Invertase immobilised on activated clay: Properties and kinetic study

Invertase was immobilised on activated ball clay. The immobilised enzyme showed maximum activity at pH 4.8–6. The thermal deactivation was significant at temperatures above 30°C. The kinetics of sucrose hydrolysis with free invertase showed substrate and product inhibition. Immobilised enzyme kinetics followed the same mechanisms. Modelling curves were plotted to explain the experimental results. The thermal deactivation followed first order kinetics. The characteristics of thermal deactivation of immobilised enzyme were studied in the range 25–50°C.     

N-Hydroxysuccinimide-terminated self-assembled monolayers on gold for biomolecules immobilisation

Pure and mixed N-hydroxysuccinimide-terminated and butanethiol monolayers were prepared on flat gold (1 1 1) surfaces with the intent of developing suitable platforms for the direct biomolecules immobilisation. The self-assembled monolayers (SAMs) were characterised by electrochemical reductive desorption of the thiolate from the gold surface. The data have shown that certain solution proportions of the two compounds yield modified electrodes exhibiting intermediate electrochemical behaviour of the corresponding pure SAMs. The reactivity of the terminal N-hydroxysuccinimide (NHS) towards amine functionalities has been tested for the covalent attachment of Dopamine. The cyclic voltammetric responses of the investigated monolayers, after contacting with a Dopamine solution, have confirmed the chemical coupling of the amine as well as the formation of mixed SAMs. The Dopamine surface coverage increased with the amount of surface NHS. Laccase was also successfully immobilised onto this modified electrodes. The electrochemical behaviour of the modified SAMs with Laccase indicates direct electron transfer between the immobilised enzyme and the gold surface. Evidence for Laccase immobilisation was also provided by atomic force microscopic measurements.     

Electroless plated gold as a support for carbon nanotube electrodes

A monolayer of -NH₂ terminated 3-aminopropyltriethoxysilane (APS) was self-assembled onto a p-type silicon (1 0 0) substrate. This amine terminated silane monolayer provided an electrostatic point of attachment for citrate stabilised gold colloid nanoparticles, which act as ‘seed’ particles for the electroless deposition of gold, creating an electrolessly deposited gold layer on silicon. A -NH₂ terminated cysteamine monolayer was then deposited onto the gold layer and carbon nanotubes, with high carboxylic acid functionality, were immobilised via a condensation reaction. A redox active molecule ferrocenemethanol was then chemically attached to the immobilised carbon nanotubes. These nanostructures were used as working electrodes in cyclic voltammetry to observe the oxidation and reduction of ferrocene. Important electrochemical parameters such as electrode kinetics, electron transfer rate and surface concentration of the redox active molecules were obtained, providing information on the ability of electroless plated gold surfaces to act as supports for carbon nanotube-based electrodes. This information has also provided insights into the behaviour of vertically aligned carbon nanotubes immobilised on nanoscale gold wires, which have been previously fabricated using atomic force microscopy.     

Properties of native and immobilised preparations of β-D-glucosidase from Aspergillus niger

The enzyme β-D-glucosidase from Aspergillus niger has been immobilised through its carbohydrate moiety on concanavalin A-Sepharose and on cyanogen bromide-activated Sepharose after aminoalkylation of the carbohydrate side chains of the enzyme. For comparison, the enzyme was also immobilised on microcrystalline cellulose through its protein moiety. High retention of activity and a decrease in K_m and V_max. were observed when β-D-glucosidase was immobilised by these methods. An increase in the thermal stability of the immobilised β-D-glucosidase preparations over the soluble enzyme was achieved if it was treated with glutaraldehyde before its adsorption on concanavalin A-Sepharose or if the enzyme immobilised on cyanogen bromide-activated Sepharose was subsequently treated with glutaraldehyde. Treatment of β-D-glucosidase immobilised on microcrystalline cellulose with glutaraldehyde hardly increased its thermal stability over the soluble enzyme.     

Factors governing the activity of lyophilised and immobilised lipase preparations in organic solvents

Active site titration and activity measurements were performed in hexane on lyophilised lipase preparations containing different amounts of phosphate buffer and lipase immobilised on porous polypropylene. Lyophilisation of Thermomyces lanuginosus lipase with large quantities of phosphate salts (200 mM) increased the specific activity fourfold, and the number of rapidly titratable active sites increased to 50 % from the 13 % observed when smaller amounts of phosphate buffer were used (20 mM) during lyophilisation. The phosphate buffer worked as an immobilisation matrix for the lipase, and the increase in specific activity was at least partly due to decreased mass transfer limitations. When lipase was immobilised on porous polypropylene, the specific activity was 770 times higher than that of the best freeze-dried preparation. At optimal enzyme loading, 93 % of the enzyme molecules were titrated at a high rate; this indicates that this adsorption on a hydrophobic surface was a very efficient means of reducing mass transfer limitations and of immobilising the enzyme in its active conformation for use in organic solvents. The variation in specific activity with water activity was found to correlate very well with the variation in titratable active sites when lipases from Burkholderia cepacia and Thermomyces lanuginosus were immobilised on porous polypropylene. The catalytic activity per competent active site was thus constant over the whole range of water activities.     

Effects of surface curvature and surface characteristics of carbon-based nanomaterials on the adsorption and activity of acetylcholinesterase

Carbon-based nanomaterials (NM) are promising candidates for a myriad of applications ranging from drug delivery to biosensing platforms. In the physiological environment, proteins can be adsorbed onto the surface of NM that can alter their structure and function. Little is known of the effect of NM on larger proteins and enzymes and an attempt has been made in this study to investigate the effect of carbon-based NM such as carbon black (CB), graphene oxide (GO) and fullerene (C₆₀) on the adsorption and activity of acetylcholinesterase (AChE), a key enzyme present in brain, blood and nervous system and a suitable neurotoxicity biomarker. Experimental and computational results showed that all the carbon-based NM tested adsorb AChE but they have different effects on the catalytic activity of the enzyme. The most efficient AChE inhibitor is CB. In contrast, AChE adsorbed on the GO surface retains its native conformation and most of its activity. As compared to GO and CB, C₆₀ was found to be an inefficient adsorbent of AChE. The distinctive adsorption pattern of NM and their inhibitory potential could be related to the surface characteristics of NM. Our studies also demonstrate the potential of GO as a substrate for immobilization of AChE.     

Flexible Assembly of an Enzyme Cascade on a DNA Triangle Prism Nanostructure for the Controlled Biomimetic Generation of Nitric Oxide

Spatial organization of multiple enzymes at specific positions for a controlled reaction cascade has attracted wide attention in recent years. Here, we report the construction of a biomimetic enzyme cascade organized on DNA triangle prism (TP) nanostructures to enable the efficient catalytic production of nitric oxide (NO) on a single microbead. Two enzymes, glucose oxidase (GOx) and horseradish peroxidase (HRP), were assembled at adjacent locations on a DNA TP nanostructure by using DNA‐binding protein adaptors with small interenzyme distances. In the cascade, the first enzyme, GOx, converts glucose into gluconic acid in the presence of oxygen. The produced H₂O₂ intermediate is rapidly transported to the second enzyme, HRP, which oxides hydroxyurea into NO and other nitroxyl species. The pH near the surface of the negatively charged DNA nanostructures is believed to be lower than that in the bulk solution; this creates an optimal pH environment for the anchored enzymes, which results in higher yields of the NO product. Furthermore, the multienzyme system was immobilized on a microbead mediated by a DNA adaptor, and this enabled the efficient catalytic generation of gas molecules in the microreactor. Therefore, this work provides an alternative route for the biomimetic generation of NO through enzyme cascades. In particular, the dynamic binding capability of the DNA sequence enabled the positions of the protein enzyme and the DNA nanostructure to be reversed, which allowed the cascade catalysis to be modulated.     

CeO(2)/rGO/Pt sandwich nanostructure: rGO-enhanced electron transmission between metal oxide and metal nanoparticles for anodic methanol oxidation of direct methanol fuel cells

Pt-based nanocomposites have been of great research interest. In this paper, we design an efficient MO/rGO/Pt sandwich nanostructure as an anodic electrocatalyst for DMFCs with combination of the merits of rigid structure of metallic oxides (MOs) and excellent electronic conductivity of reduced oxidized graphene (rGO) as well as overcoming their shortcomings. In this case, the CeO(2)/rGO/Pt sandwich nanostructure is successfully fabricated through a facile hydrothermal approach in the presence of graphene oxide and CeO(2) nanoparticles. This structure has a unique building architecture where rGO wraps up the CeO(2) nanoparticles and Pt nanoparticles are homogeneously dispersed on the surface of rGO. This novel structure endows this material with great electrocatalytic performance in methanol oxidation: it reduces the overpotential of methanol oxidation significantly and its electrocatalytic activity and stability are much enhanced compared with Pt/rGO, CeO(2)/Pt and Pt/C catalysts. This work supplies a unique MO/rGO/Pt sandwich nanostructure as an efficient way to improve the electrocatalytic performance, which will surely shed some light on the exploration of some novel structures of electrocatalyst for DMFCs.     

Amaryllidaceae Alkaloids of Norbelladine-Type as Inspiration for Development of Highly Selective Butyrylcholinesterase Inhibitors: Synthesis,Biological Activity Evaluation,and Docking Studies

Alzheimer’s disease (AD) is a multifactorial neurodegenerative condition of the central nervous system (CNS) that is currently treated by cholinesterase inhibitors and the N-methyl-d-aspartate receptor antagonist, memantine. Emerging evidence strongly supports the relevance of targeting butyrylcholinesterase (BuChE) in the more advanced stages of AD. Within this study, we have generated a pilot series of compounds (1–20) structurally inspired from belladine-type Amaryllidaceae alkaloids, namely carltonine A and B, and evaluated their acetylcholinesterase (AChE) and BuChE inhibition properties. Some of the compounds exhibited intriguing inhibition activity for human BuChE (hBuChE), with a preference for BuChE over AChE. Seven compounds were found to possess a hBuChE inhibition profile, with IC₅₀ values below 1 µM. The most potent one, compound 6, showed nanomolar range activity with an IC₅₀ value of 72 nM and an excellent selectivity pattern over AChE, reaching a selectivity index of almost 1400. Compound 6 was further studied by enzyme kinetics, along with in-silico techniques, to reveal the mode of inhibition. The prediction of CNS availability estimates that all the compounds in this survey can pass through the blood-brain barrier (BBB), as disclosed by the BBB score.     

Electron Transfer and Stability of a Quinohaemoprotein Alcohol Dehydrogenase Electrode

Quinohaemoprotein alcohol dehydrogenase from Comamonas testosteroni (QH-EDH) was immobilised in a redox polymer network of a polyvinylpyridine, partially N-complexed with osmiumbis(bipyridine)chloride. Substrate-dependent electron transfer occurred, indicating that the enzyme was active and that effective electron transport was achieved. It was shown that the enzyme molecular weight is of importance with respect to the enzyme electrode stability. Long term stability and current density of the QH-EDH electrodes were highest when the enzyme was immobilised at pH 10·0 and 4°C, followed by an additional cross-linking step with glutaraldehyde (1%) at pH 7·0. With such an electrode current densities of 40 μA cm⁻² were obtained for several primary alcohols. The affinity of the immobilised enzyme for these substrates (K_m(app) values) was similar to that of the enzyme in solution. The half-life time of the electrodes was between 50 h and 200 h depending on the time the enzyme was in contact with the substrate. When the immobilised enzyme electrode was operated at temperatures above 37°C the stability decreased. © 1997 SCI.     

The yeast alcohol dehydrogenase catalysed conversion of cinnamaldehyde to cinnamyl alcohol

The kinetics of the conversion of trans-cinnamaldehyde to trans-cinnamyl alcohol catalysed by yeast alcohol dehydrogenase (EC 1.1.1.1) have been characterised. Reaction with the free enzyme is curtailed after a short time as a result of inactivation by the substrate. It has been shown that immobilisation of the enzyme provides substantial protection against this inactivation. Use of the immobilised enzyme in a flow-through reactor further enhanced the enzyme lifetime, creating a viable synthetic process. The product cinnamyl alcohol may be recovered by continuous solvent extraction.     

Reactivity imaging of a passive ferritic FeAlCr steel

A technique named reactivity imaging is introduced. It combines optical imaging of a polycrystalline material with orientation imaging by electron back scattering diffraction (EBSD) for a determination of the crystallographic orientation map and scanning electrochemical microscopy (SECM) for a visualization of the local reactivity. Dissolving metal ions from the substrate are directly detected by the scanning Pt tip of the SECM to measure the amount of locally dissolving material. A ferritic light weight steel (alloy Fe7.5Al7Cr) with a strong anisotropic dissolution behaviour was investigated as an example. This steel shows good passivation behaviour both, in air and through anodisation. In the passive state investigated here, the difference in dissolution rate between various crystallographic orientations is only marginal. Grain boundaries on the other hand showed a higher activity as compared to the grains themselves, which is attributed to the electronic tunnelling in the grain boundaries. The results demonstrate that the rate determining step responsible for the anisotropic dissolution does not result from a deficiency in passivation but from the active dissolution kinetics.     

Production,thermal stability and immobilisation of inulinase from Fmarium oxysporum

Fusarium oxysporum produced maximum extracellular inulinase after 9 days of its growth at 25°C on a medium (pH 5.5) containing 3% fructan and 0.2% sodium nitrate. The level of this enzyme decreased on the addition of either glucose, fructose, galactose or sucrose to F. oxysporum already growing on a fructan-containing medium. A significant increase in invertase production which resulted in an increase of the invertase/inulinase (S/I) ratio, was observed on addition of inulin to this fungus growing on other carbon sources. Glycerol (10%) gave better protection to inulinase against thermal denaturation at 50°C compared to ethylene glycol and sorbitol. Inulinase immobilised in polyacrylamide gel retained 45% of its original activity. The immobilised enzyme showed a higher optimum temperature (45°C) compared to free enzyme (37°C). The immobilised enzyme after storage at 25°C for 96 h showed 58% activity. Thermal stability of entrapped inulinase increased in the presence of inulin.     

Computer simulation and SERR detection of cytochrome c dynamics at SAM-coated electrodes

In this paper we present a combined experimental and theoretical study of the heterogeneous electron transfer reaction of cytochrome c electrostatically adsorbed on metal electrodes coated with monolayers of 6-mercaptohexanoic acid. Molecular dynamics simulations and pathways calculations show that adsorption of the protein leads to a broad distribution of orientations and, thus, to a correspondingly broad distribution of electron transfer rate constants due to the orientation-dependence of the electronic coupling parameter. The adsorbed protein exhibits significant mobility and, therefore, the measured reaction rate is predicted to be a convolution of protein dynamics and tunnelling probabilities for each orientation. This prediction is confirmed by time-resolved surface enhanced resonance Raman which allows for the direct monitoring of protein (re-)orientation and electron transfer of the immobilised cytochrome c. The results provide a consistent explanation for the non-exponential distance-independence of electron transfer rates usually observed for proteins immobilized on electrodes.     

Invertase immobilised on montmorillonite: reusability enhancement and reduction in leaching

Invertase was immobilised on microporous montmorillonite K-10 via adsorption and covalent binding. The immobilised enzymes were tested for sucrose hydrolysis activity in a batch reactor. K_m for immobilised systems was greater than free enzyme. The immobilised forms could be reused for 15 continuous cycles without any loss in activity. After 25 cycles, 85% initial activity was retained. A study on leaching of enzymes showed that 100% enzyme was retained even after 15 cycles of reuse. Leaching increased with reaction temperature. Covalent binding resisted leaching even at temperatures of 70 °C.     

Oxidation decomposition of unsaturated fatty acids by singlet oxygen in phospholipid bilayer membranes

Oxidation decomposition of unsaturated fatty acids with singlet oxygen generated from a photosensitizing agent was investigated in liposome bilayer membranes under a light irradiation condition. The liposome of which the bilayer membrane was composed of L-alpha-dipalmitoylphosphatidylcholine (DPPC), protoporphyrin IX (PpIX), and an unsaturated fatty acid (oleic acid, linoleic acid, alpha-linolenic acid, or arachidonic acid) were prepared with Bangham's method. In irradiating the liposome dispersion with light ranged from 550 to 750 nm, the unsaturated fatty acid was decomposed through an oxidation reaction with singlet oxygen. The decomposition rate constant was obeyed as the following order: arachidonic acid > oleic acid > alpha-linolenic acid > linoleic acid. This result indicates that oleic acid is readily degraded despite its lower unsaturated degree. In addition, micropolarity and microfluidity of the hydrocarbon region in the liposome bilayer membrane including the unsaturated fatty acid and PpIX decreased with an increase in light irradiation time. These findings suggest that interaction among the hydrocarbon chains of DPPC in the liposome bilayer membrane is promoted by migration of the oxidized unsaturated fatty acid from the hydrocarbon region, leading to form close-packed and well-ordered orientation of the hydrocarbon chains.     

Polymer-based tubular microbots: role of composition and preparation

The influence of the composition and electropolymerization conditions upon the propulsion of new template-prepared polymer-based bilayer microtubular microbots is described. The effects of different electropolymerized outer layers, including polypyrrole (PPy), poly(3,4-ethylenedioxythiophene) (PEDOT), polyaniline (PANI), and of various inner catalytic metal surfaces (Ag, Pt, Au, Ni-Pt alloy), upon the movement of such bilayer microtubes are evaluated and compared. Electropolymerization conditions, such as the monomer concentration and medium (e.g. surfactant, electrolyte), have a profound effect upon the morphology and locomotion of the resulting microtubes. The most efficient propulsion is observed using PEDOT/Pt microbots that offer a record-breaking speed of over 1400 body lengths s(-1) at physiological temperature, which is the fastest relative speed reported to date for all artificial micro/nanomotors. An inner Pt-Ni alloy surface is shown useful for combining magnetic control and catalytic fuel decomposition within one layer, thus greatly simplifying the preparation of magnetically-guided microbots. Polymer-based microbots with an inner gold layer offer efficient biocatalytic propulsion in low peroxide level in connection to an immobilized catalase enzyme. Metallic Au/Pt bilayer microbots can also be prepared electrochemically to offer high speed propulsion towards potential biomedical applications through functionalization of the outer gold surface. Such rational template preparation and systematic optimization of highly efficient microbots hold considerable promise for diverse practical applications.     

Intrinsic kinetic constants of an immobilised glucose-isomerase

The isomerization of glucose into fructose using a commercial immobilised glucose-isomerase has been studied and an initial transitory state of the enzyme, probably due to an establishment of stationary concentration profiles, was observed. Operational conditions to minimise the effects of external and internal mass transfer were determined using immobilised enzyme particles with diameters less than 0.064 mm. Thermal enzyme deactivation of the enzyme was insignificant if it was pre-treated with cobalt. The intrinsic kinetic constants of the reversible reaction Michaelis—Menten equation were calculated, in operational conditions free of mass transfer effects.     

Reactivators of acetylcholinesterase inhibited by organophosphorus nerve agents

Since the September 11, 2001, terrorist attacks in the United States, the specter of a chemical threat against civilian populations has renewed research interest in chemical warfare agents, their mechanisms of action, and treatments that reverse their effects. In this Account, we focus specifically on organophosphorus nerve agents (OPNAs). Although some OPNAs are used as pest control, the most toxic chemicals in this class are used as chemical warfare agents in armed conflicts. The acute toxicity of OPNAs results from the irreversible inhibition of acetylcholinesterase (AChE, EC 3.1.1.7) via the formation of a covalent P-O bond at the serine hydroxyl group in the enzyme active site. AChE breaks down the neurotransmitter acetylcholine at neuronal synapses and neuromuscular junctions. The irreversible inhibition of AChE causes the neurotransmitter to accumulate in the synaptic cleft, leading to overstimulation of cholinergic receptors, seizures, respiratory arrest, and death. The current treatment for OPNA poisoning combines an antimuscarinic drug (e.g., atropine), an anticonvulsant drug (e.g., diazepam), and an AChE reactivator of the pyridinium aldoxime family (pralidoxime, trimedoxime, obidoxime, HI-6, HL?-7). Because of their high nucleophilicity, oximes can displace the phosphyl group from the catalytic serine, thus restoring the enzyme's catalytic activity. During 50 years of research in the reactivator field, researchers have synthesized and tested numerous structural modifications of monopyridinium oximes and bispyridinium oximes. In the past decade, medicinal chemists have focused their research on the more efficient bispyridinium reactivators, but all known reactivators have several drawbacks. First, due to their permanent positive charge, they do not cross the blood-brain barrier (BBB) efficiently and do not readily reactivate AChE in the central nervous system. Second, no single oxime is efficient against a wide variety of OPNAs. Third, oximes cannot reactivate "aged" AChE. This Account summarizes recent strategies for the development of AChE reactivators capable of crossing the BBB. The use of nanoparticulate transport and inhibition of P-glycoprotein efflux pumps improves BBB transport of these AChE reactivators. Chemical modifications that increased the lipophilicity of the pyridinium aldoximes, the addition of a fluorine atom and the replacement of a pyridyl ring with a dihydropyridyl moiety, enhances BBB permeability. The glycosylation of pyridine aldoximes facilitates increased BBB penetration via the GLUT-1 transport system. The development of novel uncharged reactivators that can move efficiently across the BBB represents one of the most promising of these new strategies.