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-10T 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, kcat, and kcat 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 (t1/2) 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.