Carlos Eduardo de Araújo Padilha

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.

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.

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.

Modeling and simulation of Bacillus cereus chitosanase activity during purification using expanded bed chromatography

A phenomenological model was used to describe sequentially the three steps (flowthrough, washing and elution) of expanded bed adsorption chromatography for recovery of chitosanases from Bacillus cereus. Additionally, a hybrid strategy for model parameter estimation was carried out using particle swarm optimization and Gauss-Newton algorithms. The model was validated with independent experimental data and the statistical criteria (χ2 and mean squared error tests) showed that the hybrid strategy was more promising than just the heuristic method. With the calibrated model, surface response methodology was applied to obtain the optimal operational conditions, and experiments were performed to confirm these results. Overall, a value of 41.08% for yield was obtained using 700 mM NaCl during elution. In summary, all approach employed in this work was relevant for maximizing the yield of the chromatographic process.

Recovery and purification of chitosanase produced by Bacillus cereus using expanded bed adsorption and central composite design

This study presents a system for expanded bed adsorption for the purification of chitosanase from broth extract in a single step. A chitosanase-producing strain was isolated and identified as Bacillus cereus C-01 and used to produce chitosanases. The expanded bed adsorption conditions for chitosanase purification were optimized statistically using STREAMLINE(TM) DEAE and a homemade column (2.6 × 30.0 cm). Dependent variables were defined by the quality criteria purification factor (P) and enzyme yield to optimize the chromatographic process. Statistical analyses showed that the optimum conditions for the maximum P were 150 cm/h load flow velocity, 6.0 cm settled bed height, and 7.36 cm distributor height. Distributor height had a strong influence on the process, considerably affecting both the P and enzyme yield. Optimizing the purification variables resulted in an approximately 3.66-fold increase in the P compared with the value under nonoptimized conditions. This system is promising for the recovery of chitosanase from B. cereus C-01 and is economically viable because it promotes the reduction steps.

Enhancing the recovery and concentration of polyphenols from camu-camu (Myrciaria dubia H.B.K. McVaugh) by aqueous two-phase flotation and scale-up process

ABSTRACT In this study, polyphenols obtained from camu-camu pulp residue were recovered and concentrated from aqueous two-phase flotation (ATPF) by evaluating different operating conditions, such as solvent type, pH, ammonium sulfate concentration, flotation time, air flow, and volumetric ratio. Under optimal conditions, the polyethylene glycol 400/ammonium sulfate ATPF system showed an efficiency of 81.02% and a concentration coefficient two times higher than traditional aqueous two-phase extraction. Experiments were also carried out on a semi-pilot scale, using an apparatus with a 2.5-L working volume. The results showed that the semi-pilot system was comparable to the smaller scale system.

Simultaneous recombinant 503 antigen recovery and endotoxin removal from E. coli M15 homogenate using expanded bed adsorption chromatography

ABSTRACT This study evaluated the simultaneous recovery of recombinant 503 antigen and endotoxin removal from E. coli homogenate by immobilized metal affinity- expanded bed adsorption chromatography (EBAC), using the non-ionic surfactant Triton X-114 during the washing step. The strategy employed was able to remove more than 99% of endotoxin in all conditions tested, and the lowest residual concentration obtained was 10.22 EU/mL, with a recovery of 503 antigen ranging from 41.4% to 61.1%. Therefore, the procedure of integrating the clarification, recovery of protein, and endotoxin removal into a unit operation exploiting the EBAC was successful.

Mathematical modeling of the whole expanded bed adsorption process to recover and purify chitosanases from the unclarified fermentation broth of Paenibacillus ehimensis

In this study, a general rate model was applied to the entire process of expanded bed adsorption chromatography (EBAC) for the chitosanases purification protocol from unclarified fermentation broth produced by Paenibacillus ehimensis using the anionic adsorbent Streamline® DEAE. For the experiments performed using the expanded bed, a homemade column (2.6cm×30.0cm) was specially designed. The proposed model predicted the entire EBA process adequately, giving R2 values higher than 0.85 and χ2 as low as 0.351 for the elution step. Using the validated model, a 33 factorial design was used to investigate other non-tested conditions as input. It was observed that the superficial velocity during loading and washing steps, as well as the settled bed height, has a strong positive effect on the F objective function used to evaluate the production of the purified chitosanases.

Production of recombinant 503 antigen of Leishmania infantum chagasi using cultivation in batch and fed-batch

Background Visceral leishmaniasis is an infecto-parasitarian disease caused by obligatory intracellular protozoa belonging to the genus Leishmania, and might be lethal since there is no precocious diagnosis and proper treatment. Despite the considerable effort, there is no effective and safe vaccine for human use [1]. Genetically modified microorganisms are used industrially in general for production of hormone, antibiotics and proteins. In the production of heterologous molecules, both the expression and the production step are important [2]. Therefore, the study of cultivation conditions in bioreactor plays an important role for the process viability of batch and fed-batch [3]. Thus, the aim of this work was to study strategies for production of the 503 antigen of Leishmania infantum chagasi using cultivation in batch and fed-batch.