Insight into the mechanism of thermostabilization of GH10 xylanase from Bacillus sp. strain TAR-1 by the mutation of S92 to E.

The mechanism of thermostabilization of GH10 xylanase, XynR, from Bacillus sp. strain TAR-1 by the mutation of S92 to E was investigated. Thermodynamic analysis revealed that thermostabilization was driven by the decrease in entropy change of activation for thermal inactivation. Crystallographic analysis suggested that this mutation suppressed the fluctuation of the amino acid residues at position 92-95.

Regulating acidogenesis and methanogenesis for the separated bio-generation of hydrogen and methane from saline-to-hypersaline industrial wastewater.

Given the limitations of acidogens and methanogens activities under saline environments, this work aims to optimize the main operational parameters affecting hydrogen and methane production from saline-to-hypersaline wastewater containing mono-ethylene glycol (MEG). MEG is the main contaminant in several saline industrial effluents. Anaerobic baffled reactor (ABR), as a multi-stage system, was used at different temperatures (i.e., 19-31 °C [ambient] and 35 °C), organic loading rates (OLRs) of 0.6-2.2 gCOD/L/d, and salinity of 5-35 gNaCl/L. Mesophilic conditions of 35 °C substantially promoted MEG biodegradability (92-98%) and hydrogen/methane productivity, even at elevated salinity. Hydrogen yield (HY) and methane yield (MY) peaked to 258 and 140 mL/gCODadd, respectively, at OLR 0.64 gCOD/L/d and salinity up to 20-25 gNaCl/L. An immobilized sludge ABR (ISABR), packed with polyurethane media, was further compared with classical ABR, resulting in 1.8-fold higher MY, at 35 gNaCl/L. Microbial analysis showed that introducing attached growth system (ISABR) substantially promoted methanogens abundance, which was dominated by genus Methanosarcina. Among bacterial genera, Acetobacterium was dominant, particularly in 1st compartment, representing MEG-degrading/salt-tolerant genus. At high salinity up to 35 gNaCl/L, the multi-phase and attached growth configuration can efficiently reduce the induced salt stress, particularly on methanogens, towards balanced and separated acidogenesis/methanogenesis. Overall, producing hydrogen and methane from anaerobic treatment of MEG-based saline wastewater is feasible at optimized parameters and configuration.

Increase in the thermostability of GH11 xylanase XynJ from Bacillus sp. strain 41M-1 using site saturation mutagenesis.

GH11 xylanase XynJ from Bacillus sp. strain 41M-1 has a ß-jellyroll fold composed of eight ß strands with a deep active-site cleft. We hypothesized that the thermostability of XynJ will increase if the flexibility of the ß strands in the jellyroll structure is decreased without impairing activity. To verify this hypothesis, we introduced random mutations into Tyr13-Arg104 and Gly169-Tyr194, both of which are located in the ß-jellyroll fold of XynJ, to construct a site saturation mutagenesis library. By screening 576 clones followed by site saturation mutation analysis of Thr82, T82A was selected as the most thermostable variant. In the hydrolysis of beechwood xylan at pH 7.8, the temperatures required to reduce initial activity by 50% in 15 min were 61 °C for the wild-type XynJ (WT) and 65 °C for T82A. The optimum hydrolysis temperatures were 60 °C for WT and 65 °C for T82A. There was little difference in the kcat and Km values and the pH dependence of activity between WT and T82A. Crystallographic analysis of WT and T82A revealed that thermostabilization by the T82A mutation might result from the removal of unfavorable van der Waals interactions. Thus, a highly thermostable XynJ variant was generated without impairing activity using this mutation strategy.

Increase in the thermostability of Bacillus sp. strain TAR-1 xylanase using a site saturation mutagenesis library.

Site saturation mutagenesis library is a recently developed technique, in which any one out of all amino acid residues in a target region is substituted into other 19 amino acid residues. In this study, we used this technique to increase the thermostability of a GH10 xylanase, XynR, from Bacillus sp. strain TAR-1. We hypothesized that the substrate binding region of XynR is flexible, and that the thermostability of XynR will increase if the flexibility of the substrate binding region is decreased without impairing the substrate binding ability. Site saturation mutagenesis libraries of amino acid residues Tyr43-Lys115 and Ala300-Asn325 of XynR were constructed. By screening 480 clones, S92E was selected as the most thermostable one, exhibiting the residual activity of 80% after heat treatment at 80°C for 15 min in the hydrolysis of Remazol Brilliant Blue-xylan. Our results suggest that this strategy is effective for stabilization of GH10 xylanase. ABBREVIATIONS: DNS: 3,5-dinitrosalicylic acid; RBB-xylan: Remazol Brilliant Blue-xylan.