Designing biopolymer-based artificial peroxidase for oxidative removal of dibenzothiophene from a model diesel fuel.

Naturally occurring peroxidases are important for living organisms and have manifold utility in industries. However, lack of stability in harsh reaction conditions hinders wide applicability of such enzymes. Thus, suitable alternative is vital which can endure severe reaction conditions. As a substitute of natural peroxidase, herein, biopolymer-based polyelectrolyte complexes (PECs) coordinated with Fen+ is proposed as macromolecular peroxidase mimicking systems. Three PECs were engineered via complexation of protonated chitosan and alginate with Fe2+ (Fe2+-PEC), Fe3+ (Fe3+-PEC), and Fe3O4 (Fe3O4-PEC), respectively. Computational study showed the Fe3+-PEC was highly stable with abundant electrostatic and intramolecular hydrogen bonding interactions. The versatility of the Fe-PECs as artificial peroxidase biocatalysts was probed by two types of peroxidase assays - ABTS oxidation in buffer systems (pH 4.0 and 7.0) and pyrogallol oxidation in organic solvents (acetonitrile, ethyl acetate and toluene). Overall, Fe3+-PEC showed remarkably high peroxidase activity both in aqueous buffers and in organic solvents, whereas, Fe3O4-PEC showed least catalytic activity. Finally, as a proof of concept, the ability of the biocatalyst to carry out deep oxidative desulphurization was demonstrated envisaging removal of dibenzothiophene from model fossil fuel in a sustainable way.

De Novo Iron Oxide Hydroxide, Ferrihydrite Produced by Comamonas testosteroni Exhibiting Intrinsic Peroxidase-Like Activity and Their Analytical Applications.

Natural enzyme mimics have attracted considerable attention due to leakage of enzymes and their easy denaturation during their storage and immobilization procedure. Here in this study, for the first time, a new iron oxide hydroxide, ferrihydrite - Fe1.44O0.32 (OH) 3.68 magnetic nanoparticles were synthesized by bacterial strain named Comamonas testosteroni. The characterization of the produced magnetic nanoparticles was confirmed by transmission electron microscopy (TEM), Fourier-transform spectroscopy (FTIR), X-ray diffraction (XRD), and magnetization hysteresis loops. Further, these extracted nanoparticles were proven to have biogenic magnetic behavior and to exhibit enhanced peroxidase-like activity. It is capable of catalyzing the oxidation of 3, 3', 5, 5'-Tetramethylbenzidine (TMB) by H2O2 to produce blue color (typical color reactions). Catalysis was examined to follow Michaelis-Menton kinetics and the good affinity to both H2O2 and TMB. The K m value of the Fe1.44O0.32 (OH) 3.68 with H2O2 and TMB as the substrate was 0.0775 and 0.0155 mM, respectively, which were lower than that of the natural enzyme (HRP). Experiments of electron spin resonance (ESR) spectroscopy proved that the BMNPs could catalyze H2O2 to produce hydroxyl radicals. As a new peroxidase mimetic, the BMNPs were exhibited to offer a simple, sensitive, and selective colorimetric method for determination of H2O2 and glucose and efficiently catalyze the detection of glucose in real blood samples.

Construction and in vivo assembly of a catalytically proficient and hyperthermostable de novo enzyme.

Although catalytic mechanisms in natural enzymes are well understood, achieving the diverse palette of reaction chemistries in re-engineered native proteins has proved challenging. Wholesale modification of natural enzymes is potentially compromised by their intrinsic complexity, which often obscures the underlying principles governing biocatalytic efficiency. The maquette approach can circumvent this complexity by combining a robust de novo designed chassis with a design process that avoids atomistic mimicry of natural proteins. Here, we apply this method to the construction of a highly efficient, promiscuous, and thermostable artificial enzyme that catalyzes a diverse array of substrate oxidations coupled to the reduction of H2O2. The maquette exhibits kinetics that match and even surpass those of certain natural peroxidases, retains its activity at elevated temperature and in the presence of organic solvents, and provides a simple platform for interrogating catalytic intermediates common to natural heme-containing enzymes.Catalytic mechanisms of enzymes are well understood, but achieving diverse reaction chemistries in re-engineered proteins can be difficult. Here the authors show a highly efficient and thermostable artificial enzyme that catalyzes a diverse array of substrate oxidations coupled to the reduction of H2O2.

Creation of a Thermally Tolerant Peroxidase.

An artificial peroxidase with thermal tolerance and high catalytic activity has been successfully prepared by mutagenesis of an electron transfer protein, cytochrome c552 from Thermus thermophilus. The mutant enzymes were rationally designed based on the general peroxidase mechanism and spectroscopic analyses of an active intermediate formed in the catalytic reaction. Stopped flow UV-vis spectroscopy and EPR spectroscopy with a rapid freezing sample technique revealed that the initial double mutant, V49D/M69A, which was designed to reproduce the peroxidase mechanism, formed an active oxo-ferryl heme intermediate with a protein radical predominantly localized on Tyr45 during the catalytic reaction. The magnetic power saturation measurement obtained from EPR studies showed little interaction between the oxo-ferryl heme and the tyrosyl radical. Kinetics studies indicated that the isolated oxo-ferryl heme component in the active intermediate was a possible cause of heme degradation during the reaction with H2O2. Strong interaction between the oxo-ferryl heme and the radical was achieved by replacing Tyr45 with tryptophan (resulting in the Y45W/V49D/M69A mutant), which was similar to a tryptophanyl radical found in active intermediates of some catalase-peroxidases. Compared to the protein radical intermediates of V49D/M69A mutant, those of the Y45W/V49D/M69A mutant showed higher reactivity to an organic substrate than to H2O2. The Y45W/V49D/M69A mutant exhibited improved peroxidase activity and thermal tolerance.

A novel self-assembled nano micelle as a highly efficient artificial peroxidase based on hexadecyl trimethyl ammonium bromide and cytochrome c.

A novel artificial peroxidase (AP) with highly catalytic efficiency was designed using hexadecyl trimethyl ammonium bromide (CTAB, 3 mM) nano-micelles and bovine heart cytochrome c (Cyt c, 0.5 µM) in 100 mM, pH 8.0 phosphate buffer at 25°C. The catalytic rate (kcat) and Michaelis-Menten (Km) of the AP were determined to be 0.311 ± 0.013 s(-1) and 8.64 ± 0.6 µM. The catalytic efficiency was 0.0360 ± 0.0020 µM(-1)s(-1) (about 50% the efficiency of native horseradish peroxidase). The Ultraviolet-visible spectrophotometer and Circular Dichroism techniques were applied to study the properties of the CTAB-Cyt c nano-micelle. Designed AP can be applied instead of native horseradish peroxidase.

Influence of the geometry around the manganese ion on the peroxidase and catalase activities of Mn(III)-Schiff base complexes.

The peroxidase and catalase activities of eighteen manganese-Schiff base complexes have been studied. A correlation between the structure of the complexes and their catalytic activity is discussed on the basis of the variety of systems studied. Complexes 1-18 have the general formulae [MnL(n)(D)(2)](X)(H(2)O/CH(3)OH)(m), where L(n)=L(1)-L(13); D=H(2)O, CH(3)OH or Cl; m=0-2.5 and X=NO(3)(-), Cl(-), ClO(4)(-), CH(3)COO(-), C(2)H(5)COO(-) or C(5)H(11)COO(-). The dianionic tetradentate Schiff base ligands H(2)L(n) are the result of the condensation of different substituted (OMe-, OEt-, Br-, Cl-) hydroxybenzaldehyde with diverse diamines (1,2-diaminoethane for H(2)L(1)-H(2)L(2); 1,2-diamino-2-methylethane for H(2)L(3)-H(2)L(4); 1,2-diamino-2,2-dimethylethane for H(2)L(5); 1,2-diphenylenediamine for H(2)L(6)-H(2)L(7); 1,3-diaminopropane for H(2)L(8)-H(2)L(11); 1,3-diamino-2,2-dimethylpropane for H(2)L(12)-H(2)L(13)). The new Mn(III) complexes [MnL(1)(H(2)O)Cl](H(2)O)(2.5) (2), [MnL(2)(H(2)O)(2)](NO(3))(H(2)O) (4), [MnL(6)(H(2)O)(2)][MnL(6)(CH(3)OH)(H(2)O)](NO(3))(2)(CH(3)OH) (8), [MnL(6)(H(2)O)(OAc)](H(2)O) (9) and [MnL(7)(H(2)O)(2)](NO(3))(CH(3)OH)(2) (12) were isolated and characterised by elemental analysis, magnetic susceptibility and conductivity measurements, redox studies, ESI spectrometry and UV, IR, paramagnetic (1)H NMR, and EPR spectroscopies. X-ray crystallographic studies of these complexes and of the ligand H(2)L(6) are also reported. The crystal structures of the rest of the complexes have been previously published and herein we have only revised their study by those techniques still not reported (EPR and (1)H NMR for some of these compounds) and which help to establish their structures in solution. Complexes 1-12 behave as more efficient mimics of peroxidase or catalase in contrast with 13-18. The analysis between the catalytic activity and the structure of the compounds emphasises the significance of the existence of a vacant or a labile position in the coordination sphere of the catalyst.

New conjugates of superoxide dismutase/catalase mimetics with cyclodestrins.

Mimetics of antioxidant enzymes such as superoxide dismutases (SOD) or catalases are reported as potential new drugs able to reduce oxidative stress damage. In particular, manganese(III) complexes of salen-type ligands have been studied as both SOD and catalase mimetics. In this paper, we report the synthesis of two novel conjugates of salen-type ligands with the beta-cyclodextrin, the 6-deoxy-6-[(S-cysteamidopropyl(1,2-diamino)N,N'-bis(salicylidene))]-beta-cyclodextrin and the 6-deoxy-6-[(S-cysteamidopropyl(1,2-diamino)N,N'-bis(3-methoxysalicylidene))]-beta-cyclodextrin, their spectroscopic characterization, and the synthesis and the characterization of their manganese(III) complexes. The SOD-like activity of the metal complexes was investigated by the indirect Fridovich method. The catalase like activity was tested using a Clark-type oxygen electrode. The peroxidase activity was tested using the ABTS (2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)) assay. The glycoconjugation of salen-manganese(III) complexes yields compounds with enhanced SOD activity. These complexes also show catalase and peroxidase activities higher than the simple salen complexes (EUK 113 and EUK 108).

Supramolecular mimetic peroxidase based on hemin and PAMAM dendrimers.

An acid-base interaction between hemin and PAMAM dendrimers affords supramolecular non-covalent peroxidase systems whose catalytic activity is enhanced after spontaneous electrostatic self-assembling onto a solid surface.

Direct conversion of ferrous myeloperoxidase to compound II by hydrogen peroxide: an anaerobic stopped-flow study.

Myeloperoxidase (MPO) is one of the essential components of the antimicrobial systems of polymorphonuclear neutrophils. It is unique in having a globin-like standard reduction potential of the ferric/ferrous couple. Here, it is shown that ferrous MPO heterolytically cleaves hydrogen peroxide forming water and oxyferryl MPO (compound II). The two-electron oxidation reaction follows second-order kinetics with the apparent bimolecular rate constant being (6.8+/-0.6)x10(4)M(-1)s(-1) at pH 7.0. After depletion of (micromolar) H(2)O(2) compound II slowly decays to ferric MPO, whereas upon addition of millimolar H(2)O(2) to ferrous MPO, compound III (oxyperoxidase) is formed in a sequence of two reactions involving compound II formation and its direct reaction with H(2)O(2), which also follows second-order kinetics [(78+/-2)M(-1)s(-1) at pH 7.0]. It is discussed how these reactions contribute to the interconversion of compound II and compound III and could explain the catalase activity of MPO.