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The plant originated stilbene "resveratrol" (3,4',5-trans-trihydroxystilbene) is well known for its diverse health benefits including anti-tumor, anti-inflammatory, anti-microbial, and anti-oxidant properties. Besides a significant amount of reports on different aspects of its application as prodrug in the last 50 years, still, a strategy leading to the production of the active drug is missing. The aim of this work was to evaluate the enzymatic activation of prodrug resveratrol to the effective drug piceatannol, without engaging expensive cofactors. Five different heme proteins were analyzed for the transformation of resveratrol. Kinetic parameters of resveratrol transformation and analysis of the transformed products were conducted through HPLC and GC-MS. Effect of pH and organic solvent on the transformation process had also been evaluated. Among all tested heme proteins, only a variant of cytochrome P450BM3 from Bacillus megaterium (CYPBM3F87A) was found suitable for piceatannol production. The most suitable pH for the reaction conditions was 8.5, while organic solvents did not show any effect on transformation. For resveratrol transformation, the turnover rate (k cat) was 21.7 (± 0.6) min-1, the affinity constant (K M) showed a value of 55.7 (± 16.7) µM for a catalytic efficiency (k cat/K M) of 389 min-1 mM-1. GC-MS analysis showed that the only product from resveratrol transformation by cytochrome P450BM3 is the biologically active piceatannol. The enzymatic transformation of resveratrol, an emerging compound with medical interest, to active product piceatannol by a variant of cytochrome P450BM3 in the absence of expensive NADPH cofactor is demonstrated. This enzymatic process is economically attractive and can be scaled up to cover the increasing medical demand for piceatannol.

Assuntos

RESUMO

Chitosan, a renewable biopolymer has the prospective applications in different fields due to its gelation capacity. Nanoconfiguration of chitosan through ionotropic gelation to encapsulate enzymatic activity offers numerous potential applications. In the present study, the preparation and characterization of chitosan nanoparticles loaded with versatile peroxidase are reported. Their performance in bioremediation process and the resistance enhancement against natural microbial biodegradation were studied. The average diameter of enzymatic nanoparticles was 120nm and showed a high enzyme loading capacity. The kinetic parameters of nanoparticles exhibited a slightly lower catalytic activity (kcat), similar affinity constant (Km) for hydrogen peroxide and higher Km value for the phenolic compound when compared with the free enzyme. The enzymatic nanoparticles showed higher thermostability and the same pH activity profile than those from free enzyme. Ten phenolic compounds, including pesticides, halogenated compounds, endocrine disruptors and antibacterials were transformed by the enzymatic nanoparticles. The transformation rate was lower than those obtained with free enzyme suggesting mass transfer limitations. But very importantly, the enzymatic nanoparticles showed a significant increase of the operational stability in real conditions of wastewater treatment process. Moreover, chemical modification of nanoparticles with different aldehydes still enhanced the operational stability of nanoparticulated enzymes. This enhancement of stability in real conditions and the potential use of biocatalytic nanoparticles in bioremediation processes are discussed.

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RESUMO

A novel concept with the result of enzyme stabilization against microbial degradation in real bioremediation processes was developed through the encapsulation of laccase in chitosan nanoparticles. Besides of abundant information on laccase-chitosan conjugates, we report the laccase encapsulation into nanoparticles based in chitosan. The chitosan-tripolyphosphate technique was applied for the production of morphologically homogeneous enzymatic nanoparticles, with high enzyme encapsulation efficiency, small asymmetric sizes (from 40 to 90 nm), and rough surfaces. Contrary to macroscopic immobilized enzymes, temperature and pH activity profiles of nano-sized laccase were similar to those of free enzyme. The substrate affinity constant (K M) of nano-encapsulated laccase was similar to these from free enzyme, while its activity rate constant (k cat) represented 60 % of these obtained with free enzyme. Importantly, stability of nano-encapsulated laccase against microbial degradation in soil, compost, and wastewater was significantly increased. After 24 h exposure to wastewater from a treatment plant, the laccase activity of the nanoparticles was 82.8 % of initial activity, compared with only 7.8 % retained activity for free enzyme. After 36 h incubation in compost extract, the laccase nanoparticles showed 72.4 % of the initial activity, while the free enzyme was almost completely inactivated. Finally, after 84 h incubation in soil extract, the nanoparticles and free preparations showed 57.9 and 17.3 % of the initial activity, respectively. Thus, the nanoencapsulation of enzymes able to transform pollutants is an alternative to improve the operational lifetime of enzymes in real environmental applications.

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Morpho-anatomical features in leaves, stems and unripe fruits of Solanum pseudocapsicum L., Solanaceae, were investigated by histological methods. Anatomically the plant may be characterised by the presence of uniseriate trichomes, anomocytic stomata, calcium oxalate needles in leaves while presence of oval to circular compound starch grains, angular vessels, vertically upright, uni-biseriate rays and intraxylary phloem with differentiation of internal cambium abutting marginal pith cells and protoxylem in transverse view. Development of distinct internal cambium may be considered as a characteristic feature for S. pseudocapsicum. Intraxylary secondary phloem was composed of sieve tube elements, companion cells and axial parenchyma cells.