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.

Secretion of recombinant thermo-alkali-stable endoxylanase of polyextremophilic Bacillus halodurans TSEV1 and its utility in generating xylooligosaccharides from renewable agro-residues

“…The divalent transition metals such as Pb 2+ , Hg 2+ and Cu 2+ are known to catalyze auto-oxidation of cysteine that leads to the formation of intramolecular and intermolecular disulfide bridges or to the formation of sulfenic acid [39]; this could be the probable reason for enzyme inhibition by these metal ions. The inhibition and stimulation of the enzyme activity are similar to the native xylanase and other bacterial xylanases [24,38]. The inhibition of activity in the presence of N-bromosuccinimide suggests the presence of tryptophan residues in their active/substrate binding site, a characteristic property which is conserved in microbial xylanases and other enzymes [40].…”

“…stable for 24 h in 8 M urea and 6 M guanidine-HCl. The enzymes from extremophilic microbes are known to display resistance to these chemicals [24,37]. The metal ions play an important role in catalysis as acceptors or donors of electrons and coordinating group between enzyme and substrate, or they may simply stabilize a catalytically active conformation of the enzyme [38].…”

Production of Chimeric Acidic α-Amylase by the Recombinant Pichia pastoris and Its Applications

“…Ba-Gt-amy inhibited activity in presence of Woodward’s reagent suggested that catalytic role of aspartic and glutamic acid residues. Woodward’s reagent K is a negatively charged reagent that covalently and irreversibly modifies carboxyl-containing amino acid residues (Adsul et al, 2009; Kumar and Satyanarayana, 2013). Strong inhibitory effect of NBS indicated structural and catalytic role of tryptophan residues in Ba-Gt-amy (Sharma and Satyanarayana, 2012).…”

Insight into Glycoside Hydrolases for Debranched Xylan Degradation from Extremely Thermophilic Bacterium Caldicellulosiruptor lactoaceticus

“…Other xylanases such as Syncephalastrum racemosum Cohn. (58.0 kDa) [10] and Trichoderma reesei (90.0 kDa) [11] are also homodimers, whereas the majorities are identified as monomeric proteins in solution, for instance, Glaciecola mesophila KMM 241 (43.0 kDa) [12], Cohnella laeviribosi HY-21 (42.0 kDa) [13], B.halodurans TSEV1 (40.0 kDa) [14], and Remersonia thermophila CBS 540.69 (42.0 kDa) [15].…”