Hydrolysis of Organophosphate Esters: Phosphotriesterase Activity of Metallo‐β‐lactamase and Its Functional Mimics

The phosphotriesterase (PTE) activity of a series of binuclear and mononuclear zinc(II) complexes and metallo‐β‐lactamase (mβl) from Bacillus cereus was studied. The binuclear complex 1 , which exhibits good mβl activity, shows poor PTE activity. In contrast, complex 2 , a poor mimic of mβl, exhibits much higher activity than 1 . The replacement of Cl^? ligands by OH^? is important for the high PTE activity of complex 2 because this complex does not show any catalytic activity in methanol. The natural enzyme mβl from B. cereus is also found to be an inefficient catalyst in the hydrolysis of phosphotriesters. These observations indicate that the binding of β‐lactam substrates at the binuclear zinc(II) center is different from that of phosphotriesters. Furthermore, phosphodiesters, the products from the hydrolysis of triesters, significantly inhibit the PTE activity of mβl and its functional mimics. Although the mononuclear complexes 3 and 4 exhibited significant mβl activity, these complexes are found to be almost inactive in the hydrolysis of phosphotriesters. These observations indicate that the elimination of phosphodiesters from the reaction site is important for the PTE activity of zinc(II) complexes.     

Optimization of Chlorophyllase-catalyzed Hydrolysis of Chlorophyll in Monophasic Organic Solvent Media

The effects of selected reaction parameters, including solvent hydrophobicity, initial water activity, agitation speed, temperature and enzyme concentration, on the biocatalytic efficiency of a chlorophyllase enzymatic extract from Phaeodactylum tricornutum in neat organic solvent media were investigated. The highest chlorophyllase specific activity of 322 nmol hydrolyzed chlorophyll per gram of protein per minute and bioconversion yield of 91% were obtained in the reaction mixture of hexane/2-octanone (98.3:1.7, v/v), at a controlled initial water activity of 0.90. R _O/A value, which is the ratio of the specific activity in the organic solvent to that in the aqueous/miscible organic solvent medium, was 1.5 × 10⁻³. To reduce the substrate diffusional limitations, the appropriate agitation speed and enzyme concentration were determined. The optimum temperature for maximal enzymatic activity and activation energy were 35°C and 105.0 kJ/mol, respectively. Although the catalytic efficiency of chlorphyllase in the neat organic solvent mixture was lower than that in the aqueous medium, its half-life time in the first environment at temperature ranging from 35 to 50°C was increased by 5.0 to 15.0 times.     

The ammonia freeze explosion (AFEX) process

A partially purified phophotriesterase was successfully immobilized onto nylon 6 and 66 membranes, nylon 11 powder, and nylon tubing. Up to 9000 U of enzyme activity was immobilized onto 2000 cm2 of a nylon 6 membrane where 1 U is the amount of enzyme necessary to catalyze the hydrolysis of 1.0 mumol of paraoxon/min at 25 degrees C. The nylon 66 membrane-bound phosphotriesterase was characterized kinetically where the apparent Km value for the immobilized enzyme was 0.35 mM. This is 5-6 times higher than that observed for the soluble enzyme. However, nylon immobilization limited the maximum rate of paraoxon hydrolysis to less than 10% of the value measured for the soluble enzyme. The addition of the cosolvent, methanol, resulted in an increase in the apparent Km value for paraoxon hydrolysis but concentrations up to 40% had no negative effect on the catalytic effectiveness with the soluble or immobilized phosphotriesterase. Based on the kinetic analysis, methanol appears to be a competitive inhibitor for both forms of enzyme. The nylon powder immobilized enzyme was shown to be stable for at least 20 mo. The immobilization of the phosphotriesterase onto nylon provides a practical method for the detoxification of organophosphate pesticides.     

Ultrasound irradiation and green synthesized CuO-NiO-ZnO mixed metal oxide: An efficient sono/nano-catalytic system toward a regioselective synthesis of 1-aryl-5-amino-1H-tetrazoles

Abstract

An efficient approach has been reported that 1-aryl-5-amino-1H-tetrazoles are prepared from structurally diverse N-arylcyanamides and sodium azide using sono/nano-catalytic system. The mixed metal oxide nanoparticles (NPs) (CuO, NiO, and ZnO NPs) were prepared with utilizing aqueous leaves extracts of the Rheum ribes. In this study, the effect of the reaction condition was investigated on the catalytic activity and selectivity of mixed metal oxide NPs for the synthesis of aminotetrazole derivatives. In this regard, various reaction conditions were precisely compared such as reflux and ultrasonication (US). In this study, the regioselective synthesis of 1-aryl-5-amino-1H-tetrazoles with high yield was achieved by the synergistic effect between US irradiation and prepared nanocatalyst which showed the potential of sono/nano-catalytic system in organic synthesis. Furthermore, the as-prepared nanocatalyst could be reused and recovered from the reaction mixture for several times without consequential abate in its catalytic activity and selectivity.     

Hydrogenation of substituted nitroarenes by a polymer-bound palladium(II) Schiff base catalyst

The catalytic activity of a polymer-bound palladium Schiff base catalyst was investigated toward the reduction of aryl nitro compounds under ambient temperature and pressure. The dependence of the rate of hydrogenation of o-nitroaniline and o-nitrotoluene on substrate concentration, catalyst concentration and temperature has been determined. Based on the results obtained a plausible mechanism for the hydrogenation reaction is proposed and a rate expression is deduced. The energy and entropy of activation have been evaluated from the kinetic data. The polymer-bound catalyst was found to be better than its homogeneous analog PdCl₂(NSBA) [NSBA = N-salicylidene benzylamine] for both stability and reusability. Recycling studies revealed that the catalyst could be used six times without metal leaching or significant loss in activity.     

PNPP catalytic hydrolysis by mono- and binuclear transition-metal complexes with polyether-bridged dihydroxamic acids

Polyether-bridged dihydroxamic acids and their mono- and binuclear copper(II), and cobalt(II) complexes have been synthesized and employed as models to mimic hydrolase in the catalytic hydrolysis of p-nitrophenyl picolinate (PNPP). The kinetics and the mechanism of PNPP hydrolysis have been investigated. The kinetic mathematical model of PNPP cleavage by the complexes has been proposed. The effects of the different central metal ion, mono- and binuclear metal, the pseudo-macrocyclic polyether constructed by the polyethoxy group in complexes, and reactive temperature on the rate of PNPP catalytic hydrolysis have been examined. The results show that the transition-metal dihydroxamates exhibit high catalytic activity in the PNPP hydrolysis; the rate of the PNPP hydrolysis increases with the increase in pH of the buffer solution; the catalytic activity of binuclear complexes is higher than that of mononuclear complexes; the catalytic activity of copper(II) complex is about four times that of the cobalt(II) complex; the pseudo-macrocyclic polyether can synergetically activate H₂O coordinated to the metal ion with the central metal ion together and promote the PNPP catalytic hydrolysis.     

Effects of Low-Shear Modeled Microgravity on the Characterization of Recombinant β-D-Glucuronidase Expressed in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

In this study, we used a high-aspect-ratio vessel (HARV), which could model environment of microgravity on ground to investigate for the first time the effects of low-shear modeled microgravity (LSMMG) on the characterization of recombinant β-D-glucuronidase expressed in Pichia pastoris. The β-D-glucuronidase gene (GenBank accession no. EU095019) derived from Penicillium purpurogenum Li-3 encoding β-D-glucuronidase (PGUS) was expressed in P. pastoris GS115 in two different environments of LSMMG and normal gravity (NG). Results manifested that both LSMMG and NG conditions had insignificant effects on temperature and pH activity (optimal temperature and pH were 55 and 5.0 °C, respectively) and characteristic stability of recombinant PGUS. However, the catalytic activity of recombinant PGUS expressed under LSMMG was less affected by metal ions and EDTA as compared with that of NG. Furthermore, K _m value of the recombinant PGUS expressed under LSMMG was nearly one fifth of that under NG (1.72 vs. 7.72), whereas catalytic efficiency (k _cat/K _m) of PGUS expressed under LSMMG (13.55) was 3.7 times higher than that of NG (3.61). The results initially reveal the significant alterations in catalytic properties of recombinant enzyme in response to LSMMG environment and have potential application in bioprocessing and biocatalysis.     

The activation characteristics of the decomposition of H<Subscript>2</Subscript>O<Subscript>2</Subscript> on palladium-carbon catalysts

The kinetics of catalytic decomposition of H₂O₂ on palladium-carbon catalysts with various deposited metal distributions in carrier (active carbon) porous granules was studied. The activation parameters (E _a and A ₀) of the process were calculated by the Arrhenius equation. A determining factor for the catalytic process was found to be the entropy factor (A ₀), which characterized the formation and dissociation of activated transition complexes. A quantum-chemical study of the electronic structure of palladium-carbon catalysts showed the occurrence of electron density transfer from the carbon matrix to metal clusters and collectivization of their electronic systems. This increased the donor-acceptor ability of the synthesized materials and, as a consequence, their catalytic activity.     

The Crystal Structure of a Homodimeric Pseudomonas Glyoxalase I Enzyme Reveals Asymmetric Metallation Commensurate with Half‐of‐Sites Activity

The Zn inactive class of glyoxalase I (Glo1) metalloenzymes are typically homodimeric with two metal‐dependent active sites. While the two active sites share identical amino acid composition, this class of enzyme is optimally active with only one metal per homodimer. We have determined the X‐ray crystal structure of GloA2, a Zn inactive Glo1 enzyme from Pseudomonas aeruginosa. The presented structures exhibit an unprecedented metal‐binding arrangement consistent with half‐of‐sites activity: one active site contains a single activating Ni²⁺ ion, whereas the other contains two inactivating Zn²⁺ ions. Enzymological experiments prompted by the binuclear Zn²⁺ site identified a novel catalytic property of GloA2. The enzyme can function as a Zn²⁺/Co²⁺‐dependent hydrolase, in addition to its previously determined glyoxalase I activity. The presented findings demonstrate that GloA2 can accommodate two distinct metal‐binding arrangements simultaneously, each of which catalyzes a different reaction.     

Molecular dynamics simulations of the detoxification of paraoxon catalyzed by phosphotriesterase

Combined QM(PM3)/MM molecular dynamics simulations together with QM(DFT)/MM optimizations for key configurations have been performed to elucidate the enzymatic catalysis mechanism on the detoxification of paraoxon by phosphotriesterase (PTE). In the simulations, the PM3 parameters for the phosphorous atom were reoptimized. The equilibrated configuration of the enzyme/substrate complex showed that paraoxon can strongly bind to the more solvent‐exposed metal ion Zn_β, but the free energy profile along the binding path demonstrated that the binding is thermodynamically unfavorable. This explains why the crystal structures of PTE with substrate analogues often exhibit long distances between the phosphoral oxygen and Zn_β. The subsequent S_N2 reaction plays the key role in the whole process, but controversies exist over the identity of the nucleophilic species, which could be either a hydroxide ion terminally coordinated to Zn_α or the μ‐hydroxo bridge between the α‐ and β‐metals. Our simulations supported the latter and showed that the rate‐limiting step is the distortion of the bound paraoxon to approach the bridging hydroxide. After this preparation step, the bridging hydroxide ion attacks the phosphorous center and replaces the diethyl phosphate with a low barrier. Thus, a plausible way to engineer PTE with enhanced catalytic activity is to stabilize the deformed paraoxon. Conformational analyses indicate that Trp131 is the closest residue to the phosphoryl oxygen, and mutations to Arg or Gln or even Lys, which can shorten the hydrogen bond distance with the phosphoryl oxygen, could potentially lead to a mutant with enhanced activity for the detoxification of organophosphates. © 2009 Wiley Periodicals, Inc. J Comput Chem, 2009     

Novel Metal Oxide of CuO-ZnO Nanocatalyst Efficiently Catalyzed the Synthesis of 2-Amino-4H-chromenes in Water

Novel metal oxide of CuO-ZnO nanocatalyst was prepared by a coprecipitation method and characterized by x-ray diffusion, Brunauer–Emmett–Teller surface area, scanning electron microscopy, transmission electron microscopy, and energy dispersive spectroscopy (EDS) analysis. The catalyst is efficient for the synthesis of 2-amino-4H-chromenes by one-pot condensation of aromatic aldehydes, hydroxynaphthalene derivatives, and malononitrile in water at reflux conditions. Moreover, CuO-ZnO nanocatalyst can be recycled up six times without any significant loss of its catalytic activity.     

Substrate Specificity and Thermostability of the Dehairing Alkaline Protease from <Emphasis Type="Italic">Bacillus pumilus</Emphasis>

An alkaline protease (DHAP) from Bacillus pumilus has shown great potential in hide dehairing. To get better insights on its catalytic properties for application, the substrate specificity and thermostability were investigated using five natural proteins and nine synthetic peptides. The results showed that DHAP could hydrolyze five proteins tested here in different specificity. Collagen, a component of animal skin, was more resistant to hydrolysis than casein, fibrin, and gelatin. Among the synthetic peptides, the enzyme showed activity mainly with tetrapeptide substrates with the catalytic efficiency in order of Phe>Leu>Ala at P1 site, although k _m value for AAVA-pN is much lower than that for AAPL-pN and AAPF-pN. With tripeptide substrates, smaller side-chain group (Gly) at P1 site was not hydrolyzed by DHAP. The enzyme showed good thermostability below 60 °C, and lost activity so quickly above 70 °C. The thermostability was largely dependent on metal ion, especially Ca²⁺, although other ions, like Mg²⁺, Mn²⁺, and Co²⁺, could sustain stability at certain extent within limited time. Cu²⁺, Fe²⁺, as well as Al³⁺, did not support the enzyme to retain activity at 60 °C even in 5 min. In addition, the selected metal ions could coordinate calcium in improvement or destruction of thermostability for DHAP.     

Catalytic Oxidation of Small Molecules in a Gas Phase in the Presence of Heterogenized Platinum and Palladium Complexes with Chemically Different Ligands

This work is devoted to the study of catalytic properties of the metal complexes of platinum and palladium with acetylacetone and N-allyl-N"-propylthiourea heterogenized on the surface of silica in the oxidation reactions of H₂ and CO in a gas phase. We found that the acetylacetonate complexes were not degraded under catalytic reaction conditions, whereas the metal complexes withN-allyl-N"-propylthiourea exhibited a high activity only after partial degradation of the ligand. We demonstrated that the catalytic activity of the grafted metal complexes was higher than that of traditional supported platinum and palladium catalysts with the same metal content. Taking into account the structure of active centers in Pt and Pd complexes grafted on SiO₂ and the interaction of these centers with reactants, we proposed a detailed mechanism for the catalytic action, which adequately describes the entire set of experimental data.     

Triesterase and Promiscuous Diesterase Activities of a Di‐CoII‐Containing Organophosphate Degrading Enzyme Reaction Mechanisms

The reaction mechanism for the hydrolysis of trimethyl phosphate and of the obtained phosphodiester by the di‐Co^II derivative of organophosphate degrading enzyme from Agrobacterium radiobacter P230(OpdA), have been investigated at density functional level of theory in the framework of the cluster model approach. Both mechanisms proceed by a multistep sequence and each catalytic cycle begins with the nucleophilic attack by a metal‐bound hydroxide on the phosphorus atom of the substrate, leading to the cleavage of the phosphate‐ester bond. Four exchange‐correlation functionals were used to derive the potential energy profiles in protein environments. Although the enzyme is confirmed to work better as triesterase, as revealed by the barrier heights in the rate‐limiting steps of the catalytic processes, its promiscuous ability to hydrolyze also the product of the reaction has been confirmed. The important role played by water molecules and some residues in the outer coordination sphere has been elucidated, while the binuclear Co^II center accomplishes both structural and catalytic functions. To correctly describe the electronic configuration of the d shell of the metal ions, high‐ and low‐spin arrangement jointly with the occurrence of antiferromagnetic coupling, have been herein considered.     

Differential properties of Aspergillus niger tannase produced under solid-state and submerged fermentations

Significant differences on structure, stability, and catalytic properties of tannase were found when this enzyme was produced under solid-state and submerged fermentations (SSF and SmF) by Aspergillus niger. The specific activity was 5.5 times higher on SSF than in SmF. Significant differences in isoelectric points of tannases were found. The pH optima for both types of enzyme was found at 6 and the pH stability of SSF and SmF tannase were at 6 and 5–8, respectively. The optimal temperature range was from 50 to 60 °C for SmF tannase and 60 °C for SSF tannase, and both enzyme types showed tolerance to high temperatures (60–70 °C). The SSF tannase showed a major specificity for methyl gallate substrate while SmF tannase for tannic acid. All metal ions tested, had an activity inhibition from 30–46% on SSF tannase. SDS-PAGE analysis as well as gel localization studies of both SSF and SmF purified tannases showed a single band with a molecular weight of 102 and 105 kDa, respectively. Different levels of glycosylation were found among SSF and SmF purified tannases. This is the first report about structural differences among tannase produced under SSF and SmF and this study provides basis for explanation of the stability and catalytic differences observed previously for this two tannase types.     

Synthesis and catalytic evaluation of Fe3O4/MWCNTs nanozyme as recyclable peroxidase mimetics: Biochemical and physicochemical characterization

In this research, the nanocomposite of multiwalled carbon nanotubes and magnetic metal oxide nanoparticles (Fe₃O₄/MWCNTs), as enzyme mimetic, was synthesized using an in situ chemical reduction method. The structure, composition and morphology of the prepared Fe₃O₄/MWCNT nanocomposite materials were characterized using X‐ray diffraction, FT‐IR and scanning electron microscopy with energy dispersive X‐ray spectroscopy, respectively. The magnetic properties of the nanocomposite were investigated by the vibrating sample magnetometer. A colorimetric system involving nanozyme, phenol/4‐aminoantipyrine and H₂O₂ was utilized for the determination of peroxidase mimetic catalytic assay. The obtained results confirmed that the synthesis of Fe₃O₄/MWCNTs nanostructures was successful. It was found that Fe₃O₄/MWCNTs nanohybrid exhibited peroxidase‐like activity without any pH limitation. Colorimetric data demonstrated that the prepared nanocatalyst had higher catalytic activity toward H₂O₂ than MWCNTs. The kinetic parameters of the nanozyme, K_m and V_max, were estimated to be 8.3 mm and 1.4 mm min^?1, respectively. The Fe₃O₄/MWCNTs nanostructures were also successfully applied for glucose detection. In addition, peroxidase‐like activity of the nanozyme increased in the presence of butyl‐imidazolium bromide ionic liquid. These biomimetic catalysts have some advantages, such as simplicity, stability, reusability and cost effectiveness, which makes them great candidates to be used in various fields of biotechnology applications.     

Kinetic and thermodynamic parameters,and partial characterization of the crude extract of tannase produced by Saccharomyces cerevisiae CCMB 520

Tannase can be used in different industrial sectors such as in food (juices and wine) and pharmaceutical production (trimethoprim) because it catalyses the hydrolysis of hydrolysable tannins. The aim of the current study is to assess the tannase found in the crude extract of Saccharomyces cerevisiae CCMB 520, and to set its catalytic and thermodynamic properties. The enzyme was optimally active at pH 6.0 and temperature 30 °C. Tannase was activated by Na⁺, Ca²⁺, K⁺ at 5 × 10^?3 mol/L. The half-life at 30 °C was 3465.7 min. The activation energy was 40.32 kJ/mol. The Gibbs free energy, enthalpy and entropy at 30 °C were 85.40, 48.10 and ?0.12 kJ/mol K, respectively. Our results suggest that the tannase found in the crude extract of S. cerevisiae is an attractive enzyme for industrial applications, such as for beverage manufacturing and gallic acid production, due its catalytic and thermodynamic properties (heat-stable and resistant to metal ions).     

Quantum chemical models and electronic structure of active centers of heterogeneous polymerization of olefins

Conclusions The peculiarities of the catalytic activity in olefin polymerizations which can find explanation in terms of the concepts suggested in this work are as follows. First, the low catalytic activity of the individual organometal compounds of group IV-VI transition metals is indicative [53] of the important role of the coordination state of the transition metal in AC, which, according to Cosse's model, must be octahedral (tetrahedral for individual metal-alkyl compounds MR_n). Second, the activity of a catalytic system depends essentially on the nature of the ligand environment of the metal in AC. The catalysts based on titanium halides display the highest activity. Third, the results of [19, 20] show that the highly active catalytic centers of homogeneous Ziegler-Natta's systems are “cation-like” Zr(IV) complexes Cp2Zr⁺-R. All these features find explanation in terms of the concept of the competitive contributions from the AC metal s and d orbitals to the active M-R bond. Thus a transition of AC environment from tetrahedral to octahedral may be compared with a change in transition metal AO hybridization:d ³s¹ (tetrahedron) ⇒d ³s¹ (octahedron). Translated fromZhurnal Strukturnoi Khimii, Vol. 41, No. 2, pp. 391–404, March–April, 2000.     

Study of the Enzymatic Activity of Arthrobacter ureafaciens Neuraminidase by Isothermal Titration Calorimetry

Isothermal titration calorimetry was used to determine the enzymatic activity and thermodynamic activation parameters of Arthrobacter ureafaciens sialidase with the sialyl substrates α‐2,3‐, α‐2,6‐sialyllactoses and α‐2,8‐sialic acid dimer. By monitoring the heat released during hydrolysis, the Michaelis constant (K_m), catalytic rate constant (k_cat), activation energy, activation Gibbs energy, enthalpy, and entropy for different monovalent sialyl conjugates were calculated and found to be consistent with those derived by chromatographic or colorimetric assays. The observed decreases in the activation energy and transition entropy of sialyllactoses were larger than the Michaelis activation parameters of lactose‐free di‐sialic acid because of the specific enzyme activity of A. ureafaciens sialidase.     

Rerouting and Improving Dauc-8-en-11-ol Synthase from Streptomyces venezuelae to a High Yielding Biocatalyst

The dauc-8-en-11-ol synthase from Streptomyces venezuelae was investigated for its catalytic activity towards alternative terpene precursors, specifically designed to enable new cyclisation pathways. Exchange of aromatic amino acid residues at the enzyme surface by site-directed mutagenesis led to a 4-fold increase of the yield in preparative scale incubations, which likely results from an increased enzyme stability instead of improved enzyme kinetics.