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Distal mutations enhance efficiency of free and immobilized NOV1 dioxygenase for vanillin synthesis.Journal of Biotechnology Aug 2024Protein engineering is crucial to improve enzymes' efficiency and robustness for industrial biocatalysis. NOV1 is a bacterial dioxygenase that holds biotechnological...
Protein engineering is crucial to improve enzymes' efficiency and robustness for industrial biocatalysis. NOV1 is a bacterial dioxygenase that holds biotechnological potential by catalyzing the one-step oxidation of the lignin-derived isoeugenol into vanillin, a popular flavoring agent used in food, cleaning products, cosmetics and pharmaceuticals. This study aims to enhance NOV1 activity and operational stability through the identification of distal hotspots, located at more than 9 Å from the active site using Zymspot, a tool that predicts advantageous distant mutations, streamlining protein engineering. A total of 41 variants were constructed using site-directed mutagenesis and the six most active enzyme variants were then recombined. Two variants, with two and three mutations, showed nearly a 10-fold increase in activity and up to 40-fold higher operational stability than the wild-type. Furthermore, these variants show 90-100 % immobilization efficiency in metal affinity resins, compared to approximately 60 % for the wild-type. In bioconversions where 50 mM of isoeugenol was added stepwise over 24-h cycles, the 1D2 variant produced approximately 144 mM of vanillin after six reaction cycles, corresponding to around 22 mg, indicating a 35 % molar conversion yield. This output was around 2.5 times higher than that obtained using the wild-type. Our findings highlight the efficacy of distal protein engineering in enhancing enzyme functions like activity, stability, and metal binding selectivity, thereby fulfilling the criteria for industrial biocatalysts. This study provides a novel approach to enzyme optimization that could have significant implications for various biotechnological applications.
Topics: Benzaldehydes; Mutation; Mutagenesis, Site-Directed; Enzymes, Immobilized; Dioxygenases; Eugenol; Bacterial Proteins; Protein Engineering
PubMed: 38880386
DOI: 10.1016/j.jbiotec.2024.06.012 -
Journal of Agricultural and Food... Jun 2024Headspace solid-phase microextraction coupled to gas chromatography-mass spectrometry (HS-SPME-GC-MS) offers an alternative analysis method for isoeugenol (an active...
Headspace solid-phase microextraction coupled to gas chromatography-mass spectrometry (HS-SPME-GC-MS) offers an alternative analysis method for isoeugenol (an active ingredient in fish sedatives) that avoids the use of organic solvents, simplifies sample preparation, and can be fully automated. This work focuses on developing and evaluating an HS-SPME-GC-MS method for isoeugenol in aquaculture samples and testing the stability of isoeugenol itself. Because of isoeugenol's relatively low volatility, more polar SPME fiber coatings (polyacrylate and polydimethylsiloxane/divinylbenzene) had better performance and the headspace extractions took over 30 min to reach equilibrium. Additionally, it was found that isoeugenol was relatively unstable compared to a deuterated standard (d-eugenol) in the presence of water. To address this, after the fish samples were homogenized with water, they were heated at 50 °C for 1 h prior to analysis for equilibration. By using the method developed in this work, isoeugenol's detection limits in multiple aquaculture matrices (shrimp, tilapia, and salmon) were in the low ng/g range (<15 ng/g), well below the target testing level (200 ng/g). Additionally, by adding d-eugenol as an internal standard, excellent linearity ( > 0.98), accuracy (97-99% recoveries), and precision (5-13% RSDs) were all achieved.
Topics: Gas Chromatography-Mass Spectrometry; Solid Phase Microextraction; Animals; Eugenol; Aquaculture; Tilapia; Fishes; Seafood; Food Contamination
PubMed: 38875496
DOI: 10.1021/acs.jafc.4c02318 -
Angewandte Chemie (International Ed. in... Jun 2024Herein we demonstrate an effective and facile end-capping technique for ring-opening metathesis polymerization (ROMP) using readily available allyl compounds as a new...
Herein we demonstrate an effective and facile end-capping technique for ring-opening metathesis polymerization (ROMP) using readily available allyl compounds as a new type of terminating agents. This new type of end-capping reactions, which are based on the basepromoted decomposition of ruthenocyclobutane intermediates, introduce diene moiety onto the chain end of ROMP polymers while simultaneously deactivating the ruthenium complex. These termination reactions are highly efficient, typically completing within 1 minute at 0 oC with >95% end-capping fidelity.
PubMed: 38869845
DOI: 10.1002/anie.202409534 -
Redox Biology Aug 2024Silicosis, characterized by interstitial lung inflammation and fibrosis, poses a significant health threat. ATII cells play a crucial role in alveolar epithelial repair...
BACKGROUND
Silicosis, characterized by interstitial lung inflammation and fibrosis, poses a significant health threat. ATII cells play a crucial role in alveolar epithelial repair and structural integrity maintenance. Inhibiting ATII cell senescence has shown promise in silicosis treatment. However, the mechanism behind silica-induced senescence remains elusive.
METHODS
The study employed male C57BL/6 N mice and A549 human alveolar epithelial cells to investigate silicosis and its potential treatment. Silicosis was induced in mice via intratracheal instillation of crystalline silica particles, with honokiol administered intraperitoneally for 14 days. Silica-induced senescence in A549 cells was confirmed, and SIRT3 knockout and overexpression cell lines were generated. Various analyses were conducted, including immunoblotting, qRT-PCR, histology, and transmission electron microscopy. Statistical significance was determined using one-way ANOVA with Tukey's post-hoc test.
RESULTS
This study elucidates how silica induces ATII cell senescence, emphasizing mtDNA damage. Notably, honokiol (HKL) emerges as a promising anti-senescence and anti-fibrosis agent, acting through sirt3. honokiol effectively attenuated senescence in ATII cells, dependent on sirt3 expression, while mitigating mtDNA damage. Sirt3, a class III histone deacetylase, regulates senescence and mitochondrial stress. HKL activates sirt3, protecting against pulmonary fibrosis and mitochondrial damage. Additionally, HKL downregulated cGAS expression in senescent ATII cells induced by silica, suggesting sirt3's role as an upstream regulator of the cGAS/STING signaling pathway. Moreover, honokiol treatment inhibited the activation of the NF-κB signaling pathway, associated with reduced oxidative stress and mtDNA damage. Notably, HKL enhanced the activity of SOD2, crucial for mitochondrial function, through sirt3-mediated deacetylation. Additionally, HKL promoted the deacetylation activity of sirt3, further safeguarding mtDNA integrity.
CONCLUSIONS
This study uncovers a natural compound, HKL, with significant anti-fibrotic properties through activating sirt3, shedding light on silicosis pathogenesis and treatment avenues.
Topics: Animals; Silicosis; Sirtuin 3; Cellular Senescence; Mice; Alveolar Epithelial Cells; Biphenyl Compounds; Humans; Lignans; Signal Transduction; Male; A549 Cells; Nucleotidyltransferases; Disease Models, Animal; Membrane Proteins; Mice, Inbred C57BL; DNA Damage; Allyl Compounds; Phenols
PubMed: 38865904
DOI: 10.1016/j.redox.2024.103224 -
Scientific Reports Jun 2024This study aimed to evaluate the impact of thyme and/or garlic oil administration on growth performance, immunity, antioxidant, biochemical parameters, and net farm...
This study aimed to evaluate the impact of thyme and/or garlic oil administration on growth performance, immunity, antioxidant, biochemical parameters, and net farm income of Damascus goats. Forty weaned Damascus goats were allocated into four groups. The first group was the control without oral administration, while the 2nd (Th), 3rd (Gr), and 4th (ThGr) groups were orally administrated by (2 ml/goat/day) of thyme oil, garlic oil and their mixture (1:1), respectively during the whole experiment period. The final body weight of goats orally administered oil mixture was the heaviest group, it was 10, 4.5 and 3.5% than the control, Th. and Gr. groups, respectively with better feed conversion ratio and high net farm income. Goats of ThGr. group revealed the best immunity, antioxidant and general health condition than the control group with 50% reduction of MDA. Liver (AST, 33% and ALT, 38%) and kidney (creatinine, 88%) functions improved by oils mixtures orally administration compared with the control group. LDL, triglyceride and cholesterol were reduced by 47, 33 and 21% compared with the control group, respectively. Thus, mixture oil administration (thyme and garlic at the ratio of 1:1, 2 ml/goat/day) improved growth (10%), antioxidant status (MDA 50%), liver (AST, 33% and ALT, 38%), kidney function (creatinine, 88%), the FCR (17.4%) and net farm income (21%), of Damascus goats.
Topics: Animals; Goats; Thymus Plant; Antioxidants; Plant Oils; Garlic; Animal Feed; Sulfides; Liver; Allyl Compounds
PubMed: 38849384
DOI: 10.1038/s41598-024-62417-0 -
Journal of the American Chemical Society Jun 2024Despite the synthetic versatility of difluorocarbene, its high reactivity severely regulates widespread applications of difluorocarbene in organic synthesis. Here, we...
Despite the synthetic versatility of difluorocarbene, its high reactivity severely regulates widespread applications of difluorocarbene in organic synthesis. Here, we report a copper difluorocarbene-involved catalytic coupling, representing a new mode of the difluoromethylation reaction. This method allows difluoromethylation of a wide range of readily available allyl/propargyl electrophiles with NaBHCN and low-cost difluorocarbene precursor BrCFCOK, featuring high cost-efficiency, high stereo- and regioselectivities, and high functional group tolerance, even with complex drug-like molecules. Applying the method led to the efficient synthesis of deuterated difluoromethylated compounds of medicinal interest. The resulting difluoromethylated allyl and allenyl products can serve as versatile synthons for diverse transformations, rendering the approach attractive for synthesizing complex fluorinated structures. Experimental mechanistic studies and computational calculations reveal that the formation of a difluoromethylcopper(I) intermediate through the nucleophilic attack of boron hydride on the copper(I) difluorocarbene is the key step in the reaction.
PubMed: 38847772
DOI: 10.1021/jacs.4c04949 -
Materials Horizons Jul 2024The real-time monitoring of volatile sulfur compounds is indispensable; however, it continues to pose a significant challenge due to issues such as limited performance...
The real-time monitoring of volatile sulfur compounds is indispensable; however, it continues to pose a significant challenge due to issues such as limited performance towards parts-per-billion (ppb)-level gas. Herein, a concept of synergistic sensitization effects involving single-atom gold (Au) and cerium (Ce) dopants is proposed to boost the sensing performance of allyl mercaptan, a common volatile sulfur compound. As a proof-of-concept, a chemiresistive gas sensor based on mesoporous SnO nanospheres with single-atom Au decoration and Ce dopant (denoted Au/Ce-SnO) is successfully synthesized. The synthesis of Au/Ce-SnO is achieved through the utilization of a self-template strategy, employing metal-phenolic hybrids as a precursor. The obtained materials exhibit high specific surface area (89.4 m g), and small particle size (∼86 nm). The gas sensor reveals unprecedented sensitivity (0.097 ppb) and ultra-low detection limit (0.74 ppb), surpassing all state-of-the-art allyl mercaptan gas sensors. Furthermore, a wireless gas sensor is constructed for highly selective and real-time monitoring of allyl mercaptan. The decoration of single-atom Au facilitates the adsorption and dissociation of oxygen and target gases. Simultaneously, the Ce dopant enhances the oxidation of allyl mercaptan. The sensing performance is boosted by the mesoporous framework of SnO, as well as the synergistic sensitization effects resulting from single-atom Au decoration and Ce doping, thereby facilitating its potential application in environmental and health-related domains.
PubMed: 38847138
DOI: 10.1039/d4mh00507d -
Nature Communications Jun 2024The [1,2]- and [2,3]-Stevens rearrangements are one of the most fascinating chemical bond reorganization strategies in organic chemistry, and they have been demonstrated...
The [1,2]- and [2,3]-Stevens rearrangements are one of the most fascinating chemical bond reorganization strategies in organic chemistry, and they have been demonstrated in a wide range of applications, representing a fundamental reaction tactic for the synthesis of nitrogen compounds in chemical community. However, their applicabilities are limited by the scarcity of efficient, general, and straightforward methods for generating ammonium ylides. Herein, we report a general difluorocarbene-induced tertiary amine-involved [1,2]- and [2,3]-Stevens rearrangements stemmed from in situ generated difluoromethyl ammonium ylides, which allows for the rearrangements of versatile tertiary amines bearing either allyl, benzyl, or propargyl groups, resulting in the corresponding products in one reaction under the same reaction conditions with a general way. Broad substrate scope, simple operation, mild reaction conditions and late-stage modification of natural products highlight the advantages of this strategy, meanwhile, this general rearrangement reaction is believed to bring opportunities for the transformations of nitrogen ylides and the assembly of valuable tertiary amines and amino acids. This will further enrich the reaction repertoire of difluorocarbene species, facilitate the development of reactions involving difluoromethyl ammonium salts, and provide an avenue for the development of this type of rearrangement reactions.
PubMed: 38839757
DOI: 10.1038/s41467-024-49054-x -
Angewandte Chemie (International Ed. in... Jun 2024Palladium-catalyzed cross-couplings of aryl chlorides usually call for bulky, electron-rich ligands such as phosphines or heterocyclic carbenes. We have now found that...
Palladium-catalyzed cross-couplings of aryl chlorides usually call for bulky, electron-rich ligands such as phosphines or heterocyclic carbenes. We have now found that similarly powerful cross-coupling catalysts are obtained by the reaction of palladium salts with alkynyllithium reagents. The species initially formed in this process was characterized as a dilithium tetraalkinyl palladate complex. It catalyzes the coupling of aryl chlorides with the lithium salts of various terminal alkynes to give alkynyl arenes. The isolated Li-alkynyl-Pd complex also efficiently promotes the reaction of aryl, and allyl chlorides with (hetero)aryl-, alkyl-, and allyllithium compounds as well as lithium amines. None of these reactions proceeded in the presence of palladium salts alone. The preparative utility of this approach was demonstrated by the synthesis of 49 molecules, including pharmaceutically relevant compounds.
PubMed: 38837734
DOI: 10.1002/anie.202408974 -
Scientific Reports Jun 2024Exposure to NO generated by plasma technology activates immunity in Arabidopsis through tryptophan metabolites. However, little is known about the effects of NO exposure...
Exposure to NO generated by plasma technology activates immunity in Arabidopsis through tryptophan metabolites. However, little is known about the effects of NO exposure on other plant species. Sweet basil synthesizes many valuable secondary metabolites in its leaves. Therefore, metabolomic analyses were performed at three different exposure levels [9.7 (Ex1), 19.4 (Ex2) and 29.1 (Ex3) μmol] to assess the effects of NO on basil leaves. As a result, cinnamaldehyde and phenolic acids increased with increasing doses. Certain flavonoids, columbianetin, and caryophyllene oxide increased with lower Ex1 exposure, cineole and methyl eugenol increased with moderate Ex2 exposure and L-glutathione GSH also increased with higher Ex3 exposure. Furthermore, gene expression analysis by quantitative RT-PCR showed that certain genes involved in the syntheses of secondary metabolites and jasmonic acid were significantly up-regulated early after NO exposure. These results suggest that NO exposure increases several valuable secondary metabolites in sweet basil leaves via plant defense responses in a controllable system.
Topics: Ocimum basilicum; Plant Leaves; Secondary Metabolism; Gene Expression Regulation, Plant; Metabolomics; Flavonoids; Eugenol; Oxylipins
PubMed: 38834771
DOI: 10.1038/s41598-024-63508-8