Purification of Thermostable Xylanase Xyn11

The Xyn11 sequence was identified in a computational analysis, where xylanases active under extreme conditions of temperature and alkaline pH were searched for. A synthetic DNA fragment coding for the protein sequence, optimized for translation in E. coli, was cloned in an expression vector as previously described^{25 (link)}. Cell extracts from the E. coli cultures expressing a His-tagged version of Xyn11, prepared in 20 mM phosphate buffer, pH 7.4, 10 mM imidazole, 500 mM NaCl, were heated at 85 °C for 10 min, which caused the precipitation of most of the proteins, leaving the xylanase in a clarified supernatant after centrifugation. The enzyme preparation was subjected to Ni-affinity chromatography in a 1 mL HisTrap FF column (Cytiva) mounted in an AKTA-Purifier (Cytiva). Elution was carried out with a 20 mM phosphate buffer, pH 7.4, 500 mM imidazole, 500 mM NaCl. This version of the enzyme was termed Xyn11_Ec.