Artigos
URI permanente para esta coleçãohttps://locus.ufv.br/handle/123456789/11847
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Item Thermostability improvement of Orpinomyces sp. xylanase by directed evolution(Journal of Molecular Catalysis B: Enzymatic, 2012-09) Trevizano, Larissa Mattos; Ventorim, Rafaela Zandonade; Rezende, Sebastião Tavares de; Silva Junior, Floriano Paes; Guimarães, Valéria MontezeThe methodology of directed evolution, using the mutagenic technique of error-prone PCR has been used to improve the thermostability of enzymes. This method was applied to the endo-β-1,4-xylanase from Orpinomyces strain PC-2. The constructed library of xylanase (xynA) mutants was subjected to several screening cycles in plates with azo-xylan-agarose as substrate and four thermostable mutants (M1–M4) were selected. Homology models for these thermostable mutants were constructed to identify the location of the residues changed by error-prone PCR and to investigate the effect of these mutations on the xylanase properties. Xylanase activities of the mutants and wild type were maximal at 60 °C and in the pH range of 5–7. The mutants displayed higher thermostability than the wild type XynA, where the wild type showed a half-life at 60 °C of 7.92 min, while half-life values for M1, M2, M3 and M4 were 209, 33.2, 401 and 15.3 min, respectively. Additionally, M3 and M4 presented a good performance in more extreme pH conditions. The mutants retained their ability to hydrolyze birchwood and oat spelt xylans, which are substrates presenting different degrees of branching.Item Impact of the removal of N-terminal non-structured amino acids on activity and stability of xylanases from Orpinomyces sp. PC-2(International Journal of Biological Macromolecules, 2017-08-03) Ventorim, Rafaela Zandonade; Mendes, Tiago Antônio de Oliveira; Trevizano, Larissa Mattos; Camargos, Ana Maria dos Santos; Guimarães, Valéria MontezeXylanases catalyze the random hydrolysis of xylan backbone from plant biomass and thus, they have application in the production of biofuels, Kraft pulps biobleaching and feed industry. Here, xylanases derived from Orpinomyces sp. PC-2 were engineered guided by molecular dynamics methods to obtain more thermostable enzymes. Based on these models, 27 amino acid residues from the N-terminal were predicted to reduce protein stability and the impact of this removal was validated to two enzyme con- structs: small xylanase Wild-Type (SWT) obtained from Wild-Type xylanase (WT) and small xylanase Mutant (SM2) generated from M2 mutant xylanase (V135A, A226T). The tail removal promoted increase in specific activity of purified SWT and SM2, which achieved 5,801.7 and 5,106.8 U mg^−1 of protein, respec- tively, while the WT activity was 444.1 U mg^−1 of protein. WT, SWT and SM2 showed half-life values at 50 ◦ C of 0.8, 2.3 and 29.5 h, respectively. Overall, in view of the results, we propose that the presence of non-structured amino acid in the N-terminal leads to destabilization of the xylanases and may promote less access of the substrate to the active site. Therefore, its removal may promote increased stability and enzymatic activity, interesting properties that make them suitable for biotechnological applications.Item Optimization of Endoglucanase and Xylanase activities from fusarium verticillioides for simultaneous saccharification and fermentation of sugarcane bagasse(Applied Biochemistry and Biotechnology, 2013-10-30) Almeida, Maíra N. de; Guimarães, Valéria M.; Falkoski, Daniel L.; Paes, Guilherme B. T.; Ribeiro Jr., José Ivo; Visser, Evan M.; Alfenas, Rafael F.; Pereira, Olinto L.; Rezende, Sebastião T. deEnzymatic hydrolysis is an important but expensive step in the production of ethanol from biomass. Thus, the production of efficient enzymatic cocktails is of great interest for this biotechnological application. The production of endoglucanase and xylanase activites from F. verticillioides were optimized in a factorial design (25) followed by a CCDR design. Endoglucanase and xylanase activities increased from 2.8 to 8.0 U/mL and from 13.4 to 114 U/mL, respectively. The optimal pH and temperature were determined for endoglucanase (5.6, 80 °C), cellobiase (5.6, 60 °C), FPase (6.0, 55 °C) and xylanase (7.0, 50 °C). The optimized crude extract was applied in saccharification and fermentation of sugarcane bagasse from which 9.7 g/L of ethanol was produced at an ethanol/biomass yield of 0.19.Item Characteristics of free endoglucanase and glycosidases multienzyme complex from Fusarium verticillioides(Bioresource Technology, 2013-06-08) Almeida, Maíra N.de; Falkoski, Daniel L.; Guimarães, Valéria M.; Ramos, Humberto Josué de O.; Visser, Evan M.; Maitan-Alfenas, Gabriela P.; Rezende, Sebastião T. deA novel multienzyme complex, E1 C , and a free endoglucanase, E2 (GH5), from Fusarium verticillioides were purified. The E1 C contained two endoglucanases (GH6 and GH10), one cellobiohydrolase (GH7) and one xylanase (GH10). Maximum activity was observed at 80 °C for both enzymes and they were thermostable at 50 and 60 °C. The activation energies for E1 C and E2 were 21.3 and 27.5 kJ/mol, respectively. The K M for E1 C was 10.25 g/L while for E2 was 6.58 g/L. Both E1 C and E2 were activated by Mn 2+ and CoCl 2 while they were inhibited by SDS, CuSO 4 , FeCl 3 , AgNO 4 , ZnSO 4 and HgCl 2 . E1 C and E2 presented endo-b-1,3–1,4-glucanase activity. E1 C presented crescent activity towards cellopentaose, cellotetraose and cellotriose. E2 hydrolyzed the substrates cellopentaose, cellotetraose and cellotriose with the same efficiency. E1 C showed a higher stability and a better hydrolysis performance than E2, suggesting advantages resulting from the physical interaction between proteins.Item Characterization and biotechnological application of recombinant xylanases from Aspergillus nidulans(International Journal of Biological Macromolecules, 2016-05-25) Maitan-Alfenas, Gabriela P.; Oliveira, Mariana B.; Nagem, Ronaldo A.P.; Vries, Ronald P. de; Guimarães, Valéria M.Two xylanases from Aspergillus nidulans, XlnB and XlnC, were expressed in Pichia pastoris, purified and characterized. XlnB and XlnC achieved maximal activities at 60 °C and pH 7.5 and at 50 °C and pH 6.0, respectively. XlnB showed to be very thermostable by maintaining 50% of its original activity after 49 h incubated at 50 °C. XlnB had its highest activity against wheat arabinoxylan while XlnC had the best activity against beechwood xylan. Both enzymes were completely inhibited by SDS and HgCl2. Xylotriose at 1 mg/ml also totally inibited XlnB activity. TLC analysis showed that the main product of beechwood xylan hydrolysis by XlnB and XlnC was xylotetraose. An additive effect was shown between XlnB and XlnC and the xylanases of two tested commercial cocktails. Sugarcane bagasse saccharification results showed that these two commercial enzymatic cocktails were able to release more glucose and xylose after supplementation with XlnB and XlnC.