Activity Of Immobilized Enzyme Research Articles

Immobilization of α‐chymotrypsin on poly(methyl methacrylate‐co‐acrylic acid) core‐shell latex

AbstractCore‐shell latex of poly(methyl methacrylate‐co‐acrylic acid) with surface containing functional carboxylic acids, was prepared by seed polymerization and characterized. α‐Chymotrypsin was immobilized on the surface of these latex particles by chemical bonding in aqueous medium, using a water soluble carbodiimide as coupling agent. The latex particles size before and after the attachment of the enzyme was determined using photon correlation spectroscopy. The activities of free and immobilized enzyme were studied as a function of temperature, reusability and pH. The activity for the immobilized enzyme was highest at a pH slightly higher than the optimum pH of free enzyme, which could be explained due to the polyanionic matrix as well as due to the variation in the size of latex at higher pH, which provided more mobility to the enzyme, and thus a higher activity.

Tyrosine hydroxylase activity of immobilized tyrosinase on enzacryl‐AA and CPG‐AA supports: Stabilization and properties

Frog epidermis tyrosinase has been immobilized on Enzacryl-AA (a polyacrylamide-based support) and CPG(zirclad)-Arylamine (a controlled pore glass support) in order to stabilize the tyrosine hydroxylase activity of the enzyme; in this way, the immobilized enzyme could be used to synthesize L-dopa from L-tyrosine. The activity immobilization yield Y(IME) (act) (higher than 86%), coupling efficiency (up to 90%), storage stability (no loss in 120 days), and reaction stability (t(1/2) was higher than 20 h in column reactors) were measured for tyrosinase after its immobilization. The results showed a noticeable improvement (in immobilization yield, coupling efficiency, and storage and operational stabilities) over previous reports in which tyrosinase was immobilized for L-dopa production. The activity and stability of immobilized enzyme preparations working in three different reactor types have been compared when used in equivalent conditions with respect to a new proposed parameter of the reactor (R(p)), which allows different reactor configurations to be related to the productivity of the reactor during its useful life time. The characteristic reaction inactivation which soluble tyrosinase shows after a short reaction time has been avoided by immobilization, and the stabilization was enhanced by the presence of ascorbate. However, another inactivation process appeared after a prolonged use of the immobilized enzyme. The effects of reactor type and operating conditions on immobilized enzyme activity and stability are discussed.

Trypsin-like enzyme from Streptomyces 771. Purification and properties of native and immobilized enzyme.

Electrophoretically homogeneous proteolytic enzyme with molecular weight 31,500 and pI 3.75 was obtained from a culture medium of Streptomyces 771 by chromatography on N-benzyl chitin adsorbent, subsequent chromatography on CM-cellulose, and preparative isofocusing and chromatography on Sephadex G-75. The enzyme hydrolyzes N-benzoyl-DL-arginine-p-nitroanilide N-benzoyl-DL-lysine-p-nitro-anilide N-benzoyl-DL-arginine ethyl ester, and Na-caseinate. It also exhibits pronounced thrombolytic activity. The activity of the enzyme was suppressed by soya bean inhibitor, but remained unaffected by chelating agents and phenylmethylsulfonyl fluoride. The enzyme was immobilized on aldehyde dextran, and some kinetic parameters of the immobilized enzyme were determined. The thrombolytic activity of native and immobilized enzyme was studied as well.

Renaturation studies of free and immobilized d-amino-acid oxidase

The renaturation of free and Sepharose-immobilized d-amino-acid oxidase ( d-amino-acid:oxygen oxidoreductase (deaminating), EC 1,4,3,3), after its denaturation with 6 M guanidine hydrochloride, was investigated. No reactivation, or extremely limited reactivation (⩽ 4%), was obtained with the free enzyme, in spite of various attempts including the use of dialysis or buffers containing cofactors, different types of anions, surfactants and low concentrations of denaturing agents. The main obstacle to renaturation appeared to be the interaction among denatured or partially renatured monomers giving rise to inactive aggregates. In contrast, using the immobilized enzyme approach, substantial renaturation (up to 50%) of d-amino-acid oxidase was achieved. The denaturation-renaturation process was followed by monitoring the catalytic activity as well as the intrinsic protein fluorescence. An inverse correlation was found to exist between the degree of matrix activation by CNBr and the yield of enzyme reactivation. The anions of the lyotropic series markedly influenced the reactivation, showing an effectiveness opposite to their salting-out potential (thiocyanate ⋍ iodide > chloride > phosphate ⋍ sulphate ⋍ citrate). Instead, the anions considerably increased the activity and stability of free and immobilized enzyme, according to their salting-out potential. Immobilized monomers of d-amino-acid oxidase, which in solution undergoes self-association, showed poor capacity to interact with the free enzyme: thus they appear unsuitable for analytical and preparative purposes.

Structure‐function relationships in immobilized chymotrypsin catalysis

Specific activities and the amounts of active immobilized enzyme were determined for several different preparations of alpha-chymotrypsin immobilized on CNBr-activated Sepharose 4B. Electron paramagnetic resonance (EPR) spectroscopy of free and immobilized enzyme with a spin label coupled to the active site was used to probe the effects of different immobilization conditions on the immobilized enzyme active site configuration. Specific activity of active enzyme decreased and rotational correlation time of the spin label increased with increasing immobilized enzyme loading. Enzyme immobilized using an intermediate six-carbon spacer arm exhibited greater specific activity and spin label mobility than directly coupled enzyme. The observed activity changes due to immobilization were completely consistent with corresponding active site structure alterations revealed by EPR spectroscopy.

Amperometric Sensor and Immobilized Enzyme Electrode for the Determination of Enzymatic Activities

Abstract A sensitive method for the rapid determination of activities of soluble or immobilized enzymes, based on the electrochemical detection of hydrogen peroxide is described. Kinetic studies (Vmax and KM determinations) can be performed for all H2O2 generating enzymes (i.e. most of the oxidases) using an amperometric probe with a platinum anode at a fixed potential. When associated with an immobilized glucose oxidase membrane, this sensor constitutes a glucose electrode and the activity of any hydrolase which releases glucose can be measured. There is no need for other auxiliary enzymes and no preincubation step is required. The possibility to carry out continuous analysis constitutes the main advantage of the described method.

Activation of nylon by alkaline glutaraldehyde solution for enzyme immobilisation

Nylon tube was directly activated by alkaline glutaraldehyde solution. PEI was utilised as a spacer molecule. Glucose oxidase was immobilised to the nylon tube after reactivating the spacer molecules with glutaraldehyde. On immobilising glucose oxidase there was more protein binding and higher immobilised enzyme activity when compared to immobilised enzyme tube activated by triethyloxonium salt. The optimal condition for direct glutaraldehyde activation of nylon was incubation with 18.5% (w/v) glutaraldehyde in 0.12M borate pH 9.0 for 15 min at 90 °.

Investigation of the binding mechanism of glucoamylase to alkylamine derivatives of titanium(IV)-activated porous inorganic supports

Glucoamylase (exo-1,4-α- d-glucosidase, EC 3.2.1.3) has been coupled to several porous silica matrices by a new covalent process using alkylamine derivatives of titanium(IV)-activated supports. In order to investigate the interaction of the titanium element with the silanol groups of the inorganic matrices, activation was performed at different times, using titanium(IV) chloride, either pure or as a 15% w/v solution, in 15% w/v hydrochloric acid at 25, 45 and 80°C, followed by washing with sodium acetate buffer (0.02 m, pH 4.5) or chloroform. Using pure TiCl 4, the highest activities of all preparations were obtained at 80°C and with acetate buffer washing, resulting from a higher content of titanium coating of the carrier. When activation was performed in aqueous TiCl 4 solution, followed by a drying step, the highest activity was obtained with preparations washed with chloroform, with or without amination. When reacting pure TiCl 4 with controlled pore glass (CPG) and with porous silica (Spherosil), colour formation was observed after reaction of glutaraldehyde with the aminated support. This did not happen when Celite was used as the support. As a criterion for comparison of the different immobilized enzyme preparations, the concept of an ‘instability factor’, which measures the percentage of immobilized enzyme activity due to release of enzyme into solution, is introduced. Instability factors of immobilized enzyme preparations on Celite were always higher than those obtained with the other matrices, confirming that there was no covalent coupling of the enzyme to Celite. However, when the activation was performed with aqueous TiCl 4 solution with drying, Schiff's base formation was observed in all preparations and very stable immobilized enzyme preparations were obtained. The results of the activation of controlled pore glass and porous silica with pure titanium(IV) chloride suggest the existence of a true reaction between the titanium element and the silanol groups of these carriers by formation of a bridge, Si-O-Ti, while with the titanium(IV) chloride solution in hydrochloric acid, a coating of hydrous titanium(IV) oxide is obtained.

The activity of immobilized enzymes on different krill chitin preparations

AbstractThe suitability of krill chitin, prepared by using different concentrations of KOH and HCI for deproteinization and demineralization, respectively, was investigated. The activity of enzymes immobilized on such supports depends on the degree of deproteinization of chitin, availability of amino groups, content of minerals, mesh size, structure of the surface, and conformation of the chitin molecules. It was found that invertase and amyloglucosidase achieved high activity after immobilization on chitin obtained at not too rigorous conditions of deproteinization. However, the activity of immobilized α‐amylase and diastase increased significantly with the increase in concentration of KOH used for deproteinization. High content of minerals and proteins in chitin preparation causes a loss of immobilized enzyme activity.

Immobilized Glucose Isomerase on DEAE Cellulose Beads

AbstractPurified glucose isomerase from Actinoplanes missouriensis was immobilized on porous DEAE‐cellulose beads by simple adsorption. The immobilized glucose isomerase retained over 70% of its original activity. The hinderance of immobilized enzyme activity due to pore diffusion and film diffusion was insignificant with the bead size at 35 mesh or smaller. The relative substrate flow rate can be kept at 0.04 cm/s or higher. The optimum pH of the imobilized enzyme did not change, however, the optimum pH range became broader. The broader pH profile indicated that immobilized enzymes are less sensitive to pH change in the substrate. The half life of the immobilized enzyme was at around 1,000 h at 60°C. Cobalt ions are not required for enzyme stability. The cost of using immobilized enzyme on DEAE cellulose beads should be less be than that of the whole cell immobilization system due primarily to the fact that DEAE‐cellulose beads are reusable for immobilization as well as for enzyme purification.

Immobilization of nucleoside diphosphatase at its allosteric site using immobilized derivatives of pyridoxal 5′-phosphate

3- O-Immobilized and 6-immobilized pyridoxal 5′-phosphate analogs of Sepharose were bound to the allosteric site of nucleoside diphosphatase with very high affinity. Active immobilized nucleoside diphosphatase was prepared by reduction of the Schiff base linkage between the enzyme and pyridoxal 5′-phosphate bound to Sepharose with NaBH 4. 3- O-Immobilized pyridoxal 5′-phosphate analog gave more active immobilized enzyme than the 6-analog; the immobilized enzyme on the 3- O-immobilized pyridoxal 5′-phosphate analog showed about 90% of activity of free enzyme. The immobilized enzyme thus prepared was less sensitive to ATP, an allosteric effector, and showed a higher heat stability than the free enzyme. When an assay mixture containing inosine diphosphate and MgCl 2 was passed through a column of the immobilized enzyme at 37 °C, inosine diphosphate liberated inorganic phosphate almost quantitatively. Properties of the immobilized enzyme on the pyridoxal 5′-phosphate analog were compared with those of the immobilized enzyme on CNBr-activated Sepharose.

A histochemical model dealing with an immobilized glucose oxidase-peroxidase system. The influence of diffusion limitations on histochemical results.

A histochemical model dealing with an immobilized bienzyme system (glucose oxidase-peroxidase) is presented. The model is an artificial proteic membrane obtained by a previously described co-cross-linking process. The kinetic properties of free and immobilized horseradish peroxidase were studied when 3,3'-diaminobenzidine is used as a hydrogen donor substrate. A new direct method was developed for immobilized enzyme activity measurements. Computer simulation based on experimental kinetic parameters was performed in order to discuss electron microscopy results. By changing diffusion limitations, various profiles of insoluble product were visualized inside the proteic film and no geometrical similarity was seen between enzyme distributions and insoluble osmiophilic product patterns.

A study of the beer chill-proofing behaviour of a water-insoluble papain conjugate of hydrous titanium(IV) oxide-use of hydrous titanium(IV) oxide as a novel chill-proofing agent

An active papain (EC 3.4.22.2) conjugate of hydrous titanium(IV) oxide has been found to exhibit substantial ability to chill-proof beer. However, this ability has been shown to be nearly identical to the chill-proofing abilities found to be exhibited by an inactive S-carboxymethyl derivative of the papain conjugate, which was prepared by treating the papain conjugate with bromoacetic acid, and by free hydrous titanium(IV) oxide. Further, the chill-proofing achieved by each material was observed to correlate with reductions in the absorbance of beer in both the ultraviolet and visible regions of the spectrum. On this basis, it is concluded that chill-proofing by these materials occurs solely by the adsorption of beer constituents and that this adsorption is probably non-specific. The rider to this conclusion is that the coupling of papain to hydrous titanium(IV) oxide, in order to give an active immobilized enzyme that acts as a reusable chill-proofing agent, is without efficacy. The broader significance of these observations in the development of enzymic chill-proofing agents is discussed. The potential of free hydrous titanium(IV) oxide as an adsorbent chill-proofing agent has been examined. It has been shown that the treatment of beer for 12 h at 4°C with fresh hydrous titanium(IV) oxide (200 g/hl) produces nearly complete chill-proofing. Chill-proofing is enhanced both by prolongation of treatment and by increased number of treatments. Further, beer that had been chill-proofed by hydrous titanium(IV) oxide was found to contain titanium(IV) at a concentration below the detection limit (2 p.p.m.) of the adopted colorimetric method of analysis. These results auger well for the safe commercial use of hydrous titanium(IV) oxide as a novel and effective chill-proofing agent, particularly as hydrous titanium(IV) oxide may be prepared conveniently on site from a readily available and inexpensive material, titanium(IV) chloride.

High-yield method for immobilization of enzymes.

Two types of polyethylenimine-coated glass microbeads (13-44 micrometer) were synthesized and used for the immobilization of glucose oxidase from Aspergillus niger and catalase from A. niger and beef liver. The two types of beads were distinguishable by differences in their surface topography. Immobilizations were performed by adsorption followed by treatment with glutaraldehyde. The immobilized-enzyme activities per unit support of all of the enzymes tested were compared with and found to be superior to the immobilized activities attainable on aminopropyl-activated glass microbeads. When enzyme was present in less than saturating amounts, the coated beads were able to remove 100% of the glucose oxidase activity initially present in the immobilization solution, with 78-87% of that activity expressed on the support surface. Bound glucose oxidase was more stable to thermal inactivation than native enzyme.

Hydrolysis of organophosphate insecticides by an immobilized-enzyme system.

An enzyme preparation that could detoxify parathion and eight other organophosphate pesticides was covalently bound to either porous glass or porous silica beads. This immobilized-enzyme system was examined for its use in detoxification of pesticides in production wastewaters. The kinetics of parathion hydrolysis were examined at flow rates up to 96 liter/hr and at influent substrate concentrations ranging from 10--250 mg/liter. The enzyme reactor was able to hydrolyze 95% or more of the parathion added to industrial wastewaters generated during its production, thus reducing the effluent parathion concentration to below 500 ppb. Laboratory continuous-flow experiments were conducted for 70 days with industrial wastewater and indicated no loss in immobilized-enzyme activity. The influence of pH, temperature, solvents, and detergents on enzyme stability and activity and enzyme reactor kinetics will be discussed.

Enzyme immobilization by radiation-induced polymerization of 2-hydroxyethyl methacrylate at low temperatures.

Enzyme immobilization by radiation-induced polymerization of hydrophilic glass-forming monomers, such as 2-hydroxyethyl methacrylate, was studied. Enzyme radiation damage could be sufficiently retarded at low temperatures. The immobilized enzyme activity yield was markedly higher at low temperature than at higher temperature polymerization. At low temperatures the polymerized composite had a porous structure owing to ice crystallization which depends on the monomer concentration. It was deduced that the enzyme was partially trapped on the polymer surface, partially isolated in the pore, and partially occluded inside the polymer matrix. A decrease in activity caused by enzyme leakage was observed with repeated use in enzyme reactions where the composites had a large porosity. The activity yield showed a maximum at certain optimum porosities, i.e., at optimum monomer concentrations. Continuous enzyme reaction was preferably carried out using immobilized enzyme columns.

Immobilization of neuraminidase and its potential application in the modification of cell surfaces.

Vibrio cholerae neuraminidase was immobilized on the inside of 1.0 mm inner diameter nylon tubing with retention of enzyme activity, when assayed at 37 degrees C and pH 5.5 with mucin as substrate. The stabilities of the immobilized and soluble enzymes were similar for up to 3 hr at 37 degrees C. Preliminary data indicated that immobilized neuraminidase will release sialic acid from the surface of leukemic AKR mouse thymus and spleen lymphocytes; however, the level of immobilized enzyme activity needs to be increased for practical applications. With this improvement immobilized neuraminidase could become a novel preparation for carrying out cell surface modifications with minimal enzyme contamination of the cell.

Catalytic activity of poly(acryloyl morpholine)-immobilized horseradish peroxidase in organic/aqueous solvent mixtures

The immobilization of horseradish peroxidase by covalent coupling within an expanded poly(acryloyl morpholine) gel network is described. The activity of the immobilized horseradish peroxidase was compared with that of the native enzyme in aqueous buffer and in buffered mixtures of dimethyl-formamide/water, ethanediol/water, methanol/water and tetrahydrofuran/water of varying solvent ratios at pH 6.1. On increasing the organic solvent concentration in the substrate solution, active immobilized enzyme retained its activity much better than an equivalent amount of the native enzyme. The oxidation of ferrocene (water-insoluble) and ferrocene derivatives to the corresponding ferricinium ions, was accomplished efficiently by the immobilized enzyme in buffered 50% methanol/water solution. The immobilized enzyme exhibited superior resistance to thermal denaturation.

Nonporous magnetic materials as enzyme supports: studies with immobilized chymotrypsin.

Chymotrypsin has been immobilized to several nonporous magnetic materials. Nickel particles were considered to be most suitable as immobilized enzyme supports. Chymotrypsin immobilized to nonporous magnetic supports was not fouled significantly by either whole milk or clarified yeast homogenate. AE-cellulose-chymotrypsin was rapidly fouled by both these materials and chymotrypsin immobilized to acrylic-based ion exchangers was slowly fouled. Immobilized enzyme activity was found to be inversely proportional to particle diameter for nonporous rock magnetic particles. Immobilization by adsorption and then glutaraldehyde crosslinking was used to produce controlled amounts of chymotrypsin on the particles. Esterolytic activity increased with enzyme loading but caseinolytic activity did not increase. Chymotrypsin is inhibited by metal ions from the magnetic supports. It is partially protected by use of a preliminary protein coating and may be reactivated by incubation with EDTA or BSA.

An active-site titration method for immobilized trypsin

An active-site titration method for immobilized trypsin with p-nitrophenyl p′-guanidinobenzoate (NPGB) employing a recirculation reactor system is described. Trypsin, covalently linked to 35 and 150 μm diameter porous glass particles was titrated by this procedure, analyzed for protein content, and then compared with soluble trypsin. Analysis of the titration results indicates that the amount of p-nitrophenol produced by the burst is equal to the amount of active immobilized trypsin. This active site titration determines the absolute amount of active immobilized enzyme and is preferable to either total protein analysis or kinetic assays for characterization of immobilized enzymes.