Study of protease activity from Aspergillus awamori INCQS2B.361U2/1 extracellular fraction and modification of culture medium composition to isolate a novel aspartic protease.

Aspergillus awamori was cultivated in a modified Breccia medium, and the extracellular fraction was obtained, which presented 260 ± 15 µg of protein/mg and specific protease activity of 3.87 ± 0.52 mM.min-1.mg of protein-1 using Nα-p-tosyl-L-arginine methyl ester hydrochloride (L-TAME) as substrate. This fraction showed major proteins about 104 and 44 kDa and maximal protease activity at pH 5.5, 6.5, and 9.0, suggesting that A. awamori secretes acidic, neutral, and alkaline proteases with expressive thermal stability, however, aspartic protease was the most important activity. When yeast extract was supplemented to a modified Breccia medium, A. awamori protein secretion and protease activity were maximal and the affinity chromatography on pepstatin-agarose was employed to isolate the aspartic protease activity, which was called ASPA, with approximately 75 kDa. ASPA maximal activity was obtained at pH 4.5 and 6.5, and 50 °C. Pepstatin inhibited about 80% of ASPA activity, with IC50 and Ki values of 0.154 and 0.072 µM, respectively. ASPA cleaved protein and peptides substrates with the highest activity against gelatin (95 U/mg) and good peptidase activity with KM 0.0589 mM and Vmax 1.909 mM.min-1.mg protein-1, using L-TAME as substrate. A. awamori extracellular fraction is a source of proteases with important activity, and the supplementation of modified Breccia medium increased the aspartic protease production. This enzyme presented different biochemical characteristics from the previously reported A. awamori aspartic proteases. Therefore, ASPA is an excellent candidate for biotechnological application due to its important activity and thermostability.

Untreated Chlorella homosphaera biomass allows for high rates of cell wall glucan enzymatic hydrolysis when using exoglucanase-free cellulases.

BACKGROUND: Chlorophyte microalgae have a cell wall containing a large quantity of cellulose Iα with a triclinic unit cell hydrogen-bonding pattern that is more susceptible to hydrolysis than that of the cellulose Iß polymorphic form that is predominant in higher plants. This study addressed the enzymatic hydrolysis of untreated Chlorella homosphaera biomass using selected enzyme preparations, aiming to identify the relevant activity profile for the microalgae cellulose hydrolysis. Enzymes from Acremonium cellulolyticus, which secretes a complete pool of cellulases plus ß-glucosidase; Trichoderma reesei, which secretes a complete pool of cellulases with low ß-glucosidase; Aspergillus awamori, which secretes endoglucanases and ß-glucosidase; blends of T. reesei-A. awamori or A. awamori-A. cellulolyticus enzymes; and a purified A. awamori ß-glucosidase were evaluated. RESULTS: The highest initial glucan hydrolysis rate of 140.3 mg/g/h was observed for A. awamori enzymes with high ß-glucosidase, low endoglucanase, and negligible cellobiohydrolase activities. The initial hydrolysis rates when using A. cellulolyticus or T. reesei enzymes were significantly lower, whereas the results for the T. reesei-A. awamori and A. awamori-A. cellulolyticus blends were similar to that for the A. awamori enzymes. Thus, the hydrolysis of C. homosphaera cellulose was performed exclusively by the endoglucanase and ß-glucosidase activities. X-ray diffraction data showing negligible cellulose crystallinity for untreated C. homosphaera biomass corroborate these findings. The A. awamori-A. cellulolyticus blend showed the highest initial polysaccharide hydrolysis rate of 185.6 mg/g/h, as measured by glucose equivalent, in addition to the highest predicted maximum glucan hydrolysis yield of 47% of total glucose (w/w). T. reesei enzymes showed the lowest predicted maximum glucan hydrolysis yield of 25% (w/w), whereas the maximum yields of approximately 31% were observed for the other enzyme preparations. The hydrolysis yields were proportional to the enzyme ß-glucosidase load, indicating that the endoglucanase load was not rate-limiting. CONCLUSIONS: High rates of enzymatic hydrolysis were achieved for untreated C. homosphaera biomass with enzymes containing endoglucanase and ß-glucosidase activities and devoid of cellobiohydrolase activity. These findings simplify the complexity of the enzyme pools required for the enzymatic hydrolysis of microalgal biomass decreasing the enzyme cost for the production of microalgae-derived glucose syrups.

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.

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.

Evaluation of Chlorella (Chlorophyta) as Source of Fermentable Sugars via Cell Wall Enzymatic Hydrolysis.

The cell wall of Chlorella is composed of up to 80% carbohydrates including cellulose. In this study, Chlorella homosphaera and Chlorella zofingiensis were evaluated as source of fermentable sugars via their cell wall enzymatic degradation. The algae were cultivated in inorganic medium, collected at the stationary growth phase and centrifuged. The cell pellet was suspended in citrate buffer, pH 4.8 and subjected to 24 hours hydrolysis at 50°C using a cellulases, xylanases, and amylases blend. The measurement of glucose and reducing sugars concentration in the reaction mixture supernatant, on a dry biomass base, showed hydrolysis yields of 2.9% and 5.03% glucose and 4.8% and 8.6% reducing sugars, for C. homosphaera and C. zofingiensis, respectively. However if cells were washed with chilled ethanol, cold dried, and grounded the biomass hydrolysis yields increased to 23.3% and 18.4% glucose and 24.5% and 19.3% reducing sugars for C. homosphaera and C. zofingiensis, respectively.

Cellulase production by Streptomyces viridobrunneus SCPE-09 using lignocellulosic biomass as inducer substrate.

An actinomycete strain, isolated from a soil sample under a sugar cane plantation in Brazil and identified as Streptomyces viridobrunneus SCPE-09, was selected as a promising cellulolytic strain, and tested for its ability to produce cellulases from agro-industrial residues. Sugar cane bagasse or wheat bran was tested as carbon source, and corn steep liquor tested as nitrogen source. Different concentrations of carbon and nitrogen were tested using factorial design to identify optimal cellulose production. The results showed that media containing wheat bran 2.0% (w/v) and corn steep liquid 0.19% (w/v) lead to the highest production, 2.0 U mL(-1) of CMCase, obtained on the fifth day of fermentation. The pH and temperature profile showed optimal activity at pH 4.9 and 50°C. As for thermostability, endoglucanases were most tolerant at 50°C, retaining more than 80% of maximal activity even after 2 h of incubation. Zymogram analyses using supernatant from growth under optimized conditions revealed the presence of two CMCase bands with apparent molecular masses of 37 and 119 kDa. The combination of pH tolerance and CMCase production from agro-industrial residues by S. viridobrunneus SCPE-09 offers promise for future bioethanol biotechnologies.

Identification of a Candida parapsilosis strain producing extracellular serine peptidase with keratinolytic activity.

A yeast strain isolated from feather waste from a chicken processing plant was identified as Candida parapsilosis by biochemical tests and morphological studies. The yeast was able to grow in phosphate-buffered saline supplemented with 1% native feather as the sole carbon and nitrogen source. A keratin substrate was obtained from the feathers by dimethylsulphoxide extraction. A 20-fold concentrated culture supernatant from Candida parapsilosis grown on feathers was analysed by SDS-PAGE electrophoresis containing either 1% gelatin or 1% keratin as copolymerised substrates. The presence of a single band with an approximate molecular mass of 60 kDa with gelatinolytic and keratinolytic activities was observed. This proteolytic activity was fully inhibited by phenylmethylsulphonyl fluoride. These results suggest that the extracellular enzyme belongs to the serine peptidase class. This is the first report of an extracellular serine peptidase produced by C. parapsilosis with keratinolytic activity. The role of this enzyme in yeast-host interactions is discussed.

Bioethanol from lignocelluloses: Status and perspectives in Brazil.

The National Alcohol Program--PróAlcool, created by the government of Brazil in 1975 resulted less dependency on fossil fuels. The addition of 25% ethanol to gasoline reduced the import of 550 million barrels oil and also reduced the emission CO(2) by 110 million tons. Today, 44% of the Brazilian energy matrix is renewable and 13.5% is derived from sugarcane. Brazil has a land area of 851 million hectares, of which 54% are preserved, including the Amazon forest (350 million hectares). From the land available for agriculture (340 million hectares), only 0.9% is occupied by sugarcane as energy crop, showing a great expansion potential. Studies have shown that in the coming years, ethanol yield per hectare of sugarcane, which presently is 6000 L/ha, could reach 10,000 L/ha, if 50% of the produced bagasse would be converted to ethanol. This article describes the efforts of different Brazilian institutions and research groups on second generation bioethanol production, especially from sugarcane bagasse.

Dibenzothiophene oxidation by horseradish peroxidase in organic media: effect of the DBT:H2O2 molar ratio and H2O2 addition mode.

Its is well known that in the biodesulfurization (BDS) process the low water solubility of sulfur compounds hinders its transference from the oil phase to the cells being the rate-limiting step in the metabolism of dibenzothiophenes (DBT). Thus sulfur compounds derivatives with high water solubility could be more easily transported increasing the BDS efficiency. The present work performed a stepwise evaluation of the enzymatic oxidation of DBT by horseradish peroxidase (HRP). Reactions were carried out in monophasic organic media containing 25% (v/v) acetonitrile. The following parameters were evaluated: DBT:H2O2 molar ratio (1:1-1:20); H2O2 addition mode (single or stepwise); pH (6.0-8.0) and temperature (37-50 degrees C). Best results were observed in a reaction medium at pH 8.0 presenting HRP 0.06IUml(-1), DBT 0.267mM, DBT:H2O2 molar ratio of 1:20 (stepwise hydrogen peroxide addition) and incubated at 45 degrees C for 60min. Under these conditions 60% of DBT was converted into dibenzothiophene sulfoxide (12%) and dibenzothiophene sulfone (46%). The DBT oxidation rate observed in this work, of 5mmolmin(-1)g(-1) of HRP, was 250-fold higher than the BDS rate, 20mumolmin(-1)g(-1) of catalyst. As such a combined enzyme-microbial desulfurization process could be envisaged. Products were determined by HPLC RP C-18.