Electrostatic interaction optimization improves catalytic rates and thermotolerance on xylanases.

Understanding the aspects that contribute to improving proteins' biochemical properties is of high relevance for protein engineering. Properties such as the catalytic rate, thermal stability, and thermal resistance are crucial for applying enzymes in the industry. Different interactions can influence those biochemical properties of an enzyme. Among them, the surface charge-charge interactions have been a target of particular attention. In this study, we employ the Tanford-Kirkwood solvent accessibility model using the Monte Carlo algorithm (TKSA-MC) to predict possible interactions that could improve stability and catalytic rate of a WT xylanase (XynAWT) and its M6 xylanase (XynAM6) mutant. The modeling prediction indicates that mutating from a lysine in position 99 to a glutamic acid (K99E) favors the native state stabilization in both xylanases. Our lab results showed that mutated xylanases had their thermotolerance and catalytic rate increased, which conferred higher processivity of delignified sugarcane bagasse. The TKSA-MC approach employed here is presented as an efficient computational-based design strategy that can be applied to improve the thermal resistance of enzymes with industrial and biotechnological applications.

A novel ß-glucosidase isolated from the microbial metagenome of Lake Poraquê (Amazon, Brazil).

The Amazon region holds most of the biological richness of Brazil. Despite their ecological and biotechnological importance, studies related to microorganisms from this region are limited. Metagenomics leads to exciting discoveries, mainly regarding non-cultivable microorganisms. Herein, we report the discovery of a novel ß-glucosidase (glycoside hydrolase family 1) gene from a metagenome from Lake Poraquê in the Amazon region. The gene encodes a protein of 52.9 kDa, named AmBgl-LP, which was recombinantly expressed in Escherichia coli and biochemically and structurally characterized. Although AmBgl-LP hydrolyzed the synthetic substrate p-nitrophenyl-ß-d-glucopyranoside (pNPßG) and the natural substrate cellobiose, it showed higher specificity for pNPßG (kcat/Km = 6 s-1·mM-1) than cellobiose (kcat/Km = 0.6 s-1·mM-1). AmBgl-LP showed maximum activity at 40 °C and pH 6.0 when pNPßG was used as the substrate. Glucose is a competitive inhibitor of AmBgl-LP, presenting a Ki of 14 mM. X-ray crystallography and Small Angle X-ray Scattering were used to determine the AmBgl-LP three-dimensional structure and its oligomeric state. Interestingly, despite sharing similar active site architecture with other structurally characterized GH1 family members which are monomeric, AmBgl-LP forms stable dimers in solution. The identification of new GH1 members by metagenomics might extend our understanding of the molecular mechanisms and diversity of these enzymes, besides enabling us to survey their industrial applications.