Seleção de fungos isolados do ambiente com atividade lipolítica / Isolation and selection of lipolytic enzymes-producing fungi

Neste estudo, foram isolados e selecionados os micro-organismos produtores de enzimas lipolíticas, e foi avaliada sua ação sobre diferentes substratos. A enzima foi produzida pela fermentação semi sólida com farelo de trigo a 50% de umidade e 120 horas de incubação a 35 °C. A atividade da lipase foi medida por titulação utilizando-se um sistema de reação composto por 4 mL de tampão acetato 50 mM, pH 5,6, 1 g de substrato e 1 mL de extrato bruto enzimático. Uma unidade de atividade enzimática foi definida como a quantidade de enzima necessária para liberar 1 μmol de ácido graxo. Os substratos testados foram creme de leite, óleo de soja e gordura de coco. Foram isoladas 33 linhagens de fungos, separadas em quatro grupos (A, B, C e D) de acordo com extratos enzimáticos. Os fungos do grupo C apresentaram os maiores valores para atividade lipolítica, sendo de 10,41 e 9,49 μmol/mL no creme de leite; 8,96 e 7,51 μmol/mL no óleo de soja; e 8,52 e 8,14 μmol/mL na gordura de coco. A técnica empregada neste trabalho foi eficiente para efetuar o isolamento de fungos filamentosos com elevada atividade lipolítica. O estudo das linhagens de fungos será relevante para possíveis aplicações biotecnológicas.

Properties of a new fungal ß-Galactosidase with potential application in the dairy industry

ß-Galactosidase or ß-D-galactohydrolase (EC.3.2.1.23) is an important enzyme industrially used for the hydrolysis of lactose from milk and milk whey for several applications. Lately, the importance of this enzyme was enhanced by its galactosyltransferase activity, which is responsible for the synthesis of transgalctosylated oligosaccharides (TOS) that act as functional foods, with several beneficial effects on consumers. Penicillium simplicissimum, a strain isolated from soil, when grown in semi-solid medium showed good productivity of ß-galactosidase with galactosyltransferase activity. The optimum pH for hydrolysis was in 4.0-4.6 range and the optimum pH for galactosyltransferase activity was in the 6.0-7.0 range. The optimum temperature for hydrolysis and transferase activity was 55-60§C and 50§C, respectively, and the enzyme showed high thermostability for the hydrolytic activity. The enzyme showed a potential for several industrial applications such as removal of 67 (per cent) of the lactose from milk and 84 (per cent) of the lactose from milk whey when incubated at their original pH (4.5 and 6.34, respectively) under optimum temperature conditions. When incubated with a 40 (per cent) lactose solution in 150 mM McIlvaine buffer, pH 4.5, at 55§C the enzyme converted 86.5 (per cent) of the lactose to its component monosaccharides. When incubated with a 60 (per cent) lactose solution in the same buffer but at pH 6.5 and 50§C, the enzyme can synthetize up to 30.5 (per cent) TOS, with 39.5 (per cent) lactose and 30 (per cent) monosaccharides remaining in the preparation.

Production and action pattern of inulinase from "Aspergillus niger"-245: hydrolysis of innulin from several sources

A strain of Aspergillus niger isolated from soil samples showed great capacity to produce extracellular inulinase. Although the enzyme has been synthesized in presence of monosaccharides, sucrose and sugar cane molasse, the productivity was significantly higher (p<0.05) when the microorganism was inoculated in media formulated with dahlia extract and pure inulin, as carbon sources. With regard to the nitrogen source, the best results were obtained with casein and other sources of proteic nitrogen, comparatively to the mineral nitrogen. However, statistic significance (p<0.01) only was found between the productivity obtained in the medium prepared with casein amonium sulphate. The optimum pH of the purified enzyme for inulin hydrolysis was found between 4.0 and 4.5 and the optimum tempereature at 60ºC. When treated by 30 minutes u=in this temperature no loss of activity was observed. The enzyme showed capacity to hydrolyse sucrose, raffinose and inulin from which it liberated only fructose units showing, therefore, an exo-action mechanism. Acting on inulins from several sources, the enzyme showed larger hydrolysis speed on the polissaccharide from chicory ("Cichorium intibus"), comparatively, to the inulins from dahlia ("Dahlia pinnata") and Jerusalem astichoke ("Helianthus tuberosus") roots.

Purification and characterization of β-Fructosidase with inulinase activity from Aspergillus niger - 245

Aspergillus niger - 245, a strain isolated from soil samples showed good β-fructosidase activity when inoculated in medium formulated with dahlia extract tubers. The enzyme was purified by precipitation in ammonium sulphate and percolated in DEAE-Sephadex A-50 and CM-cellulose columns, witch showed a single peack in all the purification steps, maintaining the I/S ratio between 0.32 to, 0.39. Optimum pH for inulinase activity (I) was between 4.0 - 4.5 and for invertase activity (S) between 2.5 and 5.0. The optimum temperature was 60O.C for both activities and no loss in activity was observed when it was maintained at this temperature for 30 min. The Km value was 1.44 and 5.0, respectively, for I and S and Vm value 10.48 and 30.55, respectively. The I activity was strongly inhibited by Hg2+ and Ag+ and 2 x 10-3 M of glucose, but not by fructose at the same concentration. The enzyme showed an exo-action mechanism, acting on the inulin of different origins. In assay conditions total hydrolysis of all the frutans was obtained, although it has shown larger activity on the chicory inulin than that one from artichoke Jerusalem and dahlia, in the first 30 min. The obtained results suggested that the enzyme presented good potential for industrial application in the preparing the fructose syrups.

Aspergillus niger - 245, isolado do solo mostrou boa atividade de b-frutosidase meio formulado com extrato de tubérculos de dahlia. A enzima foi purificada por precipitação em sulfato de amônia e percolada em colunas de DEAE-Sephadex A-50 e CM-celulose, produzindo um único pico em todas as fases de purificação e mantendo a relação I/S entre 0,32 a 0,39. O pH ótimo para a atividade de inulinase (I) foi encontrado entre 4,0 - 4.5 e para a atividade de invertase (S) em 2,5 e 5,0. A temperatura ótima foi de 60O.C para ambas as atividades e nenhuma perda foi observada quando mantida nesta temperatura por 30 min. Os valores de Km foram de 1,44 e 5,0, respectivamente, para I e S e os valores de Vm de 10,48 e 30,55, respectivamente. A atividade I foi fortemente inibida por Hg2+, Ag+ e 2 x 10-3 M de glicose, mas não por frutose na mesma concentração. A enzima mostrou um mecanismo de exo-ação, atuando sobre a inulina de diferentes origens. Em condições de ensaio foi obtida hidrólise total de frutanas, apesar de ter mostrado maior atividade sobre a inulina de chicória que sobre as de alcachofra de Jesrusalém e dahlia, nos primeiros 30 minutos de reação. Os resultados obtidos sugerem que a enzima apresenta bom potencial para aplicações industriais na preparação de xaropes de frutose.

Produçäo e caracterizaçäo de alfa-amilase produzida por Rhizopus sp / Production and characterization of alfa-amilase from Rhizopus sp

A strain of Rhizopus sp. screened among more than 800 filamentous fungi showed great ability to produce a thermostable alfa-amylase by solid state fermentation. The best production was obtained with a bran moisture content of 40 per cent when the enzyme activity reached 60 EU/g. of medium. During the purification procedures, a column of DEAE- Sephadex A-50 separeted the enzyme in two fractions and the larger (85 per cent of the total activity) showed optimum pH in a range from 4.0 to 5.6. Optimum temperature was found at 60-65§ C and in this range no loss of activity was observed after 60 min. of treatment in pH 5,0. Its Km and Vm are, respectively, of 5.0 mg/ml of starch and 10,01 uMol of reducing sugar/min./mg. of protein. Its molecular weight was calculated in 64.000 by gel filtration in Sephadex G-200. The dextrinization power of the enzyme was observed preferentialy on substrates compound by chain with higher ramifications, traht is: amylopectin > starch> amylose. Other aspects of the enzyme pattern action are also discussed