Cloning, overexpression, and characterization of a novel alkali-thermostable xylanase fromGeobacillussp. WBI

Abstract

An endo-β-1,4-xylanase gene xynA of a thermophilic Geobacillus sp. WBI from "hot" compost was isolated by PCR amplification. The gene encoding 407 residues were overexpressed in E. coli and purified by Ni-NTA chromatography. The purified enzyme (47 kDa) had a broad pH optimum of 6.0 to 9.0, and was active between 50 and 90 °C. The enzyme retained 100% of its activity when incubated at 65 °C for 1 h under alkaline condition (pH 10.0) and retained 75% activity at pH 11.0. The K(m) and V(max) of the enzyme were 0.9 mg ml(-1) and 0.8 µmol ml(-1) min(-1), respectively. In molecular dynamics simulation at 338 K (65 °C), the enzyme was found to be stable. At an elevated temperature (450 K) specific α-helix and β-turns of the proteins were most denatured. The denaturation was less in WBI compared with its highest homolog G. stearothermophilus T-6 xylanase with difference of six residues. The results predict that these regions are responsible for the improved thermostability observed over related enzymes. The present work encourages further experimental demonstration to understand how these regions contribute thermostability to WBI xylanase. The study noted that WBI produces a xylanase with unique characteristics, specifically alkali-thermostability.

Extremophilic Prokaryotic Endoxylanases: Diversity, Applicability, and Molecular Insights

“…WBI (pHopt. 6-9; Mitra et al, 2015) have also been well documented for alkaliphilic endoxylanases. However, xylanases of G. thermodenitrificans TSAA1 (pHopt.…”

“…The recombinant xylanases of Geobacillus sp. WBI (Mitra et al, 2015) and G. thermodenitrificans A333 (Marcolongo et al, 2015) were highly stable that can withstand the higher pH of 11.0 by retaining 97 and 75% of activity for 1 h, respectively. The endoxylanases from G. thermoleovorans (Verma and Satyanarayana, 2012) and G. thermodenitrificans TSAA1 were successfully employed for the saccharification of the agro-residues for the liberation of xylooligosaccharides.…”

“…The recombinant xylanases of Geobacillus sp. WBI ( Mitra et al, 2015 ) and G . thermodenitrificans A333 ( Marcolongo et al, 2015 ) were highly stable that can withstand the higher pH of 11.0 by retaining 97 and 75% of activity for 1 h, respectively.…”

Cloning, Expression, and Characterization of a GHF 11 Xylanase from <i>Alteromonas macleodii </i>HY35<i> </i>in <i>Escherichia col</i><i>i</i>

“…purified with Ni-NTA His-Bind metal chelating column (Mitra et al, 2015). After equilibrated with 5 column volumes of ddH2O and 30 mM of phosphate buffer (pH 7.4, 500 mM NaCl and 30 mM imidazole), the samples were applied to the Ni-NTA His-Bind metal chelating column.…”

Lignocellulolytic capacities of Geobacillus thermodenitrificans: towards consolidated bioprocessing

“…Endoxylanases from Geobacillus species belong to the GH10 family and have been described in multiple studies (Bhalla et al 2014;Canakci et al 2012;Gerasimova & Kuisiene 2012;Irfan et al 2016;Gao et al 2009;Marcolongo et al 2015;Mitra et al 2015;Verma et al 2013). These studies mainly focused on the characterization of activity ranges at higher temperatures and thermo-stability but lack detailed analyses of the mode of action of the enzyme on its substrate.…”