Everaldo Silvino dos Santos

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.

Recovery and purification of recombinant 503 antigen of Leishmania infantum chagasi using expanded bed adsorption chromatography

Visceral leishmaniasis, a disease caused by Leishmania infantum chagasi, represents a major public health problem in many areas of the world. However, there is currently no vaccine for human use. The aim of this work was to purify the 503 antigen of Leishmania i. chagasi directly from unclarified Escherichia coli feedstock through expanded bed adsorption (EBA) chromatography. Batch experiments were performed to optimize the adsorption and elution conditions of the antigen onto a STREAMLINE Chelating resin using two central composite rotatable designs (CCRD). The results showed that the optimal binding conditions of the 503 antigen were pH 8.0 in the presence of 2.4 M NaCl. For the elution of the target protein, the optimized conditions included the presence of 600.0 mM imidazole. The adsorption isothermal data of the 503 antigen were fitted to the Langmuir adsorption isotherm. The EBA experiment successfully recovered 59.2% of the 503 antigen from the unclarified E. coli homogenate with a purification factor of 6.0.

Alcoholic fermentation of Saccharomyces cerevisiae, Pichia stipitis and Zymomonas mobilis in the presence of inhibitory compounds and seawater.

Production of cellulosic ethanol and holocellulosic ethanol from vegetable or microbial biomass starts with a hydrolysate containing compounds which may produce negative effects in the enzymatic hydrolysis and fermentation stages due to the need of pretreatment of the materials. In this way, the simultaneous presence of hydroxymethylfurfural (HMF), furfural, acetic acid, levulinic acid, and formic acid in different concentrations was tested in the fermentation using Saccharomyces cerevisiae, Pichia stipitis, and Zymomonas mobilis. The substitution of freshwater by seawater in the culture medium was also analyzed. Thus, inhibitory effects were stronger in the fermentation using P. stipitis, followed by Z. mobilis and S. cerevisiae. Formic acid and acetic acid presented more significant effects among the inhibitory compounds, followed by HMF, furfural and levulinic acid. Fermentation performed in culture medium with seawater showed promising results, especially in the ethanol yield using S. cerevisiae (0.50 g ethanol/g glucose) and Z. mobilis (0.49 g ethanol/g glucose). Whereas the production of cellulosic ethanol and holocellulosic ethanol are in early stages of development on an industrial scale, and that the availability and use of freshwater may cause socio‐environmental problems for expansion of ethanol production, the use of seawater appears as an alternative to mitigate this problem.

Modeling and simulation of breakthrough curves during purification of two chitosanases from Metarhizium anisopliae using ion-exchange with expanded bed adsorption chromatography

A mathematical model was developed to predict breakthrough curves during purification of the two chitosanases from Metarhizium anisopliae by expanded bed adsorption, taking into account the axial dispersion of liquid and using Streamline DEAE and SP XL adsorbents, anion and cation exchange resins, respectively. All the experiments were performed without clarification (with cells) aiming at the reduction of unit operations in future projects of separation processes, thereby reducing capital and operating costs. Chitosanases are enzymes that hydrolyze the carbohydrate chitosan, resulting in oligosaccharides that have many remarkable biological activities, such as anti-cancer, anti-HIV and antioxidant activities. The two adsorbents had similar performance in relation to hydrodynamics and mass transfer. The results of the parametric sensitivity analysis agree with the literature, and the model was validated with an average high degree of fit (94.68%) between simulated and experimental data obtained in this work.

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.

Recovery of Rhamnolipids Produced by Pseudomonas aeruginosa Using Acidic Precipitation, Extraction, and Adsorption on Activated Carbon

Biosurfactants are produced by microorganisms, especially those of the genus Pseudomonas. This study is concerned with the recovery of rhamnolipids produced by Pseudomonas aeruginosa P029-GVIIA, using molasses as substrate. A central compound design 23 in triplicate at the central point was used to evaluate, the influence of the centrifugation time, the agitation speed, and the pH on the amount of rhamnolipids precipitated by HCl (2 N). A 24 factorial design in triplicate at the central point was used to investigate the influence of the pH (3–10), temperature (30 to 50°C), the concentration of carbon (1–3% w/v), and the agitation speed (100–200 rpm) on the adsorption of rhamnolipids to activated carbon. The tests showed that the adsorption is governed particularly by the pH and the temperature, as well as by the temperature × pH interaction. A pseudo-first order kinetic model successfully fitted the data, showing that the adsorbent had the ability to adsorb approximately 17.16 mg of rhamnolipids/gram of activated carbon.

Enriquecimento protéico da palma forrageira com Saccharomyces cerevisiae para alimentação de ruminantes

The process of protein enrichment of the forage palm (Opuntia ficus-indica Mill) using the Saccharomyces cerevisiae yeast in a semi-solid culture to improve the nutritional value of forage palm for ruminants feeding was evaluated. The yeast concentrations of 1, 2 and 3% (wet basis) in the forage palm substrate were used. The periods of incubation were of 6, 12, 24, and 36 hours. A complete randomized experimental design in a split plot arrangement with four replicates was used. The crude protein content increased from 4.4% (in natura) to 10.4% when 3% of inoculums were used and the processing period was of 6 hours. The observed protein contents for 1% of the inoculum, used for the fermentation periods of 6, 12, 24, and 36 hours were 6.1, 8.1, 8.1, and 9.2%, respectively. These values were 9.6, 9.7, 9.8, and 9.8% for 2% of the inoculum, and 10.4, 10.4, 7.9, and 7.9% for 3% of the inoculum, respectively. An alternative for ruminant feeding can be obtained by bioconversion of forage palm.

Estudio del Secado de Anacardo (Anacardium occidentale L.) mediante Secador Solar de Radiación Directa

In this work solar drying tests with direct radiation and its mathematical modeling to evaluate the kinetics of cashew-nut (Anacardium occidentale L.) drying comparing their efficiency concerned to natural solar drying board are presented. The experiments were performed using fruit slices with 1 cm and 2 cm of thickness, obtaining its water content versus time. Using a direct solar radiation dryer, the drying time was lower than that of natural drying board, therefore adding value to products. Diffusivity values obtained from water sliced fruits were similar to other data available in the literature. The dried products had good organoleptic properties, aroma, color and crunchy texture.

Enhancing enzymatic hydrolysis of coconut husk through Pseudomonas aeruginosa AP 029/GLVIIA rhamnolipid preparation

This work investigated the influence of chemical (Triton X-100) and biological surfactant preparation (rhamnolipids) in coconut husk hydrolysis that was subjected to pretreatment with acid-alkali or alkaline hydrogen peroxide. The natural and pretreated biomass was characterized using the National Renewable Energy Laboratory protocol analysis as well as X-ray diffraction and scanning electron microscopy. The results demonstrated that in terms of the total reducing sugars, there was no significant difference between the hydrolysis using Triton X-100 and rhamnolipids, regardless of the pretreatment. A cellulosic conversion value as high as 33.0% was obtained in experiments with rhamnolipids. The coconut husk was observed to be a potential biomass that could produce second generation ethanol, and the rhamnolipid preparation can be used to support for the enzymatic hydrolysis, enhancing the advantage of cellulose conversion into glucose over chemical surfactants because it is an environmentally friendly approach.

Single-step purification of chitosanases from Bacillus cereus using expanded bed chromatography.

A chitosanase-producing strain was isolated and identified as Bacillus cereus C-01. The purification and characterization of two chitosanases were studied. The purification assay was accomplished by ion exchange expanded-bed chromatography. Experiments were carried out in the presence and in the absence of cells through different expansion degree to evaluate the process performance. The adsorption experiments demonstrated that the biomass does not affect substantially the adsorption capacity of the matrix. The enzyme bound to the resin with the same extent using clarified and unclarified broth (0.32 and 0.30 U/g adsorbent, respectively). The fraction recovered exhibited 31% of the yield with a 1.26-fold increase on the specific activity concerned to the initial broth. Two chitosanases from different elution steps were recovery. Chit A and Chit B were stable at 30-60°C, pH 5.5-8.0 and 5.5-7.5, respectively. The highest activity was found at 55°C, pH 5.5 to Chit A and 50°C, pH 6.5 to Chit B. The ions Cu(2+), Fe(2+) and Zn(2+) indicated inhibitory effect on chitosanases activities that were significantly activated by Mn(2+). The methodology applied in this study enables the partial purification of a stable chitosanase using a feedstock without any pre-treatment using a single-step purification.