Luciana Pellegrini Malpiedi

Features of partitioning pattern of two pancreatic enzymatic precursors: trypsinogen and chymotrypsinogen in polyethyleneglycol-sodium citrate aqueous biphasic systems.

The partitioning behaviour of bovine trypsinogen and alpha-chymotrypsinogen, enzymatic precursors with similar physicochemical properties, was investigated in different polyethyleneglycol/sodium citrate aqueous two-phase systems. The effect of different factors such as polyethyleneglycol molecular weight, pH, tie line length and temperature was also examined. The increase of pH and the decrease of polyethyleneglycol molecular weight displaced the partitioning equilibrium of both proteins to the top phase. An enthalpy-entropy compensation pattern was observed, indicating the participation of water molecules in the partitioning mechanism. TRPz phase equilibrium showed to be more displaced to the citrate-rich phase than ChTRPz for most of the assayed systems. From a practical view, the aqueous two-phase system formed by polyethylenglycol of molecular weight 1450 and sodium citrate pH 8.20 showed the best capability for separating both proteins. When a mixture formed by equal quantities of both zymogens was partitioned in this system, significant recoveries (about 60%) were obtained. Purity values were improved significantly (84-89%) by either developing a second extractive step or increasing the top-bottom volume ratio.

Role of polymer–protein interaction on partitioning pattern of bovine pancreatic trypsinogen and alpha-chymotrypsinogen in polyethyleneglycol/sodium tartrate aqueous two-phase systems

The partitioning pattern of bovine trypsinogen (TRPz) and alpha-chymotrypsinogen (ChTRPz) was investigated in a low impact aqueous two-phase system formed by polyethyleneglycol (PEG) and sodium tartrate (NaTart) pH 5.00. ChTRPz exhibited higher partition coefficients than TRPz did in all the assayed systems. The decrease in PEG molecular weight and the increase in tie line length were observed to displace the partitioning equilibrium of both proteins to the top phase, while phase volume ratios in the range 0.5-1.5 showed not to affect protein partitioning behaviour. Systems formed by PEG of molecular weight 600 with composition corresponding to a high tie line length (PEG 12.93%, w/w and NaTart 21.20%, w/w) are able to recover most of both zymogens in the polymer-enriched phase. A crucial role of PEG-protein interaction in the partitioning mechanism was evidenced by isothermal calorimetric titrations. The major content of highly exposed tryptophan rests, present in ChTRPz molecule, could be considered to be determinant of its higher partition coefficient due to a selective charge transfer interaction with PEG molecule. A satisfactory correlation between partition coefficient and protein surface hydrophobicity was observed in systems formed with PEGs of molecular weight above 4000, this finding being relevant in the design of an extraction process employing aqueous two-phase systems.

Integrated extraction and purification of soy isoflavones by using aqueous micellar systems

In this work, an integration of solid-liquid and liquid-liquid extractions by using aqueous micellar two-phase systems was evaluated as potential tool to purify soy isoflavones. Additionally, the proposed methodology aimed to preserve the protein content of the processed soy flour. The extractive assays were performed in AMTPS formed by Triton X-114 and sodium tartrate. In order to optimize the purification process, temperature and time were evaluated as independent variables. Under optimal working conditions, i.e. 100min and 33°C of incubation, IF were purified with a recovery percentage of 93 and a purification factor of almost 10. More importantly, the obtained sample presented an aglycone proportion superior to the reported by other methodologies. These results open perspectives to the use of aqueous micellar two-phase systems as an integrative methodology to extract, concentrate and purify isoflavones.

Optimized extraction of a single-chain variable fragment of antibody by using aqueous micellar two-phase systems

In this work, the purification of a single-chain variable fragment (scFv) of an antibody by using liquid-liquid extraction in aqueous micellar two-phase systems was optimized by means of central composite design. Protein partitioning assays were performed by using the selected system composition in previous works: Triton X-114 at 4% wt/wt, yeast fermentation supernatant at 60% wt/wt, McIlvaine buffer pH 7.00. The other system component concentrations, Cibacron Blue F3GA (CB), Fabsorbent™ F1P HF (HF) and NaCl, were selected as independent variables. ScFv recovery percentage (%R) and purification factor (PF) were selected as the responses. According to the optimization process both, scFv recovery percentage and purification factor were favored with the addition of HF and NaCl in a range of concentrations around the central point of the second central composite design (HF 0.0120% w/w, CB 0.0200% w/w, NaCl 0.200% w/w). These experimental conditions allowed the concentration and pre-purification of scFv in the micelle-rich bottom phase of the systems with a recovery percentage superior to 88% and a purification factor of approximately 3.5. These results improved the previously presented works and demonstrated the convenience of using aqueous micellar two-phase systems as a first step in the purification of scFv molecules.

Continuous method to determine the trypsin inhibitor activity in soybean flour.

The determination of trypsin inhibitor (TI) activity is of importance to evaluate the nutritional value of soybean flours. An analytical method, which involves a continuous spectrophotometric rate determination for trypsin activity against the substrate N-benzoyl-DL-arginine p-nitroanilide, is proposed as an alternative to the standard discontinuous assay. Stopping the reaction with acetic acid and a centrifugation/filtration step to decrease turbidity are not required, thus reducing costs and sample preparation time. The TI activity of different flour samples, determined by both assays, demonstrated to be statistically comparable, irrespective of the TI concentration level. The coefficients of variation of the novel method did not exceed 8% at any concentration level. The curves of progress reaction showed a non-linear behavior in samples without TI. A reduction of incubation time from 10min to 2min increased the method sensitivity and extended its linear range. A more economical, faster and simpler assay was developed.

Micellar systems of aliphatic alcohol ethoxylates as a sustainable alternative to extract soybean isoflavones

Ethoxylated aliphatic surfactants belonging to the Genapol and Tergitol series were assessed as extraction systems of isoflavones. They showed good extraction properties when compared with different solvents, the Genapol X-080 exhibiting the best performance. Available commercial isoflavone pills were used, as a starting simple matrix, to determine the parameters that affect the extraction procedure. The temperature and the surfactant concentration showed to be factors that favored significantly the extraction performance. The application of optimized variables (Genapol X-080 11% m/m, pH 4.5; extraction temperature of 54 °C and extraction time of 60 min) on soybean flour (natural) allowed extracting 3.237 ± 0.173 mg of isoflavone per gram of treated flour. This result was three times what it was for methanol under identical conditions. Extraction with these micellar systems represents a sustainable alternative methodology for industrial purposes due to its low cost, biodegradability, non-toxicity and easy scaling up.