Insight into kinetics and thermodynamics of a novel hyperstable GH family 10 endo-1,4-β-xylanase (TnXynB) with broad substrates specificity cloned from Thermotoga naphthophilaRKU-10T

Abstract

Currently, hyperstable endo-1,4-β-xylanase has been the focus of attention as potent biocatalyst as well as utilization in bioconversion process. Therefore, the gene (1035 bp) of a monomeric glycoside hydrolase family 10 (GH10) endo-1,4-β-xylanase (TnXynB) from a hyperthermophilic eubacterium Thermotoga naphthophila RKU-10^T was cloned and overexpressed in a mesophilic host system. The extracellular TnXynB was purified to homogeneity with a molecular mass of 40 kDa, and showed peak activity at pH 6.0 and 95 °C temperature. Purified TnXynB has prodigious stability over a broad range of pH (5.5-8.0) and temperature (50-85 °C) even after 8 h incubation, and also revealed great tolerance toward different modulators (metal cations, surfactants and organic solvents). TnXynB exhibited great affinity towards various heteroglycans and para-nitrophenyl glycosides substrates. The values of K _m, Vmax, k_cat, and k_cat K _m^-1 were 2.75 mg mL^-1, 3146.7 μmol mg^-1 min^-1, 40,342.3 s^-1, 14,669.93 mL mg^-1 s^-1, respectively using birchwood xylan as substrate. Thermodynamic parameters for birchwood xylan hydrolysis at 95 °C as ΔS*, ΔH*, and ΔG* were -22.88 J mol^-1 K^-1, 62.44 kJ mol^-1, and 70.86 kJ mol^-1 respectively. TnXynB displayed a half-life (t_1/2) of 54.15 min at 96 °C with ΔS*_D, ΔH*_D, and ΔG*_D values of 1.074 kJ mol^-1 K^-1, 513.23 kJ mol^-1 and 116.92 kJ mol^-1, respectively. All noteworthy features of TnXynB make this new recombinant enzyme an appropriate candidate for the biodegradation of lignocellulosic substrates as well as various other industrial bioprocesses.

Keywords: Endo-1,4-β-xylanase; Hyperthermostable; Kinetic; Thermodynamics; Thermotoga naphthophila.