Search Results (454)

Naringinase is an enzyme complex composed of α-L-rhamnosidase and β-D-glucosidase, capable of deglycosylating flavonoids such as hesperidin. α-L-rhamnosidase converts hesperidin into rhamnose and hesperetin 7-O-glucoside (Hes-7-G), while β-D-glucosidase further hydrolyses Hes-7-G to hesperetin. Selective inactivation of β-D-glucosidase enables accumulation of Hes-7-G, a compound with higher water solubility and bioavailability than hesperidin or hesperetin, making it valuable for food and biotechnological applications. This study aimed to identify conditions allowing selective inhibition of β-D-glucosidase while preserving α-L-rhamnosidase activity for efficient Hes-7-G production. The effects of pH, temperature, and incubation time were investigated, together with the influence of polyols and sugars, including inositol, sucrose, glycerol, xylose, erythritol, xylitol, and sorbitol, on α-L-rhamnosidase thermostability. Among the tested additives, erythritol significantly improved α-L-rhamnosidase thermostability. The highest selectivity was achieved by incubating the enzyme in 1.4 M erythritol at 70 °C for 10 min, resulting in ~5% residual β-D-glucosidase activity and 50% α-L-rhamnosidase activity. Under these conditions, α-L-rhamnosidase activity exceeded β-D-glucosidase activity by more than 60-fold. Selective thermal inactivation of β-D-glucosidase in the presence of erythritol provides an effective strategy for producing Hes-7-G from hesperidin and may enhance flavonoid bioavailability for industrial applications. Full article

The efficacy of enzyme therapy is limited by their poor stability under physiological conditions. Thermostable enzymes, derived from extremophilic organisms or generated by advanced protein engineering, offer a revolutionary solution to this long-standing challenge. They are widely used in industrial biocatalysis. Their therapeutic applications are poorly investigated and spread across diverse disciplines. While most applications are in the preclinical stages, emerging evidence from animal models demonstrates proof-of-concept for thermostable antioxidant enzymes in cardiovascular, neurodegenerative, and inflammatory diseases. This review critically assesses the translational landscape, distinguishing between established therapeutic enzymes (e.g., asparaginase, PEGylated SOD) and emerging experimental candidates. This narrative review consolidates existing knowledge about thermostable enzyme engineering and their emerging functions as molecular therapies, particularly in oxidative stress-related diseases. This review synthesizes recent advances in structural biology, computational protein design, biomaterials engineering, and translational antioxidant strategies, highlighting how breaking down disciplinary barriers is accelerating the development of sustainable and self-regenerating antioxidant platforms. By integrating molecular precision with systems-level therapeutic design, engineered thermostable antioxidant enzymes exemplify the future of biological development, where multidisciplinary collaboration drives innovation against oxidative stress-driven pathologies. Engineered thermostable enzymes provide a versatile basis for next-generation therapeutics, with the potential to address medical needs through improved stability, targeted activity, and multifunctional design. Full article

Xylanases are enzymes used in the conversion of lignocellulosic substances to fuels, digestion of animal feed, food and textile industries and as bleaching agents in paper production. The present study evaluated the production of a thermostable xylanase of the thermophilic fungus Myceliophthora heterothallica by solid-state fermentation of agro-industrial residues (sugarcane bagasse, sugarcane straw, wheat bran, and a mixture of the three substrates (1:1:1 w/w). Different cultivation parameters for the production of the enzyme were evaluated. The highest production of xylanase occurred in the mixture of the three substrates, after 4 days of cultivation. The activity of the enzyme was higher in the following conditions: water at pH 5.0 and incubation temperature of the fungus at 40 °C, with initial substrate moisture at 80%. The enzyme presented higher activity in the pH range between 5.0 and 6.5, with a peak at pH 5.0 and over 90% stability over a wide pH range (3.5 to 9.5). The optimum temperature was 65 °C and the enzyme showed 100% stability for 1 h, up to 60 °C. The results demonstrate that agro-industrial residues are efficient substrates for xylanase production, allowing the production of an enzyme with high stability under pH and temperature variations, a feature essential for its application in industrial processes. Full article

Cyclodextrin glycosyltransferase (CGTase) is a highly valuable biocatalyst in industrial starch conversion, particularly for the synthesis of cyclic oligosaccharides. In this study, a CGTase, designated HfCGT, was cloned from Haloferax sp. and heterologously expressed in Escherichia coli. The recombinant enzyme was purified and biochemically characterized. HfCGT exhibited maximal catalytic activity at 70 °C and pH 8.0, tolerance to metal ions and EDTA, and enhanced activity in the presence of 1 M NaCl and Ca²⁺. High-performance liquid chromatography (HPLC) and high-resolution mass spectrometry (HRMS) analyses revealed that the starch products by HfCGT degradation were mainly large-ring cyclodextrins (LR-CDs) with polymerization degrees of 9 to 20. Altogether, the thermostability, haloalkaliphilic, and distinctive product profile make HfCGT a promising biocatalyst for pharmaceutical, food, and biotechnological applications. Full article

Background: Reverse transcription is a key step in emerging RNA diagnostics, but reverse transcriptase (RT) enzymes often fail in the presence of inhibitors carried over from clinical samples or introduced during RNA extraction. Here, we dissect the molecular basis of inhibitor resistance in five engineered variants (V1 to V5) of Moloney Murine Leukemia Virus RT, originally optimized for thermostability and catalytic activity. Methods: Using a systematic framework that integrates structural analysis, thermal profiling, and diagnostic benchmarking, we evaluated cDNA synthesis from 40 to 70 °C under a panel of 11 clinically relevant inhibitors. Results: Across 30 mutations assessed, a recurrent set of substitutions (E69K, E302K/R, W313F, and N454K), present in RT V1 and V4, was associated with enhanced robustness, consistent with strengthened enzyme–nucleic acid engagement, while L435G likely contributes by modulating conformational flexibility. Notably, inhibitor tolerance was maximal at moderate reaction temperatures (≈40 °C), where productive enzyme–substrate interactions best offset inhibitory stress, while the wild-type enzyme was effectively inactivated by several inhibitors under the conditions tested. Although the engineered RTs remained catalytically competent at higher temperatures, increased thermal stress may destabilize productive enzyme–nucleic acid complexes, reducing resistance under inhibitory conditions. Conclusions: Together, these findings support substrate engagement as an important determinant of RT robustness and provide practical guidance for engineering inhibitor-resistant RTs for high-sensitivity RT-qPCR. Full article

Bioprospecting for extremozymes from unique ecological niches is crucial for developing robust biocatalysts for green chemistry. Here, we report the de novo hybrid genome assembly of Penicillium crustosum CTM10622, isolated from the humid montane forest of El Feïdja National Park, Tunisia. Using Illumina NextSeq™ 500 and Nanopore PromethION 2 Solo, a highly contiguous 31.38 Mb assembly (N50 = 1.94 Mb; 98.3% BUSCOs) was achieved. This robust genomic foundation enabled the identification of an extensive hydrolase repertoire, leading to the discovery of a novel alkaline serine lipase, PCLIP, subsequently heterologously expressed in Pichia pastoris. Recombinant rPCLIP exhibited a high specific activity (15,000 U/mg at pH 10, 65 °C) and exceptional thermostability, with half-lives of 14 and 8 h at 80 and 90 °C, respectively. The enzyme’s identity as a serine lipase was confirmed by its complete inhibition by Orlistat or tetrahydrolipstatin (THL) (51 µM), PMSF (5 mM), and diisopropylfluorophosphate (DIFP) (2 mM). To determine its substrate specificity, advanced computational approaches, including convolutional neural network-based docking and explicitly solvated molecular dynamics, were employed to compare rPCLIP with its homologue PCrL, a recombinant serine alkaline lipase from Penicillium crustosum Thom P22. While rPCLIP showed optimal experimental activity toward short-chain glyceryl tributyrate, simulations revealed that long-chain trioctanoin acts as a ‘thermodynamic trap’ due to over-stabilization. Conversely, the rigid rPCrL favors tricaprylin, driven by a ‘hydrophobic engine’ effect where the solvated environment forces chain burial with minimal entropic penalty. The findings demonstrate that rPCLIP specificity is driven by a delicate interplay of geometric complementarity, Van der Waals enthalpy, and conformational entropy. Full article

Mineral waters represent unique limnological ecosystems with stable physicochemical conditions and specialised microbial communities adapted to extreme environments. Bulgarian mineral waters remain comparatively underexplored despite their considerable ecological and biotechnological significance. These studies present a systematic narrative review of microbial diversity, ecological functions, and biotechnological potential of microbial communities from Bulgarian mineral springs. A total of 233 scientific sources published between 1990 and 2026 were analysed, of which 33 focused on Bulgarian sites. Data were retrieved from major scientific databases, regional reports and grey literature. Due to strong methodological heterogeneity, a qualitative synthesis was conducted, supported by bibliometric summaries of research focus and environmental context. The available evidence demonstrates that microbial communities in Bulgarian mineral waters include diverse bacteria, archaea, cyanobacteria, and microalgae that adapt to broad thermal and geochemical gradients. These microorganisms actively participate in element cycles, form complex biofilms, and show numerous physiological adaptations to oligotrophic and extreme temperature conditions. Bulgarian systems broadly reflect global microbial patterns but exhibit additional variability linked to contrasting hydrogeological settings. Many taxa produce thermostable enzymes, antimicrobial compounds, and exopolysaccharides with significant biotechnological potential. The review identifies significant research gaps and emphasises the importance of integrated multi-omics approaches for future exploration of Bulgarian mineral water ecosystems. Full article

Starch-degrading enzymes are key biocatalysts in industrial applications, particularly when derived from thermophilic microorganisms with potential to operate under elevated temperatures. In this study, three recombinant starch-degrading enzymes were heterologously produced, purified, and biochemically characterized: an α-amylase from Geobacillus kaustophilus, and an α-glucosidase and a type I pullulanase from Geobacillus sp. G4, a thermophilic strain isolated from a geothermal field in southern Peru. The three enzymes were successfully expressed in soluble form in Escherichia coli and purified by one-step affinity chromatography. Biochemical characterization showed that α-glucosidase and α-amylase displayed optimum activity at pH 6–7, whereas pullulanase exhibited a broader pH profile, retaining high activity up to pH 9. All three enzymes reached maximum activity at 60 °C, although their thermal stability profiles differed markedly, with pullulanase showing the highest thermostability. Metal ion assays revealed enzyme-dependent effects, with pullulanase being stimulated by Ca²⁺ and Mg²⁺, while α-amylase and α-glucosidase showed limited responses to divalent ions. Kinetic analysis using soluble potato starch indicated that α-amylase had the most favorable catalytic profile, with the lowest K_m and the highest catalytic efficiency among the three enzymes. Functional hydrolysis assays demonstrated that all enzymes were active on soluble starch and pretreated potato peel, while the enzymatic mixture consistently released the highest concentration of reducing sugars. These results expand the biochemical knowledge of thermophilic amylolytic enzymes from Geobacillus and support their potential use in future enzymatic systems for the conversion of starch-rich residues. Full article

Microbial biodegradation represents a promising approach to addressing global plastic pollution, yet the metabolic pathways and environmental origins of polymer-degrading microorganisms remain incompletely characterized. This review synthesizes current knowledge on biodegradation mechanisms across major polymer classes and identifies key environmental reservoirs harboring native plastic-degrading microbiota. Biodegradation pathways differ fundamentally according to polymer chemistry. Polyesters such as PET undergo hydrolytic cleavage by PETases and MHETases, releasing terephthalic acid and ethylene glycol for assimilation via the β-ketoadipate pathway and the TCA cycle. Biodegradable polyesters (PLA, PBAT, PHAs, PCL) are similarly hydrolyzed by cutinases, lipases, and depolymerases. In contrast, polyolefins (PE, PP) and polystyrene lack hydrolyzable bonds and require oxidative attack by laccases, peroxidases, and alkane monooxygenases, followed by β-oxidation to acetyl-CoA. Three principal environmental reservoirs supply plastic-degrading microorganisms: contaminated ecosystems including landfills and the plastisphere; soil microbiota contributing ligninolytic fungi and actinomycetes; and compost environments yielding thermostable enzymes such as leaf-branch compost cutinase. Across all environments, microbial consortia demonstrate superior degradation efficiency compared to single-species cultures, reflecting the enzymatic complexity required for complete polymer mineralization. Understanding these pathways and their environmental origins provides a foundation for biological plastic waste management strategies. Full article

Bst DNA polymerase is a biotechnologically modified thermostable enzyme from the thermophilic Gram-positive bacterium Geobacillus stearothermophilus. The unique structure of Bst DNA polymerase determines its thermal stability, ability to replace a DNA strand and specificity. The high specificity of Bst DNA polymerase ensures the efficiency, sensitivity, and high rate of loop-mediated isothermal amplification (LAMP), which is widely used in vitro biotechnology. The review reveals the structural and functional features of the enzyme, its application in LAMP and methods of improvement of thermal stability (including directed evolution, site-directed mutagenesis, fusion constructs, and chemical modifications). The terminal transferase activity and ab initio synthesis are discussed regarding problems of Bst DNA polymerase and the ways to eliminate them. The questions of introducing modified nucleotides and primers to expand the diagnostic capabilities of LAMP are also discussed. Modern advances in Bst DNA polymerase engineering pave the way for the creation of reliable, thermostable, and highly specific test systems suitable for widespread diagnostic applications. Full article

This review comprehensively examines the structural architecture, catalytic mechanisms, and targeted molecular engineering of α-amylase (primarily the GH13 family), a pivotal biocatalyst in the food industry. We highlight diverse microbial sources of α-amylases and their cost-effective heterologous expression in well-characterized hosts like Bacillus subtilis and Escherichia coli. To overcome extreme operational bottlenecks—such as elevated temperatures and acidic environments—recent advances in protein engineering are critically evaluated. These strategies, including directed evolution, semi-rational design, and advanced immobilization on nanomaterials, synergistically enhance the enzyme’s thermostability, catalytic efficiency, and reusability. Furthermore, this paper synthesizes the state-of-the-art applications of engineered α-amylases across key food processing sectors, including baking, sugar refining, and brewing. By integrating structural biology with advanced material science, this review provides a targeted roadmap for developing next-generation, high-performance α-amylases to address current and future challenges in sustainable food processing. Full article

Aflatoxin B₁ (AFB₁) is a highly stable mycotoxin that can persist during conventional food processing and therefore poses a serious risk to food and feed safety. In this study, two enzymes (CotA and Prx) were heterologously expressed in Bacillus subtilis, purified by Ni–NTA affinity chromatography, and evaluated for their ability to degrade AFB₁. Both enzymes exhibited remarkable thermostability and distinct catalytic optima. CotA exhibited its highest activity at 80 °C with an AFB₁ removal of 38.4%, whereas Prx showed its highest activity at 90 °C with a removal of 82.6%. The optimal pH values were near neutral, with CotA performing best at pH 7.0 and Prx at pH 7.5, and both reactions approached maximal conversion within approximately 10 h. When the two enzymes were combined, a clear cooperative effect was observed. The mixed system achieved 91.0% AFB₁ removal at 80 °C after 10 h, with the best degradation activity occurring at a CotA to Prx ratio of 1:3. At 50 °C, neither enzyme alone caused appreciable AFB₁ degradation, but the addition of hydrogen peroxide markedly enhanced catalytic activity. Both enzymes also retained substantial activity after boiling and autoclaving. In a maize flour model, the mixed-enzyme system showed strong AFB₁ degradation capacity, and peroxide-assisted treatment further improved activity. These results establish a thermostable and peroxide-responsive enzymatic platform for AFB₁ degradation and support future development of enzyme-based detoxification strategies for food and feed applications. Product identification and toxicological validation will be needed to confirm the safety of the treated products. Full article

Fluorene poses ecological and health hazards that originate from biomass combustion and petroleum. However, some microorganisms can counter fluorene through complex enzymatic degradation pathways. This research aimed to explore the catalytic efficiency of enzymes on metabolites and their toxicity levels throughout the fluorene biodegradation pathway. Several web servers and software were used to characterize them and analyse molecular dockings between ligands and proteins. Fluorene and its metabolites have mild toxicities to the brain, lung, neurons, and kidneys, and consequent endpoints cause mutations, cancer, and ecotoxicity at different levels. The catalytic enzymes are well-folded, single-chained, medium-sized proteins that are acidic, thermostable, and with few exceptions, hydrophilic, cytoplasmic, non-allergenic, and nonvirulent, possessing multiple active sites. The ERRAT, PROCHECK, and VERIFY 3D tools successfully validated the SWISS-modelled 3D structures of proteins. Molecular docking results showed moderate binding affinities between proteins and ligands, ranging from −9.4 to −6.1 kcal/mol, indicating potential activities of the enzymes. This computational study supports the conventional fluorene degradation pathway and may provide a new avenue for further research. Full article

Ultrasound is a non-thermal technology increasingly applied in food processing to modulate enzyme activity. This study investigated the effects of ultrasonic irradiation on the activity of a commercial thermostable α-amylase. Enzyme activity was determined by quantifying reducing sugars released from starch using the 3,5-dinitrosalicylic acid method, and protein concentration was measured by the Bradford assay. A one-factor-at-a-time approach was used to evaluate the effects of ultrasonic amplitude, treatment time, enzyme concentration, incubation temperature, and calcium ion concentration. Subsequently, a Box–Behnken design was applied to analyze the combined influence of amplitude, treatment duration, temperature, and calcium concentration on residual activity. The enzyme exhibited an initial activity of 46.27 ± 3.63 U/mL under standard assay conditions. Moderate ultrasonic amplitudes enhanced activity, whereas prolonged exposure and elevated temperatures promoted inactivation. Statistical analysis showed that the incubation temperature and calcium concentration significantly influenced residual activity, and the quadratic model provided a good fit (R² = 0.94). Optimal inactivation conditions were identified at 60% amplitude, 9 min treatment, 85 °C, and 9 ppm calcium, resulting in 66.3% enzyme inactivation. These findings support the use of ultrasound-assisted processing as a controllable strategy to regulate thermostable α-amylase activity in industrial enzyme applications. Full article

Peptide deformylase (PDF) belongs to a conserved enzyme family critical for N-terminal methionine excision (NME), an essential protein maturation process in prokaryotes and eukaryotic organelles (chloroplasts, mitochondria). To explore the potential functions of OsPDFs in Oryza sativa, this study employed bioinformatics approaches and experimental validation to systematically identify and analyze the OsPDF gene family. Three OsPDF genes (OsPDF1A, OsPDF1B, OsPDF1B2) were identified in rice. These genes are exclusively distributed on chromosome 1. The biophysical properties of these proteins showed that OsPDF1A and OsPDF1B are alkaline proteins, while OsPDF1B2 is acidic, and all are hydrophilic with moderate thermostability potential. Synteny analysis revealed closer evolutionary relationships between Oryza sativa and the monocot Triticum aestivum than with dicots, reflecting conserved PDF function in gramineous plants. Analysis of cis-acting elements in the 2000 bp upstream region of OsPDF gene promoters revealed numerous elements associated with abiotic stress response and hormone regulation. Furthermore, quantitative real-time PCR (qRT-PCR) data supported these findings, indicating that OsPDF1A and OsPDF1B were upregulated under low-temperature stress, and all three OsPDF genes were transcriptionally activated by heat, salt and UV-B stresses, indicating their active involvement in rice growth, development, and abiotic stress tolerance. In summary, OsPDFs exhibit significant functions in rice’s stress adaptation, growth, and development. This study not only enhances our understanding of the OsPDF gene family’s genomic, evolutionary, and functional characteristics, but also provides new perspectives and foundational data for further exploring their regulatory mechanisms in protein maturation and abiotic stress responses, as well as their potential applications in rice stress tolerance breeding. Full article