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The valorization of lignocellulosic biomass, derived from various bio-waste materials, has received considerable attention as a sustainable approach to improve production chains while reducing environmental impact. Microbial enzymes have emerged as key players in the degradation of polysaccharides, offering versatile applications in biotechnology and industry. Among these enzymes, glycoside hydrolases (GHs) play a central role. Xylanases, in particular, are used in a wide range of applications and are essential for the production of xylose, which can be fermented into bioethanol or find use in many other industries. Currently, fungal secretomes dominate as the main reservoir of lignocellulolytic enzymes, but thermophilic microorganisms offer notable advantages in terms of enzyme stability and production efficiency. Here we present the genomic characterization of Geobacillus stearothermophilus GF16 to identify genes encoding putative enzymes involved in lignocellulose degradation. Thermostable GHs secreted by G. stearothermophilus GF16 were investigated and found to be active on different natural polysaccharides and synthetic substrates, revealing an array of inducible GH activities. In particular, the concentrated secretome possesses significant thermostable xylanase and β-xylosidase activities (5 ×10 3 U/L and 1.7 ×10 5 U/L, respectively), highlighting its potential for application in biomass valorization. We assessed the hemicellulose hydrolysis capabilities of various agri-food wastes using the concentrated secretome of the strain cultivated on xylan. An impressive 300-fold increase in xylose release compared to a commercially available cocktail was obtained with the secretome, underscoring the remarkable efficacy of this approach., Competing Interests: Declaration of Competing Interest All the authors declare no conflict of interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

A novel endo-1,4-β-xylanase-encoding gene was identified in Alicyclobacillus mali FL18 and the recombinant protein, named AmXyn, was purified and biochemically characterized. The monomeric enzyme worked optimally at pH 6.6 and 80 °C on beechwood xylan with a specific activity of 440.00 ± 0.02 U/mg and a good catalytic efficiency (k cat /K M  = 91.89 s -1 mLmg -1 ). In addition, the enzyme did not display any activity on cellulose, suggesting a possible application in paper biobleaching processes. To develop an enzymatic mixture for xylan degradation, the association between AmXyn and the previously characterized β-xylosidase AmβXyl, deriving from the same microorganism, was assessed. The two enzymes had similar temperature and pH optima and showed the highest degree of synergy when AmXyn and AmβXyl were added sequentially to beechwood xylan, making this mixture cost-competitive and suitable for industrial use. Therefore, this enzymatic cocktail was also employed for the hydrolysis of wheat bran residue. TLC and HPAEC-PAD analyses revealed a high conversion rate to xylose (91.56 %), placing AmXyn and AmβXyl among the most promising biocatalysts for the saccharification of agricultural waste., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

To fight oxidative damage due to reactive oxygen species (ROS), cells are equipped of different enzymes, among which Peroxiredoxins (Prxs) (EC 1.11.1.15) play a key role. Prxs are thiol-based enzymes containing one (1-Cys Prx) or two (2-Cys Prx) catalytic cysteine residues. In 2-Cys Prxs the cysteine residues form a disulfide bridge following reduction of peroxide which is in turn reduced by Thioredoxin reductase (Tr) /Thioredoxin (Trx) disulfide reducing system to regenerate the enzyme. In this paper we investigated on Prxs of Thermus thermophilus whose genome contains an ORF TT_C0933 encoding a putative Prx, belonging to the subfamily of Bacterioferritin comigratory protein (Bcp): the synthetic gene was produced and expressed in E. coli and the recombinant protein, TtBcp, was biochemically characterized. TtBcp was active on both organic and inorganic peroxides and showed stability at high temperatures. To get insight into disulfide reducing system involved in the recycling of the enzyme we showed that TtBcp catalically eliminates hydrogen peroxide using an unusual partner, the Protein Disulfide Oxidoreductase (TtPDO) that could replace regeneration of the enzyme. Altogether these results highlight not only a new anti-oxidative pathway but also a promising molecule for possible future biotechnological applications., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The transcription factors of the ArsR/SmtB family are widespread within the bacterial and archaeal kingdoms. They are transcriptional repressors able to sense a variety of metals and undergo allosteric conformational changes upon metal binding, resulting in derepression of genes involved in detoxification. So far, the molecular determinants of specificity, selectivity, and metal binding mechanism have been scarcely investigated in thermophilic microorganisms. TtSmtB, the only ArsR/SmtB member present in the genome of Thermus thermophilus HB27, was chosen as a model to shed light into such molecular mechanisms at high temperature. In the present study, using a multidisciplinary approach, a structural and functional characterization of the protein was performed focusing on its metal interaction and chemical-physical stability. Our data demonstrate that TtSmtB has two distinct metal binding sites per monomer and interacts with di-tri-penta-valent ions with different affinity. Detailed knowledge at molecular level of protein-metal interaction is remarkable to design metal binding domains as scaffolds in metal-based therapies as well as in metal biorecovery or biosensing in the environment., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Dtur_0462 gene from the hypertermophilic bacterium Dictyoglomus turgidum, encoding a β-glucosidase, was synthetically produced and expressed in Escherichia coli BL21(DE3)-RIL. DturβGlu was purified to homogeneity by affinity chromatography and its homotetrameric structure was determined by gel filtration. The monomer is composed by 418 amino acidic residues and showed high sequence similarity with Glycoside Hydrolases (GHs) belonging to GH1 family. The maximum activity of DturβGlu was observed at 80°C and at pH5.4. DturβGlu was stable in the range of pH5-8 and retained 70% of its activity after 2h of incubation at 70°C. Metal ions and chemical reagents differently influenced the β-glucosidase activity; furthermore, DturβGlu displays a good ethanol and glucose tolerance (K i 750mM). The enzyme is active on p-nitrophenyl-β-d-glucopyranoside (pNPGlu) (K m 0.84mM) and p-nitrophenyl-β-d-galactopyranoside (pNPGal) (K m 1.36mM) and shows a broad substrate specificity towards natural compounds as salicin, cellobiose and genistin. The ability to hydrolyze different substrates, the activation in the presence of surfactants, the good thermal resistance, and finally the high glucose and ethanol tolerance make this enzyme a good candidate for industrial applications., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The full utilization of hemicellulose sugars (pentose and exose) present in lignocellulosic material, is required for an efficient bio-based fuels and chemicals production. Two recombinant thermophilic enzymes, an endo-1,4-β-mannanase from Dictyoglomus turgidum (DturCelB) and an α-galactosidase from Thermus thermophilus (TtGalA), were assayed at 80 °C, to assess their heterosynergystic association on galactomannans degradation, particularly abundant in hemicellulose. The enzymes were tested under various combinations simultaneously and sequentially, in order to estimate the optimal conditions for the release of reducing sugars. The results showed that the most efficient degree of synergy was obtained in simultaneous assay with a protein ratio of 25% of DturCelB and 75% of TtGalA, using Locust bean gum as substrate. On the other hand, the mechanism of action was demonstrated through the sequential assays, i.e. when TtGalA acting as first to enhance the subsequent hydrolysis performed by DturCelB. The synergistic association between the thermophilic enzymes herein described has an high potential application to pre-hydrolyse the lignocellulosic biomasses right after the pretreatment, prior to the conventional saccharification step., (Copyright © 2017. Published by Elsevier Inc.)

Evaluation of vector control is crucial for improving malaria containment and, according to World Health Organization, new complementary indicators would be very valuable. In this study the IgG response to the Anopheles-specific cE5 salivary protein was tested as a tool to evaluate the efficacy of insecticide-treated nets in reducing human exposure to malaria vectors. Sera collected during a longitudinal study carried out in Angola, and including entomological and parasitological data, were used to assess the IgG response to the Anopheles gambiae cE5 in both children and adults, before and after the application of insecticide-treated nets. Seasonal fluctuation of specific IgG antibody levels according to exposure was only found in children (up to ≈ 14 years old) whose anti-cE5 IgG response dropped after bed nets installation. These results were fully consistent with previous findings obtained with the same set of sera and indicating a substantial reduction of human-vector contact shortly after nets implementation. Overall, children IgG response to the cE5 protein appeared a very sensitive biomarker, which allowed for the detection of even weak exposure to Anopheles bites, indicating it may represent a reliable additional tool to evaluate the efficacy of vector control interventions., (Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

The multiple antibiotic resistance regulator (MarR) family constitutes a significant class of transcriptional regulators whose members control a variety of important biological functions such as regulation of response to environmental stress, control of virulence factor production, resistance to antimicrobial agents, and regulation of aromatic catabolic pathways. Although the majority of MarR family members have been characterized as transcriptional repressors, a few examples of transcriptional activators have also been reported. BldR is a newly identified member of this family that has been demonstrated to act as a transcriptional activator in stress response to aromatic compounds in the crenarchaeon Sulfolobus solfataricus. In this work, we report findings on the BldR X-ray crystal structure and present a molecular modeling study on the complex that this protein forms with its cognate DNA sequence, thus providing the first detailed description of the DNA-binding mechanism of an archaeal activator belonging to the MarR family. Two residues responsible for the high binding specificity of this transcriptional regulator were also identified. Our studies demonstrated that, in Archaea, the capability of MarR family members to act as activators or repressors is not related to a particular DNA-binding mechanism but rather could be due to the position of the binding site on the target DNA. Moreover, since genes encoding MarR proteins often control transcription of operons that encode for multisubstrate efflux pumps, our results also provided important insights for the identification of new tools to overcome the microorganism's multidrug resistance.

A selective removal of humic constituents involved in ester groups was conducted by a boron trifluoride-methanol transesterification reaction after removal of structurally unbound lipids. An analytical separation of subfractions containing specific classes of compound liberated from the humic matrix simplifies their identification by gas chromatography-mass spectrometry. We compared the traditional liquid-liquid separation into phenolic and aliphatic fractions with the modern and versatile fractionation technique using solid-phase extraction (SPE) on aminopropyl-bonded phases. Our results showed that both methods ensured separation of the same compounds, such as di- and tri-hydroxyalkanoic acids, alpha-, beta- and omega-hydroxy fatty acids, alkanoic acids, alpha,omega-alkanedioic acids, n-alkanols, phenolic acids and sterols. Moreover, the SPE method not only provided a larger recovery of compounds, but involved smaller sample and solvent requirements, and larger ease and rapidity of sample handling than the traditional liquid-liquid separation. The SPE method should be thus recommended in structural studies of natural organic matter.