In silico screening and experimental analysis of family GH11 xylanases for applications under conditions of alkaline pH and high temperature

摘要

Xylanases are one of the most extensively used enzymes for biomass digestion. However, in many instances, their use is limited by poor performance under the conditions of pH and temperature required by the industry. Therefore, the search for xylanases able to function efficiently at alkaline pH and high temperature is an important objective for different processes that use lignocellulosic substrates, such as the production of paper pulp and biofuels. A comprehensive in silico analysis of family GH11 sequences from the CAZY database allowed their phylogenetic classification in a radial cladogram in which sequences of known or presumptive thermophilic and alkalophilic xylanases appeared in three clusters. Eight sequences from these clusters were selected for experimental analysis. The coding DNA was synthesized, cloned and the enzymes were produced in E. coli. Some of these showed high xylanolytic activity at pH values??8.0 and temperature??80?°C. The best enzymes corresponding to sequences from Dictyoglomus thermophilum (Xyn5) and Thermobifida fusca (Xyn8). The addition of a carbohydrate-binding module (CBM9) to Xyn5 increased 4 times its activity at 90?°C and pH??9.0. The combination of Xyn5 and Xyn8 was proved to be efficient for the saccharification of alkali pretreated rice straw, yielding xylose and xylooligosaccharides. This study provides a fruitful approach for the selection of enzymes with suitable properties from the information contained in extensive databases. We have characterized two xylanases able to hydrolyze xylan with high efficiency at pH??8.0 and temperature??80?°C.