Graphene Oxide Modulating the Bioelectronic Properties of Penicillinase Immobilized in Lipid Langmuir-Blodgett Films.

In this paper, graphene oxide was incorporated in penicillinase-lipid Langmuir monolayers and transferred to solid supports as Langmuir-Blodgett (LB) films so that the enzyme catalytic properties could be evaluated. Adsorption of penicillinase and graphene oxide on dimyristoylphosphatidic acid (DMPA) monolayers at the air-water interface was investigated by tensiometry, vibrational spectroscopy, and Brewster angle microscopy. The LB films were characterized by quartz crystal microbalance, infrared spectroscopy, luminescence spectroscopy, and atomic force microscopy. Enzyme activity was studied with UV-vis spectroscopy, and the feasibility of the supramolecular device nanostructured as ultrathin films was essayed as an optical sensor device. The presence of graphene oxide in the enzyme-lipid LB film not only tuned the catalytic activity of penicillinase but also helped conserve its enzyme activity after weeks. These results may be related not only to the molecular architecture provided by the film but also to the synergism between the compounds on the active layer, leading to a molecular architecture that allowed a fast analyte diffusion owing to a suitable molecular accommodation which also preserved the penicillinase activity. This work then demonstrates the feasibility of employing LB films composed of lipids, graphene oxide, and enzymes as optical devices for biosensing applications as a proof-of-concept experiment.

Entrapment of surfactant modified lipase within zeolitic imidazolate framework (ZIF)-8.

Mostly, enzyme activity is reduced after immobilization of enzyme within MOF due to unfavourable conformational changes occurred during the immobilization procedure. In this context, lipase was activated by surfactants (in order to get a highly active enzyme) followed by encapsulation within zeolitic imidazolate framework (ZIF)-8 via one-pot facile self-assembly method. The prepared lipase-SDS ZIF-8 exhibited 250% enhanced activity compared to native form. The prepared biocomposite was characterized and analysed by X-ray diffraction (XRD), Fourier transform infrared (FT-IR) and scanning electron microscopy (SEM). Thermo-stability was determined for prepared lipase-SDS ZIF-8 biocomposite in the range of 50-70 °C, which showed more than two-folds enhanced stability in terms of half-life. Further, immobilized lipase retained 76% of residual activity even after six repetitive cycles and, it showed 91% residual activity after twenty days of long term storage. Finally, lipase-SDS ZIF-8 was tested for chemical stability in polar denaturing solvents which showed excellent stability as compared to free lipase.

Evaluation of thrombin inhibitory activity of catechins by online capillary electrophoresis-based immobilized enzyme microreactor and molecular docking.

An online capillary electrophoresis (CE)-based thrombin (THR) immobilized enzyme microreactor (IMER) method was established to screen THR inhibitors in this study. S-2366 was used as chromogenic substrate for determination of THR activity and other kinetic constants. After continuously run for 50 times, the prepared IMER could still remain 89% of the initial immobilized enzyme activity. The Michaelis-Menten constant (Km) of immobilized THR was measured as 0.514 mmol/L and the half-maximal inhibitory concentration (IC50) and inhibition constant (Ki) of argatroban on THR were determined as 78.07 and 26.53 nmol/L, respectively, which indicated that CE-based THR IMER was successfully established and could be applied to screen THR inhibitors. Then the prepared IMER was used to investigate the inhibitory potency on THR of four main catechins in green tea including epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG), and epigallocatechin gallate (EGCG). The results showed that ECG and EGCG had good THR inhibition activity and their inhibition rates at concentration of 200 µmol/L were 53.2 ±â€¯3.8% and 55.8 ±â€¯2.6%, respectively, which was in consistent with the results of microplate reader assay. Additionally, molecular docking results showed that the benzopyran groups of ECG and EGCG were inserted into the THR active pocket and interacted with residues LYS60F, TRP60D, TRY60A, IEU99, GLY216, HIS57 and SER195, but EC and EGC did not. Therefore, the developed CE-based THR IMER is reliable method for measuring THR inhibitory activity of natural inhibitors.

Glutaraldehyde cross-linked glutamate oxidase coated microelectrode arrays: selectivity and resting levels of glutamate in the CNS.

Glutaraldehyde is widely used as a cross-linking agent for enzyme immobilization onto microelectrodes. Recent studies and prior reports indicate changes in enzyme activity and selectivity with certain glutaraldehyde cross-linking procedures that may jeopardize the performance of microelectrode recordings and lead to falsely elevated responses in biological systems. In this study, the sensitivity of glutaraldehyde cross-linked glutamate oxidase-based microelectrode arrays to 22 amino acids was tested and compared to glutamate. As expected, responses to electroactive amino acids (Cys, Tyr, Trp) were detected at both nonenzyme-coated and enzyme-coated microelectrodes sites, while the remaining amino acids yielded no detectable responses. Electroactive amino acids were effectively blocked with a m-phenylene diamine (mPD) layer and, subsequently, no responses were detected. Preliminary results on the use of poly(ethylene glycol) diglycidyl ether (PEGDE) as a potentially more reliable cross-linking agent for the immobilization of glutamate oxidase onto ceramic-based microelectrode arrays are reported and show no significant advantages over glutaraldehyde as we observe comparable selectivities and responses. These results support that glutaraldehyde-cross-linked glutamate oxidase retains sufficient enzyme specificity for accurate in vivo brain measures of tonic and phasic glutamate levels when immobilized using specific "wet" coating procedures.

Preparation of a dual-enzyme co-immobilized capillary microreactor and simultaneous screening of multiple enzyme inhibitors by capillary electrophoresis.

A CE method based on a dual-enzyme co-immobilized capillary microreactor was developed for the simultaneous screening of multiple enzyme inhibitors. The capillary microreactor was prepared by co-immobilizing adenosine deaminase and xanthine oxidase on the inner wall at the inlet end of the separation capillary. The enzymes were first immobilized on gold nanoparticles, and the functionalized gold nanoparticles were then assembled on the inner wall at the inlet end of the separation capillary treated with polyethyleneimine. With the developed CE method, the substrates and products were baseline separated within 3 min. The activity of the immobilized enzyme can be directly detected by measuring the peak height of the products. A statistical parameter Z' factor was recommended for evaluation of the accuracy of a drug screening system. In the present study, it was calculated to be larger than 0.5, implying a good accuracy. Finally, screening a small compound library containing two known enzyme inhibitors and 20 natural extracts by the proposed method was demonstrated. The known inhibitors were identified, and some natural extracts were found to be positive for two-enzyme inhibition by the present method.

Activation of Leishmania (Leishmania) amazonensis arginase at low temperature by binuclear Mn2+ center formation of the immobilized enzyme on a Ni2+ resin.

In Leishmania, arginase is responsible for the production of ornithine, a precursor of polyamines required for proliferation of the parasite. In this work, the activation kinetics of immobilized arginase enzyme from L. (L.) amazonensis were studied by varying the concentration of Mn2+ applied to the nickel column at 23 degrees C. The intensity of the binding of the enzyme to the Ni2+ resin was directly proportional to the concentration of Mn2+. Conformational changes of the enzyme may occur when the enzyme interacts with immobilized Ni2+, allowing the following to occur: (1) entrance of Mn2+ and formation of the metal bridge; (2) stabilization and activation of the enzyme at 23 degrees C; and (3) an increase in the affinity of the enzyme to Ni2+ after the Mn2+ activation step. The conformational alterations can be summarized as follows: the interaction with the Ni2+ simulates thermal heating in the artificial activation by opening a channel for Mn2+ to enter.

Immobilized butyrylcholinesterase in the characterization of new inhibitors that could ease Alzheimer's disease.

Focus of this work was the development and characterization of a new immobilized enzyme reactor (IMER) containing human recombinant butyrylcholinesterase (rBChE) for the on-line kinetic characterization of specific, pseudo-irreversible and brain-targeted BChE inhibitors as potential drug candidates for Alzheimer's disease (AD). Specifically, a rBChE-IMER containing 0.99 U of covalently bound target enzyme was purposely developed and inserted into a HPLC system connected to a UV-vis detector. Selected reversible cholinesterase inhibitors, (-)-phenserine and (-)-cymserine analogues, were then kinetically characterized by rBChE-IMER, and by classical in solution assays and their carbamoylation and decarbamoylation constants were determined. The results support the elucidation of the potency, inhibition duration, mode of action and specific structure/activity relations of these agents and allow cross-validation of the two assay techniques.

[Immobilization of Candida sp. lipase on resin D301].

We immobilized Candida sp. lipase onto seven kinds of industrial adsorption and ion exchange resins. By determining the activity of each immobilized enzyme, the weakly basic anionic exchange resin of D301 showed the best results for the immobilization of Candida sp. lipase. Comparing the scanning electron micrographs of D301 with Novozym 435 (immobilized Candida antarctica lipase B from Novo Nordisk Corp.), we selected D301 as a carrier for the immobilization of Candida sp. lipase. And we pretreated the resin D301 with the bifunctional agent glutaraldehyde and crosslinked it with Candida sp. lipase. The optimal conditions for the immobilization of Candida sp. lipase were as follows: 8 mL of the amount of 5% glutaraldehyde solution, five hours of the time pretreated D301 with glutaraldehyde, 1.0 g/L the concentration of Candida sp. lipase used, pH of the phosphate buffered, 6.0 and 10 hours of time for immobilization, respectively. The activity of immobilized enzyme was over 35 U/mg and the efficiency of immobilization was around 3.5 Ul(mg x h).

Effect of surface concentration on secondary and tertiary conformational changes of lysozyme adsorbed on silica nanoparticles.

Kinetics of tertiary conformation of lysozyme adsorbed on 90 nm silica nanoparticles was inferred using tryptophan fluorescence for different surface concentrations (0.24 to 0.92 mg/m(2)), pH (4, 7 and 9), ionic strength (10 and 100 mM), 2,2,2-trifluoroethanol (TFE) (5, 15 and 30%) and Dithiothreitol (DTT) (0.5 mg/ml) concentrations. A rapid initial unfolding, followed by a much slower refolding and subsequent unfolding, were observed with the extent of unfolding being higher at lower surface concentration, higher ionic strengths, higher TFE and DTT concentrations and at pH 9. The rate of unfolding was found to be higher at lower surface concentrations, pH 4, higher ionic strengths, higher TFE and DTT concentrations. In contrast, earlier results showed that beta lactoglobulin unfolded slower and exhibited only an initial rapid and a subsequent slow unfolding phase. Circular Dichroism spectra showed that alpha helix content was lower for adsorbed lysozyme compared to bulk with a corresponding increase in beta sheet and random coil. This decrease in alpha helix was found to be more pronounced at lower surface concentrations. DTT decreased alpha helix with a corresponding increase in random coil while TFE was found to have negligible effect on secondary structure.

Effects of organic solvents on immobilized lipase in pectin microspheres.

Lipase from Brevibacillus agri 52 was found stable up to 90% diethylenglycol (DEG), glycerol (GLY), and 1,2 propanediol (1,2 PRO) at 37 degrees C for 1 h and the stability was reduced only approximately 20% after 12 h incubation, but in 40% dimethylsulfoxide (DMSO), lipase activity was stable only for 1 h. Inhibition of the biocatalysts with dimethylformamide (DMF) was detected at 20% solvent concentration. In water immiscible systems, the stability of lipase in n-hexane, n-tetradecane and n-heptane resembles the water activity, but in the presence of isobutanol, 1-hexanol, and butylbutirate, the stability was significantly reduced. Lipase 52 precipitates in the presence of 50% acetone or ethanol/water mixtures, but enzymatic activity was partially recovered by adding 20% GLY, DEG, 1,2 PRO, or DMSO to the reaction mixture. Furthermore, by increasing DEG in 70% DMF/DEG mixtures, the lipase activity was protected. Encapsulation of lipase in pectin gels cross-linked with calcium ions brings three to four times more enzymatic activity in 70% water miscible organic solvents compared to aqueous systems.

Inactivators in competition. How to deal with them ... and not!

A method is described to determine the values of the equilibrium (K) and rate (k(2)) constants for enzyme inactivations which occur according to two-step pathways involving a first non-covalent complex and a covalent, irreversibly inactivated adduct. The method rests on a competition between a reference compound [R] for which the k(2) and K values are already known and another inactivator [C]. During the experiments, the disappearance of the reference compound or the appearance of the ER(*) adduct is monitored. The analysis shows that under conditions where the k(2) and K values for the competing substrate can be determined, the measured apparent first-order rate constant for the disappearance of the reference compound is not the sum of the rate constants obtained for each inactivator in the absence of the other. The method can be used to determine the K and k(2) constants when an adequate reference compound is available, in particular, for the interactions between beta-lactam antibiotics and penicillin-binding proteins. The precautions which must be taken to avoid large errors on the estimation of the parameters of the competing inactivator are discussed. Examples found in the literature are discussed where an erroneous simplified equation has been utilised, thus yielding incorrect values for k(2) and K. Interestingly, the correct values can be calculated on the basis of the published results which do not contain the raw experimental data. But some of the values should be considered with a lot of caution since the experiments have not been performed under optimal conditions.

High temperature peroxidase activities of HRP and hemoglobin in the galleries of layered Zr(IV)phosphate.

Horseradish peroxidase, and met hemoglobin, when intercalated in the galleries of alpha-Zr(IV) phosphate, show peroxidase activities at elevated temperatures (86-90 degrees C) and the rates increased to 2-3.6 times the rates observed at room temperature.

Stabilization of activity of oxidoreductases by their immobilization onto special functionalized glass and novel aminocellulose film using different coupling reagents.

Glucose oxidase (GOD), horseradish peroxidase (HRP), and lactate oxidase (LOD) were covalently immobilized on special NH(2)-functionalized glass and on a novel NH(2)-cellulose film via 13 different coupling reagents. The properties of these immobilized enzymes, such as activity, storage stability, and thermostability, are strongly dependent on the coupling reagent. For example, GOD immobilized by cyanuric chloride on the NH(2)-cellulose film loses approximately half of its immobilized activity after 30 days of storage at 4 degrees C or after treatment at 65 degrees C for 30 min. In contrast, GOD immobilized by L-ascorbic acid onto the same NH(2)-cellulose film retains 90% of its initial activity after 1 year of storage at 4 degrees C and 92% after heat treatment at 65 degrees C for 30 min. Unlike GOD, in the case of LOD only immobilization on special NH(2)-functionalized glass, e.g., via cyanuric chloride, led to a stabilization of the enzyme activity in comparison to the native enzyme. The operational stability of immobilized HRP was up to 40 times higher than that of the native enzyme if coupling to the new NH(2)-cellulose film led to an amide or sulfonamide bond. Regarding the kinetics of the immobilized enzymes, the coupling reagent plays a minor role for the enzyme substrate affinity, which is characterized by the apparent Michaelis constant (K(M,app)). The NH(2)-functionalized support material as well as the immobilized density of the protein and/or immobilized activity has a strong influence on the K(M,app) value. In all cases, K(M,app) decreases with increasing immobilized enzyme protein density and particularly drastically for GOD.

Coupled rotation within single F0F1 enzyme complexes during ATP synthesis or hydrolysis.

F0F1 ATP synthases are the smallest rotary motors in nature and work as ATP factories in bacteria, plants and animals. Here we report on the first observation of intersubunit rotation in fully coupled single F0F1 molecules during ATP synthesis or hydrolysis. We investigate the Na+-translocating ATP synthase of Propionigenium modestum specifically labeled by a single fluorophore at one c subunit using polarization-resolved confocal microscopy. Rotation during ATP synthesis was observed with the immobilized enzyme reconstituted into proteoliposomes after applying a diffusion potential, but not with a Na+ concentration gradient alone. During ATP hydrolysis, stepwise rotation of the labeled c subunit was found in the presence of 2 mM NaCl, but not without the addition of Na+ ions. Moreover, upon the incubation with the F0-specific inhibitor dicyclohexylcarbodiimide the rotation was severely inhibited.

Efficient chemoenzymatic synthesis of (S)- and (R)-5-(1-aminoethyl)-2-(cyclohexylmethoxy)benzamide: key intermediate for Src-SH2 inhibitor.

A facile chemoenzymatic synthesis of both the S and R forms of 5-(1-aminoethyl)-2-(cyclohexylmethoxy)benzamide a key intermediate of non-peptidic Src SH2 inhibitors is described. Both the enantiomers were synthesized in high optical purity (>99% ee) by reduction followed by lipase-mediated acylation of the precursor 6 in one-pot. Immobilized Pseudomonas cepacia lipase offered high degree of enantioselectivity with spontaneity.

Peptide synthesis in organic media with subtilisin 72 immobilized on poly(vinyl alcohol)-cryogel carrier.

Serine proteinase subtilisin 72 was covalently attached to the beads of poly(vinyl alcohol)-cryogel, a macroporous hydrogel prepared by the freeze-thaw technique. The immobilized enzyme was examined as a catalyst in the synthesis of protected peptides Z-Ala-Ala-Xaa-Phe-pNA (Xaa = Leu, Glu, Lys) in acetonitrile/dimethylformamide mixtures. Immobilized subtilisin catalyzed with high yield the formation of peptide bonds between Phe-pNA and acyl donors including those with free carboxylic group and non-protected C-terminal basic and acidic amino acid residues.

Activity and stability of chemically modified Candida antarctica lipase B adsorbed on solid supports.

The effect of various covalent chemical modifications on the transesterification activity and stability of adsorbed lipase B from Candida antarctica (CALB) was studied in 2-butanone and o-xylene. CALB species modified with either polyethylene glycol 2000 monomethyl ether (MPEG), polyethylene glycol 300 mono-octyl ether (OPEG) or n-octanol (OCT) were used in combination with a hydrophobic (Accurel) and a hydrophilic (Duolite) support. The thermostabilities of adsorbed CALB in both solvents, and that of free CALB in o-xylene were not influenced by the modifications. In contrast, the thermostability of free CALB in 2-butanone decreased 2.5-fold after MPEG modification and increased 1.5-fold after modification with OPEG and n-octanol, compared to that of native CALB. The activities of the native and modified CALB species were up to 9-fold higher after adsorption onto Accurel than those of the corresponding free enzymes. Adsorption of these enzyme species onto Duolite only resulted in a 2- to 3-fold increase in the activity of OPEG- and OCT-modified CALB. The modified CALB species adsorbed onto Accurel show similar or up to 2-fold lower activities than do native adsorbed CALB species, while 1.5- to 6-fold higher activities were found for modified CALB species adsorbed onto Duolite. We propose that hydrophobic modifiers induce conformational changes of CALB during adsorption on a hydrophobic support whereas all three modifiers protect CALB from structural alterations during adsorption onto a hydrophilic support.

Brain myo-inositol monophosphatase: activity of the single subunit in a dimeric enzyme.

Reversible dissociation of the dimeric structure of brain myo-inositol monophosphatase into subunits was attained by the addition of guanidine-HCl (4M). The molecular mass of the subunits (29 KDa) was determined by HPLC chromatography. Separation of the processes of refolding and association of the monomeric species was achieved by attaching the protein subunits to a rigid matrix (Affi-gel 15). The matrix-bound monomer is determined to be catalytically active, indicating that monomeric subunit of the phosphate is capable of conducting independent catalysis.

Immobilized rhodanese: some aspects of anion inhibition kinetics and modulation by cations.

The rat liver rhodanese (thiosulphate: cyanide sulfurtransferase EC 2.6.1.1) has been immobilized on polyacrylamide gels. The immobilized enzyme had a pH optimum of 7.4 and Km values of 3.25 mM and 1.12 mM for S2O3(2-) and KCN, respectively. The enzyme was competitively inhibited by NaNO2 and CH3COONa and noncompetitively by amyl-nitrite. A modulation of activity was observed in the presence of Ca2+, Zn2+, and Cu2+. The results are discussed in line with the detoxicating function of liver rhodanese.

Permeation of 6-nitro-3-phenylacetamide benzoic acid (NIPAB) and hydrolysis by penicillin acylase immobilized in emulsion liquid membranes.

The effects of various commercial and model surfactants of different structure and hydrophilicity were studied on water-in-oil (w/o) emulsion stability, potassium cation leakage and permeation of 6-nitro-3-phenylacetamide benzoic acid in a model system using Penicillin acylase (EC 3.5.1.11) immobilized in a liquid membrane. Both emulsion stability, potassium leakage and permeation of organic substances depend upon hydrophilicity of surfactants. Hydrophilic surfactants may be used to stabilize emulsions only in mixtures with hydrophobic emulsifiers. Additions of small quantities of hydrophilic surfactants to the system in which permeation occurs together within an enzymatic process may be advantageous. Both the rate of permeation and potassium transfer significantly increase when hydrophilic surfactants are present. There was no relationship observed between potassium cation transfer from the internal phase and emulsion stability in the storage test.