RESUMO
BACKGROUND: ß-Xylosidases are glycoside hydrolases (GHs) that cleave xylooligosaccharides and/or xylobiose into shorter oligosaccharides and xylose. Aspergillus nidulans is an established genetic model and good source of carbohydrate-active enzymes (CAZymes). Most fungal enzymes are N-glycosylated, which influences their secretion, stability, activity, signalization, and protease protection. A greater understanding of the N-glycosylation process would contribute to better address the current bottlenecks in obtaining high secretion yields of fungal proteins for industrial applications. RESULTS: In this study, BxlB-a highly secreted GH3 ß-xylosidase from A. nidulans, presenting high activity and several N-glycosylation sites-was selected for N-glycosylation engineering. Several glycomutants were designed to investigate the influence of N-glycans on BxlB secretion and function. The non-glycosylated mutant (BxlBnon-glyc) showed similar levels of enzyme secretion and activity compared to the wild-type (BxlBwt), while a partially glycosylated mutant (BxlBN1;5;7) exhibited increased activity. Additionally, there was no enzyme secretion in the mutant in which the N-glycosylation context was changed by the introduction of four new N-glycosylation sites (BxlBCC), despite the high transcript levels. BxlBwt, BxlBnon-glyc, and BxlBN1;5;7 formed similar secondary structures, though the mutants had lower melting temperatures compared to the wild type. Six additional glycomutants were designed based on BxlBN1;5;7, to better understand its increased activity. Among them, the two glycomutants which maintained only two N-glycosylation sites each (BxlBN1;5 and BxlBN5;7) showed improved catalytic efficiency, whereas the other four mutants' catalytic efficiencies were reduced. The N-glycosylation site N5 is important for improved BxlB catalytic efficiency, but needs to be complemented by N1 and/or N7. Molecular dynamics simulations of BxlBnon-glyc and BxlBN1;5 reveals that the mobility pattern of structural elements in the vicinity of the catalytic pocket changes upon N1 and N5 N-glycosylation sites, enhancing substrate binding properties which may underlie the observed differences in catalytic efficiency between BxlBnon-glyc and BxlBN1;5. CONCLUSIONS: This study demonstrates the influence of N-glycosylation on A. nidulans BxlB production and function, reinforcing that protein glycoengineering is a promising tool for enhancing thermal stability, secretion, and enzymatic activity. Our report may also support biotechnological applications for N-glycosylation modification of other CAZymes.
RESUMO
Several epidemiological and experimental studies demonstrate that modulation of inflammatory response and oxidative stress by natural phytochemicals is a promising strategy to prevent and treat many chronic inflammatory diseases. Camu-camu is an Amazonian fruit with a high content of antioxidants, especially phenolic compounds and vitamin C. In the present study we evaluated the in vivo effects of chronic ingestion of raw extracts derived from camu-camu (Myrciaria dubia McVaugh) frozen pulp on plasma lipid profile and oxidative stress in streptozotocin-induced diabetic rats. Oral administration of camu-camu raw extracts significantly increased plasma antioxidant activity, reduced triacylglycerol and total cholesterol and lipid peroxidation in the plasma of streptozotocin-induced diabetic rats. However, no effect was observed on glucose metabolism of diabetic rats, probably due to the severity of this model.
RESUMO
Foods provide essential and bioactive compounds with health-promoting properties such as antioxidant, anti-inflammatory, and hypocholesterolemic activities, which have been related to vitamins A, C, and E and phenolic compounds such as flavonoids. Therefore, the aim of this work was to identify potential sources of bioactive compounds through the determination of flavonoids and ellagic acid contents and the in vitro antioxidant capacity and alpha-glucosidase and alpha-amylase inhibitory activities of Brazilian native fruits and commercial frozen pulps. Camu-camu, cambuci, uxi, and tucuma and commercial frozen pulps of cambuci, cagaita, coquinho azedo, and araca presented the highest antioxidant capacities. Cambuci and cagaita exhibited the highest alpha-glucosidase and alpha-amylase inhibitory activities. Quercetin and kaempferol derivatives were the main flavonoids present in most of the samples. Ellagic acid was detected only in umbu, camu-camu, cagaita, araca, and cambuci. According to the results, native Brazilian fruits can be considered as excellent sources of bioactive compounds.