Characterization and comparison of thermostability of purified beta-glucosidases from a mesophilic Aureobasidium pullulans and a thermophilic Thermoascus aurantiacus

The thermophilic fungus Thermoascus aurantiacus 179-5 and the mesophilic Aureobasidium pullulans ER-16 were cultivated in corn-cob by solid state fermentation for P-glucosidase production. After fermentation both enzymes were purified. The beta-glucosidases produced by the strains A. pullulans and T aurantiacus were most active at pH 4.0-4.5 and 4.5, with apparent optimum temperatures at 80 and 75 degrees C, respectively. Surprisingly, the enzyme produced by the mesophilic A. pullulans was stable over a wider range of pH (4.5-9.5 against 4.5-6.5) and more thermostable (98% after 1 h at 75 degrees C against 98% after 1 h at 70 degrees C) than the enzyme from the thermophilic T. aurantiacus. The t((1/2)) at 80 degrees C were 90 and 30 min for A. pullulans and T. aurantiacus, respectively. beta-Glucosidase thermoinactivation followed first-order kinetics and the energies of denaturation were 414 and 537 kJ mol(-1) for T. aurantiacus and A. pullulans, respectively. The result showed that beta-glucosidase obtained from the mesophilic A. pullulans is more stable than that obtained from the thermophilic T. aurantiacus. (C) 2007 Elsevier Ltd. All rights reserved.

Pectin methylesterase activity and ascorbic acid content from guava fruit, cv. Predilecta, in different phases of development

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Screening of filamentous fungi for production of enzymes of biotechnological interest

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Meta-análise da digestibilidade ileal de aminoácidos e minerais em suínos alimentados com dietas contendo enzimas

O objetivo deste trabalho foi avaliar, por meio da meta-análise, o efeito da fitase e da xilanase sobre a digestibilidade ileal aparente (DIa) de aminoácidos, cálcio e fósforo, em suínos em fase de crescimento. A base de dados consistiu de 21 artigos publicados entre 1998 e 2009, no total de 82 tratamentos e 644 suínos. A meta-análise foi realizada por análise gráfica, de correlação, de variância-covariância. As concentrações de fósforo fítico e as frações fibra em detergente neutro, fibra em detergente ácido e lignina em detergente ácido, nas dietas, apresentaram correlações baixas e negativas com a DIa do cálcio, fósforo e aminoácidos. A adição de fitase às dietas aumentou em 2% a DIa da arginina, em 14% a do cálcio e em 34% a do fósforo. A DIa da arginina, fenilalanina, isoleucina e lisina foi 3,3% superior em suínos alimentados com dietas com xilanase, em relação às dietas sem a enzima. O fósforo fítico e as fibras, nas dietas, reduzem a DIa do cálcio, do fósforo e dos aminoácidos essenciais. O uso de fitase e xilanase, nas dietas, melhora o aproveitamento de cálcio, fósforo e alguns aminoácidos. No entanto, o excesso de cálcio e fósforo nas dietas reduz a ação da fitase sobre a digestibilidade ileal dos nutrientes.

Xilanase e β-glucanase na digestibilidade aparente de nutrientes do triticale pela Tilápia-do-nilo

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%). O delineamento experimental foi inteiramente ao acaso, com cinco tratamentos e três repetições. O nível de substituição da dieta-referência foi 50,0% 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%; da proteína bruta 88,19; 88,39; 90,52; 92,05 e 88,34%, da energia bruta 75,93; 71,31; 81,78; 80,27 e 78,62%, 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.

Xylanases from fungi: properties and industrial applications

Xylan is the principal type of hemicellulose. It is a linear polymer of beta-D-xylopyranosyl units linked by (1-4) glycosidic bonds. In nature, the polysaccharide backbone may be added to 4-O-methyl-alpha-D-glucuronopyranosyl units, acetyl groups, alpha-L-arabinofuranosyl, etc., in variable proportions. An enzymatic complex is responsible for the hydrolysis of xylan, but the main enzymes involved are endo-1,4-beta-xylanase and beta-xylosidase. These enzymes are produced by fungi, bacteria, yeast, marine algae, protozoans, snails, crustaceans, insect, seeds, etc., but the principal commercial source is filamentous fungi. Recently, there has been much industrial interest in xylan and its hydrolytic enzymatic complex, as a supplement in animal feed, for the manufacture of bread, food and drinks, textiles, bleaching of cellulose pulp, ethanol and xylitol production. This review describes some properties of xylan and its metabolism, as well as the biochemical properties of xylanases and their commercial applications.

Purification and biochemical characterization of an endoxylanase from Aspergillus versicolor

An endoxylanase (beta-1,4-xylan xylanohydrolase, EC 3.2.1.8) was purified from the culture filtrate of a strain of Aspergillus versicolor grown on oat wheat. The enzyme was purified to homogeneity by chromatography on DEAE-cellulose and Sephadex G-75. The purified enzyme was a monomer of molecular mass estimated to be 19 kDa by SDS-PAGE and gel filtration. The enzyme was glycoprotein with 71% carbohydrate content and exhibited a pI of 5.4. The purified xylanase was specific for xylan hydrolysis. The enzyme had a K-m of 6.5 mg ml(-1) and a V-max of 1440 U (mg protein)(-1). (C) 1998 Federation of European Microbiological Societies. Published by Elsevier B.V. B.V. All rights reserved.

Influence of temperature on the properties of the xylanolytic enzymes of the thermotolerant fungus Aspergillus phoenicis

This study reports on the effects of growth temperature on the secretion and some properties of the xylanase and beta-xylosidase activities produced by a thermotolerant Aspergillus phoenicis. Marked differences were observed when the organism was grown on xylan-supplemented medium at 25 degreesC or 42 degreesC. Production of xylanolytic enzymes reached maximum levels after 72 h of growth at 42 degreesC; and levels were three- to five-fold higher than at 25 degreesC. Secretion of xylanase and beta-xylosidase was also strongly stimulated at the higher temperature. The optimal temperature was 85 degreesC for extracellular and 90 degreesC for intracellular beta-xylosidase activity, independent of the growth temperature. The optimum temperature for extracellular xylanase increased from 50 degreesC to 55 degreesC when the fungus was cultivated at 42 degreesC. At the higher temperature, the xylanolytic enzymes produced by A. phoenicis showed increased thermo stability, with changes in the profiles of pH optima. The chromatographic profiles were distinct when samples obtained from cultures grown at different temperatures were eluted from DEAE-cellulose and Biogel P-60 columns.

Partial purification, heat stability and kinetic characterization of the pectinmethylesterase from Brazilian guava, Paluma cultivars

Pectinmethylesterase (PME) was extracted from guava fruit (Psidium guajava L.), cultivar Paluma, by 70% ammonium sulphate saturation and partially purified by gel filtration on Sephadex G100. Gel filtration showed PME isoenzymes with different values of molecular mass. Two samples were examined: concPME (70% saturation by ammonium sulphate) and Iso4 PME (one of the isoforms from gel filtration with the greatest specific activity). Optimum pH of the enzyme (for both samples) was 8.5 and optimum temperature ranged from 75 and 85 degrees C. The optimum sodium chloride concentration was 0.15 M. The K-M and V-max ranged from 0.32 to 0.23 mg m1(-1) and 244 to 53.2 mu mol/min, respectively, for concPME and Iso4PME. The activation energies (E-a) were 64.5 and 103 kJ/mol, respectively, for concPME and Iso4PME. Guava PME, cv Paluma, is a very thermostable enzyme, showing great heat stability at all temperatures studied. (c) 2005 Elsevier Ltd. All rights reserved.

Production and characterization of alpha-amylase from Rhizopus sp

A strain of Rhizopus sp. screened among more than 800 filamentous fungi showed great ability to produce a thermostable alpha-amylase by solid state fermentation. The best production was obtained with a bran moisture content of 40% when the enzyme activity reached 60 EU/g. of medium. During the purification procedures, a column of DEAE-Sephadex A-50 separated the enzyme in two fractions and the larger (85% of the total activity) showed optimum pH in a range from 4.0 to 5.6. Optimum temperature was found at 60-65 degrees C and in this range no loss of activity was observed after 60 min. of treatment in pH 5,0. Its K-m and V-m 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 chains with higher ramifications, that is: amylopectin > starch > amylose. Other aspects of the enzyme pattern action are also discussed.

Improvement of Aspergillus niger glucoamylase thermostability by directed evolution

Directed evolution was used to improve the thermostability of Aspergillus niger glucoamylase (GA) expressed in Saccharomyces cerevisiae. A starch-plate assay developed to screen GA mutants for thermostability gave results consistent with those of irreversible thermoinactivation kinetic analysis. Several thermostable multiply-mutated GAs were isolated and characterized by DNA sequencing and kinetic analysis. Three new GA mutations, T62A, T290A and H391Y, have been identified that encode GAs that are more thermostable than wild-type GA, and that improve thermostability cumulatively. These individual mutations were combined with the previously constructed thermostable site-directed mutations D20C/A27C (forming a disulficle bond), S30P, and G137A to create a multiply-mutated GA designated THS8. THS8 GA is substantially more thermostable than wild-type GA at 8OoC, with a 5.1 kJ/mol increase in the free energy of therrnoinactivation, making it the most thermostable Aspergillus niger GA mutant characterized to date. THS8 GA and the singly-mutated GAs have specific activities and catalytic efficiencies (k(cat)/K-m) similar to those of wild-type GA.

Role of the surface state and structural feature in the thermoreversible sol-gel transition of a zirconyl aqueous precursor modified by sulfuric acid

The sols produced by admixture of ZrOCl2 acidified solutions to hot H2SO4 aqueous solutions were studied to clarify the effects of Cl- and SO42- ions on the kinetic stability of nanoparticles and to obtain some new evidence concerning the mechanism of a thermoreversible sol-gel transition observed in this system. The study of suspensions prepared with different molar ratios R-S = [Zr]/[SO42-] and R-Cl = [Zr]/[Cl-] revealed domains of composition of formation of thermoreversible gels, thermostable sols, and powder precipitation. The effects of R-S and R-Cl on the structural features of nanoparticles and on the particle solution interface were systematically analyzed for samples of thermoreversible and thermostable sol domains. Small-angle X-ray scattering measurements revealed the presence of small fractal aggregates in all samples of thermoreversible domains, while compact packing aggregates of primary particles are present in the thermostable sol. Extended X-ray absorption fine structure and elemental chemical analysis revealed that irrespective of the nominal value of R-S and R-Cl all studied samples of the thermoreversible domain are constituted by a well-defined compound possessing an inner core made of hydroxyl and oxo groups bridging together zirconium atoms surrounded on the surface by complexing sulfate ligands. zeta potentials of powders extracted by freeze-drying from the thermoreversible gel revealed a point of surface charge inversion attributed to the specific adsorption of SO42- ion. Thermoreversible gel formation is rationalized by considering the effect of the specific adsorption on the electrical double-layer repulsion together with the temperature dependency of the physical chemical properties of ions in solution.

Purification and characterization of a cyclomaltodextrin glucanotransferase from Paenibacillus campinasensis strain H69-3

A cyclomaltodextrin glucanotransferase (E.C. 2.4.1.19) from a newly isolated alkalophilic and moderately thermophilic Paenibacillus campinasensis strain H69-3 was purified as a homogeneous protein from culture supernatant. Cyclomaltodextrin glucanotransferase was produced during submerged fermentation at 45 degrees C and purified by gel filtration on Sephadex G50 ion exchange using a Q-Sepharose column and ion exchange using a Mono-Q column. The molecular weight of the purified enzyme was 70 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the pI was 5.3. The optimum pH for enzyme activity was 6.5, and it was stable in the pH range 6.0-11.5. The optimum temperature was 65 degrees C at pH 6.5, and it was thermally stable up to 60 degrees C without substrate during 1 h in the presence of 10 mm CaCl2. The enzyme activity increased in the presence of Co2+, Ba2+, and Mn2+. Using maltodextrin as substrate, the K-m and K-cat were 1.65 mg/mL and 347.9 mu mol/mg.min, respectively.

Farelo de trigo e complexo enzimático na alimentação de poedeiras semipesadas na fase de produção

The study was carried out with the objective to evaluate the effects of the inclusion of the wheat bran (WB) with or without supplementation of an enzymatic complex (EC) on the performance of semi-heavy hens in the egg-production phase. A total of 288 Lohmann Brown pullets were used, distributed to a completely randomized design in 4 x 2 factorial arrangement, composed by four WB levels (0, 3, 6 and 9%) in the ration and enzymatic complex supplementation (0 or 100g/100 kg diet), with eight treatments and six replicates of six birds. The enzymatic complex contained the enzymes beta-galactosidase, galactomananase, xilanase and alpha-glucanase. Feed intake, final body weight, egg production, egg weight, egg mass, egg mass feed conversion or egg dozen feed conversion was not affected by WB inclusion in the diets. Egg shell specific gravity deteriorated as WB levels increase in the diets. None of the characteristics was affected by the enzymatic complex supplementation, except for egg weight, that improved from 62.74 to 64.28 g. Then, the use up to 9.0% of wheat bran in the ration is recommended for semi-heavily chickens in the production phase. The supplementation of alpha-galactosidase, galactomannanase, xylanase and alpha-glucanase improve egg weight.