Search Results (2,014)

Comamonas testosteroni is an aerobic, Gram-negative bacterium belonging to the class of β-proteobacteria that is naturally present in soils, wastewater and sludge. It has recently gained popularity for its ability to act as a biocatalyst for the degradation of microplastics and other complex organics. Microplastics are globally considered as ubiquitous pollutants due to the increased use of polymers (plastics) which break down over time. In the urban water cycle, the drinking water treatment plants and the wastewater treatment plants are the first and last barriers to microplastics pollution, respectively. While conventional water and wastewater treatment has seen continuous technological improvements in producing cleaner effluents, industry technology adoption for the targeted removal of microplastics has been minimal. Therefore, the treatment of microplastics in soils and wastewater is of growing interest, and understanding C. testosteroni may provide insight into biological treatment and degradation of these pollutants. This review provides a summary of (1) favorable microbiological and environmental properties of C. testosteroni that lend themselves to bioremediation; (2) evidence of the bacterium’s ability to degrade microplastics, steroids, and organic pollutants; (3) implementation potential in the wastewater treatment process train; and (4) challenges and limitations in its application for microplastics biodegradation. Overall, while treatment applications of C. testosteroni through inoculation of media such as soil and wastewater are mentioned, further research into C. testosteroni concentrations found typically at wastewater treatment facilities would be beneficial. Full article

The enzymatic valorization of agarose, a major polysaccharide in red algae, is critical for its application in the food, pharmaceutical, and biotechnology industries. In this study, a gene encoding a thermostable α-neoagarobiose hydrolase, aga2457, was cloned from an epiphytic bacterium associated with Indonesian macroalgae. Unlike typical mesophilic GH117 enzymes, recombinant Aga2457 displayed a higher optimal temperature at 50 °C and retained 55% activity after 12 days of incubation at 50 °C. The enzyme specifically hydrolyzes neoagarobiose into D-galactose and 3,6-anhydro-L-galactose, thereby facilitating the complete depolymerization of agarose. Combined molecular dynamics (MD) simulations and site-directed mutagenesis revealed that residues P253, N256, and Q285 are pivotal for substrate recognition and active site stability. These findings highlight Aga2457 as a robust biocatalyst for industrial agar processing and provide structural insights for the rational design of thermostable agarolytic enzymes. Full article

Microbial cutinases are promising biocatalysts for polymer recycling. Here, we investigated the structural basis of catalytic activation in a thermophilic cutinase from Chaetomium thermophilum (CtCut). Differential scanning calorimetry revealed a three-state thermal unfolding pathway (Tm = 66.4 °C and 69.5 °C), indicating hierarchical stability. To capture distinct conformational states while avoiding affinity-tag artifacts, we employed both tag-free and tagged constructs. We determined apo-structures of wild-type and S136A mutant CtCut at 1.7 Å resolution and a complementary inhibitor complex at 2.65 Å. In the apo-state, a chloride ion coordinated the electrostatically pre-organized active site, while the catalytic H204 adopted a solvent-exposed, inactive loop conformation. In the inhibitor complex, p-nitrophenol displaced the chloride, establishing a characteristic oxyanion hole network. Concomitantly, the “lid” loop transitioned to an open state, with H204 exhibiting pronounced conformational heterogeneity across eight independent molecules. These complementary structures provide structural evidence for conformational dynamics of the catalytic lid loop, consistent with the conformational cycling model previously proposed for a mesophilic homolog. Full article

We report on the isolation and comprehensive genomic and biochemical characterization of Aspergillus fumigatus VP2T, a thermophilic filamentous fungus recovered from Himalayan Forest soil with exceptional lignocellulolytic capacity. Whole-genome sequencing revealed a 32.1 Mb genome encoding 12,675 predicted genes, including an extensive repertoire of >300 carbohydrate-active enzymes (CAZymes). Notably, the genome harbors multiple auxiliary activity enzymes, including AA9-family lytic polysaccharide monooxygenases and several cellobiose dehydrogenases (CDHs), supporting oxidative–hydrolytic synergism during biomass degradation. Submerged fermentation using a cellulose–wheat bran–rice straw substrate induced high enzyme titers, including 33 U/mL endoglucanase and 131 U/mL CDH, exceeding activities commonly reported for both native and engineered fungal strains. Although exoglucanase (0.02 U/mL) and xylanase (14.22 U/mL) activities were comparatively modest, the strain VP2T demonstrated superior hydrolysis of untreated rice straw, achieving a 1.89-fold increase in saccharification efficiency relative to the commercial enzyme cocktail Cellic^® CTec2. Scanning electron microscopy confirmed extensive disruption of lignocellulosic architecture, consistent with enhanced enzyme accessibility and oxidative fiber loosening. Collectively, genomic evidence and functional assays identify A. fumigatus VP2T as a redox-optimized, moderately thermophilic biocatalyst suited for low-pH lignocellulose conversion. This study highlights the value of exploring thermophilic fungal biodiversity to discover native strains with inherent oxidative capacity, offering promising alternatives to pretreatment-intensive biorefinery processes and informing the rational development of tailored enzyme systems. Full article

Whole-cell biocatalysis presents a sustainable and efficient approach for the selective reduction in α,β-unsaturated bonds in flavonoid derivatives. This study investigates the capability of yeast strains from the Yarrowia clade to catalyze the chemoselective reduction of 4′-methoxychalcone (1a) to its dihydro derivative. All tested strains exhibited similarly high hydrogenation activity, indicating a broadly conserved enoate reductase function within the clade. Among them, Yarrowia lipolytica KCh 71, previously reported and well characterized in the literature, was selected for preparative-scale transformation of a diverse series of synthetic methoxychalcones bearing additional methoxy groups in positions C-2, C-3, C-4, C-5, and C-6 of ring B. All derivatives were effectively converted into the corresponding dihydrochalcones, with yields ranging from 62% to 92%. Among the tested derivatives, the 2′,4′,6′-trimethoxy chalcone (7a) did not undergo biotransformation under our conditions, whereas mono- and di-methoxy derivatives (2a–6a) were efficiently reduced. These results confirm the broad substrate tolerance, high efficiency, and potential scalability of Y. lipolytica KCh 71, supporting its potential as a whole-cell biocatalyst for the sustainable synthesis of bioactive dihydrochalcones. The consistently high hydrogenation activity observed across 21 tested strains suggests the involvement of evolutionarily conserved enoate reductases. Bioinformatic analysis supports that the Yarrowia clade possesses a robust complement of Old Yellow Enzymes (OYE), providing a reliable enzymatic basis for the observed chemoselective reductions. All Yarrowia tested strains showed the same general transformation type, although the extent and rate of conversion differed among strains, and Y. lipolytica KCh 71 was one of the most tolerant. The broad reduction in α,β-unsaturated chalcones is consistent with the action of flavoenzymatic ene-reductases, particularly Old Yellow Enzyme (OYE)–like reductases. Bioinformatic analysis of Yarrowia genomes reveals putative OYE homologs, supporting this mechanistic interpretation, although the specific enzymes were not identified in this study. Full article

Phosphatidylserine (PS), a valuable phospholipid, is widely used in food, pharmaceutical and cosmetic industries. Its enzymatic synthesis, catalyzed by phospholipase D (PLD) via transphosphatidylation under mild conditions, has drawn considerable attention. However, the industrial use of free PLD is limited by poor stability, difficult recovery, and high cost. To address these challenges, a ternary composite carrier—integrating the flexibility of chitosan, the stability of cellulose, and the macroporosity of agarose—was constructed for immobilizing the PLD from Streptomyces antibioticus (saPLD). The resulting saPLD@chitosan–cellulose–agarose biocatalyst demonstrated enhanced immobilization efficiency, catalytic performance, and stability across varying pH and temperatures. After eight consecutive batches of usage, the PS yield of saPLD@chitosan–cellulose–agarose reached over 60% of that from the first batch. Thus, this study established a new method for preparing immobilized saPLD, and developed a robust and promising platform for the efficient and sustainable production of PS. Full article

This work presents the synthesis, characterization, and application of zirconium oxide (ZrO₂)-based catalysts, modified with macro (silica nanospheres, NSP-SiO₂) and mesopore templates (Pluronic 123), impregnated with tungstophosphoric acid (TPA), in the catalytic pyrolysis of tomato agro-industrial residues. The NSP-SiO₂ (SXX) and P123 (PYY) amount mainly influences the ZrO₂SXXPYY-specific surface area (S_BET) and average pore diameter (D_p). ³¹P MAS NMR and FT-IR characterization results show that TPA (H₃PW₁₂O₄₀) was partially transformed into [P₂W₂₁O₇₁]⁶⁻ and [PW₁₁O₃₉]⁷⁻ during the synthesis steps. The acidic properties of ZrO₂SXXPYY samples containing 25 and 50 wt% of TPA (ZrO₂SXXPYYT25 and ZrO₂SXXPYYT50, respectively) are dependent on both the TPA content and the support nature. Bio-oil composition and product selectivity were strongly influenced by the textural and acid-based properties of the catalysts. Notably, non-catalytic pyrolysis favored pathways leading to C2 compounds, with a high content of acetic acid and hydroxyacetone. In contrast, the use of catalysts promoted the formation of higher molecular weight oxygenated compounds (C5–C6), specifically furans, aldehydes, and ketones. Full article

Lignin valorization is a central objective of modern biorefinery research. This study investigates the catalytic depolymerization of two technical lignins, kraft lignin from beech hardwood and natron lignin from annual plants, via two complementary routes: analytical catalytic pyrolysis (Py-GC/MS, 300–600 °C) and hydrogenolysis (250–310 °C, Ru/C, isopropanol/H₂). In Py-GC/MS experiments, noble-metal catalysts on carbon supports (Ru/C, Pd/C, RuPd/C) were screened. Relative compound distributions revealed phenolic derivatives as the dominant products, with Ru/C yielding the highest conversion for lignin from annual plants at 500 °C and Pd/C proving most selective for hardwood lignin at 400 °C. Hydrogenolysis was optimized through a five-level, three-factor central composite design, varying temperature, residence time, and catalyst loading. Lignin conversion ranged from 64 to 83 wt% and bio-oil yield from 69 to 89 wt%. A regression model identified optimal conditions at 295 °C, 32 min, and 17 wt% Ru/C. Catalyst regeneration via solvent washing, H₂O₂ oxidation, and controlled thermal treatment resulted in only an 8% decrease in lignin conversion. The results demonstrate that lignin origin, catalyst type, and depolymerization pathway jointly govern product selectivity, highlighting clear strategies for targeted phenolic compound production. Full article

The development of thermostable and pH-robust endo-polygalacturonases (endo-PGases) is crucial for industrial applications such as food processing. This study aimed to engineer the thermostability of an acidic, thermophilic endo-PGase (PoxaEnPG28B) by rigidifying its flexible regions. We employed an integrated computational strategy combining molecular dynamics (MD) simulations at elevated temperatures with in silico analyses of unfolding free-energy changes to identify and design stabilizing mutations. This approach successfully yielded the mutant D249K, which exhibited a 5 °C higher optimal temperature (70 °C) and a 68.8% longer half-life at 55 °C, and it retained over 76.8% activity at 75 °C. Notably, D249K maintained the wild-type’s optimal pH (5.0) and broad pH stability (3.0–8.0). Although it is not the absolute top performer in every single metric, D249K achieves the best overall balance between thermostability and pH robustness among all reported thermophilic endo-PGases. MD simulations revealed that its enhanced stability sems from reduced global and local flexibility and a more compact structure. In juice extraction applications, D249K increased yields by up to 98.5%, significantly surpassing the wild-type. This study demonstrates the efficacy of MD-guided flexible region engineering for the GH28 family and presents D249K as a highly promising industrial biocatalyst. Full article

Cell-free protein synthesis has become a powerful tool for producing functional proteins, circumventing many limitations of live-cell systems. Platforms based on wheat germ extract are favored for their high efficiency in translating and folding complex eukaryotic proteins. To overcome the energy limitation common in such systems, we engineered an Escherichia coli strain to function as a self-renewing ATP source. This strain co-expresses a three-enzyme cascade—adenosine kinase, adenylate kinase, and acetate kinase—that efficiently converts adenosine and acetyl phosphate into ATP. Using the lysate from this biocatalyst to energize an optimized wheat germ extract, we established a high-performance cell-free synthesis platform. This integrated system supported the robust production of multiple recombinant proteins. As a key demonstration, we synthesized human vascular endothelial growth factor 165, which exhibited full biological activity. The cell-free-produced VEGF₁₆₅ significantly stimulated the proliferation of human umbilical vein endothelial cells (HUVECs) and human skin fibroblasts (HSFs). It also potently induced angiogenic responses, including the formation of extensive, interconnected capillary-like networks by HUVECs in vitro and accelerated cell migration in scratch-wound assays. Our work establishes a scalable and efficient platform for on-demand production of bioactive eukaryotic proteins, highlighting its considerable potential for advancing regenerative medicine and related therapeutic applications. Full article

UDP-glycosyltransferases (UGTs) represent a large multigene family that play a central role in glycosylating a highly diverse array of natural products, underscoring their critical importance in various biological processes. However, the functional roles of a substantial majority of UGTs remain to be elucidated. In the present study, we characterized the glycosyltransferase UGT78G3, a member of the UGT78 glycosyltransferase family in the model legume Medicago truncatula. Amino-acid sequence analysis revealed a conserved PSPG motif at the C-terminus of UGT78G3. Liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) analysis demonstrated that UGT78G3 catalyzes the formation of kaempferol 3-O-glucoside in vitro. However, neither UGT78G3 overexpression nor CRISPR/Cas9-mediated mutagenesis resulted in significant changes to the endogenous levels of kaempferol 3-O-glucoside, indicating that UGT78G3 does not play a predominant role in the biosynthesis of kaempferol 3-O-glucoside in vivo. Our findings identify a putative glycosyltransferase in M. truncatula and provide a target for biocatalyst design aimed at synthesizing flavonoid glucosides. Full article

Testosterone is a vital steroid hormone with important physiological roles and broad clinical significance, serving as a central molecular precursor in the synthesis of many pharmacologically active steroids. Testosterone is traditionally produced through complex chemical synthesis routes that involve hazardous reagents, harsh conditions, and produce significant toxic waste. In recent decades, growing regulatory requirements and environmental sustainability goals have spurred the development of alternative biotechnological methods that use microbial biotransformation. This review offers a comparative analysis of chemical and biological methods for producing testosterone, focusing on microbial steroid biotransformation pathways and the key enzymatic steps involved in testosterone biosynthesis. It examines key advances in sterol breakdown, pathway engineering, and enzyme driven modifications, including the roles of 17β-hydroxysteroid dehydrogenases and cytochrome P450 monooxygenases. The performance, specificity, and environmental impacts of bacterial and fungal cells as cell factories, especially Mycolicibacterium and Aspergillus species, are critically analyzed within the framework of modern green chemistry principles. Overall, by combining molecular insights with process considerations, this review illustrates how microbial platforms could complement and gradually transform traditional chemical synthesis methods, promoting a shift toward more sustainable steroid hormone production through engineered biocatalysts. Full article

Catalytic ozonation often suffers from a low ozone utilization rate and incomplete mineralization of organic pollutants. To address these challenges, we designed and prepared a novel catalyst via a one-step thermal polymerization method, anchoring single-atom manganese on a glucose-derived carbon network-modified C₃N₅ framework (Mn/C-C₃N₅). Aberration-corrected high-angle annular dark-field scanning transmission electron microscopy (AC-HAADF-STEM) on an FEI Titan Themis Z microscope confirmed the atomic dispersion of Mn sites, while Raman spectroscopy using a Renishaw inVia Reflex laser micro-Raman spectrometer verified the successful incorporation of a graphitic carbon network within the C₃N₅ matrix. Moreover, electrochemical analyses, including electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) performed on a Bio-Logic SP-150 electrochemical workstation, demonstrated that the integration of the conductive carbon matrix substantially enhanced the interfacial charge transfer capability. The optimized Mn/C-C₃N₅ catalyst demonstrated exceptional performance in phenol mineralization, achieving a 97% total organic carbon (TOC) removal within 60 min, a remarkable improvement compared to pristine C₃N₅ (30%). Furthermore, the catalyst exhibited excellent operational stability, preserving more than 95% of its original activity over five repeated runs. Mechanistic investigations, including electron paramagnetic resonance (EPR) spectroscopy and radical quenching experiments, revealed that the Mn/C-C₃N₅ system accelerated the generation of multiple oxidizing radicals (•O₂⁻, ¹O₂, and •OH), with •OH identified as the predominant reactive species responsible for complete mineralization. This work establishes an integrated catalytic platform and provides fundamental insights into electronic structure modulation for designing advanced oxidation catalysts. Full article

Biochar-based catalysts have emerged as sustainable alternatives for biodiesel production, achieving high yields (up to 99%) from various feedstocks. This study aimed to utilize Spirulina-derived biochar as a bifunctional green catalyst for biodiesel synthesis from waste cooking oil (WCO) through transesterification and assess its green performance metrics. Biochar synthesized by carbonization (324 °C) was modified with calcium and sulfuric acid, featuring dual acid-base sites. Energy dispersive spectra revealed impregnation of calcium (11.11%) compared to the raw biomass (2.34%), followed by peaks of methoxy group and methylene group, and with methylene and β-carbonyl protons shown by nuclear magnetic spectroscopy. Thus, the biochar catalyst tested on WCO achieved a 93.27% yield under optimized conditions (65 °C, 1:15 methanol-to-oil ratio, 3% catalyst, 3.5 h) via central composite design. Catalyst reusability was maintained over four cycles with an average biodiesel yield (90%). Further, green metrics validate their eco-friendliness with a single-cycle reaction mass efficiency (RME) of 60.8%. When the initial catalyst mass is amortized over four cycles, the cumulative biodiesel yield per initial catalyst input reaches the equivalent of 243% of a single-batch theoretical yield (catalyst productivity = 3.12 g FAME/g catalyst). E-Factor at 0.67 (reduced to 0.17) and mass intensity at 1.68 (down to 0.42), contrasting with business-as-usual scenarios such as sulfuric acid catalysis (RME 70.0%, E-Factor 0.25) using 8.85 g H₂SO₄ vs. ~5 g H₂SO₄/kg biochar. Our results demonstrate that bio-based catalysts minimize non-benign inputs, supporting a circular economy from algal waste. Full article

The transition to sustainable energy systems has intensified the search for renewable alternatives to reduce greenhouse gas emissions and reliance on fossil fuels. In this context, microalgae have emerged as a promising third-generation feedstock for biofuel production due to their rapid development, high lipid content, and ability to grow in wastewater without competing with freshwater resources. In this study, the hydrotreatment of biocrudes derived from C. vulgaris, T. obliquus, and a mixed microalgal culture cultivated in domestic wastewater is investigated. Catalytic upgrading was applied using sulphided CoMo/Al₂O₃ (sCoMo) and Pt/Al₂O₃ catalysts. The results demonstrated that catalytic upgrading enhanced the upgraded bio-oils’ quality compared to non-catalysed reactions, confirming the crucial role of catalysts in improving bio-oil properties. Compared with the Pt catalyst, sCoMo produced higher yields of upgraded bio-oil, greater enrichment in carbon and hydrogen, and higher heating value (HHV), while effectively enhancing nitrogen and oxygen removal. However, when compared with the non-sulphided CoMo, the sulphiding treatment did not significantly improve denitrogenation and treated oil yields. The highest fraction of components within the jet fuel boiling range (37.7%) was obtained using a Pt catalyst, while the non-catalysed process yielded the lowest (26.6%). In this sense, catalytic upgrading of microalgae-based biocrude represents an important step towards the production of advanced and environmentally sustainable fuels. Full article