Caracterización de una enzima xilanasa obtenida mediante reconstrucción ancestral de secuencias

Abstract

Accelerated economic and demographic growth has promoted the development of agricultural, timber and paper industries, as well as their respective residues generation. Such by-products are mainly composed of lignocellulose, a recalcitrant polymer considered the most bioavailable source of biomass. Despite this, its potential has not been exploited enough, reason of why, part of science focus on lignocellulosic enzymes characterization. Some of them are xylanases belonging to the GH30 family, a catalytically diverse group, products of which, can be useful in value-added compounds elaboration, such as biofuels, polymers, prebiotics, among others. Additionally, there is some interest in enzymatic engineering of these proteins to reveal their behavior or improve their characteristics through rational design. However, ancestral reconstructions of sequences have also been used, under the idea that ancestors of existing enzymes possess improved properties related with promiscuity and resistance to extreme conditions such as temperature. Thus, the present study describes ancestral reconstruction of a GH30 xylanase, expressed in Escherichia coli BL21 and subsequently purified by IMAC, whose enzyme activity was tested in beechwood xylan. The ancestor, called Xyn_AN4, displays optimal activity at 55 Celsius degrees and pH 4.7 and has structural features similar to GH30-7 subfamily. Furthermore, its enzymatic activity is comparable to that observed in specific glucuronoxylanases and bifunctional xylanases, suggesting a possible connection with the catalyzed reaction.