Electrospray deposition of starch-containing laccase: A green technique for low-cost and eco-friendly biosensors.

Recently a laccase-based biosensors with unprecedented reuse and storage capabilities in the detection of catechol compound has been manufactured using ambient Electrospray Deposition (ESD) technique. These biosensors showed to be reused up to 63 measurements on the same electrode just prepared at room temperature and pressure. In this new work the reasons behind such a high-performance functioning have been investigated by analysing the commercial sample of laccase with different chemical physics methods: Electrophoresis, Fourier Transform Infrared Spectroscopy, X-ray Fluorescence and Nuclear Magnetic Resonance Spectroscopy.

Tailor-made alkaliphilic and thermostable fungal laccases for industrial wood processing.

Background: During the kraft process to obtain cellulosic pulp from wood, most of the lignin is removed by high-temperature alkaline cooking, released in the black liquors and usually incinerated for energy. However, kraft lignins are a valuable source of phenolic compounds that can be valorized in new bio-based products. The aim of this work is to develop laccases capable of working under the extreme conditions of high temperature and pH, typical of the industrial conversion of wood into kraft pulp and fibreboard, in order to provide extremophilic biocatalysts for depolymerising kraft lignin, and enzyme-assisted technologies for kraft pulp and fibreboard production.

Influence of Nanoaggregation Routes on the Structure and Thermal Behavior of Multiple-Stimuli-Responsive Micelles from Block Copolymers of Oligo(ethylene glycol) Methacrylate and the Weak Acid [2-(Hydroxyimino)aldehyde]butyl Methacrylate.

In this work, we compare nanoaggregation driven by pH-induced micellization (PIM) and by the standard solvent displacement (SD) method on a series of pH-, light-, and thermosensitive amphiphilic block copolymers. Specifically, we investigate poly(HIABMA)--poly(OEGMA) and poly(HIABMA)--poly(DEGMA--OEGMA), where HIABMA = [(hydroxyimino)aldehyde]butyl methacrylate, OEGMA = oligo(ethylene glycol)methyl ether methacrylate, and DEGMA = di(ethylene glycol)methyl ether methacrylate. The weakly acidic HIA group (p ≈ 8) imparts stability to micelles at neutral pH, unlike most of the pH-responsive copolymers investigated in the literature.

Unusually Chemoselective Photocyclization of 2-(Hydroxyimino)aldehydes to Cyclobutanol Oximes: Synthetic, Stereochemical, and Mechanistic Aspects.

Photocyclization of carbonyl compounds (known as the Norrish-Yang reaction) to yield cyclobutanols is, in general, accompanied by fragmentation reactions. The latter are predominant in the case of aldehydes so that secondary cyclobutanols are not considered accessible via the straightforward Norrish-Yang reaction. A noteworthy exception has been reported in our laboratory, where cyclobutanols bearing a secondary alcohol function were observed upon UV light irradiation of 2-(hydroxyimino)aldehydes (HIAs).

Structural and biochemical insights into an engineered high-redox potential laccase overproduced in Aspergillus.

Fungal laccases have great potential as biocatalysts oxidizing a variety of aromatic compounds using oxygen as co-substrate. Here, the crystal structure of 7D5 laccase (PDB 6H5Y), developed in Saccharomyces cerevisiae and overproduced in Aspergillus oryzae, is compared with that of the wild type produced by basidiomycete PM1 (Coriolopsis sp.), PDB 5ANH.

2-(Hydroxyimino)aldehydes: Photo- and Physicochemical Properties of a Versatile Functional Group for Monomer Design.

In the context of our research on stimuli-responsive polymers bearing the 2-(hydroxyimino)aldehyde (HIA) group, we have explored the photochemical behavior and physicochemical properties of a number of HIAs. Interpretation of the experimental data is supported by quantum mechanical calculations. HIAs are expected to undergo photoisomerization, chelate metal ions, yield hydrogen-bonded dimers or oligomers, exhibit relatively low pK s, and form >C=NO radicals through OH hydrogen abstraction or oxidation of the oximate ion.

Kinetics and mechanistic study of competitive inhibition of thymidine phosphorylase by 5-fluoruracil derivatives.

In a previous investigation, cationic liposomes formulated with new 5-FU derivatives, differing for the length of the polyoxyethylenic spacer that links the N(3) position of 5-FU to an alkyl chain of 12 carbon atoms, showed a higher cytotoxicity compared to free 5-FU, the cytotoxic effect being directly related to the length of the spacer. To better understand the correlation of the spacer length with toxicity, we carried out initial rate studies to determine inhibition, equilibrium and kinetic constants (KI, KM, kcat), and get inside inhibition activity of the 5-FU derivatives and their mechanism of action, a crucial information to design structural variations for improving the anticancer activity. The experimental investigation was supported by docking simulations based on the X-ray structure of thymidine phosphorylase (TP) from Escherichia coli complexed with 3'-azido-2'-fluoro-dideoxyuridin.

Concerted electron/proton transfer mechanism in the oxidation of phenols by laccase.

This study aimed to assess structural requirements in the enzyme/substrate interactions that are responsible for tuning the enzymatic reactivity. To better assess the role of the aspartic residue in the substrate-binding pocket of basidiomycete-type laccases, we compared the catalytic efficiency of wild-type enzymes to that of a mutant in which carboxylic acid residue Asp206 was changed to alanine. Oxidation efficiency towards phenolic substrates by laccases of Trametes villosa, Trametes versicolor and a T.

The Rabe amination after a century: direct addition of N-heterocycles to carbonyl compounds.

A catalytic version of the Rabe electrophilic amination is presented. This kind of reaction was originally employed in 1918 in a key step for the conversion of quinotoxine to quinine. Ketones and α-substituted aldehydes give the corresponding α-aminated carbonyl compounds in moderate yield.

A remarkably simple α-oximation of aldehydes via organo-SOMO catalysis.

A novel α-oximation reaction of unactivated aldehydes has been achieved in excellent yields by reaction with NaNO(2)-FeCl(3) couple and in the presence of pyrrolidine as organocatalyst.

How is the reactivity of laccase affected by single-point mutations? Engineering laccase for improved activity towards sterically demanding substrates.

In spite of its broad specificity among phenols, Trametes versicolor laccase hardly succeeds in oxidizing hindered substrates. To improve the oxidation ability of this laccase towards bulky phenolic substrates, we designed a series of single-point mutants on the basis of the amino-acid layout inside the reducing substrate active site known from the crystal structure of the enzyme. Site-directed mutagenesis has addressed four phenylalanine residues in key positions 162, 265, 332, and 337 at the entrance of the binding pocket, as these residues appeared instrumental for docking of the substrate.

Chemoselective C-4 aerobic oxidation of catechin derivatives catalyzed by the Trametes villosa laccase/1-hydroxybenzotriazole system: synthetic and mechanistic aspects.

Catechin derivatives were oxidized in air in the presence of the Trametes villosa laccase/1-hydroxybenzotriazole (HBT) system in buffered water/1,4-dioxane as reaction medium. The oxidation products, flavan-3,4-diols and the corresponding C-4 ketones, are bioactive compounds and useful intermediates for the hemisynthesis of proanthocyanidins, plant polyphenols which provide beneficial health properties for humans. Determinations of oxidation potentials excluded that catechin derivatives could be directly oxidized by laccase Cu(II), while it resulted in the H-abstraction from benzylic positions being promptly promoted by the enzyme in the presence of the mediator HBT, the parent species producing in situ the reactive intermediate benzotriazole-N-oxyl (BTNO) radical.

Hydrogen atom abstraction from C-H bonds of benzylamides by the aminoxyl radical BTNO: a kinetic study.

The aminoxyl radical BTNO (benzotriazole-N-oxyl; >N-O*) is generated from HBT (1-hydroxybenzotriazole; >N-OH) by oxidation with a Ce(IV) salt. BTNO presents a broad absorption band with lambda(max) 474 nm that lends itself to investigate the kinetics of H-abstraction from H-donor substrates by spectrophotometry. Thus, rate constants (k(H)) of H-abstraction by BTNO from CH(2)-groups alpha to the nitrogen atom in X-substituted-(N-acetyl)benzylamines (X-C(6)H(4)CH(2)NHCOCH(3)) have been determined in MeCN solution at 25 degrees C.

Hydrogen abstraction and electron transfer with aminoxyl radicals: synthetic and mechanistic issues.

Aminoxyl radicals (R(2)NO(*)) are a valuable class of reactive intermediates with interesting synthetic and reactivity properties. This Minireview summarizes salient synthetic results obtained in radical oxidations using aminoxyl radicals, and then focuses on reactivity issues arising from recent literature surveys. The structural and reactivity features of the aminoxyl radical and substrate provides a possible explanation of the double reactivity of the aminoxyl radicals.

An assessment of the relative contributions of redox and steric issues to laccase specificity towards putative substrates.

Laccases catalyze the one-electron oxidation of a broad range of substrates coupled to the 4 electron reduction of O2 to H2O. Phenols are typical substrates, because their redox potentials (ranging from 0.5 to 1.

Kinetic study of the hydrogen abstraction reaction of the benzotriazole-N-oxyl radical (BTNO) with H-donor substrates.

[Reaction: see text]. The aminoxyl radical (>N-O*) BTNO (benzotriazole-N-oxyl) has been generated by the oxidation of 1-hydroxybenzotriazole (HBT; >N-OH) with a Ce(IV) salt in MeCN. BTNO presents a broad absorption band with lambda(max) 474 nm and epsilon 1840 M(-1) cm(-1), and spontaneously decays with a first-order rate constant of 6.

Kinetics of oxidation of benzyl alcohols by the dication and radical cation of ABTS. Comparison with laccase-ABTS oxidations: an apparent paradox.

Laccase, a blue copper oxidase, in view of its moderate redox potential can oxidise only phenolic compounds by electron-transfer. However, in the presence of ABTS (2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) as a redox mediator, laccase reacts with the more difficult to oxidise non-phenolic substrates, such as benzyl alcohols. The role of ABTS in these mediated oxidations is investigated.

Spectrophotometric, EPR and kinetic characterisation of the >N-O* radical from 1-hydroxybenzotriazole, a key reactive species in mediated enzymatic oxidations.

Characterisation of the aminoxyl (>N-O*) radical BTNO, generated from 1-hydroxybenzotriazole (HBT) by the one-electron oxidant CAN (a Ce(IV) salt), confirms BTNO as the reactive intermediate in oxidations run with the laccase/HBT system.

Promoting laccase activity towards non-phenolic substrates: a mechanistic investigation with some laccase-mediator systems.

The oxidation of benzyl alcohols with the enzyme laccase, under mediation by appropriate mediator compounds, yields carbonylic products, whereas laccase can not oxidise these non-phenolic substrates directly. The oxidation step is performed by the oxidised form of the mediator (Med(ox)), generated on its interaction with laccase. The Med(ox) can follow either an electron transfer (ET) or a radical hydrogen atom transfer (HAT) route of oxidation of the substrates.

Escherichia coli flavohemoglobin is an efficient alkylhydroperoxide reductase.

Escherichia coli flavohemoglobin (HMP) is shown to be capable of catalyzing the reduction of several alkylhydroperoxide substrates into their corresponding alcohols using NADH as an electron donor. In particular, HMP possesses a high catalytic activity and a low Km toward cumyl, linoleic acid, and tert-butyl hydroperoxides, whereas it is a less efficient hydrogen peroxide scavenger. An analysis of UV-visible spectra during the stationary state reveals that at variance with classical peroxidases, HMP turns over in the ferrous state.

Stereochemistry of the Vinylic S(RN)1 Reaction of Triarylvinyl Halides. The Structure of the Intermediate alpha-Arylvinyl Radical.

Evidence for the intermediacy of a vinyl radical in the vinylic S(RN)1 reaction (S(RN)1(V)) of 2-anisyl-1,2-diphenylvinyl bromide 2 is obtained. The photostimulated S(RN)1(V) reaction of pinacolone enolate ion with (E)-2 and (Z)-2, which are used as stereoindicators, gives complete loss of the original stereochemistry of the two precursors in the substituted and hydrodehalogenated products; i.e.