RESUMO
Two experiments were performed to evaluate the hematological and blood biochemistry parameters, biometry of digestive organs, enzyme activities, protein content and absolute weight of the pancreas of broilers fed pre-starter and pre-starter diets supplemented or not with amylase from Aspergillus awamori. In total, 120 male Cobb chicks were housed in heated cages in each experiment. A completely randomized experimental design, with two treatments (feed with and without amylase) and six replicates per treatment of 10 birds each was applied. The data were subjected to analysis of variance using the F-test at 5% probability level. The dietary amylase addition did not affect hematological and blood biochemistry parameters and the biometry of the gastrointestinal tract of 7- and 21-d-old broilers, nor the absolute weight, enzyme activities or protein concentration of the pancreas of 7-d-old broilers. However, the inclusion of amylase in the diet reduced amylase activity and pancreatic protein concentration in 21-d-old broilers. The application of amylase to broiler chicken pre-starter and starter feeds is not justified given the pancreatic amylase activity and protein concentrations.(AU)
Assuntos
Animais , Galinhas/crescimento & desenvolvimento , Dieta/veterinária , Sangue , Aditivos Alimentares/uso terapêutico , Suplementos Nutricionais/análise , Fenômenos Fisiológicos da Nutrição Animal/fisiologia , Aspergillus , Trato Gastrointestinal/fisiologia , Criação de Animais Domésticos/métodos , Fenômenos Químicos , Testes Hematológicos/veterináriaRESUMO
Two experiments were performed to evaluate the hematological and blood biochemistry parameters, biometry of digestive organs, enzyme activities, protein content and absolute weight of the pancreas of broilers fed pre-starter and pre-starter diets supplemented or not with amylase from Aspergillus awamori. In total, 120 male Cobb chicks were housed in heated cages in each experiment. A completely randomized experimental design, with two treatments (feed with and without amylase) and six replicates per treatment of 10 birds each was applied. The data were subjected to analysis of variance using the F-test at 5% probability level. The dietary amylase addition did not affect hematological and blood biochemistry parameters and the biometry of the gastrointestinal tract of 7- and 21-d-old broilers, nor the absolute weight, enzyme activities or protein concentration of the pancreas of 7-d-old broilers. However, the inclusion of amylase in the diet reduced amylase activity and pancreatic protein concentration in 21-d-old broilers. The application of amylase to broiler chicken pre-starter and starter feeds is not justified given the pancreatic amylase activity and protein concentrations.
Assuntos
Animais , Aditivos Alimentares/uso terapêutico , Aspergillus , Dieta/veterinária , Fenômenos Fisiológicos da Nutrição Animal/fisiologia , Galinhas/crescimento & desenvolvimento , Sangue , Suplementos Nutricionais/análise , Trato Gastrointestinal/fisiologia , Criação de Animais Domésticos/métodos , Fenômenos Químicos , Testes Hematológicos/veterináriaRESUMO
Humicola grisea var. thermoidea is a deuteromycete which secretes a large spectrum of hydrolytic enzymes when grown on lignocellulosic residues. This study focused on the heterologous expression and recombinant enzyme analysis of the major secreted cellulase when the fungus is grown on sugarcane bagasse as the sole carbon source. Cellobiohydrolase 1.2 (CBH 1.2) cDNA was cloned in Pichia pastoris under control of the AOX1 promoter. Recombinant protein (rCBH1.2) was efficiently produced and secreted as a functional enzyme, presenting a molecular mass of 47 kDa. Maximum enzyme production was achieved at 96 h, in culture medium supplemented with 1.34 % urea and 1 % yeast extract and upon induction with 1 % methanol. Recombinant enzyme exhibited optimum activity at 60 °C and pH 8, and presented a remarkable thermostability, particularly at alkaline pH. Activity was evaluated on different cellulosic substrates (carboxymethyl cellulose, filter paper, microcrystalline cellulose and 4-para-nitrophenyl ß-D-glucopyranoside). Interestingly, rCBH1.2 presented both exoglucanase and endoglucanase activities and mechanical agitation increased substrate hydrolysis. Results indicate that rCBH1.2 is a potential biocatalyst for applications in the textile industry or detergent formulation.
Assuntos
Celulose 1,4-beta-Celobiosidase/metabolismo , Celulose/metabolismo , Proteínas Fúngicas/metabolismo , Fungos Mitospóricos/metabolismo , Proteínas Recombinantes/metabolismo , Clonagem Molecular/métodos , Meios de Cultura/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Fungos Mitospóricos/enzimologia , TemperaturaRESUMO
The effect of tunicamycin, an inhibitor of protein N-glycosylation, was studied in non-growing mycelium of Trichoderma harzianum induced to secrete N-acetyl-beta-D-glucosaminidase by the addition of N-acetylglucosamine. Tunicamycin (30 microg ml(-1)) had no significant effect on growth of the fungus, or on the total protein secreted or specific activity of N-acetyl-beta-D-glucosaminidase. However, in the presence of the inhibitor an underglycosylated form of the enzyme was produced. The apparent molecular masses for this and the native enzyme were 110 and 124 kDa, respectively. Both forms of the enzyme showed the same optimum pH and temperature, but the underglycosylated form was more sensitive to inactivation by both high temperature (60 degrees C) and the proteolytic enzyme trypsin.
Assuntos
Acetilglucosaminidase/metabolismo , Antibacterianos/farmacologia , Trichoderma/efeitos dos fármacos , Tunicamicina/farmacologia , Acetilglucosamina/farmacologia , Acetilglucosaminidase/antagonistas & inibidores , Acetilglucosaminidase/química , Estabilidade Enzimática , Glicosilação , Concentração de Íons de Hidrogênio , Temperatura , Trichoderma/enzimologiaRESUMO
AIMS: The chitinolytic activity of an actinomycete, isolated from a tropical acidic ferrasol (FAO) under cerrado (savanna) vegetation, is reported. METHODS AND RESULTS: Selection of the strain was based on spot inoculation on solid colloidal chitin medium. The use of chemotaxonomic, morphological and physiological procedures placed it in the Streptomyces genus, but identification to species level could not be achieved. A protein with endochitinase activity was isolated and purified from the supernatant fluid by concentration, precipitation, hydrophobic interaction, gel filtration and adsorption procedures. The molecular size of the purified chitinase was estimated by gel filtration to be 70 kDa, and its pI was 6.1. The enzyme had temperature and pH optima of 40 degrees C and 8.0, respectively, and showed thermal (30-70 degrees C) and pH (4-9) stabilities. Antifungal activity of the selected strain was observed following in vitro experiments using growing cells, crude extract or the purified endochitinase, and by detecting growth inhibition of the tested phytopathogenic fungi. CONCLUSION: Strain Streptomyces RC 1071 could not be placed into any known species, suggesting a new taxon. The purified endochitinase presented similar molecular weight, optimum temperature and pH activity, and stability of other endochitinolytic enzymes reported in the literature. In all three in vitro experiments performed, inhibition of growth of the phytopathogenic fungi used as test organisms was observed. SIGNIFICANCE AND IMPACT OF THE STUDY: Some of the endochitinase characteristics such as thermal stability, as well as pH tolerance, are very interesting for biotechnological purposes. In addition, due to its antifungal activity, Streptomyces RC 1071 seems promising for use in biological control.
Assuntos
Quitinases/isolamento & purificação , Streptomyces/enzimologia , Aspergillus/efeitos dos fármacos , Extratos Celulares/farmacologia , Eletroforese , Estabilidade Enzimática , Temperatura Alta , Doenças das Plantas/microbiologia , Microbiologia do SoloRESUMO
The effect of carbon sources on the level of beta-1,3-glucanases in the culture filtrates of Trichoderma harzianum (Tc) was investigated. Enzyme activity was detected in all carbon sources, but highest levels were found when laminarin and purified cell walls were used. Three isoforms of beta-1,3-glucanase were produced during growth of the fungus on purified cell walls. Two isoforms were produced on chitin, chitosan, N-acetylglucosamine and laminarin, while only one was detected when the fungus was grown on cellulose and glucose. A 36-kDa beta-1,3-glucanase (GLU36) was secreted from T. harzianum (Tc) grown on all carbon sources tested as demonstrated by Western blot analysis. We found that a significant increase in the level of GLU36 in the culture filtrate follows glucose exhaustion, suggesting that this enzyme is controlled by carbon catabolite repression.
Assuntos
Regulação Fúngica da Expressão Gênica , Trichoderma/enzimologia , beta-Glucosidase/biossíntese , beta-Glucosidase/genética , Western Blotting , Carbono/metabolismo , Meios de Cultura , Eletroforese em Gel de Poliacrilamida , Glucana 1,3-beta-Glucosidase , Trichoderma/genética , Trichoderma/crescimento & desenvolvimentoRESUMO
A beta-1,3-glucanase, from culture filtrates of Trichoderma harzianum, was purified in sequential steps by gel filtration, hydrophobic interaction and ion exchange chromatography. A typical procedure provided 69-fold purification with 0.32% yield. The molecular mass of the protein was found to be approximately 29 kDa, as estimated by SDS-PAGE on a 10% slab gel. The K(M) and V(max) values for beta-1,3-glucanase, using laminarin as substrate, were 1. 72 mg ml(-1) and 3.10 U ml(-1), respectively. The pH optimum for the enzyme was pH 4.4 and maximum activity was obtained at 50 degrees C. The enzyme was strongly inhibited by HgCl(2) and SDS. These results suggest that each beta-1,3-glucanase produced by T. harzianum is different and is probably encoded by different genes.
Assuntos
Trichoderma/enzimologia , beta-Glucosidase/isolamento & purificação , beta-Glucosidase/metabolismo , Quitina/metabolismo , Meios de Cultura , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Glucana 1,3-beta-Glucosidase , Concentração de Íons de Hidrogênio , Temperatura , Trichoderma/crescimento & desenvolvimentoRESUMO
A Trichoderma sp. isolate, hereafter called T6, produces a 46-kDa endochitinase (CHIT 46) which had been shown to drastically affect in vitro the cell walls of the phytopathogens Sclerotium rolfsii and Rhizoctonia solani. We attempted to gain insight into its properties. The CHIT 46 N-terminal amino acid sequence shares a very high homology with other fungal chitinases. Western blot analysis using polyclonal antibodies anti-CHIT 46 revealed that this enzyme is immunologically distinct from other proteins produced by the same Trichoderma isolate T6, but is immunologically identical with proteins having equivalent molar mass, probably chitinases, produced by other Trichoderma spp. isolates. In addition, the antibodies revealed also that a substantial amount of this enzyme is secreted into the culture medium 2 d after the Trichoderma isolate T6 comes into contact with chitin.
Assuntos
Quitinases/química , Quitinases/farmacologia , Proteínas Fúngicas/química , Proteínas Fúngicas/farmacologia , Rhizoctonia/efeitos dos fármacos , Trichoderma/enzimologia , Animais , Western Blotting , Parede Celular/efeitos dos fármacos , Quitinases/classificação , Quitinases/genética , Quitinases/imunologia , Eletroforese em Gel de Poliacrilamida , Proteínas Fúngicas/classificação , Proteínas Fúngicas/genética , Proteínas Fúngicas/imunologia , Camundongos , Homologia de Sequência , Trichoderma/crescimento & desenvolvimentoRESUMO
The non-immunoglobulin component of human milk responsible for the inhibition of Escherichia coli cell adhesion (haemagglutination) mediated by colonisation factor antigen 1 (CFA1) was determined by chromatographic fractionation of human whey proteins with Sephadex G-200, DEAE cellulose and heparin-sepharose. Pure free secretory component (fSC) and pure lactoferrin (Lf) were isolated and both compounds inhibited the haemagglutination induced by E. coli CFA1+. The lowest concentrations of purified fSC and Lf able to inhibit the haemagglutination induced by E. coli strain TR50/3 CFA1+ were 0.06 mg/ml and 0.1 mg/ml respectively. Commercially available lactoferrin from human milk and transferrin from human serum, which has a great structural analogy to lactoferrin, also inhibited the haemagglutination. The lowest concentrations of the commercial lactoferrin and transferrin able to inhibit the haemagglutination induced by E. coli TR50/3 CFA1+ were 0.03 mg/ml and 0.4 mg/ml, respectively. These results indicate that fSC and Lf may be important non-specific defence factors against enterotoxigenic E. coli infections.