate the effects of the phenolic compounds syringic acid and vanillic acid on the enzymatic kinetics of the before mentioned recombinant endoxylanase. For this purpose, the recombinant enzyme was purified and then incubated for 30 minutes with the phenolic compounds at a concentration of 20 mM. Samples of these solutions were used to perform various enzymatic reactions at 60 °C for 5 minutes, using increasing substrate concentrations. Based on these reactions, the kinetic parameters were calculated and reaction graphs were constructed. The values of the Michaelis constant (Km) and the maximum reaction rate (Vmax) were obtained by non-linear parameter fitting using the QtiPlot software version 0.9.9.11 (©Ion Vasilief 2004–2017) for Linux. To evaluate thermostability, samples prepared in the same manner were incubated at temperatures of 50, 60, 70, and 75 °C for 15, 30, 60, 120, and 180 minutes, followed by overnight incubation at 4 °C to allow for the reversal of any changes in the secondary and tertiary structures. Enzymatic activity was determined and compared to a control sample that was not subjected to thermal incubation. The optimum temperature was determined by measuring the enzymatic activity at different incubation temperatures (40, 50, 60, 70, 80, 90, and 95 °C) for 10 minutes. Based on the obtained results, an Arrhenius plot was constructed. The results indicated that the tested phenolic compounds had a positive impact on the kinetics of the xylanase, increasing Vmax and keeping Km relatively unchanged. No significant differences were observed in the optimum temperature compared to the control group, as well as in thermostability, although the residual activity showed an increase. Palavras-chave: phenolic compounds, recombinant enzyme, xylanase Agência de fomento:FAPESP | |||||