Magnetic separation and high reusability of chloroperoxidase entrapped in multi polysaccharide micro-supports

Enzyme immobilization on magnetic supports represents a great advantage for the industrial application of enzymatic catalysis since it allows an easy recovery of the catalyst, avoiding any contamination of the product by residual enzyme. Iron oxide nanoparticles are very useful for this purpose. Using a polymer to diminish the interaction between the magnetic cores themselves, can improve the colloidal stability of the support and prevent any interaction with the environment that would affect both support properties and enzyme stability. For this reason, in this work different magnetic micro-supports, based on polydopamine-coated iron oxide nanoparticles with a multi polysaccharide shell, have been developed. These supports have been used to immobilize chloroperoxidase, a very interesting enzyme, able to catalyze many reactions of large-scale interest, but whose application is limited by its sensitivity to reaction conditions. The multi polysaccharide shells of the supports were obtained through a combination of chitosan and alginate. An in-depth analysis of physicochemical and catalytic properties of all the developed magnetic supports is reported. CPO was successfully immobilized with an efficiency of entrapment between 92% and 100% in the case of supports with chitosan in the interior or outer shell respectively. A very good chemical stability of the support under reaction conditions was observed in the case of an interior shell of alginate and an outer coating of chitosan, together with an excellent reusability of the immobilized enzyme, that was recycled to catalyze up to 25 consecutive reaction cycles

Understanding the role of temperature in structural changes of choline chloride/glycols deep eutectic solvents

Deep eutectic solvents (DESs) represent a new “green” alternative to classic organic solvents; however, their high viscosity often limits their utilization. The physicochemical properties of DESs can be tuned by adding cosolvents, by varying the operating temperature, or both, but their structure has to be preserved. In this work, three pure DESs based on choline chloride (ChCl) mixed with diethylene glycol (DEG), triethylene glycol (TEG) or polyethylene glycol 200 (PEG 200), as well as ChCl/TEG/water mixtures were studied. Particular attention was paid to the effect of temperature on some properties, i.e. density, conductivity, and viscosity, and on the structural characteristics of pure DESs and DES/water blends in the range 303–353 K. All the pure eutectic solvents followed both Arrhenius law and Walden's rule and showed very similar activation energy and poor ionicity, indicating the formation of intimate ion pairs. Multinuclear NMR analysis, such as the determination of the chemical shift of 1H, 13C, 35Cl and linewidths at half height of 14N and 35Cl, as a function of temperature highlighted the conservation of the characteristic supramolecular interactions up to 353 K. However, the temperature effects on the physicochemical properties of DES/water mixtures changed differently depending on the water content. Excess properties and NMR investigations highlighted the strengthening of the hydrogen bond interactions up to a 10% level of added water and their progressive weakening at a higher degree of hydration. These results also showed that temperature only slightly affects the structure of both DES and DES/water systems but can drastically increase their transport properties

1H-NMR-relaxation and colorimetry for evaluating nanopolymeric dispersions as stone protective coatings

Non-destructive and non-invasive assessment of new nanopolymeric dispersions for protecting stone materials was accomplished; the effectiveness of five formulations was tested regarding commercial products widely used in conservation on Lecce, Montescaglioso and Matera stones. We observed the specimens’ wettability during capillary water absorption up to saturation by nuclear magnetic transverse relaxation time measurements, using a surface and portable probe. The comparison between treated and the untreated ones, concerning their specific hydration behavior, allowed establishing the effects of the polymer films. Finally, color evaluations were performed to inspect differences induced by treatments. The data analysis enabled to select the most satisfactory protective coating for each lithotype

Effect of water addition on choline chloride/glycol deep eutectic solvents: Characterization of their structural and physicochemical properties

In the present study the effect of water on the physicochemical properties and on the structural features of three deep eutectic solvents (DESs) were evaluated. The DESs are formed by choline chloride (ChCl) and glycols with a different number of oxyethylene units: diethylene glycol (DEG), triethylene glycol (TEG) and poly(ethylene glycol) 200 (PEG 200). From the dilution effect on the physicochemical properties of DESs it has emerged that even a small amount of water could increase fluidity, conductivity and polarity of the DESs. The effect of water addition on the nanostructural changes of DESs was also investigated by FTIR and NMR spectroscopies. The results clearly demonstrated that there are strong interactions between the two components of the DES and dilution with water caused the interactions weaken gradually up to 50% (w/w) water added, but with the supramolecular structures to some extent preserved. Subsequent water additions led to the disappearance of such interactions until they disappeared completely at around 75% (w/w). Moreover, NMR studies showed that the H-bonding interactions between ChCl and DEG are less strong than those between ChCl and both TEG and PEG 200, indicating that the number of oxyethylene units may be important. The changes observed in the physicochemical properties and on the nanostructural features of the mixtures can lead to the realization of liquid designed for tailored applications

alpha-Chymotrypsin superactivity in quaternary ammonium salt solution: kinetic and computational studies

As previously reported, quaternary ammonium salts with bulky hydrophobic portions provoke a superactivation of alpha-chymotrypsin in aqueous solution: this is the case of the surfactant cetyltributylammonium bromide (CTBABr) and the corresponding salt tetrabutylammonium bromide (TBABr). In order to achieve a broader knowledge of the enzyme-additive interactions, in this paper the activity and stability of alpha-chymotrypsin were tested in the presence of additives with slightly modified bulky ammonium groups. The effect of three additives with a benzylic group as substituent (benzyltrimethylammonium bromide (BzTMABr), benzyltributylammonium bromide (BzTBABr) and benzyldodecyldimethylammonium bromide (BzDDABr)) was investigated. A significant increase in instantaneous activity, but a deactivation of enzyme, faster than in pure buffer, was observed. Moreover, two novel dicationic salts, (1, 8-bis(tributylammonium)octane dibromide (bisBOAB) and 1, 4-bis(tributylammonium)xylene dibromide (bisBAB)) were designed and synthesized in order to evaluate the effect of two tributylammonium head groups with a different spacer. BisBOAB provoked superactivation and stabilization effects in a way similar to the "homologue" TBABr, but at lower concentration. In contrast, when the benzyl group was constrained within the spacer structure, the obtained superactivity was lower than in the presence of a more flexible hydrocarbon chain spacer, and enzyme deactivation was faster. Molecular modelling studies allowed us to rationalize the hypotheses derived from kinetic evidence. The results confirmed that the improvement in the catalytic properties observed in the presence of additives with a bulky, hydrophobic ammonium head group could be addressed to an increase in the overall hydrophobicity of the alpha-chymotrypsin catalytic site