Penicillium Vitale Research Articles

Structure, recombinant expression and mutagenesis studies of the catalase with oxidase activity fromScytalidium thermophilum

Scytalidium thermophilum produces a catalase with phenol oxidase activity (CATPO) that catalyses the decomposition of hydrogen peroxide into oxygen and water and also oxidizes various phenolic compounds. A codon-optimized catpo gene was cloned and expressed in Escherichia coli. The crystal structures of native and recombinant S. thermophilum CATPO and two variants, H82N and V123F, were determined at resolutions of 2.7, 1.4, 1.5 and 1.9 Å, respectively. The structure of CATPO reveals a homotetramer with 698 residues per subunit and with strong structural similarity to Penicillium vitale catalase. The haem component is cis-hydroxychlorin γ-spirolactone, which is rotated 180° with respect to small-subunit catalases. The haem-binding pocket contains two highly conserved water molecules on the distal side. The H82N mutation resulted in conversion of the native d-type haem to a b-type haem. Kinetic studies of the H82N and V123F mutants indicate that both activities are likely to be associated with the haem centre and suggest that the secondary oxidase activity may be a general feature of catalases in the absence of hydrogen peroxide.

X-ray diffraction study of Penicillium Vitale catalase in the complex with aminotriazole

The three-dimensional structure of the enzyme catalase from Penicillium vitale in a complex with the inhibitor aminotriazole was solved and refined by protein X-ray crystallography methods. An analysis of the three-dimensional structure of the complex showed that the inhibition of the enzyme occurs as a result of the covalent binding of aminotriazole to the amino-acid residue His64 in the active site of the enzyme. An investigation of the three-dimensional structure of the complex resulted in the amino-acid residues being more precisely identified. The binding sites of saccharide residues and calcium ions in the protein molecule were found.

Evaluation of amperometric glucose biosensors based on glucose oxidase encapsulated within enzymatically synthesized polyaniline and polypyrrole

In this article potential and suitability of enzymatically synthesized conducting polymers polyaniline (PANI) and polypyrrole (PPY) for fabrication of enzymatic amperometric glucose biosensors were evaluated. The polymerisation of these polymers was induced by catalytic activity of glucose oxidase (GOx) from Penicillium vitale cross-linked by glutaraldehyde (GA) on the graphite rod electrode (GOx-electrode) surface. The main precursors for initiation of polymerisation reactions were hydrogen peroxide as an initiator of polymerisation reaction and β- d-gluconic acid as a medium, which reduced the pH towards acidic one is the most suitable for the formation of PANI and PPY. During the polymerisation reactions the immobilized GOx was self-encapsulated within formed PANI or PPY layers in order to form GOx/PANI- and GOx/PPY-modified electrodes (GOx/PANI-electrode and GOx/PPY-electrode, respectively). Kinetic properties of GOx, which is acting as a biocatalyst in GOx/PANI- and GOx/PPY-electrodes, were studied and results were compared with GOx-electrode. The results show that in both GOx/PANI- and GOx/PPY-electrodes self-encapsulated GOx exhibited different parameters of catalysed reaction kinetics due to increasing diffusion limitations if compared with that of the GOx-electrode and it allowed the detection of glucose in a wider concentration interval. Moreover, both GOx/PANI- and GOx/PPY-electrodes exhibited good operational stability and reproducibility of analytical signal. The electrochemical characteristics of formed PANI and PPY in the GOx/PANI- and GOx/PPY-electrodes were also determined. In addition, the influence of temperature, pH and common interfering compounds on the steady-state current response of modified electrodes were investigated and discussed.

Proton Transfer Drives Protein Radical Formation in Helicobacter pylori Catalase but Not in Penicillium vitale Catalase

Heme catalases prevent cells from oxidative damage by decomposing hydrogen peroxide into water and molecular oxygen. Here we investigate the factors that give rise to an undesirable side reaction competing with normal catalase activity, the migration of a radical from the heme active site to the protein in the principal reaction intermediate compound I (Cpd I). Recently, it has been proposed that this electron transfer reaction takes place in Cpd I of Helicobacter pylori catalase (HPC), but not in Cpd I of Penicillium vitale catalase (PVC), where the oxidation equivalent remains located on the heme active site. Unraveling the factors determining the different radical locations could help engineer enzymes with enhanced catalase activity for detection or removal of hydrogen peroxide. Using quantum mechanics/molecular mechanics metadynamics simulations, we show that radical migration in HPC is facilitated by the large driving force (-0.65 eV) of the subsequent proton transfer from a histidine residue to the ferryl oxygen atom of reduced Cpd I. The corresponding free energy in PVC is significantly smaller (-0.19 eV) and, as we argue, not sufficiently high to support radical migration. Our results suggest that the energetics of oxoferryl protonation is a key factor regulating radical migration in catalases and possibly also in hydroperoxidases.

Enzymatically synthesized polyaniline layer for extension of linear detection region of amperometric glucose biosensor

In this article a new method for fabrication of enzymatic electrodes suitable for design of amperometric glucose biosensor and/or anode of biofuel cell powered by glucose is presented. Glucose oxidase (GOx) E.C. 1.1.3.4. from Penicillium vitale was immobilized on the carbon rod electrode by cross-linking it with glutaraldehyde (GOx-electrode). Catalytic activity of immobilized GOx was exploited for polymerisation of aniline by taking a high concentration of hydrogen peroxide produced during the catalytic action of immobilized GOx and locally lowered pH due to the formation of gluconic acid; it created optimal conditions for the polymerisation of aniline. The GOx layer was self-encapsulated within formed polyaniline (PANI) matrix (GOx/PANI-electrode). Properties of the GOx/PANI-electrode have been studied and results were compared with GOx-electrode. The results show that the upper detection limit of glucose using GOx-electrode was dramatically changed by the formation of PANI layer. An increase in the upper detection limit, optimal pH region for operation and stability of GOx based electrode modified by PANI was detected when comparing that of an unmodified GOx-electrode.

The Molecular Mechanism of the Catalase Reaction

Catalases are ubiquitous enzymes that prevent cell oxidative damage by degrading hydrogen peroxide to water and oxygen (2H(2)O(2) --> 2 H(2)O + O(2)) with high efficiency. The enzyme is first oxidized to a high-valent iron intermediate, known as Compound I (Cpd I) which, in contrast to other hydroperoxidases, is reduced back to the resting state by further reacting with H(2)O(2). By means of hybrid QM/MM Car-Parrinello metadynamics simulations, we have investigated the mechanism of the reduction of Compound I by H(2)O(2) in Helicobacter pylori catalase (HPC) and Penicillium vitale catalase (PVC). We found that the Cpd I-H(2)O(2) complex evolves to a Cpd II-like species through the transfer of a hydrogen atom from the peroxide to the oxoferryl unit. To complete the reaction, two mechanisms may be operative: a His-mediated (Fita-Rossmann) mechanism, which involves the distal His as an acid-base catalyst mediating the transfer of a proton (associated with an electron transfer), and a direct mechanism, in which a hydrogen atom transfer occurs. Independently of the mechanism, the reaction proceeds by two one-electron transfers rather than one two-electron transfer, as has long been the lore. The calculations provide a detailed view of the atomic and electronic reorganizations during the reaction, and highlight the key role of the distal residues to assist the reaction. Additional calculations on the in silico HPC His56Gly mutant and gas-phase models provide clues to understand the requirements for the reaction to proceed with low barriers.

Polyaniline synthesis catalysed by glucose oxidase

A novel method for synthesis of polyaniline (PANI) in aqueous media based on application of oxidizing-enzyme glucose oxidase (GOx) is reported. Hydrogen peroxide was produced during catalytic reaction of oxidizing-enzyme glucose oxidase from Penicillium vitale and initiated the polymerization of aniline. The increase in optical absorbance in the range of 340–700nm was exploited for the monitoring of PANI polymerization process. The role of GOx in the formation of PANI, influence of the initial concentrations of GOx, and glucose and aniline monomer on the aniline polymerization rate was studied. The study of pH influence on polymerization rate showed that PANI polymerization was occurring in a broad pH range from the pH 2.0 to 9.0. Optimal polymerization/oligomerization temperature was found to be at 37°C, which is also optimal for GOx-catalysed enzymatic reaction. After 10 days of continuous GOx-catalysed polymerization PANI appeared as colloid-microparticles visible by an optical microscope.

The Structures and Electronic Configuration of Compound I Intermediates of Helicobacter pylori and Penicillium vitale Catalases Determined by X-ray Crystallography and QM/MM Density Functional Theory Calculations

The structures of Helicobacter pylori (HPC) and Penicillium vitale (PVC) catalases, each with two subunits in the crystal asymmetric unit, oxidized with peroxoacetic acid are reported at 1.8 and 1.7 A resolution, respectively. Despite the similar oxidation conditions employed, the iron-oxygen coordination length is 1.72 A for PVC, close to what is expected for a Fe=O double bond, and 1.80 and 1.85 A for HPC, suggestive of a Fe-O single bond. The structure and electronic configuration of the oxoferryl heme and immediate protein environment is investigated further by QM/MM density functional theory calculations. Four different active site electronic configurations are considered, Por*+-FeIV=O, Por*+-FeIV=O...HisH+, Por*+-FeIV-OH+ and Por-FeIV-OH (a protein radical is assumed in the latter configuration). The electronic structure of the primary oxidized species, Por*+-FeIV=O, differs qualitatively between HPC and PVC with an A2u-like porphyrin radical delocalized on the porphyrin in HPC and a mixed A1u-like "fluctuating" radical partially delocalized over the essential distal histidine, the porphyrin, and, to a lesser extent, the proximal tyrosine residue. This difference is rationalized in terms of HPC containing heme b and PVC containing heme d. It is concluded that compound I of PVC contains an oxoferryl Por*+-FeIV=O species with partial protonation of the distal histidine and compound I of HPC contains a hydroxoferryl Por-FeIV-OH with the second oxidation equivalent delocalized as a protein radical. The findings support the idea that there is a relation between radical migration to the protein and protonation of the oxoferryl bond in catalase.

Glyoxyl agarose as a new chromatographic matrix

At alkaline pHs, glyoxyl agarose is able to immobilize most of the proteins contained in a crude extract. However, due to its special immobilization features, at pH 7.0 only proteins that contain at least two exposed low p K amino groups in the same plane were immobilized (β-galactosidase from Escherichia coli, catalase from bovine liver, and IgG from rabbit). However, with many other proteins, even multimeric ones, immobilization could not be achieved (e.g.: glucose oxidase from Aspergillus niger and Penicillium vitale; catalase from Micococcus sp., A. niger and bovine liver; alcohol oxidase from Pichia pastoris, Hansenula sp. and Candida boidinii, β-galactosidase from Thermus sp., etc.). Elution of the attached proteins under mild conditions was not simple, if the number of protein-support bonds was very high, only boiling in SDS allowed the elution of the proteins. However, using glyoxyl agarose 4BCL with only 20 μmol of aldheyde groups/g of support, proteins could be fully eluted by competition with amino compounds (e.g., Tris buffer). In this first approach, we have tried to take advantage of this specific immobilization at pH 7.0 to purify multimeric proteins, using a β-galactosidase from E. coli as a model. The enzyme could be eluted from the support using Tris–HCl buffer as eluting agent, with a high yield (80%) and a high purification factor (32).

Biphasic character of fungal catalases inhibition with hydroxylamine in presence of hydrogen peroxide

The kinetics of hydrogen peroxide decomposition with fungal catalases, i.e. Aspergillus niger (ANC), Penicillium vitale (PVC), Scytalidium thermophilum (STC), exhibits biphasic character in the presence of hydroxylamine (HA). HA is inactive in the initial phase of the hydrogen peroxide decomposition. In the second phase, at pH 7.2, 30 °C and 1 mM H 2O 2 the concentration of the inhibitor that results in 50% inhibition was estimated as 25, 40 and 261 nM for STC, PVC and ANC, respectively. In acidic solutions, the inhibition decreases with an apparent p K a 5.3–5.8 that is related to HA protonation. The inhibition of ANC at pH 7.2 is reversible since dialysis of the samples restores activity and spectral properties of ferricatalase. Hydrazine (HZ) does not inhibit ANC even at the pH values larger than p K a of HZ protonation. During the reaction of ANC with HA and H 2O 2, catalase intermediate is generated showing maximum absorbance at 428 nm with a little bit smaller extinction compared to the maximal absorbance of ferricatalase. The scheme of catalase inhibition includes an intermediate, possibly nitric oxide-ferrocatalase. Good agreement with the experimental data is achieved if the kinetic constants estimated from the non-stationary hydrogen peroxide decomposition model are used for the calculations of the inhibition of catalases at the second phase, and for the kinetics of intermediate formation.

Taxonomic position and nitrogen-containing secondary metabolites of the fungusPenicillium vitale pidoplichko et bilai apud bilai

The type strain Penicillium vitale Pidoplichko et Bilai apud Bilai 1961 VKM F-3624 was found to considerably differ from a sibling species P. janthinellium (syn. P. simplicissimum) in some physiological and morphological features (growth rates at different temperatures, the size of philiades, and the shape of conidia), as well as in the pattern of the nitrogen-containing secondary metabolites produced (roquefortine, 3,12-dihydroroquefortine, meleagrin, aurantioclavine, indole-3-acetic acid, and N-acetyltryptamine). The data obtained suggest that P. vitale represent an independent species.

Interaction of catalase with cationic hydrogels: Influence of pH, kinetics of process and isotherms of adsorption

The adsorption interaction of catalase Penicillium vitale with cationic polymer hydrogels prepared from the vinyl ether of monoethanolamine was studied at various pHs of phosphate buffer solution, interaction times and enzyme concentrations. The hydrogels behaved as anion exchange swellable gel adsorbents. The formation of stable polymer–protein complexes with high enzymatic activity has been shown. The dependence of the interaction degree of protein macromolecules on swelling coefficients of gel has been found. The isotherms of protein adsorption indicate the thermodynamical irreversibility of the process. Copyright © 1999 John Wiley & Sons, Ltd.

Исследование первичной структуры каталазы гриба Penicillium vitale. 5. Реконструкция полипептидной цепи и сравнение ее с другими каталазами

Исследование первичной структуры каталазы гриба Penicillium vitale. 5. Реконструкция полипептидной цепи и сравнение ее с другими каталазами

Исследование первичной структуры каталазы гриба Penicillium vitale. 3. Пептиды, полученные расщеплением каталазы протеиназой из Staphylococcus aureus V8

Исследование первичной структуры каталазы гриба Penicillium vitale. 3. Пептиды, полученные расщеплением каталазы протеиназой из Staphylococcus aureus V8

Исследование первичной структуры каталазы гриба Penicillium vitale. 2. Триптические пептиды модифицированной по остаткам лизина каталазы

Исследование первичной структуры каталазы гриба Penicillium vitale. 2. Триптические пептиды модифицированной по остаткам лизина каталазы

Аминокислотная последовательность вокруг остатка цистеина каталазы гриба Penісillium vitale

Аминокислотная последовательность вокруг остатка цистеина каталазы гриба Penісillium vitale

Immobilization of glucose oxidase via carbohydrate moiety onto hydrazide derivative of polyacrylate-coated porous glass

The hydrazide derivative of polyacrylate-coated glass was used for the immobilization of glucose oxidase (GO) from Penicillium vitale after periodate oxidation of its carbohydrate moiety. Periodate oxidation of the carbohydrate residues did not influence catalytic activity of the enzyme. Immobilized GO is extremely stable both at 4°C and 25°C for extended period.

Structure of the Heme d of Penicillium vitale and Escherichia coli Catalases

A heme d prosthetic group with the configuration of a cis-hydroxychlorin gamma-spirolactone has been found in the crystal structures of Penicillium vitale catalase and Escherichia coli catalase hydroperoxidase II (HPII). The absolute stereochemistry of the two heme d chiral carbon atoms has been shown to be identical. For both catalases the heme d is rotated 180 degrees about the axis defined by the alpha-gamma-meso carbon atoms, with respect to the orientation found for heme b in beef liver catalase. Only six residues in the heme pocket, preserved in P. vitale and HPII, differ from those found in the bovine catalase. In the crystal structure of the inactive N201H variant of HPII catalase the prosthetic group remains as heme b, although its orientation is the same as in the wild type enzyme. These structural results confirm the observation that heme d is formed from protoheme in the interior of the catalase molecule through a self-catalyzed reaction.

Crystal structure of catalase HPII from Escherichia coli.

Catalase is a ubiquitous enzyme present in both the prokaryotic and eukaryotic cells of aerobic organisms. It serves, in part, to protect the cell from the toxic effects of small peroxides. Escherichia coli produces two catalases, HPI and HPII, that are quite distinct from other catalases in physical structure and catalytic properties. HPII, studied in this work, is encoded by the katE gene, and has been characterized as an oligomeric, monofunctional catalase containing one cis-heme d prosthetic group per subunit of 753 residues. The crystal structure of catalase HPII from E. coli has been determined to 2.8 A resolution. The asymmetric unit of the crystal contains a whole molecule, which is a tetramer with accurate 222 point group symmetry. In the model built, that includes residues 27-753 and one heme group per monomer, strict non-crystallographic symmetry has been maintained. The crystallographic agreement R-factor is 20.1% for 58,477 reflections in the resolution shell 8.0-2.8 A. Despite differences in size and chemical properties, which were suggestive of a unique catalase, the deduced structure of HPII is related to the structure of catalase from Penicillium vitale, whose sequence is not yet known. In particular, both molecules have an additional C-terminal domain that is absent in the bovine catalase. This extra domain contains a Rossmann fold but no bound nucleotides have been detected, and its physiological role is unknown. In HPII, the heme group is modified to a heme d and inverted with respect to the orientation determined in all previously reported heme catalases. HPII is the largest catalase for which the structure has been determined to almost atomic resolution.

Строение некоторых пептидов, полученных расщеплением каталазы гриба Penicillium vitale стафилококковой протеиназой

Строение некоторых пептидов, полученных расщеплением каталазы гриба Penicillium vitale стафилококковой протеиназой