Crystallization of Scytalidium thermophilum Xylanase by Vapor Diffusion

The main heteropolymers of the hemicellulosic fraction of plant biomass are xylan, mannan, galactans and arabinans [1]. Xylan is a complex molecule mainly consisting of a five-carbon sugar D-xylose. Due to the complex structure of xylan, synergistic action of different hemicellulase enzymes is required for complete hydrolysis. Endoxylanases are one of the most important enzymes in this enzyme group. Microbial xylanase producers are mainly bacteria and fungi [2]. Filamentous fungi produce extracellular xylanase which makes fungal xylanases favorable at industrial scale such as animal feed production, manufacture of bread, food and drinks, pharmaceutical and chemical applications, textiles, pulp and paper. For crystallographic purposes, xylanase from Scytalidium thermophilum was purified to homogeneity by a two-step column chromatography technique including gel filtration and anion exchange, 21.8 fold with 9.6% yield. Xylanase crystallization was screened using ready-to-use screening kits by sitting drop vapor diffusion technique in 96-well plates. After dye test of observed crystals, xylanase-crystal forming conditions were optimized in 24-well plates by hanging drop vapor diffusion technique. So far, best crystals, having potential for X-ray structure studies, were obtained using ammonium citrate dibasic and sodium acetate trihydrate at 18°C at 3 mg/ml protein concentration as shown in Figure 1.