RESUMO
Trichoderma species are known for their ability to produce lytic enzymes, such as exoglucanases, endoglucanases, chitinases, and proteases, which play important roles in cell wall degradation of phytopathogens. ß-glucanases play crucial roles in the morphogenetic-morphological process during the development and differentiation processes in Trichoderma species, which have ß-glucans as the primary components of their cell walls. Despite the importance of glucanases in the mycoparasitism of Trichoderma spp., only a few functional analysis studies have been conducted on glucanases. In the present study, we used a functional genomics approach to investigate the functional role of the gluc31 gene, which encodes an endo-ß-1,3-glucanase belonging to the GH16 family in Trichoderma harzianum ALL42. We demonstrated that the absence of the gluc31 gene did not affect the in vivo mycoparasitism ability of mutant T. harzianum ALL42; however, gluc31 evidently influenced cell wall organization. Polymer measurements and fluorescence microscopy analyses indicated that the lack of the gluc31 gene induced a compensatory response by increasing the production of chitin and glucan polymers on the cell walls of the mutant hyphae. The mutant strain became more resistant to the fungicide benomyl compared to the parental strain. Furthermore, qRT-PCR analysis showed that the absence of gluc31 in T. harzianum resulted in the differential expression of other glycosyl hydrolases belonging to the GH16 family, because of functional redundancy among the glucanases.
Assuntos
Antibiose/genética , Parede Celular/enzimologia , Parede Celular/metabolismo , Endo-1,3(4)-beta-Glucanase/metabolismo , Trichoderma/enzimologia , Trichoderma/metabolismo , Ascomicetos/metabolismo , Benomilo/farmacologia , Parede Celular/química , Parede Celular/efeitos dos fármacos , Quitina/metabolismo , Endo-1,3(4)-beta-Glucanase/genética , Fusarium/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Genômica , Microscopia de Fluorescência , Filogenia , Rhizoctonia/metabolismo , Trichoderma/efeitos dos fármacos , Trichoderma/patogenicidade , beta-Glucanas/metabolismoRESUMO
Xyloglucan-specific endo-ß-1,4-glucanases (Xegs, EC 3.2.1.151) exhibit high catalytic specificity for ß-1,4 linkages of xyloglucan, a branched hemicellulosic polysaccharide abundant in dicot primary cell walls and present in many monocot species. In nature, GH12 Xegs are not associated with carbohydrate-binding modules (CBMs), and here, we have investigated the effect of the fusion of the xyloglucan-specific CBM44 on the structure and function of a GH12 Xeg from Aspergillus niveus (XegA). This fusion presented enhanced catalytic properties and conferred superior thermal stability on the XegA. An increased k cat (chimera, 177.03 s(-1); XegA, 144.31 s(-1)) and reduced KM (chimera, 1.30 mg mL(-1); XegA, 1.50 mg mL(-1)) resulted in a 1.3-fold increase in catalytic efficiency of the chimera over the parental XegA. Although both parental and chimeric enzymes presented catalytic optima at pH 5.5 and 60 °C, the thermostabilitiy of the chimera at 60 °C was greater than the parental XegA. Moreover, the crystallographic structure of XegA together with small-angle X-ray scattering (SAXS) and molecular dynamics simulations revealed that the spatial arrangement of the domains in the chimeric enzyme resulted in the formation of an extended binding cleft that may explain the improved kinetic properties of the CBM44-XegA chimera.
Assuntos
Aspergillus/enzimologia , Endo-1,3(4)-beta-Glucanase/química , Endo-1,3(4)-beta-Glucanase/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Glucanos/metabolismo , Xilanos/metabolismo , Sequência de Aminoácidos , Aspergillus/química , Aspergillus/genética , Endo-1,3(4)-beta-Glucanase/genética , Proteínas Fúngicas/genética , Glucanos/química , Cinética , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Engenharia de Proteínas , Estrutura Terciária de Proteína , Espalhamento a Baixo Ângulo , Especificidade por Substrato , Difração de Raios X , Xilanos/químicaRESUMO
The mangrove ecosystem is an unexplored source for biotechnological applications. In this unique environment, endemic bacteria have the ability to thrive in the harsh environmental conditions (salinity and anaerobiosis), and act in the degradation of organic matter, promoting nutrient cycles. Thus, this study aimed to assess the cellulolytic activities of bacterial groups present in the sediment from a mangrove located in Ilha do Cardoso (SP, Brazil). To optimize the isolation of cellulolytic bacteria, enrichments in two types of culture media (tryptone broth and minimum salt medium), both supplemented with 5% NaCl and 1% of cellulose, were performed. Tests conducted with the obtained colonies showed a higher occurrence of endoglycolytic activity (33 isolates) than exoglycolytic (19 isolates), and the degradation activity was shown to be modulated by the presence of NaCl. The isolated bacteria were clustered by BOX-PCR and further classified on the basis of partial 16S rRNA sequences as Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, Firmicutes or Bacteroidetes. Therefore, this study highlights the importance of studies focusing on the endemic species found in mangroves to exploit them as novel biotechnological tools for the degradation of cellulose.(AU)
Assuntos
Endo-1,3(4)-beta-Glucanase , Áreas Alagadas , Biofilmes , Tolerância ao SalRESUMO
A bifunctional enzyme has been created by fusing two Bacillus subtilis enzymes: the ß-1,3-1,4-glucanase (BglS, EC 3.2.1.73) that hydrolyzes plant cell wall ß-glucans and the copper-dependent oxidase laccase (CotA, EC 1.10.3.2) that catalyzes the oxidation of aromatic compounds with simultaneous reduction of oxygen to water. The chimeric laccase/ß-1,3-1,4-glucanase was created by insertion fusion of the bglS and cotA genes, and expressed in Escherichia coli. The affinity-purified recombinant chimeric enzyme showed both laccase and glucanase activities, with a maximum laccase activity at pH 4.5 and 75°C that showed a V(max) 30% higher than observed for the parental laccase. The maximum glucanase activity in the chimeric enzyme was at pH 6.0 and 50°C, with a slight reduction in V(max) by â¼10% compared with the parental glucanase. A decreased K(M) resulted in an overall increase in the K(cat)/K(M) value for the glucanase activity of the chimeric enzyme. The hydrolytic activity of the chimera was 20% higher against natural milled sugarcane bagasse as compared with equimolar mixtures of the separate parental enzymes. Molecular dynamics simulations indicated the approximation of the two catalytic domains in the chimeric enzyme, and the formation of an inter-domain interface may underlie the improved catalytic function.
Assuntos
Bacillus subtilis/enzimologia , Celulose/metabolismo , Endo-1,3(4)-beta-Glucanase/metabolismo , Lacase/metabolismo , Engenharia de Proteínas/métodos , Saccharum/metabolismo , Celulose/química , Endo-1,3(4)-beta-Glucanase/química , Endo-1,3(4)-beta-Glucanase/genética , Cinética , Lacase/química , Lacase/genética , Simulação de Dinâmica Molecular , Oxirredução , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharum/químicaRESUMO
The production of xylanase, -xylosidase, ferulic acid esterase and -glucosidase by Aspergillus awamori 2B.361 U2/1, a hyper producer of glucoamylase and pectinase, was evaluated using selected conditions regarding nitrogen nutrition. Submerged cultivations were carried out at 30 ºC and 200 rpm in growth media containing 30 g wheat bran/L as main carbon source and either yeast extract, ammonium sulfate, sodium nitrate or urea, as nitrogen sources, in all cases it was used a fixed molar carbon to molar nitrogen concentration of 10.3. The use of poor nitrogen sources favored the accumulation of xylanase, -xylosidase and ferulic acid esterase to a peak concentrations of 44,880, 640 and 118 U/L, respectively, for sodium nitrate and of 34,580, 685 and 170 U/L, respectively, for urea. However, the highest -glucosidase accumulation of 10,470 U/L was observed when the rich organic nitrogen source yeast extract was used. The maxima accumulation of filter paper activity, xylanase, -xylosidase, ferulic acid esterase and -glucosidase by A. awamori 2B.361 U2/1 was compared to that produced by Trichoderma reesei Rut-C30. The level of -glucosidase was over 17-fold higher for the Aspergillus strain, whereas the levels of xylanase and -xylosidase were over 2-fold higher. This strain also produced ferulic acid esterase (170 U/L), which was not detected in the T. reesei culture.(AU)
Assuntos
Aspergillus , Trichoderma , Endo-1,3(4)-beta-Glucanase , Hidrólise , XilosidasesRESUMO
This study investigated the enzymatic treatment of soy slurry using Viscozyme L to hydrolyze the carbohydrates. The optimum temperature of Viscozyme L action was 55 °C. The increase of glucose and galactose content in tofu (1.36 and 0.19 g/100 g, respectively) confirmed the Viscozyme activity on soy slurry when compared to the control. The treated tofu had more total phenolics than the control (173 and 161 mg gallic acid equivalents/100 g freeze-dried tofu, respectively) and higher antioxidant activity by the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt and 1,1-diphenyl-2-picryhydrazyl,2,2-diphenyl-1-picryhydrazyl radical tests. Total reducing sugar (glucose equivalents) content in treated tofu was approximately four times higher than that in the control under the optimum conditions (30 Fungal Beta-Glucanase units/10 g solids, 55 °C, 30 min). The tofus differed in the sensory analysis for soy odor and surface uniformity, but there was no preference for one over the other.
Assuntos
Antioxidantes/análise , Carboidratos da Dieta/análise , Endo-1,3(4)-beta-Glucanase/metabolismo , Manipulação de Alimentos/métodos , Proteínas Fúngicas/metabolismo , Complexos Multienzimáticos/metabolismo , Alimentos de Soja/análise , Antioxidantes/metabolismo , Aspergillus/enzimologia , Brasil , Fenômenos Químicos , Carboidratos da Dieta/metabolismo , Preferências Alimentares , Galactose/análise , Galactose/metabolismo , Glucose/análise , Glucose/metabolismo , Humanos , Fenômenos Mecânicos , Odorantes , Fenóis/análise , Fenóis/metabolismo , Sensação , Leite de Soja/química , Leite de Soja/metabolismo , PaladarRESUMO
Avaliou-se o efeito de diferentes níveis do composto enzimático Natugrain Blend L®, que contém endo-xilanase e endo-beta-glucanase, sobre a digestibilidade dos nutrientes e a energia do triticale pela tilápia-do-nilo. O método para a determinação da digestibilidade foi o indireto, utilizando-se o óxido de crômio III (0,10 por cento). O delineamento experimental foi inteiramente ao acaso, com cinco tratamentos e três repetições. O nível de substituição da dieta-referência foi 50,0 por cento pelo triticale. Os tratamentos foram 0,0; 150,0; 300,0; 450,0 e 600,0mg kg-1 de Natugrain Blend L, que contém 800 unidades g-1 de endo-1,3(4)-β-glucanase (BGU) e 36.600 unidades g-1 de endo-1,4-β-xylanase (EXU). Os coeficientes de digestibilidade aparente foram: da matéria seca, 76,42; 74,01; 83,39; 82,97 e 78,34 por cento; da proteína bruta 88,19; 88,39; 90,52; 92,05 e 88,34 por cento, da energia bruta 75,93; 71,31; 81,78; 80,27 e 78,62 por cento, respectivamente, para os níveis de inclusão na dieta 0,0; 150,0; 300,0; 450,0 e 600,0mg kg-1 de Natugrain Blend L.Os resultados demonstram que 300mg kg-1 do complexo de enzimas foi suficiente para aumentar o coeficiente de digestibilidade aparente da matéria seca. O composto de enzimas pode ser utilizado para aumentar a eficiência de aproveitamento dos nutrientes do triticale.(AU)
The effects of different levels of Natugrain Blend L® enzymatic compound on triticale nutrients and energy digestibility by Nile tilapia were evaluated. The digestibility was indirectly determined with chromic oxide III (0.10 percent) as an external marker. The level of substitution by triticale in the reference diet was 50 percent. The treatments were 0, 150, 300, 450, and 600g kg of Natugrain Blend L, which contains 800 units g-1 of endo-1,3(4)-β-glucanase (BGU) and 36.600 units g-1 of endo-1,4-β-xylanase (EXU). The apparent digestibility coefficients were: dry matter 76.42, 74.01, 83.39, 82.97, and 78.34 percent; crude protein 88.19, 88.39, 90.52, 92.05, and 88.34 percent; crude energy 75.93, 71.31, 81.78, 80.27, and 78.62 percent, respectively to inclusion levels of 0.0, 150.0, 300.0, 450.0, and 600.0mg kg-1 of Natugrain Blend L in diet. Results demonstrated that 300mg kg-1 of enzymes were enough to increase the dry matter apparent digestibility coefficient and energy. The enzyme compound can be used to increase the efficiency of triticale feed utilization.(AU)
Assuntos
Animais , Xilano Endo-1,3-beta-Xilosidase/administração & dosagem , Xilano Endo-1,3-beta-Xilosidase , Endo-1,3(4)-beta-Glucanase/administração & dosagem , Endo-1,3(4)-beta-Glucanase , Análise de Alimentos/métodos , Ciclídeos/fisiologia , Grão Comestível/efeitos adversosRESUMO
Trichoderma species are readily isolated from Brazilian cerrado soil by conventional methods and some of them were characterized as Trichoderma koningii. The effect of carbon source on the production of beta-1,3-glucanases in the culture filtrates of a specific Trichoderma koningii strain (ALL 13) was investigated. Enzyme activity was detected in all carbon sources tested and only one band of beta-1,3-glucanase was detected in non-denaturing PAGE. This enzyme was purified by Sephacryl S-200 gel filtration and Phenyl Sepharose CL 4B chromatography. A typical procedure provided 105-fold purification with 13.4% yield. The molecular weight of the purified enzyme was 75 kDa as estimated by SDS-PAGE. The enzyme hydrolyzed laminarin in an endo-like fashion to form small oligosaccharides and glucose. The Km and Vmax values for beta-1,3-glucanase, using laminarin as substrate, were 0.148 mg.mL-1 and 0.159 U.min-1, respectively. The pH optimum for the enzyme was pH 4.6 and maximum activity was obtained at 50 degrees C. Hg2+ inhibited the purified enzyme.