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

Abstract

Background 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. Results 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. Conclusions 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.

pH-Induced structural changes in xylanase GH11 from Thermoanaerobacterium saccharolyticum

“…Endo-xylanases (EC 3.2.1.8) cleaves the β-1,4 glycosidic bonds in the xylan backbone, converting the polymeric xylan into xylose or xylooligosaccharides. 1 In the carbohydrate-active enzyme database (CAZy), various glycoside hydrolase (GH) families (GH 5,7,8,9,10,11,12,16,26,30,43,44,51,and 62) are classified into xylanase. 2,3 Each of these xylanase families exhibits distinct amino acid sequence, amino acid length, functional domains or enzymatic activity.…”

Screening New Xylanase Biocatalysts from the Mangrove Soil Diversity

“…A characterization of the mangrove-derived Xyn11-29 was conducted after its production in P. pastoris and its purification. Concerning its general properties, Xyn11-29 was shown to possess only a catalytic module without CBM, as is the case for 80% of xylanases (family GH11) from eukaryotic organisms [ 24 ]. Xyn11-29 has the average size for a xylanase of around 20 kDa and possesses the two key catalytic residues characteristic of the xylanases (Glu120 and Glu217) [ 25 ].…”

The bone-degrading enzyme machinery: From multi-component understanding to the treatment of residues from the meat industry

“…The demineralized bone, which now had turned elastic, was then cleaned to remove non-collagenous proteins and then freeze dried in liquid nitrogen, lyophilized to fully eliminate the water, and finally pulverized with a coffee grinder (Moulinex Super Junior S). For the degradation test a protocol commonly employed for preparation of sugar-polymers for biomass degradation tests, was adapted [46] . Briefly, 1 g of pulverized dry bone material thus obtained was added to a 100 ml Erlenmeyer flask containing 60 ml pre-warmed buffer (50 mM Britton and Robinson buffer, pH 7.0) at 60 °C.…”