title: Biochemical and thermodynamic characteristics of thermo-alkali-stable xylanase from a novel polyextremophilic Bacillus halodurans TSEV1.
abstract: The purified extracellular xylanase of polyextremophilic Bacillus halodurans TSEV1 has been visualized as a single band on SDS-PAGE and eluted as single peak by gel filtration, with a molecular mass of 40 kDa. The peptide finger print and cloned xylanase gene sequence analyses indicate that this enzyme belongs to GH family 10. The active site carboxyl residues are mainly involved in catalysis, while tryptophan residues are involved in substrate binding. The enzyme is optimally active at 80 °C and pH 9.0, and stable in the pH range of 7.0-12.0 with T 1/2 of 35 min at 80 °C (pH 9.0). Activation energy for birch wood xylan hydrolysis is 30.51 kJ mol(-1). The K m, V max and k cat (birchwood xylan) are 2.05 mg ml(-1), 333.33 μmol mg(-1 )min(-1) and 3.33 × 10(4) min(-1), respectively. The pKa1 and pKa2 of ionizable groups of the active site that influence V max are 8.51 and 11.0. The analysis of thermodynamic parameters for xylan hydrolysis suggests this as a spontaneous process. The enzyme is resistant to chemical denaturants like urea and guanidinium-HCl. The site-directed mutagenesis of catalytic glutamic acid residues (E196 and E301) resulted in a complete loss of activity. The birch wood xylan hydrolyzate contained xylobiose and xylotriose as the main products without any trace of xylose, and the enzyme hydrolyzes xylotetraose and xylopentaose rapidly to xylobiose. Thermo-alkali-stability, resistance to various chemical denaturants and mode of action make it a useful biocatalyst for generating xylo-oligosaccharides from agro-residues and bleaching of pulp in paper industries.
authors.affiliation: Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110021, India. vikash.udsc@gmail.com
authors.forename: Vikash
authors.initials: V
authors.lastname: Kumar
authors.forename: T
authors.initials: T
authors.lastname: Satyanarayana
mesh_terms: D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D013379:Substrate Specificity; D013816:Thermodynamics; D014934:Wood / Q000737:chemistry; D014990:Xylans / Q000737:chemistry; D014995:Xylosidases / Q000737:chemistry* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D014990:Xylans; D014995:Xylosidases