High Catecholase Activity Research Articles

Catalytic properties of variously immobilized mushroom tyrosinase: A kinetic study for future development of biomimetic amperometric biosensors

Mushroom tyrosinase was immobilized by direct embedding into electrode material (modified carbon paste electrode), incorporation of cross-linked enzyme aggregates into a polymer membrane (glassy carbon electrode covered by thin layer of Nafion®), and covalent attachment using self-assembled monolayers (gold electrode with the chemically bound enzyme). Both, standard UV–Vis spectrophotometry and amperometry in a batch configuration are presented as complementary methods to study the tyrosinase enzyme kinetics, whose catecholase activity results in electroactive products (ortho-quinones). Due to higher sensitivity of amperometric detection, evident advantage in the enzyme consumption was obtained. Prepared amperometric tyrosinase biosensors were characterized using cyclic voltammetry and atomic force microscopy. The Michaelis constant values of immobilized and unbound tyrosinase (free enzyme solution) towards dopamine and catechol were compared. The apparent Michaelis constant values for immobilized tyrosinase are significantly lower than the declared value of 0.840 mmol L−1 dopamine for the unbound enzyme. The enzyme tyrosinase arranged in self-assembled monolayer serves as an efficient sensor due to low apparent Michaelis constant of 0.061 mmol L−1 dopamine and high maximum reaction velocity of 0.458 μA s−1. This fact reflects the ideal arrangement of enzyme molecules causing high availability of the binding site. Tris-glycine sodium dodecyl sulphate polyacrylamide gel electrophoresis and atomic force microscopy clarified that the protein of molecular weight 25 kDa is bound preferably on chemically modified gold electrode. A sensor prepared by the immobilization of tyrosinase on gold electrode results in higher catecholase activity towards dopamine than in case of CPE and GC electrodes, where enzyme is immobilized physically.

Trinuclear copper and mononuclear nickel complexes of oxime containing Schiff bases: Single crystal X-ray structure, catecholase and phenoxazinone synthase activity, catalytic study for the homocoupling of benzyl amines

Here we report the synthesis and characterisation of one trinuclear copper complex, [Cu3L3O]ClO4 (1), and one nickel complex, [Ni(L′H)2(dmso)2](ClO4)2) (2), with the Schiff base ligands (3Z)-3-((Z)-(1-(thiophen-2-yl)ethylidene)hydrazono)butan-2-one oxime (LH) and 1-(pyridin-2-yl)ethylidene)hydrazono)butan-2-one oxime (ĹH). Single crystal X-ray structures have been described for the ligand LH and the complexes 1 and 2. 1 shows high catecholase activity (kcat = 8.14 × 102 h−1 in MeOH) using 3,5-di-tert-butylcatechol as the substrate in methanol medium. 2 shows phenoxazinone synthase activity with a moderate value of kcat = 63.11 h−1 in dimethyl sulfoxide. This is the first report for phenox-azinone synthase activity by a nickel complex containing a ligand with oxime functionality. 1 has also been tested as a catalyst for the synthesis of benzylimine and substituted benzylimines from the corresponding amines. Substrates with electron withdrawing halo-substituted benzylamines undergo quantitative conversions to the corresponding benzylimines, whereas electron donating groups on the aromatic ring yielded lower conversions.

Catecholase activity of a self-assembling dimeric Cu(II) complex with distant Cu(II) centers

Catecholase activity and its mechanistic investigation of a self-assemblying dimeric Cu(II) complex, Cu2L2, where L is N,N′-(ethane-1,2-diyldi-o-phenylene)-bis(pyridine-2-carboxamidide), is presented. The complex was constructed by a spontaneous self-assembly of the organic ligand and Cu(II) ion. X-ray crystallography of Cu2L2(CH3OH)2·2CH3OH, found that the coordination geometry of the Cu(II) site was a distorted square pyramid and two Cu(II) ions were apart by 6.992Å. Though the metal centers are distant, Cu2L2 showed high catecholase activity when 3,5-di-tert-butylcatechol (3,5-H2dtbc) was treated with Cu2L2 in the presence of air at basic condition with kcat of 720h−1 in Michaelis–Menten model. Information collected from the UV–vis kinetic, electron paramagnetic resonance (EPR), and high-resolution electrospray ionization mass spectrometry (ESI MS) measurements on the reaction mixtures could lead the suggestion of the mechanistic pathway of the catecholase activity of Cu2L2 in which each Cu(II) site oxidized 3,5-H2dtbc to 3,5-di-tert-butyl-1,2-benzosemiquinonate anion radical (3,5-dtbq−). Subsequently, 3,5-di-tert-butyl-o-quinone (3,5-dtbq) was formed via both disproportionation and air oxidation of 3,5-dtbq−. Cu2L2 could be reduced to either one- or two-electron reduced state. The reduced complexes were reoxidized by dioxygen molecule accompanying with the generation of hydrogen peroxide.

Catalytic oxidation of 3,5-di- tert-butylcatechol by a manganese(III) 18-azametallacrown-6 compound: Synthesis, crystal structure, fluorescence, magnetic and kinetic investigation

A new macrocyclic hexanuclear manganese(III) 18-azametallacrown-6 compound, [Mn 6(ashz) 6(CH 3OH) 3(H 2O) 3] · 3H 2O · 3DMF ( 1), has been prepared using a trianionic pentadentate ligand N-acetylsalicylhydrazide (ashz 3−) and characterised by various techniques such as elemental analysis, IR, UV-vis and fluorescence spectroscopy, cyclic voltammetry and X-ray diffraction. Six ashz 3− ligands connect six metal ions to form the cyclic skeleton based on the M–N–N–M linkage. Due to the meridional coordination of the ligand to the Mn 3+ ion, the ligand enforces the stereochemistry of the Mn 3+ ions as a propeller configuration with alternating Δ/Λ forms. The kinetic studies on catecholase activity of 1 for the oxidation of 3,5-di- tert-butylcatechol (3,5-DTBC) by O 2 were done using UV–Vis absorption spectra method. Compound 1 has been evaluated as a model system for the catechol oxidase enzyme and it is found that the compound shows high catecholase activity. It exhibits the activity with a turnover number of 270 h −1. A kinetic treatment on the basis of the Michaelis–Menten model has been applied. The magnetic susceptibility (300–5 K) study indicates antiferromagnetic exchange interactions with J = −2.6 cm −1 between the adjacent Mn 3+ ions.

Characterization and changes in polyphenol oxidase from eggplant fruit ( Solanum melongena L.) during storage at low temperature

Polyphenoloxidase (PPO) from eggplant fruit was characterized, and its catecholase activity studied during storage of fruit at 0 and 10 °C. The high catecholase activity was observed using 4-methylcatechol as substrate at pH 6 and 30 °C and activation with sodium dodecyl sulphate (SDS) was unnecessary. The soluble PPO fraction was the most thermostable, as well as the most active, form of the enzyme. Fruits stored at 10 °C were undamaged, whereas those kept at 0 and 5 °C experienced chilling injury from days 6 and 8, respectively, as indicated by the decrease of lightness ( L 0) of pulp tissue. During exposure of fruits at 10 °C, the activities of soluble and insoluble PPO fractions increased, whereas, at 0 °C, PPO activity of both fractions decreased and maintained lower levels when browning of pulp tissue was observed. Soluble PPO activity at 0 °C was directly related to the value for L 0.

Structure, magnetic properties and catecholase activity study of oxo-bridged dinuclear copper(II) complexes

Six alkoxy-bridged dinuclear copper(II) complexes with catecholase-like sites, [Cu(L 1)(NCS)] 2 ( 1), [Cu(L 2)(CH 3CO 2)] 2·2H 2O ( 2), [Cu(L 3)(NCO)] 2 ( 3), [Cu(L 4)(NCS)] 2 ( 4), [Cu(L 5)(NCS)] 2 ( 5), and [Cu(L 4)(Cl)] 2 ( 6), where HL 1=2-(hydroxymethyl)-pyridine, HL 2=6-methyl-2-pyridine-methanol, HL 3=2-(2-hydroxyethyl)-pyridine, HL 4=1-dimethylamino-2-methyl-1-propanol, and HL 5=1-dimethylamino-2-propanol, respectively), have been prepared and characterized. The single crystal X-ray analysis show that the structures of complexes 1– 6 are dimeric with two adjacent copper(II) atoms bridged by pairs of μ-oxy atoms from the alkoxyl group of the ligands. Magnetic susceptibility measurements in the temperature range 2–300 K for complexes 1– 6 indicate significant antiferromagnetic coupling between the copper(II) atoms. The kinetic studies on catecholase activity of complexes for the oxidation of 3,5-di- tert-butylcatechol by O 2 was studied and it found that the complexes with the bond distance of Cu(II)⋯Cu(II) located near 2.98 to ∼2.99 Å show high catecholase activity. The reaction rate constant for the catecholase activity was obtained by Michaelis–Menten model.

Effects of dietary ascorbic acid on larvae of the Egyptian cotton leafworm, Spodoptera littoralis

Ascorbic acid deficiency symptoms of Spodoptera littoralis larvae were studied. The moulting cycle failure and smaller weight gain of the ascorbic acid-deprived larvae resulted from a smaller head capsule per instar which, in turn, impaired ecdysis and mouth-part function for food ingestion. This caused the larvae to die of hunger. Ascorbic acid was shown to be associated with the moulting stage. Haemolymph ascorbic acid was depleted at the pharate stage with concomitant appearance in the moulting fluid, where active, soluble O-diphenoloxidase was also determined. This enzyme was active against catechol, dopa, dopamine, and N-acetyl dopamine. The high catecholase activity of this enzyme was inhibited in vitro by ascorbates and sulfhydryl compounds. On the other hand, these sulfhydryl-reducing compounds, cysteine, and glutathione-SH, did not replace the dietary effect of ascorbic acid to prevent moulting cycle failure, nor were they detected in the moulting fluid. It appears that the insufficient cuticle expansion in the ascorbic acid-deficient larvae is due to premature hardening of the cuticle. The possible function of ascorbic acid to control cuticle melanization and sclerotization is discussed.

Catecholase Activity in Tissue Cultures of Claviceps litoralis

An acetone powder catecholase preparation was obtained from the pigmented tissues of Claviceps litoralis . Determined by a monometric method, the catecholase activity was found to be low in younger, less pigmented tissues, and higher in older, more pigmented tissues. It reached a maximum shortly after the peak of the growth period and declined slightly thereafter. The maximum catecholase activity occurred in twenty‐eight to thirty‐five days of growth. The catecholase activity of the powder preparation was found to be proportional to the concentration of the powder enzyme preparation, and was inhibited by sodium diethyldithiocarbamate and by potassium cyanide. The powder preparation of young tissues showed some ability to oxidize tyrosine and dopa, but at a very low rate in comparison with catechol oxidation. There was no oxidation of tyrosine and dopa by enzyme preparations from the older tissues which possessed high catecholase activity.