TL;DR
The results demonstrate that XynA is a highly efficient thermostable xylanase suitable for mild, eco-friendly functional modification of xylan-based biomaterials.
Lipases obtained from thermophilic microorganisms are attracting great interest in the industrial field due to their stability under high-temperature conditions and their wide range of industrial applications. In this study, thermophilic bacterial strains were isolated from water and mud samples collected from Erzurum Ilica Hot Springs (Turkey). An isolate producing a potent extracellular lipase was selected, named IO3, and identified by 16S rRNA sequencing. Sequencing analysis revealed that this isolate showed 99% similarity to Aeribacillus pallidus. The lipase enzyme purified from the isolate was obtained using two comparative strategies. The conventional multi-stage chromatographic approach, which includes ammonium sulfate precipitation, ion exchange chromatography, and gel filtration chromatography, provided 9.3-fold purification with a 3.18% recovery. However, the alternative Three-Phase Partitioning (TPP) system provided rapid, single-step recovery with a significantly higher yield of 38.79%, although the purification fold remained 0.65 under the tested conditions. The molecular weight of the purified A. pallidus IO3 lipase was determined to be approximately 33.88 kDa by SDS-PAGE. The enzyme showed optimum activity at pH 8.0 and 50 °C and maintained considerable thermal stability; the enzyme retained significant activity levels (45-69%) even at high temperatures such as 70 °C and 80 °C after an incubation period of 120 minutes. Among the tested metal ions, Fe2+, Fe³+, Cu2+, and Zn2+ enhanced enzyme activity, while the enzyme showed stability in the presence of Li+ and Cu2+ ions. Lipase activity also increased in the presence of surfactants, while chloroform enhanced enzyme activity by 22-338% depending on solvent concentration. The enzyme showed the highest activity toward p-nitrophenyl palmitate. The enzyme was completely inhibited by DTNB, IAA, and EDTA. In addition, the enzyme retained low activity in the presence of β-mercaptoethanol. The Km and Vmax values were calculated as 0.46 mM and 51.44 µmol·min-1·mg-1, respectively, using nonlinear regression analysis. Overall, these findings indicate that A. pallidus IO3 lipase is a thermostable and chemically tolerant enzyme with potential applicability in industrial biocatalysis and detergent-related processes.
BSEG2 is highly thermostable, tolerant of high salt and ionic liquids, and notably resistant to cellobiose inhibition up to 200 mM, suggesting potential to reduce freshwater demand in biomass processing and a strong candidate for simplified, cost-effective enzyme formulations for glucose production from lignocellulosic feedstocks.
Local enrichment of acidic residues on the PlGH3 surface could generate a negative electrostatic potential, which enables adaptation to high-salt and alkaline environments, thereby sustaining the enzyme's catalytic activity under such extreme conditions.
In this study, the glucansucrase-encoding gene (dsr) from Weissella confusa P2 was cloned and heterologously expressed in Escherichia coli BL21(DE3). A 4542 bp fragment encoding 1510 amino acids was obtained, and the recombinant enzyme exhibited a molecular weight of approximately 161 kDa with a theoretical pI of 4.74. After Ni-IDA affinity chromatography, the specific activity reached 300.19 U/mg with a purification fold of 18.43. Enzymatic characterization revealed that the recombinant glucansucrase displayed optimal activity at pH 5.5 and 30 °C, and retained over 70% relative activity after 48 h incubation at 10-30 °C. The enzyme was significantly activated by Ca2+, while strongly inhibited by Ni2+, Cd2+, SDS, EDTA, DTT, and high concentrations of organic solvents. Kinetic analysis using sucrose as substrate yielded Km of 0.56 mM and Vmax of 4.57 U/mg, indicating exceptionally high substrate affinity. Using this recombinant enzyme, exopolysaccharide (EPS) was synthesized in vitro, achieving a yield of 59.23 ± 0.21 g/L, representing an 18.79% improvement over EPS synthesis by the wild-type strain. These findings enrich the genetic resources of glucansucrase, provide a promising biocatalyst for efficient EPS production, and establish a foundation for the application of glucansucrase and its polysaccharides products in the food and biotechnology industries.
Xylan, the principal component of hemicellulose, requires the concerted action of endo- and exo-acting xylanolytic enzymes for its complete depolymerization into fermentable sugars. This study reports the biochemical and thermodynamic characterization of a glycoside hydrolase family 10 endo-β-1,4-xylanase from the thermophilic bacterium Geobacillus stearothermophilus CECT43 (GsXynA), cloned and heterologously expressed in Escherichia coli BL21(DE3) and purified to homogeneity by immobilized metal-ion affinity chromatography. The 338-residue enzyme behaved as a monomer and showed maximal activity on xylan substrates at pH 6.5 and 75 °C, retaining over 90% residual activity between pH 5 and 9 and remaining stable up to 70 °C, after 60 min of incubation. Kinetic and thermodynamic analyses of thermal inactivation monitored by enzymatic activity measurements revealed an enthalpy-driven denaturation process, while circular dichroism indicated that this irreversible unfolding is kinetically, not thermodynamically, controlled. GsXynA hydrolyzed beechwood xylan mainly to xylobiose and xylotetraose, consistent with mixed endo and exo activity, and was uncompetitively inhibited by 4-hydroxybenzoic acid. Combined action with the β-xylosidase GsXynB2 markedly increased D-xylose release, reaching a 53.23% hydrolysis yield and a degree of synergy of 3.79 after 24 h. These results demonstrate that GsXynA is a thermostable, versatile xylanase with promising potential for lignocellulosic biomass bioconversion.
