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.

Virulence factors in Escherichia coli strains isolated from pigs in the Ribeirao Preto region, State of Sao Paulo, Brazil.

Three-hundred faecal swabs were obtained from pigs with diarrhoea in farms located in different areas of the Ribeirao Preto region in the State of Sao Paulo. One-hundred Escherichia coli strains were isolated and tested for production of thermolabile (TL) and thermostable (STRa and STb) enterotoxins, and for the presence of colonization factors F4, F5 and F6. The strains were also tested for sensitivity to 14 antibiotics and chemotherapeutic agents. Twenty-four Escherichia coli strains produced enterotoxin STb, 5 produced LT and 3 produced STa. In the mannose-resistant haemagglutination reaction, one strain reacted positively with sheep, chicken, horse and human red blood cells and another reacted positively with guinea pig, sheep, chicken, horse and human red cells. However, both strains were negative for colonization factors F4, F5 and F6 when submitted to the slide agglutination test. All Escherichia coli strains were resistant to at least one antibiotic, the highest percentages being obtained for resistance to penicillin, tetracycline and cephalotin. In addition to the importance of the virulence factors normally encountered in enterotoxigenic Escherichia coli strains from pigs, the present results show the possible existence of new colonization factors other than F4, F5 and F6 participating in E. coli-induced pigs colibacillosis in the Ribeirao Preto region.

Production, characterization and properties of polysaccharide depolymerizing enzymes from a strain of Curvularia inaequalis

Xylanase, β-glucosidase, β-xylosidase, endoglucanase and polygalacturonase production from Curvularia inaequalis was carried out by means of solid-state and submerged fermentation using different carbon sources. β-Glucosidase, β-xylosidase, polygalacturonase and xylanase produced by the microorganisms were characterized. β-Glucosidase presented optimum activity at pH 5.5 whereas xylanase, polygalacturonase and β-xylosidase activities were optimal at pH 5.0. Maximal activity of β-glucosidase was determined at 60°C, β-xylosidase at 70°C, and polygalacturonase and xylanase at 55°C. These enzymes were stable at acidic to neutral pH and at 40-45°C. The crude enzyme solution was studied for the hydrolysis of agricultural residues.

Purification and characterization of xylanases from Aspergillus giganteus

A strain of Aspergillus giganteus cultivated in a medium with xylan produced two xylanases (xylanase I and II) which were purified to homogeneity. Their molar mass, estimated by SDS-PAGE, were 21 and 24 kDa, respectively. Both enzymes are glycoproteins with 50°C temperature optimum; optimum pH was 6.0-6.5 for xylanase I and 6.0 for xylanase II. At 50°C xylanase I exhibited higher thermostability than xylanase II. Hg2+, Cu 2+ and SDS were strong inhibitors, 1,4-dithiothreitol stimulated the reaction of both enzymes. Both xylanases are xylan-specific; kinetic parameters indicated higher efficiency in the hydrolysis of oat spelts xylan. In hydrolysis of this substrate, xylotriose, xylotetraose and larger xylooligosaccharides were released and hence the enzymes were classified as endoxylanases.

Purification and biochemical characterization of two xylanases produced by Aspergillus caespitosus and their potential for kraft pulp bleaching

Two extracellular xylanases produced by the thermotolerant fungus Aspergillus caespitosus grown in sugar cane bagasse were purified and characterized. Estimated molecular masses were 26.3 and 27 kDa (xyl I); 7.7 and 17.7 kDa (xyl II) for gel filtration and SDS-PAGE, respectively. Optimal temperature for both xylanases was 50-55°C. Optimal pH was 6.5-7.0 for xyl I, and 5.5-6.5 for xyl II. The thermostability (T half) at 55°C was 27.3 min (xyl I) and >90 min (xyl II). Xylanase activity was inhibited by several ions. β-mercaptoethanol activated 59 and 102% xyl I and xyl II activities, respectively. These enzymes preferentially hydrolyzed birchwood xylan, and the K m and V max values were 2.5 mg/ml and 1679 U/mg protein (xyl I), and 3.9 mg/ml and 113 U/mg protein (xyl II). The action of both xylanases mainly that of xyl II, on kraft pulp reduced kappa number and increased pulp viscosity. © 2004 Elsevier Ltd. All rights reserved.

Production of saccharogenic and dextrinogenic amylases by Rhizomucor pusillus A 13.36

A newly-isolated thermophilic strain of the zygomycete fungus Rhizomucor pusillus 13.36 produced highly active dextrinogenic and saccharogenic enzymes. Cassava pulp was a good alternative substrate for amylase production. Dextrinogenic and saccharogenic amylases exhibited optimum activities at a pH of 4.0-4.5 and 5.0 respectively and at a temperature of 75°C. The enzymes were highly thermostable, with no detectable loss of saccharogenic or dextrinogenic activity after 1 h and 6 h at 60°C, respectively. The saccharogenic activity was inhibited by Ca2+ while the dextrinogenic was indifferent to this ion. Both activities were inhibited by Fe2+ and Cu2+ Hydrolysis of soluble starch by the crude enzyme yielded 66% glucose, 19.5% maltose, 7.7% maltotriose and 6.6% oligosaccharides. Copyright © 2005, The Microbiological Society of Korea.

A specific short dextrin-hydrolyzing extracellular glucosidase from the thermophilic fungus Thermoascus aurantiacus 179-5

The thermophilic fungus Thermoascus aurantiacus 179-5 produced large quantities of a glucosidase which preferentially hydrolyzed maltose over starch. Enzyme production was high in submerged fermentation, with a maximal activity of 30 U/ml after 336 h of fermentation. In solid-state fermentation, the activity of the enzyme was 22 U/ml at 144 h in medium containing wheat bran and 5.8 U/ml at 48 h when cassava pulp was used as the culture medium. The enzyme was specific for maltose, very slowly hydrolyzed starch, dextrins (2-7G) and the synthetic substrate (α-PNPG), and did not hydrolyze sucrose. These properties suggest that the enzyme is a type II α-glucosidase. The optimum temperature of the enzyme was 70°C. In addition, the enzyme was highly thermostable (100% stability for 10 h at 60°C and a half-life of 15 min at 80°C), and stable within a wide pH range. Copyright © 2006, The Microbiological Society of Korea.

Avaliação da capacidade imunoestimulante e da estabilidade de um probiótico empregado em rações de cães

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Fontes de carbono de baixo custo para a produção de xilanase termoestável por aspergillus niger

A strain of the flamentous fungus Aspergillus niger was isolated and shown to possess extracellular xylanolytic activity. These enzymes have biotechnological potential and can be employed in various industries. This fungus produced its highest xylanase activity in a medium made up of 0.1% CaCO3, 0.5% NaCl, 0.1% NH4Cl, 0.5% corn steep liquor and 1% carbon source, at pH 8.0. A low-cost hemicellulose residue (powdered corncob) proved to be an excellent inducer of the A. niger xylanolytic complex. Filtration of the crude culture medium with suspended kaolin was ideal for to clarify the extract and led to partial purifcation of the xylanolytic activity. The apparent molecular mass of the xylanase was about 32.3 kDa. Maximum enzyme activity occurred at pH 5.0 and 55-60oC. Apparent Km was 10.41 ± 0.282 mg/mL and Vmax was 3.32 ± 0.053 U/mg protein, with birchwood xylan as the substrate. Activation energy was 4.55 kcal/mol and half-life of the crude enzyme at 60oC was 30 minutes. Addition of 2% glucose to the culture medium supplemented with xylan repressed xylanase production, but in the presence of xylose the enzyme production was not affected.

Enzyme effects on extruded diets for dogs with soybean meal as a substitute for poultry by-product meal

The effects of exogenous enzymes supplementation on kibble diets for dogs formulated with soybean meal (SBM) as a substitute for poultry by-product meal (PM) was investigated on nutrient digestibility, fermentation products formation, post-prandial urea response and selected faecal bacteria counts. Two kibble diets with similar compositions were used in two trials: PM-based diet (28.9% of PM; soybean hulls as a fibre source) and SBM-based diet (29.9% of SBM). In experiment 1, the SBM diet was divided into three diets: SBM-0, without enzyme addition; SBM-1, covered after extrusion with 7500U protease/kg and 45U cellulase/kg; and SBM-2, covered with 15000U protease/kg and 90U cellulase/kg. In experiment 2, the SBM diet was divided into three diets: SBM-0; SBM-1, covered with 140U protease/kg; 8U cellulase/kg, 800U pectinase/kg, 60U phytase/kg, 40U betaglucanase/kg and 20U xylanase/kg; and SMB-2, covered with 700U protease/kg, 40U cellulase/kg, 4000U pectinase/kg, 300U phytase/kg, 200U betaglucanase/kg and 100U xylanase/kg. Each experiment followed a block design with six dogs per diet. Data were submitted to analysis of variance and means compared by orthogonal and polynomial contrasts (p<0.05). In both experiments, nutrients and energy digestibility did not differ between diets (p>0.05). SBM consumption resulted in increased faecal moisture and production (p<0.05), without effect on faecal score. Higher concentration of propionate, acetate and lactate, and lower ammonia and pH were found in the faeces of dogs fed SBM (p<0.05). Higher post-prandial urea was verified in dogs fed SBM (p<0.05). In experiment 2, the addition of enzymes increased faecal concentration of propionate, acetate and total short-chain fatty acid (p<0.05) and tended to reduce post-prandial urea concentration (p=0.06). Although with similar digestibility, SBM shows a worse utilization of absorbed amino acids than the PM. Soybean oligosaccharides can beneficially change gut fermentation product formation. Enzymes can increase the gut fermentation activity and improve the SBM proteic value. © 2013 Blackwell Verlag GmbH.

Enzyme use in kibble diets formulated with wheat bran for dogs: Effects on processing and digestibility

Recently, there is an interest in technologies that favour the use of coproducts for animal nutrition. The effect of adding two enzyme mixtures in diets for dogs formulated with wheat bran (WB) was evaluated. Two foods with similar compositions were formulated: negative control (NC; without WB) and test diet (25% of WB). The test diet was divided into four treatments: without enzyme (positive control), enzyme mixture 1 (ENZ1; added before extrusion β-glucanase, xylanase, cellulase, glucoamylase, phytase); enzyme mixture 2 (ENZ2; added before extrusion the ENZ1 more α-amylase); enzyme mixture 2 added after the extrusion (ENZ2ex). ENZ1 and ENZ2 were used to evaluate the enzyme effect on extruder pre-conditioner (processing additive) and ENZ2ex to evaluate the effect of enzyme supplementation for the animal. Digestibility was measured through total collection of faeces and urine. The experiment followed a randomized block design with five treatments (diets) and six dogs per diet, totalling 30 dogs (7.0 ± 1.2 years old and 11.0 ± 2.2 kg of body weight). Data were submitted to analysis of variance and means compared by Tukey's test and orthogonal contrasts (p < 0.05). Reducing sugars showed an important reduction after extrusion, suggesting the formation of carbohydrate complexes. The apparent total tract digestibility (ATTD) of dry matter, organic matter, crude protein, acid-hydrolysed fat and energy was higher in NC than in diets with WB (p < 0.001), without effects of enzyme additions. WB diets resulted in higher faecal production and concentration of short-chain fatty acids (SCFA) and reduced pH and ammonia concentration (p < 0.01), with no effect of enzyme addition. The enzyme addition did not result in improved digestibility of a diet high in non-starch polysaccharides; however, only ATTD was measured and nutrient fermentation in the large intestine may have interfered with the results obtained. WB modified fermentation product formation in the colon of dogs. © 2013 Blackwell Verlag GmbH.

Caracterização bioquímica de uma serino-protease produzida pelo fungo termofílico Myceliophthora sp

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)