In order to overcome the lack of characterization on the relative hydrophobicity of aqueous biphasic systems (ABS), the partition of three alkaloids as alternative probes, was evaluated in a series of biocompatible ABS composed of cholinium-based salts or ionic liquids (ILs) and polyethylene glycol (PEG). The caffeine partitioning in ABS was firstly addressed to infer on the effect of the phase-forming components composition. In all systems, caffeine preferentially concentrates in the lower water content PEG-rich phase. Additionally, a linear dependence between the logarithmic function of the partition coefficients and the water content ratio was found. To confirm this linear dependency, the partition coefficients of caffeine, theobromine and theophylline were determined in other ABS formed by different cholinium-based salts/ILs. In most systems, it is shown that all alkaloids partition to the most hydrophobic phase. To support the experimental results, COSMO-RS (Conductor-like Screening Model for Real Solvents) was used to compute the screening charge distributions of both phaseforming components of ABS and alkaloids, the excess enthalpy of mixing and the activity coefficients at infinite dilution. It is here demonstrated that the partition trend of alkaloids can be used to address the relative hydrophobicity of the coexisting phases in polymer-salt/-IL ABS.

The objectives of this work were to produce fructooligosaccharides (FOSs) by using the microorganism Bacillus subtilis natto CCT 7712 and to employ these FOSs as a functional ingredient in cassava starch edible films, which were characterized according to their microstructure, mechanical and barrier properties. The produced FOSs could be easily dissolved, resulting in homogeneous filmogenic solutions, which were easily manipulated to obtain films by casting. FOSs were added in different concentrations (0, 1, 5 and 10g/100g solids), and glycerol was used as a plasticizer (20g/100g solids). All formulations resulted in films that had a good appearance and were easily removable from the plates without bubbles or cracks. The FOSs exerted a plasticizing effect on the starch films and decreased their glass transition temperature. The addition of FOSs resulted in higher solubility and elongation and a decreased water vapor permeability of the films. FOSs were shown to be a promising ingredient for use in edible starch films.

L-asparaginase or ASNase (L-asparagine aminohydrolase, E.C.3.5.1.1) is an enzyme clinically accepted as an antitumor agent to treat acute lymphoblastic leukemia (ALL) and lymphosarcoma through the depletion of L-asparagine (L-Asn) resulting in cytotoxicity to leukemic cells. ASNase is also important in the food industry, preventing acrylamide formation in processed foods. Several quantification techniques have been developed and used for the measurement of the ASNase activity, but standard pharmaceutical quality control methods were hardly reported, and in general, no official quality control guidelines were defined. To overcome this lack of information and to demonstrate the advantages and limitations, this work properly compares the traditional colorimetric methods (Nessler; L-aspartic acid β-hydroxamate (AHA); and indooxine) and the high-performance liquid chromatography (HPLC) method. A comparison of the methods using pure ASNase shows that the colorimetric methods both overestimate (Nessler) and underestimate (AHA and indooxine) the ASNase activity when compared to the values obtained with HPLC, considered the most precise method as this method monitors both substrate consumption and product formation, allowing for overall mass-balance. Correlation and critical analysis of each method relative to the HPLC method were carried out, resulting in a demonstration that it is crucial to select a proper method for the quantification of ASNase activity, allowing bioequivalence studies and individualized monitoring of different ASNase preparations. Graphical abstract ᅟ.

BACKGROUND l‐Asparaginase (ASNase) is an important biopharmaceutical for the treatment of acute lymphoblastic leukemia (ALL); however, with some restrictions due to its high manufacturing costs. Aqueous biphasic systems (ABS) have been suggested as more economical platforms for the separation/purification of proteins, but a full understanding of the mechanisms behind the ASNase partition is still a major challenge. Polymer/salt‐based ABS with different driving‐forces (salting‐out and hydrophilicity/hydrophobicity effects) were herein applied to control the partition of commercial ASNase. RESULTS The main results showed the ASNase partition to the salt‐ or polymer‐rich phase depending on the ABS studied, with extraction efficiencies higher than 95%. For systems composed of inorganic salts, the ASNase partition was controlled by the polyethylene glycol (PEG) molecular weight used. Cholinium‐salts‐based ABS were able to promote a preferential ASNase partition to the polymer‐rich phase using PEG‐600 and to the salt‐rich phase using a more hydrophobic polypropylene glycol (PPG)‐400 polymer. It was possible to select the ABS composed of PEG‐2000 + potassium phosphate buffer as the most efficient to separate the ASNase from the main contaminant proteins (purification factor = 2.4 ± 0.2), while it was able to maintain the enzyme activity for posterior application as part of a therapeutic. CONCLUSION Polymer/salt ABS can be used to control the partition of ASNase and adjust its purification yields, demonstrating the ABS potential as more economic platform for the selective recovery of therapeutic enzymes from complex broths. © 2019 Society of Chemical Industry

l-Asparaginase (ASNase) is a high value enzyme for the pharmaceutical and food industries. Although ionic liquids (ILs) have been recognized as promising solvents or additives for biocatalysis, there are very few studies on the effect of ILs on the activation of ASNase. To address this, the enzymatic activity of a commercial ASNase in aqueous ILs was evaluated. ASNase was exposed for up to 24 h to aqueous solutions (0.001–0.050 mol·mol–1) of cholinium ([Ch])-based ILs, at three temperatures (25, 37, and 50 °C). At 25 °C, [Ch]-based IL aqueous solutions enhanced the biocatalytic activity of ASNase, with a maximum increase of the relative ASNase activity (of ca. 250%) achieved by adding cholinium chloride ([Ch]­Cl). Spectroscopic and calorimetric analyses were performed to understand the effect of [Ch]Cl on the ASNase structure, but no significant changes were observed. Although all [Ch]-based ILs enhanced the relative ASNase activity, the positive effects were diminished with increasing the anion alkyl chain length, i.e. from acetate to hexanoate. The increase of temperature to 50 °C caused a negative effect in the catalytic behavior of ASNase, leading, in most cases, to ASNase inactivation. Overall it was evident that [Ch]-based ILs are highly efficient biocatalytic additives to enhance ASNase activity in water (a 2-fold increase by adding less than 0.050 mol·mol–1). This simple and useful approach can be applied to increase the sustainability of several ASNase biocatalytic applications, especially in food processes.