RESUMO
OBJECTIVES: Keratinases are proteolytic enzymes that emerge as an alternative for dealing with the disposal of chicken feathers. In this study, we aimed to investigate the keratin-degrading enzymes secreted by the fungus Coriolopsis byrsina and their partial biochemical characterization to adapt their use for keratin decomposition, detergent additive applications, and collagen degradation. RESULTS: We observed the secretion of different proteolytic enzymes that possessed caseinolytic activity that peaked at pH 7.0-9.0 and 60-70 °C and at pH 10.5 and 55-60 °C, and keratinolytic activity that reached a maximum at pH 7.0-7.5 and 40-55 ºC and at pH 9.0 and 55 °C. Keratinolytic activity was maintained at approximately 63% of residual activity for 1 h at 50 °C. The caseinolytic activity at pH 10.5 remains stable until 1 h at 50 °C, and this is in contrast to the activity at pH 8.5, where the residual activity was 50%. Caseinolytic activity was inhibited only by PMSF, while keratinolytic activity was inhibited by PMSF and EDTA. When investigating the application of C. byrsina peptidases as an additive to commercial detergent, we observed an egg stain removal performance that was similar to that demonstrated by the commercial detergent. CONCLUSIONS: Based on their activity and stability at alkaline pH, these enzymes appear to be attractive candidates for use in the detergent industry. Additionally, the collagenolytic activity of these enzymes potentially allows for their use in a wide array of industrial sectors that require collagenolytic enzymes, such as for the production of collagen hydrolysates from residues derived from the meat industry.
Assuntos
Plumas/química , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Polyporaceae/crescimento & desenvolvimento , Animais , Técnicas de Cultura Celular por Lotes , Caseínas/química , Estabilidade Enzimática , Fermentação , Proteínas Fúngicas/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Polyporaceae/enzimologia , TêxteisRESUMO
In this paper, we present the responses of the white-rot fungus Perenniporia medulla-panis to iron availability with regard to alterations in growth, expression of cellular proteins, Fe3+-reducing activity, and Fe3+ chelators production. Iron supplementation stimulated fungal growth but did not result in a significant increase in biomass production. Catechol and hydroxamate derivatives were produced mainly under iron deficiency, and their productions were repressed under iron supplementation conditions. Perenniporia medulla-panis showed several cellular proteins in the range of 10-90 kDa. Some of them showed negative iron-regulation. Iron-supplemented medium also repressed both cell surface and extracellular Fe3+-reducing activities; however, the highest cell surface activity was detected at the initial growth phase, whereas extracellular activity increased throughout the incubation period. No significant production of chelators and extracellular Fe3+-reducing activity were observed within the initial growth phase, suggesting that the reduction of Fe3+ to Fe2+ is performed by ferrireductases.
Assuntos
Compostos Férricos/metabolismo , Ferro/metabolismo , Polyporaceae/enzimologia , Polyporaceae/crescimento & desenvolvimento , FMN Redutase/metabolismo , Compostos Férricos/química , Compostos Ferrosos/química , Compostos Ferrosos/metabolismo , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Quelantes de Ferro/metabolismo , Oxirredução , Polyporaceae/metabolismoRESUMO
A laccase from Pycnoporus sanguineus was purified by two steps using phenyl-Sepharose columm. A typical procedure provided 54.1-fold purification, with a yield of 8.37%, using syringaldazine as substrate. The molecular weight of the purified laccase was 69 and 68 kDa as estimated by 12% (w/v) SDS-PAGE gel and by gel filtration, respectively. The K (m) values for the substrates ABTS, syringaldazine, and guaiacol were 58, 8.3, and 370 muM, respectively. The enzyme's pH optimum for syringaldazine was 4.2 and optimal activity was 50 degrees C. The enzyme showed to be thermostable because when kept at 50 degrees C for 24 and 48 h it retained 93 and 76% activity. This laccase was inhibited by L: -cysteine, beta-mercaptoethanol, NaN(3), NaF, and HgCl(2).
Assuntos
Cromatografia/métodos , Interações Hidrofóbicas e Hidrofílicas , Lacase/isolamento & purificação , Polyporaceae/enzimologia , Biotecnologia , Meios de Cultura , Estabilidade Enzimática , Temperatura Alta , Concentração de Íons de Hidrogênio , Cinética , Lacase/química , Lacase/metabolismo , Polyporaceae/crescimento & desenvolvimento , Especificidade por Substrato , TemperaturaRESUMO
A Box-Wilson central composite design was applied to optimize copper, veratryl alcohol and l-asparagine concentrations for Trametes trogii (BAFC 212) ligninolytic enzyme production in submerged fermentation. Decolorization of different dyes (xylidine, malachite green, and anthraquinone blue) by the ligninolytic fluids from the cultures was compared. The addition of copper stimulated laccase and glyoxal oxidase production, but this response was influenced by the medium N-concentration, with improvement higher at low N-levels. The medium that supported the highest ligninolytic production (22.75 U/ml laccase, 0.34 U/ml manganese peroxidase, and 0.20 U/ml glyoxal oxidase) also showed the greatest ability to decolorize the dyes. Only glyoxal oxidase activity limited biodecoloration efficiency, suggesting the involvement of peroxidases in the process. The addition of 1-hydroxybenzotriazole (a known laccase mediator) to the ligninolytic fluids increased both their range and rate of decolorization. The cell-free supernatant did not decolorize xylidine, poly R-478, azure B, and malachite green as efficiently as the whole broth, but results were similar in the case of indigo carmine and remazol brilliant blue R. This indicates that the mycelial biomass may supply other intracellular or mycelial-bound enzymes, or factors necessary for the catalytic cycle of the enzymes. It also implies that this fungus implements different strategies to degrade dyes with diverse chemical structures.
Assuntos
Oxirredutases do Álcool/metabolismo , Corantes/metabolismo , Microbiologia Industrial/métodos , Lacase/metabolismo , Lignina/metabolismo , Polyporaceae/enzimologia , Meios de Cultura , Fermentação , Proteínas Fúngicas/metabolismo , Polyporaceae/crescimento & desenvolvimentoRESUMO
Trametes trogii was grown in a liquid synthetic medium containing different carbon and nitrogen sources. Enzymatic activities of cellulases (endoglucanase, exoglucanase and beta-glucosidase) were measured in culture supernatants. Organic nitrogen sources were the most favourable for growth and cellulase production. Increasing nitrogen concentrations also increased cellulase production. Among carbon sources, crystalline cellulose, cellobiose and a mixture of carboxymethylcellulose and cellobiose induced maximal endoglucanase production. The optimal concentration of the carbon source was 10 g/l.
Assuntos
Celulase/metabolismo , Polyporaceae/enzimologia , beta-Glucosidase/metabolismo , Carbono/administração & dosagem , Carbono/metabolismo , Meios de Cultura , Relação Dose-Resposta a Droga , Glucana 1,3-beta-Glucosidase , Nitrogênio/administração & dosagem , Nitrogênio/metabolismo , Polyporaceae/crescimento & desenvolvimentoRESUMO
The effect of ethanol and tunicamycin on synthesis and secretion of galactose oxidase was studied in resting cells of Dactylium dendroides. Ethanol promoted an overall decrease in both intra- and extracellular enzyme levels to the same extent that it inhibited [14C]glucosamine incorporation into total protein. The carbohydrate content of the intracellular enzyme was also depressed (44%) with a simultaneous decrease in O-Ser linked oligosaccharides. The intracellular galactose oxidase obtained after exposure of mycelia to ethanol plus tunicamycin lost 86% of its carbohydrate moieties, whereas the extracellular form lost only 35%. In both cases, residual sugar moieties were not eliminated by mild alkaline treatment. These data suggest that ethanol affects O-glycosylation of galactose oxidase. O-Underglycosylation did not affect the S0.5 values for galactose but diminished the molar catalytic activity. The absence of O-Ser/Thr-linked saccharides turned the intracellular enzyme into a form more susceptible to proteolysis than that devoid of N-linked sugars (tunicamycin-treated). O-Underglycosylation had a significant effect on the renaturation-reactivation of the enzyme after denaturation with 2.4 M Gdn-HCl.
Assuntos
Basidiomycota/enzimologia , Etanol/farmacologia , Galactose Oxidase/biossíntese , Glucosamina/metabolismo , Polyporaceae/enzimologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Galactose Oxidase/genética , Glicosilação , Cinética , Leucina/metabolismo , Polyporaceae/efeitos dos fármacos , Polyporaceae/crescimento & desenvolvimento , Tunicamicina/farmacologiaRESUMO
The purpose of the present study was to determine whether incubation of the edible mushroom Pleurotus ostreatus in barley straw for 45 or 60 days, proved to be a means of increasing the nutritive value and digestibility of the straw for ruminant animals. In this respect, the following determinations were performed in untreated barley straw (control), and in incubated barley straw with the mushroom strain mentioned previously, for 45 or 60 days: pH, moisture, crude protein, ash, hemicellulose, cellulose, lignin, gross energy and in vitro digestibility of the dry matter. Results showed that crude protein percentage remained constant (p less than or equal to 0.05) in all treatments (means 2.67%), increasing the ash content of the straw incubated for 60 days. The hemicellulose and cellulose percentages diminished significantly (p less than or equal to 0.05) in the straw incubated for 45 or 60 days (16.74, 32.24, 17.43, 32.41% respectively) than in the control straw (24.54, 40.15%). The lignin percentages increased, although not significantly in the straw incubated for 45 or 60 days with respect to the control straw (8.36; 9.10, 9.06%, respectively). Energy values were lower for the straw incubated 45 or 60 days (2.70; 2.74 Kcal/g) than for the control straw (2.80 Kcal/g), without difference in the in vitro dry matter digestibility by incubating the straw for 45 or 60 days with Pleurotus ostreatus and the control (56.04; 52.65; 53.06% respectively). It is concluded that the Pleurotus ostreatus strain used in this study was unable to delignify the straw, because of its lack of fenoloxidases, enzymes which are necessary for lignin biodegradation.(ABSTRACT TRUNCATED AT 250 WORDS)