Biochemical characterization and biological properties of mycelium extracts from Lepista sordida GMA-05 and Trametes hirsuta GMA-01: new mushroom strains isolated in Brazil.

The objective of this study was to evaluate the antioxidant activity, determine and quantify the phenolic compounds and other compounds, and evaluate the cellular cytotoxicity of mycelium extracts of two new Basidiomycete mushrooms strains isolated in Brazil and identified as Lepista sordida GMA-05 and Trametes hirsuta GMA-01. Higher amounts of proteins, free amino acids, total and reducing carbohydrates, and phenolic compounds as chlorogenic, ferulic, caffeic, and gallic acids were found in extracts of T. hirsuta and L. sordida. Protocatechuic acid was found only in aqueous extracts of L. sordida. The TLC of the extracts showed the predominance of glucose and smaller amounts of xylose. It was observed through UPLC-MS higher amounts of phenolic compounds. The aqueous extract from T. hirsuta had the most noteworthy results in the antioxidant assays, especially the ABTS test. The cytotoxic activity was evaluated using two different cell lineages and showed higher toxicity for L. sordida in macrophages J774-A1. However, in Vero cells, it was 12.6-fold less toxic when compared to T. hirsuta. Thus, both mushrooms show potential as functional foods or additives, presenting phenolic content, antioxidant activity, and low cytotoxic activity in the tested cells.

Structural model and functional properties of an exo-polygalacturonase from Neosartorya glabra.

A purified exo-polygalacturonase of Neosartorya glabra (EplNg) was successfully characterized. EplNg native presented 68.2 kDa, with 32% carbohydrate content. The deglycosylated form showed 46.3 kDa and isoelectric point of 5.4. The identity of EplNg was confirmed as an exo-polygalacturonase class I (EC 3.2.1.67) using mass spectrometry and Western-Blotting. Capillary electrophoresis indicated that only galacturonic acid was released by the action of EplNg on sodium polypectate, confirming an exoenzyme character. The structural model confers that EplNg has a core formed by twisted parallel ß-sheets structure. Among twelve putative cysteines, ten were predicted to form disulfide bridges. The catalytic triad predicted is composed of Asp223, Asp245, and Asp246 aligned along with a distance in 4-5 Å, suggesting that EplNg probably does not perform the standard inverting catalytic mechanism described for the GH28 family. EplNg was active from 30 to 90 °C, with maximum activity at 65 °C, pH 5.0. The Km and Vmax determined using sodium polypectate were 6.9 mg·mL-1 and Vmax 690 µmol·min-1.mg-1, respectively. EplNg was active and stable over a wide range of pH values and temperatures, confirming the interesting properties EplNg and provide a basis for the development of the enzyme in different biotechnological processes.

Production of Omegas-6 and 9 from the Hydrolysis of Açaí and Buriti Oils by Lipase Immobilized on a Hydrophobic Support.

This paper describes a bioprocess to obtain omegas-6 and 9 from the hydrolysis of Açaí (Euterpe oleracea Martius) and Buriti (Mauritia flexuosa) oils by lipases immobilized on octyl-sepharose. For this, oils and butters were initially selected as the carbon source which resulted in higher production of lipases in Beauveria bassiana and Fusarium oxysporum cultures. The carbon source that provided secretion of lipase by B. bassiana was Açaí oil, and for F. oxysporum, Bacuri butter. Lipases obtained under these conditions were immobilized on octyl-sepharose, and both, the derivatives and the crude extracts were biochemically characterized. It was observed that the immobilization promoted an increase of stability in B. bassiana and F. oxysporum lipase activities at the given temperatures and pH. In addition, the immobilization promoted hyperactivation of B. bassiana and F. oxysporum lipase activities being 23.5 and 11.0 higher than free enzyme, respectively. The hydrolysis of Açaí and Buriti oils by the derivatives was done in a biphasic (organic/aqueous) system, and the products were quantified in RP-HPLC. The results showed the potential of these immobilized lipases to obtain omegas-6 and 9 from Brazilian natural oils. This work may improve the enzymatic methodologies for obtaining foods and drugs enriched with fatty acids.

Purification and functional properties of a novel glucoamylase activated by manganese and lead produced by Aspergillus japonicus.

Microbial amylases are used to produce ethanol, glucose and can be applied in textiles products, detergents and other industries. This study aimed to determine the best carbon source concentration to induce the amylase production by A. japonicus, and its purification and biochemical characterization. For that, this fungus was cultivated in Khanna medium, pH 5.5, for 4 days, at 25°C, in static condition, supplemented with potato starch and maltose in different concentrations. The fungal crude enzymatic extract was purified in a unique elution in DEAE-cellulose column and the molecular mass was determined as 72kDa. The optimum temperature and pH was 65°C and 5.0, respectively. Amylase remained 75% of its activity after one hour at 50°C and was stable in the pH range 3.0-7.0. The analysis of the end-products by thin layer chromatography showed only glucose formation, which characterizes the purified enzyme as a glucoamylase. Amylopectin was the best substrate for the enzyme assay and Mn+2 and Pb+2 were good glucoamylase activators. This activation, in addition to the biochemical characteristics are important results for future biotechnological applications of this glucoamylase in the recycling and deinking process by the paper industries.

Purification and partial characterization of an exo-polygalacturonase from Paecilomyces variotii liquid cultures.

An extracellular polygalacturonase (PG) produced from Paecilomyces variotii was purified to homogeneity through two chromatography steps using DEAE-Fractogel and Sephadex G-100. The molecular weight of P. variotii PG was 77,300 Da by gel filtration and SDS-PAGE. PG had isoelectric point of 4.37 and optimum pH 4.0. PG was very stable from pH 3.0 to 6.0. The extent of hydrolysis of different pectins by the purified enzyme was decreased with an increase in the degree of esterification. PG had no activity toward non-pectic polysaccharides. The apparent K(m) and V(max) values for hydrolyzing sodium polypectate were 1.84 mg/mL and 432 micromol/min/mg, respectively. PG was found to have temperature optimum at 65 degrees Celsius and was totally stable at 45 degrees Celsius for 90 min. Half-life at 55 degrees Celsius was 50.6 min. Almost all the examined metal cations showed partial inhibitory effects under enzymatic activity, except for Na(+1), K(+1), and Co(+2) (1 mM) and Cu(+2) (1 and 10 mM).

Cyclodextrin glycosyltransferase from Bacillus licheniformis: optimization of production and its properties

Cyclodextrin glycosyltransferase (EC 2.4.1.19) is an enzyme that produces cyclodextrins from starch via an intramolecular transglycosylation reaction. An alkalophilic Bacillus strain, isolated from cassava peels, was identified as Bacillus licheniformis. CGTase production by this strain was better when potato starch was used as carbon source, followed by cassava starch and amylopectin. Glucose and amylose, on the other hand, acted as synthesis repressors. When the cultivation was supplemented with sodium ions and had the pH adjusted between 6.0 and 9.0, the microorganism maintained the growth and enzyme production capacity. This data is interesting because it contradicts the concept that alkalophilic microorganisms do not grow in this pH range. After ultrafiltration-centrifugation, one protein of 85.2 kDa with CGTase activity was isolated. This protein was identified in plates with starch and phenolphthalein. Determination of the optimum temperature showed higher activities at 25°C and 55°C, indicating the possible presence of more than one CGTase in the culture filtrate. Km and Vmax values were 1.77 mg/mL and 0.0263 U/mg protein, respectively, using potato starch as substrate.

Ciclodextrina glicosiltransferase (EC 2.4.1.19) é uma enzima que produz ciclodextrinas a partir de amido via transglicosilação intramolecular. Uma cepa de Bacillus alcalofílico, isolada de cascas de mandioca, foi identificada como Bacillus licheniformis. A produção de CGTase por esta cepa foi melhor quando amido de batata foi utilizado como fonte de carbono, seguido por amido de mandioca e amilopectina. Glicose e amilose, por outro lado, atuaram como repressor de síntese desta enzima. Quando o cultivo foi suplementado com íons sódio e teve o pH ajustado entre 6,0 e 9,0, o microrganismo manteve a capacidade de crescimento e de produção da enzima. Este dado é interessante pois contraria o conceito de que microrganismos alcalofílicos não apresentam crescimento nesta faixa de pH. Após ultrafiltração-centrifugação, uma proteína de 85,2 kDa com atividade de CGTase foi isolada. Esta proteína foi identificada em placas contendo amido e fenolftaleína. A determinação da temperatura ótima mostrou atividades mais elevadas em 25°C e 55°C, indicando a possível presença de mais de uma CGTase no filtrado de cultura. Valores de Km e Vmax foram 1,77 mg/mL e 0,0263 U/mg proteína, respectivamente, usando amido de batata como substrato.