Digestive proteinases from the marine fish processing wastes of the South-West Atlantic Ocean: Their partial characterization and comparison.

Fish processing generates plenty of waste that is directly discarded in open-air dumps and water sources, or treated in the same way as urban solid waste, causing serious pollution problems. The waste represents a significant source of high-value bioproducts with potential applications in different industrial processes such as the production of feed, fertilizers, biodiesel and biogas, detergent additives and cosmetics. The objective of this study was to characterize and compare specific activities under different pH values and temperature conditions of acid and alkaline proteinases and viscera yield from the following fish species: Argentine hake Merluccius hubbsi, Brazilian flathead Percophis brasiliensis, Brazilian codling Urophycis brasiliensis and Stripped weakfish Cynoscion guatucupa. Individuals were fished off the coast of Mar del Plata (Argentina) by a commercial fleet and the viscera were immediately extracted and kept on ice until use. Stomach proteinases from four species had the highest activity at pH 2, with stability in the range of pH 2-4. The optimum pH was 11.5 from intestinal enzymes of C. guatucupa, M. hubbsi and P. brasiliensis and 9.5 from intestinal enzymes of U. brasiliensis. Alkaline proteinases from all species were highly stable in the range of 7-11.5. The optimum temperature of stomach proteinases from the four species studied were 30 and 50°C, with stability at 10 and 30°C during 150 min. The optimum temperature of intestinal enzymes from the tested species were 50°C with high stability at 10 and 30°C during 150 min. Alkaline proteinase from all species and acid proteinases from C. guatucupa were inactive at 70°C after 150 min, while there was a residual activity lower than 5% at 80°C on pre-incubated stomach enzymes of M.hubbsi, P. brasiliensis and U. brasiliensis after 5, 10 and 20 min, respectively. Digestive proteinases recovered in this study could be appropriate for technological usage, reducing manufacturing costs, obtaining revenue from fishery wastes, and contributing to the reduction of environmental pollution.

Production, concentration and partial characterization of an enzymatic extract produced by an Aspergillus niger mutant in solid state fermentation.

An enzymatic extract from Aspergillus niger 3T5B8 was produced by Solid State Fermentation (SSF) in aerated columns, using wheat bran as substrate. A combination of extracts produced using three different process conditions varying temperature, pH and aeration formed the final extract (Mixture). The Mixture was concentrated by an ultrafiltration process that partially purified and provided an efficient recovery of the enzymatic activities of xylanase (88.89%), polygalacturonase (89.3%), ß-glucosidase (93.15%), protease (98.68%) and carboxymethylcellulase (CMCase) (98.93%). SDS-PAGE analysis showed 15 visible protein bands in the crude and concentrated Mixture with molecular weights ranging from 15.1 to 104.6 kDa. Thin layer chromatography confirmed the effective action of ß-glucosidase and xylanase hydrolysis activities over cellobiose and xylan, respectively. A central composite design (CCD) with two variables and four replicates at the center points was used to determine the optimal temperature and pH for CMCase and ß-glucosidase. The optimal temperature was 78.9 °C and pH 3.8 for CMCase and 52.8 °C and pH 4.8 for ß-glucosidase, respectively.

Purification of a thermostable antinociceptive lectin isolated from Andira anthelmia.

Andira anthelmia (tribe Dalbergieae), a plant from Brazilian Amazon, possesses a seed lectin that was purified by affinity chromatography in sepharose-mannose. This novel Dalbergieae lectin, named AAL, agglutinated rabbit erythrocytes treated with trypsin. The hemagglutinating activity of AAL was maintained after incubation at a wide range of temperature (40 to 70 °C) and pH, was shown to be dependent on divalent cations, and was inhibited by d-mannose and d-sucrose. AAL showed an electrophoretic profile in sodium dodecyl sulfate-polyacrylamide gel electrophoresis similar to other lectins of the tribe Dalbergieae, presenting a double band of molecular weight with approximately 20 kDa and other minor bands of 17, 15, and 13 kDa, being the smaller fragment glycosylated. AAL injected by intravenous route in mice showed antinociceptive activity in two behavioral tests (writhing and formalin). In the writhing test induced by acetic acid, AAL showed inhibitory effect at 0.01 mg/kg (68%), 0.1 mg/kg (46%) and 1 mg/kg (74%). In the formalin test, AAL (0.1 mg/kg) inhibited by 48% the licking time in the inflammatory phase, an effect that was recovered by the lectin association with mannose. In conclusion, AAL presents analgesic effect involving the lectin domain via peripheral mechanisms of inflammatory nociception. This activity highlights the importance of lectins as tools to be used for understanding the interaction of protein-carbohydrate in processes associated to inflammatory pain. Copyright © 2015 John Wiley & Sons, Ltd.

Biotechnological potential of agro-industrial waste in the synthesis of pectin lyase from Aspergillus brasiliensis.

This study aims at investigating pectin lyase bioproduction in submerged fermentation with synthetic medium and agro-industrial residues, using the filamentous fungus Aspergillus brasiliensis. The maximum pectin lyase activity in a synthetic medium (42 g/l pectin, 40 g/l yeast extract, and 0.02 g/l iron sulfate) was 31 U/ml, and 46 U/ml in the agro-industrial medium (160 g/l orange peel, 150 g/l corn steep liquor, and 300 g/l parboiled rice water), obtained over 60 and 124 h of bioproduction, 180 r/min, 30 ℃, pHinitial 5.5, and 5·106 spores/ml, respectively. Partial characterization of pectin lyase crude enzyme extract obtained from the synthetic medium and the one made of agro-industrial residues showed optimum conditions at pH of 5.5 and 4.5 and temperatures of 37 and 55 ℃, respectively. The Ed obtained was 3.13 and 9.15 kJ/mol, and the half-life time (t1/2) was 5.71 and 80 h at 55 ℃ for pectin lyase produced in synthetic and agro-industrial medium, respectively.

Generation and Partial Characterization of Rabbit Monoclonal Antibody to Pyroglutamate Amyloid-ß3-42 (pE3-Aß).

N-terminally truncated pyroglutamate amyloid-ß (Aß) peptide starting at position 3 represents a significant fraction of Aß peptides (pE3-Aß) in amyloid plaques of postmortem brains from patients with Alzheimer's disease (AD) and older persons with Down syndrome (DS). Studies in transgenic mouse models of AD also showed that pE3-Aß is a major component of plaques, and mouse monoclonal antibody to pE3-Aß appears to be a desirable therapeutic agent for AD. Since small peptides do not typically elicit a good immune response in mice, but do so favorably in rabbits, our aims were to generate and partially characterize a rabbit monoclonal antibody (RabmAb) to pE3-Aß. The generated RabmAb was found to be specific for pE3-Aß, since it showed no reactivity with Aß16, Aß40, Aß42, Aß3-11, and pE11-17 Aß peptides in an enzyme linked immunosorbent assay (ELISA). The isotype of the antibody was found to be IgG class. The antibody possesses high affinity to pE3-Aß with dissociation constant (KD) for the antibody of 1 nM. The epitope of the antibody lies within the sequence of pE3-FRHD. In dot blotting, the optimal detection of pE3-Aß was at an antibody concentration of 0.5 µg/ml. The threshold of pE3-Aß detection was 2 fmol. The antibody was sensitive enough to detect 10 pg/ml of pE3-Aß in sandwich ELISA. pE3-Aß was detected in AD and DS brain extracts in ELISA and immunoblotting. Immunohistological studies showed immunolabeling of plaques and blood vessels in brains from patients with AD, and DS showing AD pathology. Thus, the antibody can be widely applied in AD and DS research, and therapeutic applications.

Newly Isolated Penicillium sp. for Cellulolytic Enzyme Production in Soybean Hull Residue

Abstract (1) Background: The aim of this study was to evaluate the production and partial characterization of xylanase and avicelase by a newly isolated Penicillium sp. in solid-state fermentation, using soybean hulls as substrate. (2) Methods: Temperature, time, number of spores, and substrate moisture on xylanase and avicelase bioproduction were evaluated, maximizing activity with 30°C, 1x106 spores/g substrate, 14 and 7 days of fermentation with 70 and 76% substrate moisture contents, for xylanase and avicelase, respectively. (3) Results: Different solvents, temperatures, and agitation in the enzymatic extraction were evaluated, obtaining higher activities, 430.77 and 26.77 U/g for xylanase and avicelase using 30 min extraction and 0.05 M citrate buffer solution (pH 4.5 ), respectively at 60°C and 175 rpm and 50°C and 125 rpm. The optimum pH and temperature for enzymatic activity determination were 5.3 and 50°C. Enzyme extract stability was evaluated, obtaining higher stability with pH between 4.5 and 5.5, higher temperature of up to 40°C. The kinetic thermal denaturation (Kd), half-life time, D-value, and Z-value were similar for both enzymes. The xylanase Ed value (89.1 kJ/mol) was slightly lower than the avicelase one (96.7 kJ/mol), indicating higher thermostability for avicelase. (4) Conclusion: In this way, the production of cellulases using alternative substrates is a way to reduce production costs, since they represent about 10% of the world demand of enzymes, with application in animal feed processing, food production and breweries, textile processing, detergent and laundry production, pulp manufacturing and the production of biofuels.