Avaliação da termoestabilidade, atividade e resistência a ambientes ácidos de uma enzima de interesse biotecnológico via dinâmica molecular

Abstract

The lignocellulosic ethanol production by enzymatic route has gained space among the industrial processes in order to replace the traditional acid treatment. Xylanases (E.C. 3.2.1.8) constitute a class of enzymes present in the enzymatic cocktail used for this purpose and have great industrial / commercial interest due to their high versatility in several known processes. In this context, some properties of biotechnological interest of a GH11 family xylanase were evaluated via molecular dynamics (MD) simulations. In a first study, we performed MD simulations of a xylanase produced by Bacillus subtilis (XynA_WT) and a quadruple mutant (Gln7His, Gly13Arg, Ser22Pro and Ser179Cys) which display an higher optimum catalytic temperature (20ºC) in relation to native. MD results suggest possible strategies for engineering GH11 xylanases to produce thermostable enzymes. Mutations in regions that exhibit reduced flexibility should preserve rigidity, but their substitution may be chosen to favorably affect other properties, such as solvation or hydrophobic interactions. The data do not only explain the thermostability effect of a GH11 xylanase observed in previous experiments of direct evolution, but also provide information for the planning of other thermostable GH11 mutants by rational design. In a second study, the target was a chimera formed between a xylanolytic domain (XynA) and a xylose binding protein (XBP) which had an experimental catalytic efficiency almost 3.5 times higher than the same non-chimerized xylanase. The factors responsible for this discrepancy were understood by MD simulations. The results suggested the formation and stabilization of a protein-protein interface between the two domains in the presence and absence of xylose in the active site of XBP. Interaction Potential Energy values (IPE) as a function of time show a greater stabilization in the interactions of this interface for xylose bound structure compared to the xylose free one. Structural parameters such as flexibility and volume of the active site were also evaluated. In general, the results suggest that the chimera displays greater rigidity in relation to free xylanase, and in particular the thumb region, which controls active site exposure, demonstrates a significant reduction in flexibility. Finally, MD simulations at different pHs were performed in order to understand the drastic decrease in the catalytic activity of this xylanase (native and chimeric form) in acidic environments, and, thus, to aggregate information to increase its resistance in such conditions. Data of side chain protonation states, open-close amplitude, gyration radius and solvent accessible surface are important analyzes to provide insights for elucidation of the mechanism of stabilization of this chimeras in acid environment.The thesis reinforces the descriptive and predictive capacities of the MD simulations in the biotechnological development of GH11 xylanases.