Free Enzyme In Solution Research Articles

Studies on the formation of bioconjugates of Endoglucanase with colloidal gold

We report herein the formation of Endoglucanase–colloidal gold bioconjugates under enzyme friendly conditions. The bioconjugates were formed by simple mixing of the enzyme molecules with colloidal gold at pH 5, centrifugation and subsequent redispersion in appropriate buffer solutions. The bioconjugates in solution were characterized by UV–vis, fluorescence spectroscopy and biocatalytic activity measurements. The films of the bioconjugate material obtained by solvent evaporation on suitable substrates were further analysed by transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy. TEM analysis showed the gold particles to be in a close-packed structure with well-defined separations indicating effective conjugation with the enzyme molecules. Fluorescence spectroscopy and biological activity measurements of the bioconjugate clearly showed the intactness of the tertiary structure of the enzyme. The bioconjugate material exhibited an increased half-life of the enzyme at 60 °C and pH 9 relative to that of the free enzyme in solution, thus highlighting the role of colloidal gold particles in stabilizing the enzyme molecules.

Radiation sterilization of enzyme hybrids with biodegradable polymers

Ionizing radiations, which have already been utilized for the sterilization of medical supplies as well as gas fumigation, should be the final candidate to decontaminate “hybrid” biomaterials containing bio-active materials including enzymes because irradiation induces neither heat nor substances affecting the quality of the materials and our health. In order to check the feasibility of 60Co-gamma rays on these materials, we selected commercial proteases including papain and bromelain hybridized with commercial activated chitosan beads and demonstrated that these enzyme-hybrids suspended in water showed the significant radiation durability of more than twice as much as free enzyme solution at 25-kGy irradiation. Enhanced thermal and storage stability of the enzyme hybrids were not affected by the same dose level of irradiation, either, indicating that commercial irradiation sterilization method is applicable to enzyme hybrids without modification.

On the preparation, characterization, and enzymatic activity of fungal protease-gold colloid bioconjugates.

We present herein details pertaining to the preparation of bioconjugates of colloidal gold with aspartic protease from the fungus Aspergillus saitoi (F-prot) and their characterization and enzymatic activity. Simple mixing of the colloidal gold and protein solutions under protein-friendly conditions (pH = 3) followed by centrifugation (to remove uncomplexed gold nanoparticles and protein molecules) results in the formation of the fungal protease-gold nanoparticle conjugates. The protein-gold nanoparticle bioconjugate was redispersed in buffer solution and indicated the formation of efficient bioconjugates with intact native protein structures. The bioconjugates in solution were characterized by UV-vis spectroscopy, fluorescence spectroscopy, and biocatalytic activity measurements while drop-dried bioconjugate films on Si (111) substrates were characterized by scanning electron microscopy (SEM), energy dispersive analysis of X-rays (EDAX), and X-ray diffraction (XRD) measurements. Microscopy images do show some aggregate formation, but the intactness of the native structure of the enzyme in the bioconjugate material was verified by fluorescence and biocatalytic activity measurements. The enzyme retains substantial biocatalytic activity in the bioconjugate material and was comparable to that of free enzyme in solution.

Enhanced Temperature and pH Stability of Fatty Amine−Endoglucanase Composites: Fabrication, Substrate Protection, and Biological Activity

Endoglucanase, one part of the multicomponent enzyme cellulase, is an extremely important enzyme in the textile industry (biopolishing), the pulp and paper industry (de-inking operations and recycling of used paper), and the detergent industry. One major problem in such applications is the inactivation of the enzyme at elevated temperatures and pH conditions. We present herein details of the encapsulation of this enzyme in thermally evaporated fatty amine films and studies of the enzymatic activity of the biocomposite film under different pH and temperature conditions. Substrate protection of the enzyme by carboxymethyl cellulose was essential to stabilize the enzyme against inactivation in the lipid matrix. The optimum operation temperature shifted to higher values relative to that of the free enzyme in solution. The enhanced stability at high temperatures coupled with significant catalytic activity of the enzyme−lipid biocomposite films at elevated pH conditions indicate immediate application of the biocomposite film in high temperature−high pH industrial applications.

Pepsin−Gold Colloid Conjugates: Preparation, Characterization, and Enzymatic Activity

Pepsin−colloidal gold conjugates were prepared by a simple protein-friendly process and the enzymatic activity of the bioconjugates is reported. The pepsin−gold conjugates are obtained by mixing colloidal gold and protein solutions at pH = 3 and, thereafter, centrifugation, washing, and redispersion of the pepsin−gold conjugate material in water. The bioconjugates in solution were characterized by UV−vis spectroscopy, fluorescence spectroscopy, and biocatalytic activity measurements while films of the bioconjugate material obtained by solvent evaporation on suitable substrates were further analyzed by scanning electron microscopy (SEM), energy dispersive analysis of X-rays (EDAX), transmission electron spectroscopy (TEM), Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). While TEM and SEM measurements showed aggregates of the enzyme/colloidal gold conjugates, the intactness of secondary and tertiary structures of the enzyme, as determined by FTIR and fluorescence spectroscopies and confirmed by biocatalytic activity measurements, clearly indicates that the enzyme is stable in its natural state and is possibly stabilized by the colloidal gold particles. The enzyme in the pepsin−Au bioconjugate retained substantial biocatalytic activity and was more stable than the free enzyme in solution.

Electrochemical reduction of oxygen on glassy carbon: catalysis by catalase

Catalase was investigated as a possible catalyst of the electrochemical reduction of oxygen on glassy carbon electrodes. Four different electrode ∣ enzyme interfaces were tested: free enzyme in solution, enzyme immobilised on the electrode surface with bovine serum albumin and glutaraldehyde, or with Nafion®, and enzyme adsorbed from solution in dimethylsulfoxide. The experimental data were analysed with a theoretical model. The model was based on two successive two-electron electrochemical reactions: reduction of oxygen to hydrogen peroxide and reduction of hydrogen peroxide to water, which were coupled to the diffusion of oxygen and hydrogen peroxide, and to the catalase-catalysed disproportionation of peroxide in solution. In most cases, the presence of catalase in solution or on the electrode surface provoked only a moderate current increase. The model demonstrated that disproportionation of hydrogen peroxide in solution fully explained this current increase. On the contrary, when catalase was adsorbed from dimethylsulfoxide on the surface of electrodes that did not undergo any electrochemical pre-treatment, a high current increase was obtained. In this case, catalase efficiently catalysed oxygen reduction via direct electron transfer with the electrode. The effects of the electrochemical pre-treatment on oxygen reduction and on the occurrence of catalase-catalysed direct electron transfer were discussed.

Amperometric detection of histamine with a methylamine dehydrogenase polypyrrole-based sensor.

Methylamine dehydrogenase (MADH) has been immobilized in a polpyrrole (PPy) film on an electrode surface and used as an amine sensor for the determination of primary amines. Its response to histamine has been characterized in detail. The PPy film containing MADH was formed electrochemically on a gold minielectrode (1-mm diameter) in the presence of ferricyanide. The film was then coated with Nafion. This enzyme electrode did not require any additional cofactors and was not sensitive to oxygen. It exhibited a maximum response current to histamine at applied potentials of 0.24-0.33 V and at pH 7.5-8.5. This MADH-PPy sensor exhibited a response time of less than 3 s. The immobilized MADH on the electrode exhibited Michaelis-Menten behavior similar to that of the free enzyme in solution with a Km value of 1.3 mM. This sensor could be used to reliably detect histamine over a concentration range from approximately 25 microM to 4 mM. This is the first example of a biosensor that uses an immobilized enzyme that possesses the tryptophan tryptophylquinone prosthetic group.

Measurement of Catalytic Reaction Kinetics for Adsorbed Enzyme Monolayers

We present a new assay based on total internal reflection fluorescence (TIRF) to quantify the catalytic activity of adsorbed enzyme monolayers on macroscopically flat surfaces. The need for such an assay derives from a general shortage of assay methods that are sufficiently sensitive to measure reaction kinetics for just a single monolayer of enzymes. The assay is based on the enzymatic conversion of a soluble, nonfluorescent fluorogenic substrate reagent to a soluble, highly fluorescent product. The reaction occurs at the solid–liquid interface where the enzymes are adsorbed. Fluorogenic substrates are introduced to the adsorbed layer by convective diffusion from solutions undergoing steady laminar slit flow. The exponentially decaying evanescent wave that is produced by total internal reflection serves as a “spectroscopic ruler” to resolve the spatial concentration profile of fluorescent products in solution near the interface. By measuring the steady-state fluorescence signal as a function of the Peclet number that characterizes mass transfer conditions in the experiment, it is possible to determine the enzymatic reaction rate. Here we present the development of the method and its application to a test system of β-galactosidase adsorbed to methylated silica surfaces. Compared to the enzymatic rate constants for this enzyme in free solution, adsorption decreased the Michaelis–Menten rate constantkcatby a factor of 10 and increased the equilibrium binding constantKmby a factor of 4.5. Thus the intrinsic activity of the enzyme, as represented by the ratiokcat/Km, decreased 45-fold due to adsorption.

1H, 13C, and 15N resonance assignments of Fusarium solani pisi cutinase and preliminary features of the structure in solution.

Essentially complete (96%) sequence-specific assignments were made for the backbone and side-chain 1H, 13C, and 15N resonances of Fusarium solani pisi cutinase, produced as a 214-residue heterologous protein in Escherichia coli, using heteronuclear NMR techniques. Three structural features were noticed during the assignment. (1) The secondary structure in solution corresponds mostly with the structure from X-ray diffraction, suggesting that both structures are globally similar. (2) The HN of Ala32 has a strongly upfield-shifted resonance at 3.97 ppm, indicative of an amide-aromatic hydrogen bond to the indole ring of Trp69 that stabilizes the N-terminal side of the parallel beta-sheet. (3) The NMR data suggest that the residues constituting the oxyanion hole are quite mobile in the free enzyme in solution, in contrast to the existence of a preformed oxyanion hole as observed in the crystal structure. Apparently, cutinase forms its oxyanion hole upon binding of the substrate like true lipases.

Binding of tissue plasminogen activator to endothelial cells: The effect on functional properties. Localization of a ligand in the B-chain of tPA

The binding of 125I-labelled tissue plasminogen activator (tPA), the tPA A- or B-chain to endothelial cells (EC) were studied in suspensions of cultured human umbilical vein EC (HUVEC) or immortalized microvascular EC (HMEC). By determinations of the concentration-dependent binding it was shown that both the A-chain and the B-chain, which were isolated after partial reduction of two-chain tPA, contain ligands for binding to EC. The affinity for the B-chain was much higher than for the A-chain according to Scatchard analysis (Kd 24 and 515 nM, respectively), whereas the number of binding sites was higher for the A-chain than for the B-chain (Bmax 8×10 5 and 1.2×10 5, respectively). There were no cross interactions between the A- and B-chains and their binding sites. The binding of tPA to EC induced an almost 100-fold increase of the activation rate when compared to the same amount of enzyme in free solution, which in contrast to the fibrin-induced stimulation was not inhibited by antibodies against fibrin. The enzymatic activity of the B-chain was much less affected by the association to the cells. Both tPA and the tPA B-chain were largely protected against inhibition by an excess plasminogen activator type-1 (PAI-1) when bound to EC, whereas the same amount of free tPA was totally inactivated. The competition studies strongly indicated that an N-terminal segment in the B-chain, AKHRRSPGER, may be the ligand part of the B-chain. It is interesting to note that this polypeptide segment also participates in a binding site for PAI-1, necessary for effective inhibition. This implies a possible competition between PAI-1 and a tPA-receptor for binding of tPA. High molecular weight urokinase had no quenching effect on the binding of the B-chain to EC.

Biofunctional membranes Part IV. Active-site structure and stability of an immobilized enzyme, papain, on modified polysulfone membranes studied by electron paramagnetic resonance and kinetics

Biofunctional membranes are entities in which biological molecules (or cells) are attached to polymeric supports cast in the form of porous membranes. Such membranes are gaining increased importance in applications of enzymatic catalysis or synthesis (bioreactors), separations (affinity membranes), and chemical analysis (biosensors). However, fundamental studies of the active site of immobilized biomolecules have been rare. In this study, electron paramagnetic resonance (EPR) spin-labeling techniques using a short, active-site specific spin label have been employed to study the properties of a model enzyme, papain, immobilized on a fully-hydrated, modified polysulfone membrane. The EPR properties of the immobilized enzyme and reaction rate results using the amidase activity of papain and N-benzoyl- dl-arginine-4-nitroanilide hydrochloride as substrate are compared with that of the free enzyme in solution. The major findings in this study are: (1) Immobilization does change the active-site conformation of papain. The spin label at the active site of the immobilized papain has slower motion than that of the free papain in solution. (2) There are two major subpopulations of immobilized enzyme on modified polysulfone membranes: subpopulation A has faster spin-label motion at the active site than subpopulation D, suggesting that the enzyme in subpopulation A may have a more open active-site cleft than that of the subpopulation D. (3) The active-site conformation of subpopulation D of the immobilized papain is insensitive to the pH of the bulk solution, while that of subpopulation A has a similar response to pH changes as that of free papain in solution, suggesting that subpopulation A may be the active form of the immobilized enzyme while subpopulation D is the denatured form. (4) The pH-dependent curve of the amidase activity of the immobilized papain has a similar bell shape as that of the free papain in solution. (5) Subpopulation A is converted into subpopulation D upon addition of denaturants (urea or guanidine hydrochloride), which further confirms the assumption that the former subpopulation is the active form of the immobilized enzyme and the latter subpopulation is the denatured form. Moreover, denaturation at elevated temperature can also convert subpopulation A into subpopulation D. (6) By paramagnetic relaxation mechanisms, which require close proximity of the paramagnetic species and the spin label, K 3Fe(CN) 6 can broaden the EPR signal of spin label bound to subpopulation A but not that of subpopulation D, suggesting that subpopulation A is accessible to the substrate. More direct evidence that subpopulation A is the active form of the immobilized enzyme and more accessible to substrate than subpopulation D was obtained by immobilizing papain on modified polysulfone membranes first followed by spin labeling. In this way, only the active subpopulation A was labeled by the spin label. (7) The K m(app) values of the immobilized papain are larger than that of papain in solution, while V max(app) is smaller. (8) The enzyme is highly reusable and has very high storage stability after immobilization. (9) Papain immobilized on polysulfone membranes has much higher thermal stability and stability toward denaturants (urea or guanidine hydrochloride) than that of free papain in solution. In this study the polymeric membrane provided three functions: sites for covalent coupling of enzymes; enhanced enzyme stability; and substrate partitioning. This paper reports the first development of an effective method to acquire EPR spectra of fully-hydrated, spin-labeled enzymes immobilized on polymeric membranes. All these findings indicate that the EPR spin-labeling technique shows great promise as a powerful method for studying membrane-immobilized enzyme systems.

Modeling of amperometric biosensors by a finite-volume method

A numerical model has been developed to analyze the transient response of amperometric enzyme biosensors. These sensors were composed of an inert metallic electrode covered by an enzyme layer which was either a free enzyme solution, confined by a semipermeable membrane, or an immobilized enzyme linked to one of the sides of a polymeric membrane placed against the electrode. The cases where the enzyme was linked to the outside and inside of the membrane are presented. The finite-volume method was used to solve the partial derivative equations coupling the diffusive mass transfer to enzymatic kinetics. This method makes it possible to adapt the discretization space grid easily to the geometry of the integration domain. The performance of the method is discussed by comparing it with the steady state analytical response and with the implicit finite-difference method.

Enzymatic Kinetic Change of Ascorbate Oxidase Loaded into Liposomes Induced by Microwave Fields Exposure

AbstractEnzymatic kinetic of enzyme Ascorbate Oxidase (AAO) loaded into liposomes has been studied during microwave (MW) fields irradiation at temperature of 25°C. DPPC:Chol (7:3 mole ratio) unilamellar vesicles of an average diameter of 110 nm were used. At the working frequency of 2.45 GHz, MW exposure at 2.8 mW/g and 5.6 mW/g Specific Absorption Rate (SAR) were investigated. At both SARs above cited, the free enzyme in solution did not show any effects induced by MW exposure. On the other hand, the enzyme loaded into liposomes exhibits a significant decrease of 13% (p<0.005, n=42) in the reaction velocity induced by MW exposure at 5.6 mW/g in comparison to control. At SAR of 2.8 mW/g no significant decrease was obtained.

Poly(Methyl Methacrylate) as a matrix for immobilization of lipase

Poly(methyl methacrylate) (PMMA) was found to be suitable for the immobilization of lipase fromCandida rugosa. The best result based on hydrolytic activity was obtained by adsorption of the purified unbuffered enzyme solution onto PMMA beads without any modification of the beads. Prolonged exposure of the protein to the beads increased its adsorption but the expressed activity decreased after 1 h of exposure. The magnitude of the immobilized activity also varied with the size of the beads. Immobilization of the lipase shifted its optimal reaction temperature from 37 to 45°C. The immobilized enzyme is also more stable than the free enzyme in solution. The operational half-life of the immobilized lipase packed in a column and assayed in a closed system is 40 d.

Thermal, operational, and storage stability of immobilized carbonic anhydrase in membrane lungs.

The significant improvement in CO2 removal rates from blood through membrane lungs with EMCO and ECCO2R is achievable by means of the immobilization of carbonic anhydrase (CA) onto the membrane surface. The practical application of this technology requires that the enzyme be maintained for long periods without loss of activity. Thus, studies were performed to evaluate the thermal, operational, and storage stability of CA in a cellulose nitrate-encapsulated, silicone rubber membrane-immobilized form. Cellulose nitrate microcapsules containing 1000 micrograms/ml CA were prepared using a modified version of the method of Chang, and immobilized onto a 0.06 m2 section of commercial silicone rubber membrane material. The extent of enzyme activity in free solution and in the encapsulated form was determined after long-term storage at 4 degrees C, 37 degrees C, and 50 degrees C. Likewise, in an in vitro test circuit, CO2 removal efficiency in both CA treated and untreated membrane lungs was measured for extended time periods of 10 hr over a 10 day period. The thermal stability tests showed a significantly greater degree of retained enzyme activity in the encapsulated form, over the free enzyme in solution, at all temperatures. This was especially evident at higher temperatures and when the enzyme was stored for extended periods. In the operational stability tests, the CO2 removal efficiency of the treated membrane was not degraded, and stayed significantly higher than the untreated membrane for extended time periods. This further illustrates the potential for the use of the immobilized enzyme, carbonic anhydrase, for improved CO2 removal efficiency.

Tetrameric structure of mitochondrially bound rat brain hexokinase: A crosslinking study

Rat brain hexokinase (ATP: d-hexose-6-phosphotransferase; EC 2.7.1.1) was derivatized with sulfosuccinimidyl-2-( m-azido- o-nitrobenzamido) ethyl-1,3′-dithiopropionate (SAND), a photosensitive and cleavable crosslinking agent. The catalytic activity and mitochondrial binding properties of the enzyme were only marginally affected by reaction with SAND. When the derivatized enzyme was bound to liver mitochondria, photolysis resulted in extensive formation of a single crosslinked species with estimated molecular mass 460 kDa. This was determined to contain only hexokinase and thus represents a tetramer of the 116 kDa (apparent molecular mass in gel system used) monomeric enzyme. Although small amounts of tetramer were detected after photolysis of relatively high concentrations of derivatized enzyme in free solution, tetramer formation was greatly enhanced when the enzyme was bound to mitochondria. No evidence of dimeric or trimeric structures was seen even when only a small fraction of the available binding sites on the mitochondrial membrane were occupied. It is thus concluded that tetramer formation is closely linked with binding of the enzyme to the outer mitochondrial membrane and, more specifically, to the pore structure through which metabolites traverse this membrane. It is speculated that a tetrameric structure surrounding the mitochondrial pores may facilitate interactions between the hexokinase reaction and oxidative phosphorylation, mediated by the adenine nucleotides which are common intermediates in these reactions.

A composite membrane bioreactor: Part II. Possible theoretical model

Although we have shown in a previous paper (L. D'Ilario and M.M. Steffan, J. Mol. Catal., 47 (1988) 7) that the entrapment of enzyme molecules between cellulose membranes did not cause variations in the working of the biocatalyst, the total efficiency of the composite membrane bioreactor was clearly dependent upon the membrane porosity through the increase of the ‘apparent’ Michaelis-Menten constant, compared with that of the free enzyme in solution. In the present paper an attempt is made to work out a theoretical model of the bioreactor which, based on the diffusional characteristics of the system and of the kinetic properties of the enzyme, would allow prediction of the efficiency of the system. A model is proposed, based on the thermodynamics of the irreversible processes. The experimental data on the biotransformation carried out in our ‘composite membrane’ reactor are compared with the theoretical ones suggested in the present paper.

Reversibility and competition in the adsorption of Trichoderma reesei cellulase components

Adsorption reversibility and competition between fractionated components of the Trichoderma reesei cellulase system were studied. Specific endoglucanase (EGI), nonspecific endoglucanases (EGII, EGIII), and cellobio-hydrolase (CBHI) were previously grouped according to their hydrolytic function. At 5 degrees C, direct evidence of exchange between adsorbed and free enzyme was obtained for each component using [(3)H] and [(14)C] radiolabeled tracers. No release of bound enzymes was detected upon dilution of the free enzyme solution. In simultaneous adsorption of enzyme pairs, CBHI was shown to predominate adsorption. Endoglucanase EGI was preferentially adsorbed over EGII and EGIII. Sequential adsorption studies have shown that interaction between enzyme components largely determines the degree of their adsorption. Evidence suggests that both common and distinct adsorption sites exist and that their occupation depends on which components are involved. Predominance in adsorption by any one of the enzyme components is decreased at 50 degrees C. Light microscopy and monitoring of sugar production during cellulose hydrolysis provided evidence that reduction in the ionic strength decreases the adsorption predominance of CBHI and enhances the synergism between the cellulase components.

Flow kinetics of immobilized beta-glucosidase.

The enzyme beta-glucosidase was attached covalently to the inner surface of nylon tubing. Flow kinetic studies were carried out at a range of temperatures, pH values, flow rates, and substrate concentrations. Various tests showed that the extent of diffusion control was negligible. At 25 degrees C the Michaelis constant was 33.4 mM, not greatly different from the value for the enzyme in free solution. The pH dependence was similar to that for the free enzyme. The Arrhenius plots showed inflexions at about 22 degrees C, as with the free enzyme, the changes in slope being small at the pH optimum of about 5.9 and becoming much more pronounced as the pH is increased or decreased. The immobilized enzyme is more stable than the free enzyme, both on storage at low and higher temperatures, and its reuse stability is greater.

A study on heat-resistance of microencapsulated glucose oxidase.

Polyurea microcapsules containing glucose oxidase were prepared and their thermodurability was examined. Microencapsulated glucose oxidase was found to be more stable to heat than the enzyme in free solution. This stability was enhanced with an increase in the amount of enzyme entrapped in the microcapsules.