Industrial applications of immobilized nano-biocatalysts.

Immobilized enzyme-based catalytic constructs could greatly improve various industrial processes due to their extraordinary catalytic activity and reaction specificity. In recent decades, nano-enzymes, defined as enzyme immobilized on nanomaterials, gained popularity for the enzymes' improved stability, reusability, and ease of separation from the biocatalytic process. Thus, enzymes can be strategically incorporated into nanostructured materials to engineer nano-enzymes, such as nanoporous particles, nanofibers, nanoflowers, nanogels, nanomembranes, metal-organic frameworks, multi-walled or single-walled carbon nanotubes, and nanoparticles with tuned shape and size.

Searching for an absolute kinetic scale of antioxidant activity against lipid peroxidation.

The inhibition properties of a number of antioxidants against peroxidation, started by a 2,2'-azobis[2-(2-imidazolin-2-yl)propane] radical initiator, of linoleic acid in sodium dodecyl sulfate micelles, have been determined in terms of oxygen consumption by a Clark electrode in an oxygen-tight cell. For the 31 antioxidants investigated at variable concentrations, the experimental results well fit the kinetic equation for competitive reactions. The ratio between the initial rates, monitored in the absence and in the presence of antioxidants, depends linearly on their concentration.

A novel enzyme with spermine oxidase properties in bovine liver mitochondria: identification and kinetic characterization.

The uptake of spermine into mammalian mitochondria indicated the need to identify its catabolic pathway in these organelles. Bovine liver mitochondria were therefore purified and their capacity for natural polyamine uptake was verified. A kinetic approach was then used to determine the presence of an MDL 72527-sensitive enzyme with spermine oxidase activity in the matrix of bovine liver mitochondria.

Immobilized papain on gold nanorods as heterogeneous biocatalysts.

Papain, a thiol protease present in the latex of Carica papaya, is an enzyme which exhibits broad proteolytic activity, and, for this reason, it is utilized in a variety of industrial applications. Immobilization of papain on gold nanoparticles highly preserves its activity and enhances the stability, allowing the reuse of the linked enzyme many times without any significant loss of its catalytic performance. In particular, kcat and KM values remain substantially unchanged, while immobilized form shows a higher activity on a wider pH range retains 80 % residual activity also at 90 °C and shows higher functionality than the free form when incubated for long time (1 h) at 90 °C and at extreme pH values (3 and 12).

Photoprotective characteristics of natural antioxidant polyphenols.

Fourteen natural polyphenols belonging to the classes of stilbenes, flavonoids and hydroxycinnamic acid derivatives, have been investigated in order to verify the combination of their photoprotective characteristics with their antioxidant properties. To this purpose, sun protection factor (SPF), UVA/UVB ratio and critical wavelengths (λc), have been considered to evaluate photoprotection capacity, while inhibition of lipid peroxidation has been adopted as a reliable measure of the antioxidant properties. The results obtained indicate that a large number of these natural phenol derivatives show both antioxidant activity and photoprotective characteristics and, as a consequence, they could be interesting components for pharma-photoprotection formulations.

Enzyme immobilization: an update.

Compared to free enzymes in solution, immobilized enzymes are more robust and more resistant to environmental changes. More importantly, the heterogeneity of the immo-bilized enzyme systems allows an easy recovery of both enzymes and products, multiple re-use of enzymes, continuous operation of enzymatic processes, rapid termination of reactions, and greater variety of bioreactor designs. This paper is a review of the recent literatures on enzyme immobilization by various techniques, the need for immobilization and different applications in industry, covering the last two decades.

Antioxidant properties of Brazilian tropical fruits by correlation between different assays.

Four different assays (the Folin-Ciocalteu, DPPH, enzymatic method, and inhibitory activity on lipid peroxidation) based on radically different physicochemical principles and normally used to determine the antioxidant activity of food have been confronted and utilized to investigate the antioxidant activity of fruits originated from Brazil, with particular attention to more exotic and less-studied species (jurubeba, Solanum paniculatum; pequi, Caryocar brasiliense; pitaya, Hylocereus undatus; siriguela, Spondias purpurea; umbu, Spondias tuberosa) in order to (i) verify the correlations between results obtained by the different assays, with the final purpose to obtain more reliable results avoiding possible measuring-method linked mistakes and (ii) individuate the more active fruit species. As expected, the different methods give different responses, depending on the specific assay reaction. Anyhow all results indicate high antioxidant properties for siriguela and jurubeba and poor values for pitaya, umbu, and pequi.

Purification and characterization of a novel thermostable luciferase from Benthosema pterotum.

A novel luciferase from Benthosema pterotum, collected from Port of Jask, close to Persian Gulf, was purified for the first time, using Q-Sepharose anion exchange chromatography. The molecular mass of the novel enzyme, measured by SDS-PAGE technique, was about 27 kDa and its Km value is 0.4 μM; both values are similar to those of other coelenterazine luciferases.

Charge binding of rhodamine derivative to OH- stabilized nanomaghemite: universal nanocarrier for construction of magnetofluorescent biosensors.

Superparamagnetic nanoparticles (20-40 nm) of maghemite, γ-Fe(2)O(3), with well-defined stoichiometric structure, are synthesized by the borohydride reduction of ferric chloride at an elevated temperature (100°C) followed by thermal treatment of the reaction product. Prepared maghemite nanoparticles reveal excellent colloidal stability for a long time without the necessity for any additional surface modification. These colloidal features are due to surface stabilizing OH(-) groups, which act as charge barriers preventing a particle aggregation and enabling a reversible binding of various oppositely charged organic substances.

Do mammalian amine oxidases and the mitochondrial polyamine transporter have similar protein structures?

Polyamine transport across the mitochondria membrane occurs by a specific, common uniporter system and appears controlled by electrostatic interactions as for polyamine oxidative deamination by bovine serum and mitochondrial matrix amine oxidases was found. In fact in all the cases, while the catalytic constants or the maximum uptake rate values show little changes with the number of the positive charges of the substrates, Michaelis-Menten constant values demonstrate exponential dependence, confirming that electrostatic forces control the docking of the substrate into the enzyme active site or polyamine channel. By the treatment of the kinetic data in terms of Gibbs equation or Eyring theory, the contribution of each positive charge of the polyamine to the Gibbs energy values for the oxidative deamination of polyamines by two mammalian amine oxidase and for polyamine transport, are obtained.

Propolis as potential cosmeceutical sunscreen agent for its combined photoprotective and antioxidant properties.

Propolis, bee glue, and its main polyphenolic components show high antioxidant activity as found measuring their inhibitory action on lipid peroxidation of linoleic acid (LA) in sodium dodecyl sulfate (SDS) micelles. Furthermore, these substances evidence effectiveness as broad spectrum UVB and UVA photoprotection sunscreens, as it results by measurements of sun protection factor (SPF), the universal indicator related primarily to UVB radiations, and of the two parameters giving an indication of the UVA absorbance properties, i.e.

Preliminary kinetic characterization of a copper amine oxidase from rat liver mitochondria matrix.

Kinetic measurements of a novel copper-dependent amine oxidase, purified from rat liver mitochondria matrix, were carried out using various substrates in a large pH (5.6-10.2) and ionic strength range (5-200 mM), in order to study the docking of substrates to the enzyme and, as a consequence, to verify the physicochemical characteristics of the active site.

Correlations between polyphenolic composition and antioxidant activity of Venetian propolis.

Four propolis samples have been picked up in the Venetian region, from different orography and habitative density areas with the purpose to: (i) evaluate propolis' antioxidant activity, measured by inhibition of lipid peroxidation; (ii) determine the polyphenolic components--flavonoids and caffeic acid derivatives--which give antioxidant activity to propolis; (iii) verify the potential correlations between antioxidant activity, polyphenolic content, that has been determined by Folin-Ciocalteu, enzymatic, DPPH quenching, TEAC-like assays, and spectroscopic characteristics of propolis and (iv) correlate chemical structure and antioxidant efficacy of each of the major components. The possible localization of the lipophylic components of propolis into the phospholipidic bilayer by thermal analysis (DSC) and spin label EPR techniques has also been investigated.

Cysteine enhances activity and stability of immobilized papain.

Immobilization of papain on Sepharose 6B in the presence of different concentrations of cysteine affected the enzyme activity depending on cysteine concentration. The maximum specific activity was observed when papain was immobilized with 200 mM cysteine. The immobilization process brought significant enhancement of stability to temperature and extreme pH values with respect to free papain.

Novel copper amine oxidase activity from rat liver mitochondria matrix.

Copper containing amine oxidases (Cu-AO) represent a heterogeneous class of enzymes classified as EC 1.4.3.

Antioxidant properties of resveratrol and piceid on lipid peroxidation in micelles and monolamellar liposomes.

The antioxidant activities of trans-resveratrol (trans-3,5,4'-trihydroxystilbene) and trans-piceid (trans-5,4'-dihydroxystilbene-3-O-beta-D-glucopyranoside), its more widespread glycosilate derivative, have been compared measuring their inhibitory action on peroxidation of linoleic acid (LA) and the radical scavenging ability towards different free radicals (such as DPPH) and radical initiators. It has been found that the two stilbenes have similar antioxidant capacity, while the comparison with BHT (2,6-di-tert-butyl-4-methylphenol) and alpha-tocopherol (vitamin E, vit. E), taken as reference, points out a slower but prolonged protective action against lipid peroxidation.

Interaction of isopropylthioxanthone with phospholipid liposomes.

Isopropylthioxanthone (ITX) is a highly lipophilic molecule which can be released in foods and beverages from the packages, where it is present as photoinitiator of inks in printing processes. Recently it was found in babies milk, and its toxicity cannot be excluded. The structure of the molecule suggests a possible strong interaction with the lipid moiety of biological membranes, and this is the first study of its effects on phospholipid organization, using differential scanning calorimetry (DSC) and spin labelling techniques.

Interaction of fluoxetine with phosphatidylcholine liposomes.

Fluoxetine (Prozac) is one of the latest of a new generation of antidepressants, approved by FDA in 2002. The interactions of fluoxetine with multilamellar liposomes of pure phosphatidylcholine (PC) or containing cholesterol 10% molar were studied as a function of the lipid chain lengths, using differential scanning calorimetry and spin labelling EPR techniques. The DSC profiles of the gel-to-fluid state transition of liposomes of DMPC (C14:0) are broadened and shifted towards lower temperatures at increasing dopant concentrations and, with less than 10% fluoxetine, any detectable transition is destroyed.

New enzymatic method for the determination of total phenolic content in tea and wine.

A new spectrophotometric enzymatic method for the determination of total phenol content in tea and wine has been developed. The method is based on the peroxidase-catalyzed oxidation, by hydrogen peroxide, of phenols to phenoxyl radicals, which can react with aromatic substrates to form intensely colored adducts. In comparison with the widely used Folin-Ciocalteu method, this method appears to be more specific and more rapid and as a whole is not affected by the common interfering substances such as ascorbate, citrate, and sulfite.

Evaluation of the antioxidant properties of propofol and its nitrosoderivative. comparison with homologue substituted phenols.

Propofol (2,6-diisopropylphenol), some substituted phenols (2,6-dimethylphenol and 2,6-ditertbutylphenol) and their 4-nitrosoderivatives have been compared for their scavenging ability towards 1,1-diphenyl-2-picrylhydrazyl and for their inhibitory action on lipid peroxidation. These products were also compared to the classical antioxidants butylated hydroxytoluene and butylated hydroxyanisole. When measuring the reactivity of the various phenolic derivatives with 1,1-diphenyl-2-picrylhydrazyl the following order of effectiveness was observed: butylated hydroxyanisole > propofol > 2,6-dimethylphenol > 2,6-di-tertbutylphenol > butylated hydroxytoluene.

Correlation between fluidising effects on phospholipid membranes and mitochondrial respiration of propofol and p-nitrosophenol homologues.

Nitrosopropofol (2-6-diisopropyl-4-nitrosophenol) has dramatic consequences for respiration, ATP synthesis and the transmembrane potential of isolated rat liver mitochondria at concentrations at which propofol (2-6-diisopropylphenol) does not cause any apparent effects. These results correlate well with the observation that nitrosopropofol is also a stronger perturbing agent of phospholipid membranes. In this paper we verify the possible biological activity of different phenols and nitrosophenols on mitochondrial respiration.

Electrostatic compared with hydrophobic interactions between bovine serum amine oxidase and its substrates.

A steady-state kinetic study of bovine serum amine oxidase activity was performed, over a wide range of pH values (5.4-10.2) and ionic strength (10-200 mM), using various (physiological and analogue) substrates as specific probes of the active-site binding region.

Binding of cations of group IA and IIA to bovine serum amine oxidase: effect on the activity.

In this paper, we report on the presence of cation binding areas on bovine serum amine oxidase, where metal ions of the groups IA and IIA, such as Na(+), K(+), Cs(+), Mg(2+), and Ca(2+), bind with various affinities. We found a cation-binding area that influences the enzyme activity if occupied, so that the catalytic reaction may be altered by some physiologically relevant cations, such as Ca(2+) and K(+). This binding area appears to be localized inside the enzyme active site, because some of these cations act as competitive inhibitors when highly charged amines, such as spermine and spermidine, are used as substrates.

Effects of nitrosopropofol on mitochondrial energy-converting system.

Nitrosopropofol (NOPR) is a relatively stable compound obtained from the reaction between the general anesthetic 2,6 diisopropylphenol (propofol) and nitrosoglutathione (GSNO) and bearing a more acidic phenol group than propofol. It interfered with mitochondrial energetic metabolism in a concentration-dependent manner. Concentrations as high as 100 or 200 microM disrupted both oxidative phosphorylation and electron transport.

Different effects of propofol and nitrosopropofol on DMPC multilamellar liposomes.

The mechanisms of reaction of propofol with nitrosoglutathione lead to the formation of an active species which was identified, and then synthesised, as 2,6-diisopropyl-4-nitrosophenol. In the present work, we demonstrate the in vitro formation of 2,6-diisopropyl-4-nitrosophenol, then we discuss the interaction of propofol and 2,6-diisopropyl-4-nitrosophenol with dimyristoylphosphatidylcholine and egg yolk phosphatidylcholine multilamellar liposomes using differential scanning calorimetry and spin labelling techniques. It was demonstrated that both molecules are highly lipophylic and absorb almost entirely in the lipid phase.