Nicoletta Spreti

Enzyme activity and stability control by amphiphilic self-organizing systems in aqueous solutions

The interaction of surfactants with proteins in aqueous solutions has been the subject of many investigations to understand the interactions between membrane proteins and lipids, structurally similar to synthetic surfactants. The effect of surfactant on enzyme structure and activity is the result of chemically selective interactions that may be influenced both by the enzyme structure and by the chemistry of the surfactant. For many years, surfactants have been considered as non-specific denaturants of proteins, even if in the literature several of them are reported to enhance activity and/or stability of some enzymes: the detergent can interact with the enzyme and cause a conformational change to a more active form and/or stabilize its native folded structure. Although the surfactant head group seems to have a determining role, other structural features of the detergent are also important in influencing the catalytic properties of an enzyme, i.e. head group size and its hydrophobic/hydrophilic balance. Up to now it is very difficult to predict the molecular features of the surfactant and an extensive investigation on the relationship between the surfactant chemical structure and the catalytic properties of enzyme is still required.

α-Chymotrypsin superactivity in aqueous solutions of cationic surfactants

Abstract α-Chymotrypsin (α-CT) activity was tested in aqueous media with the following cetyltrialkylammonium bromide surfactants in the series methyl, ethyl, propyl and butyl, different in the head group size, and for the sake of comparison also with the anionic sodium n -dodecyl sulfate and the zwitterionic myristyldimethylammonium propanesulfonate. N -glutaryl- l -phenylalanine p -nitroanilide hydrolysis rate was monitored at surfactant concentration above the critical micellar one. Only some cationic surfactants gave superactivity and the head group size had a major weight. The highest superactivity was measured in the presence of cetyltributylammonium bromide. The effect of both nature and concentration of three different buffers was also investigated. There is a dependence of enzyme superactivity on buffer type. Michaelis–Menten kinetics were found. The binding constants of substrate with micellar aggregates were determined in the used buffers and the effective improvement of reaction rate (at the same free substrate concentration in the medium) was calculated. k cat significantly increased while K m was little changed after correction to free substrate concentration. The ratio of k cat to K m was between 12 and 35 times higher than in pure buffer, depending on buffer and surfactant concentrations. The increase of α-CT activity (30%) was less important in the presence of 1×10 −2 M tetrabutylammonium bromide, a very hydrophobic salt, unable to micellise. Fluorescence spectra showed differences of enzyme conformation in the presence of various surfactants.

Stabilization of Chloroperoxidase by Polyethylene Glycols in Aqueous Media: Kinetic Studies and Synthetic Applications

Chloroperoxidase (CPO) is one of the most versatile of the heme peroxidase enzymes for synthetic applications. Despite the potential use of CPO, commercial processes have not been developed because of the low water solubility of many organic substrates of synthetic interest and the limited stability due to inactivation by H2O2. CPO catalytic properties have been studied in aqueous solutions in the presence of short‐chain poly(ethylene glycol)s (PEGs), and the sulfoxidation of thioanisole, as model substrate, has been investigated. The addition of PEGs allows a better substrate solubilization in the reaction mixture and the enzyme to retain more of its initial activity, with respect to pure buffer. Kinetic studies were performed to optimize the experimental conditions, and complete enantioselective conversion to the ( R)‐sulfoxide (ee = 99%) was observed in the presence of PEG 200 and tri(ethylene glycol). The relevant stabilization of chloroperoxidase due to the presence of PEGs allows the enzyme to convert the substrate with significant product yields even after 10 days, with a consequent increase in enzyme productivity. This is a promising result in view of industrial application of the enzyme.

α-chymotrypsin superactivity in cetyltrialkylammonium bromide-rich media

Abstractα-Chymotrypsin (α-CT) activity was tested with N-glutaryl-l-phenylalanine p-nitroanilide in buffered media with added cationic surfactants. The effect of the commercial cetyltrimethylammonium bromide (CTABr) was compared with that of three other surfactants with ethyl (CTEABr), propyl (CTPABr), and butyl (CTBABr) head groups. These were synthesized and purified in this laboratory. Surfactant head groups provided distinct environments that largely modulated the catalytic performance. Larger alkyl head group hydrophobicity led to a marked enhancement of α-CT activity. CTBABr-rich media induced the highest superactivity.Kinetic measurements were performed in Tris-HCl buffer at a surfactant concentration either below or above CMC, and α-CT superactivity occurred in both media. Positive interactions between the enzyme and surfactants happened independently of thesupramolecular organization of the medium. The reaction followed the Michaelis-Menten kinetics. The substrate to micelle aggregates binding constant was used to calculate the substrate concentration available for catalysis. The kcat to km ratio was in CTBABr-rich media always higher than in pure buffer and depended on the surfactant concentration. α-CT superactivity depended on the pH value of buffer solution. Enzyme inactivation followed a single-step mechanism in pure buffer and a series mechanism in the presence of a surfactant. The rate of activity decay obeyed a first-order kinetics.

Effects of Surfactants on the Stabilization of the Bovine Lactoperoxidase Activity

Bovine lactoperoxidase (LPO) is taken as a model protein of mammalian peroxidases to investigate the activity and the stability of the enzyme in the presence of different surfactants. The cationic benzalkonium chloride (Bz) has proved efficient in preserving the enzymatic activity for over 10 days, while the native enzyme completely lost its activity within 3−4 days. The presence of Bz allows the enzyme to preserve its secondary structure for a long time, as shown in CD spectra, and creates a more hydrophobic environment for the enzyme, as indicated in fluorescence studies. Moreover, this surfactant at a concentration of 0.01% (0.3 mM) increases the lactoperoxidase activity in the first 2 h of incubation at 37 °C. Both hydrophobic and electrostatic interactions of the cationic surfactant seem to be responsible for the enzyme activation and stabilization, and this is a promising result in view of industrial applications of enzymes.

A New In Vitro Model of Lignin Biosynthesis

A strictly coordinate sequence of radical and ionic steps appears to be the mechanism by which oligolignols are generated. A synthetic lignin was produced under micellar conditions [Eq. (1)], and the beginning of the polymerization process was studied by electrospray ionization mass spectrometry. CTA=cetyltrimethylammonium ion.

Room temperature deep eutectic solvents of (1S)-(+)-10-camphorsulfonic acid and sulfobetaines: hydrogen bond-based mixtures with low ionicity and structure-dependent toxicity

Twelve novel deep eutectic solvents (DESs) were prepared and characterized in this work. They are mixtures of (1S)-(+)-10-camphorsulfonic acid (CSA) and differently structured sulfobetaines (SBs) with aliphatic, aromatic and amphiphilic moieties. They are liquids at room temperature, their melting points span, in fact, from −5° to 19 °C, so we can name these mixtures RTDESs (room temperature deep eutectic solvents). These zwitterionic DESs were characterized in terms of their viscosity, conductivity (and therefore ionicity via Walden plots), density, surface tension and toxicity on eukaryotic model cells. The collected data suggest that the interaction between CSA and the SBs can be ascribed as a hydrogen bond instead of a proton transfer, therefore they are not ionic liquids. To our knowledge, their position on the Walden plot, in the left portion close to the diagonal, has not yet been observed for other DESs or ionic liquid systems and indicates the low ionicity of these mixtures. A FTIR-based bioassay was performed to determine the toxicity of these mixtures on eukaryotic model cells (Saccharomyces cerevisiae). The DESs showed merely a dehydrating effect on the cells, similar to that produced by CaCl2, a low cell toxicity salt. This supports these DESs as promising green media. Amphiphilic SBs DESs showed a stronger effect on the cells and a structure-activity trend can be described for this class. A preliminary study on the use of these novel DESs as Bronsted catalyst media was accomplished by the use of one of them in chalcone synthesis, which showed promising catalytic and recycling capabilities.

Decarboxylation of 6-nitrobenzisoxazole-3-carboxylate ion in dichloromethane : the possible role of reverse micelles

Abstract Spontaneous decarboxylation of 6-nitrobenzisoxazole-3-carboxylate ion ( 1 ) in dichloromethane with added base is catalyzed by cationic surfactants ( R NMe 3 Br, R = n -C 8 H 17 , n -C 12 H 25 , and n -C 16 H 33 ) by EtNMe 3 Br and n -BuNMe 3 Br, by R 4 NBr ( R = Et, n -Bu, n -C 8 H 17 , and n -C 12 H 25 ), and by PhCH 2 N R 3 Br ( R = Et, n - Bu). The teraalkylammonium and benzylammonium bromides are better catalysts than the n -alkylammonium bromides. Reaction in Bu 4 NBr and CTABr ( n -C 16 H 33 NMe 3 Br) is inhibited by water, and conductivity and 1 H NMR spectrometry show that CTABr plus water form “water pool” reverse micelles. Reaction in the absence of quaternary ammonium salts is faster when the base is tetramethylguanidine rather than Et 3 N, probably because Et 3 N + H deactivates 1 by hydrogen bonding. This difference disappears in CTABr, which disrupts the hydrogen bonding. In the absence of water, reaction probably occurs in small ion clusters of quaternary ammonium bromides which are gradually transformed into less catalytically effective water pool reverse micelles on addition of water.

Lignin Chemistry: Biosynthetic Study and Structural Characterisation of Coniferyl Alcohol Oligomers Formed In Vitro in a Micellar Environment

Model coniferyl alcohol lignin (the so-called dehydrogenative polymerisate, DHP) was produced in water under homogeneous conditions guaranteed by the presence of a micellised cationic surfactant. A complete study of the activity of the enzymatic system peroxidase/H(2)O(2) under our reaction conditions was reported and all the reaction products up to the pentamer were characterised by (1)H NMR spectroscopy and ESI mass spectrometry. Our system, and the molecules that have been generated in it, represent a closer mimicry of the natural microenvironment since an enzyme, under micellar conditions, reproduces the cell system better than in buffer alone. On the basis of the oligomers structures a new biosynthetic perspective was proposed that focused attention on a coniferyl alcohol dimeric quinone methide as the key intermediate of the reaction. A formal, strictly alternate sequence of a radical and an ionic step underlines the reaction, thus generating ordered oligolignols structures. Alternatively to other model lignins, our olignols present a lower degree of radical coupling between oligomeric units. This offers a closer biosynthetic situation to the observation of a low rate of radical generation in the cell wall.

Influence of sulfobetaines on the stability of the Citrobacter diversus ULA-27 β-lactamase

The activity and stability of β‐lactamase from Citrobacter diversus ULA‐27 have been investigated in the presence of different ionic and zwitterionic surfactants. All the sulfobetaine surfactants tested allow the enzyme to retain its full activity, but the best stabilizing effect is greatly dependent on their structure. Very little variations on the monomer headgroup can significantly reduce enzyme deactivation or speed up the loss of activity with respect to buffer alone. The whole hydrophobic/hydrophilic balance on the headgroup seems to have a determining role in preserving β‐lactamase activity and structure. The presence of zwitterionic surfactants stabilizes the protein conformation toward denaturation by urea and low‐temperature inactivation. Similar experiments were performed in the presence of other two zwitterionic surfactants, an amine oxide, dimethylmyristylamine oxide (DMMAO) and a carboxybetaine, cetyldimethylammonium methanecarboxylate (CB1–16). The former stabilizes the enzyme even better than the sulfobetaines, the latter quickly deactivates it. Therefore, the factors responsible for β‐lactamase stabilization are dependent not only on the zwitterionic nature of the surfactant headgroup but also specific interactions between the surfactant and the protein may be important.