RESUMEN
OBJECTIVE: To enhance adipic acid production, a computer-aided approach was employed to engineer the adipyl-CoA synthetase from Thermobifida fusca by combining sequence analysis, protein structure modeling, in silico site-directed mutagenesis, and molecular dynamics simulation. RESULTS: Two single mutants of T. fusca adipyl-CoA synthetase, E210ßN and E210ßQ, achieved a specific enzyme activity of 1.95 and 1.84 U/mg, respectively, which compared favorably with the 1.48 U/mg for the wild-type. The laboratory-level fermentation experiments showed that E210ßN and E210ßQ achieved a maximum adipic acid titer of 0.32 and 0.3 g/L. In contrast, the wild-type enzyme yielded a titer of 0.15 g/L under the same conditions. Molecular dynamics (MD) simulations revealed that the mutants (E210ßN and E210ßQ) could accelerate the dephosphorylation process in catalysis and enhance enzyme activity. CONCLUSIONS: The combined computational-experimental approach provides an effective strategy for enhancing enzymatic characteristics, and the mutants may find a useful application for producing adipic acid.
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Adipatos/metabolismo , Diseño Asistido por Computadora , Hidrolasas/biosíntesis , Ligasas/biosíntesis , Adipatos/química , Catálisis , Cefalosporinas/farmacología , Escherichia coli/genética , Hidrolasas/química , Ligasas/química , Mutagénesis Sitio-Dirigida , Especificidad por SustratoRESUMEN
Novel sulfur reagents N-vinylthio phthalimides (N-VTPs) have been employed as modular reagents for vinylthiolation, enabling the construction of aggregation-induced emission (AIE)-active tetraaryldivinyl sulfides (TADVSs) and diarylvinyl sulfides (DAVSs). Notably, TADVSs with sulfur insertion to ethene stators are reported as AIE luminogens (AIEgens) for the first time, and the corresponding photophysical properties and aggregated confirmation have been detailed for the demonstration of the AIE effect. A water-soluble TADVS with a quinolinium salt was prepared for cell imaging.
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Differentiation between similarly reactive sites in molecules represents an ongoing challenge of organic synthesis. Herein we described one kind of versatile reagents, N-thiohydroxy succinimide esters (NTSEs), serving as both acyl and acylthio surrogates for the diverse synthesis of ketones, thioesters, amides, and acyl disulfides by selective cleavage of similarly reactive C-S and N-S bonds.
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Ésteres , Succinimidas , Amidas , Ésteres/química , Indicadores y Reactivos , Cetonas/químicaRESUMEN
ß-Conglycinin, composed of three subunits (α', α and ß), is the main allergen of soy protein which can cause severe allergic reactions, such as diarrhea, decreased growth performance and even death. Among them, the ß subunit is more stable and difficult to remove, being one of the main nutritional inhibitors, which can be used to evaluate the concentration of ß-conglycinin. However, there is no effective, accurate method for its ß subunit rapid detection. Herein, we have successfully selected a high affinity ß subunit aptamer (Kd = 6.9 nM) and developed a highly sensitive aptasensor. The aptasensor displayed high specificity and the ß subunit at a concentration of 70-350 nM could be detected with a detection limit of 4.48 nM (3S/N). In addition, the recoveries of ß subunit were more than 90%, demonstrating its practical properties for complicated conditions such as food quality control and disease diagnosis, without requiring expensive and sophisticated equipment.
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Globulinas , Proteínas de Soja , Alérgenos , Antígenos de Plantas , Proteínas de Almacenamiento de Semillas , Glycine maxRESUMEN
This study aimed to elucidate the antimicrobial efficacy of carboxymethylated ß-glucan (CMG) against Staphylococcus aureus and the possible underlying mechanisms. ß-Glucan (BG) was extracted from naked barley bran, then ultrafiltrated to obtain four fractions (BG-1, BG-2, BG-3, BG-4). Five types of CMG were prepared using an amidation reaction and characterized by FTIR spectroscopy. The underlying mechanisms were studied using changes in the transmembrane electrical potential, pH gradient, and intracellular ATP, confocal laser scanning microscopy, and electron microscopy. The data showed that CMG-2 had the highest antibacterial activity. CMG-2 induced membrane permeabilization, morphological changes, and structural disruption in S. aureus cells, with the formation of blebs and microspheres, the disruption of cell walls and membranes, and cell lysis. Our findings provide insight into the antimicrobial mechanism of CMG against S. aureus, which will provide a foundation for developing CMG as a novel antibacterial agent in the food and biomedical fields.
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Antibacterianos/farmacología , Hordeum/química , Staphylococcus aureus/efectos de los fármacos , beta-Glucanos/farmacología , Antibacterianos/síntesis química , Antibacterianos/química , Pruebas de Sensibilidad Microbiana , Tamaño de la Partícula , Propiedades de Superficie , beta-Glucanos/síntesis química , beta-Glucanos/químicaRESUMEN
MicroRNAs (miRs) are endogenous non-coding RNAs that serve key functions in a wide range of biological processes, including cell growth, development, apoptosis and carcinogenesis. However, the association between miR-187 and B-cell lymphoma 6 (BCL6) has yet to be fully investigated in lymphoma cell apoptosis. The present study hypothesized that a post-translational mechanism may exist for BCL6 expression, which is regulated by miR-187 in lymphoma cells. The present study demonstrated that the expression of miR-187 in diffuse large B-cell lymphoma (DLBCL) cells was significantly decreased, and its expression was negatively correlated with BCL6 expression. It was also observed that miR-187 directly binds to the 3'-untranslated region of BCL6 mRNA and subsequently suppresses the expression of BCL6. Additionally, the induced expression of miR-187 significantly promoted DLBCL cell apoptosis in vitro. The drug sensitivity of human DLBCL SUDHL2 cells was increased following induction of miR-187 overexpression via an miR-187 mimic. In conclusion, the results of the present study suggest that the modulation of miR-187 expression in DLBCL cells may improve the sensitivity of chemotherapy through BCL6 targeting.
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APETALA2/Ethylene-Responsive Factor (AP2/ERF) gene family is plant specific transcription factor. It plays critical roles in development process, tolerance to biotic and abiotic stresses, and responses to plant hormones. However, limited data are available on the contributions of AP2/ERF gene family in barley (Hordeum vulgare L.). In the present study, 121 HvAP2/ERF genes in barley were identified by using bioinformatics methods. A total of 118 HvAP2/ERF (97.5%) genes were located on seven chromosomes. According to phylogenetic classification of AP2/ERF family in Arabidopsis, HvAP2/ERF proteins were divided into AP2 (APETALA2), RAV (Related to ABI3/VP), DREB (dehydration responsive element binding), ERF (ethylene responsive factors) and soloist sub families. The analysis of duplication events indicated that tandem repeat and segmental duplication contributed to the expansion of the AP2/ERF family in barley. HvDREB1s/2s genes displayed various expression patterns under abiotic stress and phytohormone. Taken together, the data generated in this study will be useful for genome-wide analysis to determine the precise role of the HvAP2/ERF gene during barley development, abiotic stress and phytohormone responses with the ultimate goal of improving crop production.