RESUMEN
Combining nanomaterials in varying morphology and functionalities gives rise to a new class of composite materials leading to innovative applications. In this study, we designed a heterostructured hybrid material consisting of two-dimensional bismuth nanosheets augmented by molecularly imprinted networks. Antibiotic overuse is now one of the main concerns in health management, and their monitoring is highly desirable but challenging. So, for this purpose, the resulting composite interface was used as a transducer for quartz crystal microbalances. The main objective was to develop highly selective mass-sensitive sensor for chloramphenicol. Morphological investigation revealed the presence of ultrathin, square shaped nanosheets, 2-3â¯nm in height and further supplemented by imprinted polymers. Sensor responses are described as the decrease in the frequency of microbalances owing to chloramphenicol re-binding in the templated cavities, yielding a detection limit down to 0.74 µM. This sensor demonstrated a 100 % specific detection of chloramphenicol over its interfering and structural analogs (clindamycin, thiamphenicol, and florfenicol). This composite interface offers the advantage of selective binding and excellent sensitivity due to special heterostructured morphology, in addition to benefits of robustness and online monitoring. The results suggest that such composite-based sensors can be favorable platforms, especially for commercial prospects, to obtain selective detection of other biomolecules of clinical importance.
Asunto(s)
Materiales Biomiméticos/química , Bismuto/química , Cloranfenicol/análisis , Nanoestructuras/química , Polímeros/química , Compuestos de Tungsteno/química , Impresión Molecular , Tamaño de la Partícula , Tecnicas de Microbalanza del Cristal de Cuarzo , Propiedades de SuperficieRESUMEN
Chemical modification of carboxyl groups of glucoamylase from a mesophilic fungus, Fusarium solani, was carried out using ethylenediamine as nucleophile in the presence of water-soluble 1-ethyl-3(3-dimethylaminopropyl)carbodiimide. Modification brought about a dramatic enhancement of catalytic activity and thermal stability of glucoamylase. Temperature and pH optima of ethylenediamine-coupled glucoamylase (ECG) increased as compared with those of native enzyme. The specificity constant (k(cat)/K(m)) of native, ECG-2, ECG-11, and ECG-17 was 136, 173, 225, and 170, respectively, at 55 degrees C. The enthalpy of activation (Delta H*) and free energy of activation (Delta G*) for soluble starch hydrolysis were lower for the chemically modified forms. All of the modified forms were stable at higher temperatures and possessed high Delta G* against thermal unfolding. The effects of alpha-chymotrypsin and subtilisin on the modified forms were activating as compared with native. Moreover, denaturation of ECG-2, ECG-11, and ECG-17 in urea at 4 mol x L(-1) also showed an activation trend. A possible explanation for the thermal denaturation of native and increased thermal stability of ECG-2, ECG-11, and ECG-17 at higher temperatures is also discussed.
Asunto(s)
Fusarium/enzimología , Glucano 1,4-alfa-Glucosidasa/metabolismo , Sitios de Unión , Activación Enzimática , Estabilidad de Enzimas , Glucano 1,4-alfa-Glucosidasa/química , Concentración de Iones de Hidrógeno , Temperatura , TermodinámicaRESUMEN
Purified glucoamylase (GA) from Fusarium solani was chemically modified by cross-linking with aniline hydrochloride in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) for 1 [aniline-coupled glucoamylase-1 (ACG-1)], 7 (ACG-7), and 13 min (ACG-13). The aniline coupling of GA had a profound enhancing effect on temperature, pH optima, and pK (a)'s of active site residues. The specificity constants (K (cat)/K (m)) of native, ACG-1, ACG-7, and ACG-13 were 136, 244, 262, and 208 at 55 degrees C for starch, respectively. The enthalpy of activation (DeltaH*) and free energy of activation (DeltaG*) for soluble starch hydrolysis were lower for the chemically modified forms compared to native GA. Proteolysis of ACGs by alpha-chymotrypsin and subtilisin resulted in activation.
Asunto(s)
Compuestos de Anilina/metabolismo , Fusarium/enzimología , Glucano 1,4-alfa-Glucosidasa/metabolismo , Termodinámica , Compuestos de Anilina/química , Glucano 1,4-alfa-Glucosidasa/química , Glucano 1,4-alfa-Glucosidasa/aislamiento & purificación , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Péptido Hidrolasas/metabolismo , Almidón/metabolismo , TemperaturaRESUMEN
We wish to report the attainment of the highest ever T(opt) by introducing approximately two aromatic rings through chemical modification of surface carboxyl groups in carboxymethylcellulase from Scopulariopsis sp. with concomitant decrease in V(max), K(m), and optimum pH! This extraordinary enhancement in thermophilicity of aniline-coupled CMCase (T(opt) = 122 degrees C) by a margin of 73 degrees C as compared with the native enzyme (T(opt) = 49 degrees C) is the highest reported for any mesophilic enzyme that has been modified either through chemical modification or site-directed mutagenesis. It is also reported for the first time that aniline coupled CMCase (ACC) is simultaneously thermostable in aqueous as well as water-miscible organic solvents. The T(opt) of native CMCase and ACC were 25 and 90 degrees C, respectively, in 40% (v/v) aqueous dioxan. The modified enzyme was also stabilized against irreversible thermal denaturation. Therefore, at 55 degrees C, ACC had a half-life of 136 min as compared with native CMCase whose half-life was only 5 min. We believe that the reasons for this elevated thermostability and thermophilicity are surface aromatic-aromatic interactions and aromatic interactions with the sugar backbone of the substrate, respectively.