Synthesis of disaccharides using ß-glucosidases from Aspergillus niger, A. awamori and Prunus dulcis.

OBJECTIVE: Glucose conversion into disaccharides was performed with ß-glucosidases from Prunus dulcis (ß-Pd), Aspergillus niger (ß-An) and A. awamori (ß-Aa), in reactions containing initial glucose of 700 and 900 g l-1. RESULTS: The reactions' time courses were followed regarding glucose and product concentrations. In all cases, there was a predominant formation of gentiobiose over cellobiose and also of oligosaccharides with a higher molecular mass. For reactions containing 700 g glucose l-1, the final substrate conversions were 33, 38, and 23.5% for ß-An, ß-Aa, and ß-Pd, respectively. The use of ß-An yielded 103 g gentiobiose l-1 (15.5% yield), which is the highest reported for a fungal ß-glucosidase. The increase in glucose concentration to 900 g l-1 resulted in a significant increase in disaccharide synthesis by ß-Pd, reaching 128 g gentiobiose l-1 (15% yield), while for ß-An and ß-Aa, there was a shift toward the synthesis of higher oligosaccharides. CONCLUSION: ß-Pd and the fungal ß-An and ß-Aa ß-glucosidases present quite dissimilar kinetics and selective properties regarding the synthesis of disaccharides; while ß-Pd showed the highest productivity for gentiobiose synthesis, ß-An presented the highest specificity.

Efficient production of lignocellulolytic enzymes xylanase, ß-xylosidase, ferulic acid esterase and ß-glucosidase by the mutant strain Aspergillus awamori 2B.361 U2/1.

The production of xylanase, ß-xylosidase, ferulic acid esterase and ß-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 °C 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, ß-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 ß-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, ß-xylosidase, ferulic acid esterase and ß-glucosidase by A. awamori 2B.361 U2/1 was compared to that produced by Trichoderma reesei Rut-C30. The level of ß-glucosidase was over 17-fold higher for the Aspergillus strain, whereas the levels of xylanase and ß-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.

Use of cellobiohydrolase-free cellulase blends for the hydrolysis of microcrystalline cellulose and sugarcane bagasse pretreated by either ball milling or ionic liquid [Emim][Ac].

This study investigated the requirement of cellobiohydrolases (CBH) for saccharification of microcrystalline cellulose and sugarcane bagasse pretreated either by ball milling (BM) or by ionic liquid (IL) [Emim][Ac]. Hydrolysis was done using CBH-free blends of Pyrococcus horikoshii endoglucanase (EG) plus Pyrococcus furiosus ß-glucosidase (EGPh/BGPf) or Optimash™ BG while Acremonium Cellulase was used as control. IL-pretreated substrates were hydrolyzed more effectively by CBH-free enzymes than were the BM-pretreated substrates. IL-treatment decreased the crystallinity and increased the specific surface area (SSA), whereas BM-treatment decreased the crystallinity without increasing the SSA. The hydrolysis of IL-treated cellulose by EGPh/BGPf showed a saccharification rate of 3.92 g/Lh and a glucose yield of 81% within 9h. These results indicate the efficiency of CBH-free enzymes for the hydrolysis of IL-treated substrates.

Efficient production of lignocellulolytic enzymes xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by the mutant strain Aspergillus awamori 2B.361 U2/1

The production of xylanase, β-xylosidase, ferulic acid esterase and β-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, β-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 β-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, β-xylosidase, ferulic acid esterase and β-glucosidase by A. awamori 2B.361 U2/1 was compared to that produced by Trichoderma reesei Rut-C30. The level of β-glucosidase was over 17-fold higher for the Aspergillus strain, whereas the levels of xylanase and β-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.

Efficient production of lignocellulolytic enzymes xylanase, -xylosidase, ferulic acid esterase and -glucosidase by the mutant strain Aspergillus awamori 2B.361 U2/1

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)

Amino acids interference on the quantification of reducing sugars by the 3,5-dinitrosalicylic acid assay mislead carbohydrase activity measurements.

This study evaluated the interference of the amino acids tryptophan, cysteine, histidine, tyrosine, hydroxyproline, leucine, proline, serine, glycine, valine, glutamic acid, phenylalanine, and methionine on the measurement of reducing sugars using a phenol-free 3,5-dinitrosalicylic acid (DNS) reagent. It was found that in reaction mixtures containing 20mM of either tryptophan, cysteine, histidine, tyrosine, or hydroxyproline the measurement of 3.7 mM glucose was overestimated by 76%, 50%, 35%, 18%, and 10%, respectively. The amino acids valine, glutamic acid, and phenylalanine did not affect the DNS reaction, while methionine decreased the color development by 5%. The measurement of glucose, xylose, arabinose, and cellobiose at the 3.7-12.4 mM range in the presence of 20 mM cysteine resulted in an overestimated concentration of 34.8-50%. Enzymatic assays for measuring xylanolytic and filter paper activity (FPAse) were conducted in the presence of 20-60 mM cysteine, and compared to cysteine-free assays. In the presence of cysteine, the measured xylanase activity increased threefold and the FPAse activity increased twofold due to the overestimation of the reducing sugar concentrations in the assays. The interference from cysteine was reduced to a maximum of 8.6% when a DNS reagent containing phenol was used.

Sugarcane bagasse enzymatic hydrolysis: rheological data as criteria for impeller selection.

The aim of this work was to select an efficient impeller to be used in a stirred reactor for the enzymatic hydrolysis of sugar cane bagasse. All experiments utilized 100 g (dry weight)/l of steam-pretreated bagasse, which is utilized in Brazil for cattle feed. The process was studied with respect to the rheological behavior of the biomass hydrolysate and the enzymatic conversion of the bagasse polysaccharides. These parameters were applied to model the power required for an impeller to operate at pilot scale (100 l) using empirical correlations according to Nagata [16]. Hydrolysis experiments were carried out using a blend of cellulases, ß-glucosidase, and xylanases produced in our laboratory by Trichoderma reesei RUT C30 and Aspergillus awamori. Hydrolyses were performed with an enzyme load of 10 FPU/g (dry weight) of bagasse over 36 h with periodic sampling for the measurement of viscosity and the concentration of glucose and reducing sugars. The mixture presented pseudoplastic behavior. This rheological model allowed for a performance comparison to be made between flat-blade disk (Rushton turbine) and pitched-blade (45°) impellers. The simulation showed that the pitched blade consumed tenfold less energy than the flat-blade disk turbine. The resulting sugar syrups contained 22 g/l of glucose, which corresponded to 45% cellulose conversion.

Purification and characterization studies of a thermostable ß-xylanase from Aspergillus awamori.

This study presents data on the production, purification, and properties of a thermostable ß-xylanase produced by an Aspergillus awamori 2B.361 U2/1 submerged culture using wheat bran as carbon source. Fractionation of the culture filtrate by membrane ultrafiltration followed by Sephacryl S-200 and Q-Sepharose chromatography allowed for the isolation of a homogeneous xylanase (PXII-1), which was 32.87 kDa according to MS analysis. The enzyme-specific activity towards soluble oat spelt xylan, which was found to be 490 IU/mg under optimum reaction conditions (50°C and pH 5.0-5.5), was 17-fold higher than that measured in the culture supernatant. Xylan reaction products were identified as xylobiose, xylotriose, and xylotetraose. K (m) values (mg ml(-1)) for soluble oat spelt and birchwood xylan were 11.8 and 9.45, respectively. Although PXII-1 showed 85% activity retention upon incubation at 50 °C and pH 5.0 for 20 days, incubation at pH 7.0 resulted in 50% activity loss within 3 days. PXII-1 stability at pH 7.0 was improved in the presence of 20 mM cysteine, which allowed for 85% activity retention for 25 days. This study on the production in high yields of a remarkably thermostable xylanase is of significance due to the central role that this class of biocatalyst shares, along with cellulases, for the much needed enzymatic hydrolysis of biomass. Furthermore, stable xylanases are important for the manufacture of paper, animal feed, and xylooligosaccharides.

Evaluation of holocellulase production by plant-degrading fungi grown on agro-industrial residues.

Agaricus brasiliensis CS1, Pleurotus ostreatus H1 and Aspergillus flavus produced holocellulases when grown in solid and submerged liquid cultures containing agro-industrial residues, including sugar cane bagasse and dirty cotton residue, as substrates. These isolates proved to be efficient producers of holocellulases under the conditions used in this screening. Bromatological analysis of agro-industrial residues showed differences in protein, fiber, hemicellulose, cellulose and lignin content. Maximal holocellulase activity (hemicellulase, cellulase and pectinase) was obtained using solid-state cultivation with 10% substrate concentration. In this case, remarkably high levels of xylanase and polygalacturonase activity (4,008 and 4,548 IU/l, respectively) were produced by A. flavus when grown in media containing corn residue, followed by P. ostreatus H1 with IU/l values of 1,900 and 3,965 when cultivated on 5% and 10% sugar cane bagasse, respectively. A. brasiliensis CS1 showed the highest reducing sugar yield (11.640 mg/ml) when grown on medium containing sugar cane bagasse. A. brasiliensis was also the most efficient producer of protein, except when cultivated on dirty cotton residue, which induced maximal production in A. flavus. Comparison of enzymatic hydrolysis of sugar cane bagasse and dirty cotton residue by crude extracts of A. brasiliensis CS1, P. ostreatus H1 and A. flavus showed that the best reducing sugar yield was achieved using sugar cane bagasse as a substrate.