Production of the prolyl endoprotease (PEP) from Aspergillus sp. FSDE 16 by solid-state fermentation (SSF) and use for producing a gluten-free beer.

Beer is a beverage that contains gluten and cannot be consumed by people with celiac disease. In this context, the enzyme prolyl endoprotease (PEP) can be used to reduce the gluten content in beer. The present study aimed to produce the PEP from Aspergillus sp. FSDE 16 using solid-state fermentation with 5 conditions and comparing with a similar commercial enzyme produced from Aspergillus niger in the production of a gluten-free beer. The results of the performed cultures showed that during the culture, the most increased protease activity (54.46 U/mL) occurred on the 4th day. In contrast, for PEP, the highest activity (0.0356 U/mL) was obtained on the 3rd day of culture in condition. Regarding beer production, cell growth, pH, and total soluble solids showed similar behavior over the 7 days for beers produced without enzyme addition or with the addition of commercial enzyme and with the addition of the enzyme extract produced. The addition of the enzyme and the enzyme extract did not promote changes, and all the beers produced showed similar and satisfactory results, with acid pH between 4 and 5, total soluble solids ranging from 4.80 to 5.05, alcohol content ranging from 2.83% to 3.08%, and all beers having a dark character with deep amber and light copper color. Gluten removal was effectively using the commercial enzyme and the enzyme produced according to condition (v) reaching gluten concentrations equal to 17 ± 5.31 and 21.19 ± 11.28 ppm, respectively. In this way, the production of the enzyme by SSF and its application in the removal of gluten in beer was efficient.

Enhancing the expression of multi-antigen chimeric TGAGS/BST protein from Toxoplasma gondii in Escherichia coli BL 21 Star during batch cultivation.

Toxoplasmosis, despite advances in science and technology, is a disease that requires attention since there is no vaccine capable of immunizing humans and animals against all isolated types of Toxoplasma gondii. Thus, the use of chimeric proteins, which can contain multiple antigens, is a very promising alternative for the process of obtaining a vaccine and diagnostic test for toxoplasmosis due to the great diversity of antigens presented by T. gondii. In this context, the present study evaluates batch culture strategies in the production of the multi-antigenic chimeric protein TgAGS/BsT from Toxoplasma gondii. Several exploratory cultures were initially carried out to observe the kinetic behavior of E. coli BL21 Star in five different medium compositions without the addition of IPTG (inducer). Cultures of E. coli B21 Star were carried out with 1.0 mM IPTG at different times of initiation of induction (0.5, 1, and 6 h) to evaluate the effects on cell growth, production of the protein of interest, culture pH, and acetic acid formation. The results showed that among the culture media evaluated, 2xTY and TB supplemented with glycerol had the best cell concentration values of 3.42 ± 0.05 g/L and 5.48 ± 0.05 g/L, respectively. In the assays induced by IPTG, a higher expression of TgAGS/BsT protein was observed, with induction beginning within 6 h of culture, with a maximum concentration of protein of interest of 1.82 ± 0.02 g/L for the 2xTY and 2.49 ± 0.03 g/L for the TB medium. In addition, later induction by IPTG provided greater stability of plasmid pET-TgAGS, remaining with values above 90% at the end of culture.

Bati Butter as a Potential Substrate for Lipase Production by Aspergillus terreus NRRL-255.

This study evaluated bati butter (Ouratea parviflora) as a substrate for lipase production by solid-state fermentation (SSF) using Aspergillus terreus NRRL-255. A gas chromatograph with a flame ionization detector determined the bati butter fatty acid profile. Lipase production and spore count were optimized using a 32 experimental design and evaluated using the response surface methodology. Moreover, the crude enzyme extract was evaluated against different pH, temperature, and activating and inhibitors reagents. Regarding the fatty acids identified, long-chain accounted for 78.60% of the total lipids. The highest lipase production was obtained at 35 °C and 120 h of fermentation, yielding 216.9 U g-1. Crude enzyme extract presented more significant activity at 37 °C and pH 9. ß-Mercaptoethanol increased the enzyme activity (113.80%), while sodium dodecyl sulfate inactivated the enzyme. Therefore, bati butter proved to be a potential substrate capable of inducing lipase production by solid-state fermentation.

Chitooligosaccharides antagonize the cytotoxic effect of glucosamine.

Chitooligosaccharides (COS) are partially hydrolyzed compounds derived from chitosan that exhibit a number of biological activities, including antitumor, antibacterial and antifungal properties. In this work, we examined the cytotoxicity of pure COS and oligomers A, B and C (solutions composed of different amounts of COS) produced by enzymatic hydrolysis using a crude enzyme extract produced by the fungus Metarhrizium anisopliae. The antiproliferative effect of these molecules was analyzed using tumor cell lines (HepG2 and HeLa cells) and in a normal cell line (3T3). The antioxidant activity was analyzed in several in vitro experiments. Glucosamine showed higher toxicity (approximately 92%) to all cell lines studied. However, the oligomers obtained after hydrolysis demonstrated no toxic effects on the normal cells (3T3). Furthermore, we showed that a small amount of other COS can decrease the cytotoxic effect of glucosamine against 3T3 cells, indicating that glucosamine could be used as an antitumor drug in the presence of other COS. In addition, different effects were found in antiproliferative assays, which depended on the COS composition in the oligomers (A, B and C), showing that a combination of them may be essential for developing antineoplastic drugs. Superoxide anion scavenging was the main antioxidant activity demonstrated by the COS and oligomers. This activity was also dependent on the oligomer composition of the chitosan hydrolysates. Further work will identify the ideal proportions of COS and glucosamine for maximizing the effects of these biological activities.

Influence of culture medium on the production of eif antigen from Leishmania chagasi in recombinant Escherichia coli

With the advent of recombinant DNA technology, recombinant protein expression has become an important tool in the study of the structure, function and identification of new proteins, especially those with therapeutic functions. Escherichia coli has been the predominant prokaryote used in genetic engineering studies due to the abundance of information about its metabolism. Despite significant advances in molecular biology and immunology of infections, there are as yet no prophylactic drugs capable of preventing visceral leishmaniasis. It is therefore important to identify specific antigens in order to develop vaccines and diagnostic kits against this disease. The objective of this study was to evaluate the influence of culture medium on the production of eIF antigen from Leishmania chagasi in recombinant Escherichia coli. An induction procedure using IPTG was carried out in a series of trials, to observe the influence of culture medium (2xTY, TB) under expression of the recombinant eIF protein. Results showed that recombinant protein expression was associated to growth and that the highest eIF antigen expression was obtained in the 2xTY medium.

Recuperação de proteína do soro de leite bovino usando cromatografia de interação hidrofóbica em leito expandido / Protein recovery from whey using hydrophobic chromatography

O presente trabalho teve por objetivo estudar a adsorção das proteínas do soro de queijo tipo “coalho” para a resina de interação hidrofóbica Streamline TMPhenyl usando o leito na forma expandida. Foi utilizada uma coluna de 2,6 cm de diâmetro por 30 cm de altura, acoplada a uma bomba peristáltica. Uma amostra de solução de soro (250 mL) foi aplicada a uma coluna contendo a resina Streamline TM Phenyl (25 mL) previamente equilibrada em tampão Tris/HCI (50 mM, pH 7,0) com NaCl ( 1,0 M), após lavagem a eluição foi conduzida a temperatura ambiente em tampão Tris/HCI (50 mM, pH 7,0) e altura de leito fixo de 5,0 cm. Os resultados mostraram que foi possível recuperar 37% das proteínas inicialmente contidas no soro em uma única etapa.