Purification, bio-chemical characterization, homology modeling and active site binding mode interactions of thermo-alkali-tolerant β-1,4 endoxylanase from Coprinus cinereus LK-D-NCIM-1369

• Purification of crude xylanase from Coprinus cinereus LK-D-NCIM-1369 produced under solid-state fermentation conditions.
• Biochemical characterization
• Peptide mass fingerprint analysis
• Homology modeling
• Docking study of modeled xylanase by PatchDock

Present study aims at purifying and characterizing crude xylanase isolated from Coprinus cinereus LK-D-NCIM-1369 under solid-state fermentation conditions. Crude xylanase is purified by (NH4)2SO4 precipitation, carboxymethyl cellulose cation-exchange and gel filtration chromatography with Superdex-200 column. Purified xylanase from C. cinereus shows 54.3% similarity with β-1,4 endoxylanase from C. cinerea okayama7#130 with accession number gi|169855830 based on MALDI-TOF/TOF. The purified xylanase—a monomeric protein with molecular weight 20.1 kD shows maximum activity at 60 °C and pH 7.0 and stable over pH 5.0–9.0 and temperature up to 70 °C. The xylanase shows Km and V values of 3.26 mg/mL and 909.09 µm/min/mg for birchwood xylan. A sequence of 186 amino acids of C. cinerea okayama7#130 gi|169855830 is retrieved from NCBI database and its 3-D model is generated on the basis of crystal structure of xylanase 1XNK-A from Chaetomium thermophilum with the help of online server SWISS-MODEL workspace. The model is verified and validated on SAVES and PROCHECK programmes, respectively. Ramachandran plot reveals that the total residues in allowed and generously allowed regions are 99.4% and 0.6% respectively. Overlapping of xylanase with the template of 1XNK-A stipulates the amino acid residues Asn35, Tyr68, Arg113, Ser118, Tyr168 and Glu174 constitute active site of the enzyme.