Preparation of some immobilized linked enzyme systems and their use in the automated determination of disaccharides

1. Glucose oxidase (EC 1.1.3.4), amyloglucosidase (EC 3.2.1.3), invertase (EC 3.2.1.26) and beta-galactosidase (EC 3.2.1.23) were covalently attached via glutaraldehyde to the inside surface of nylon tube. 2. The linked enzyme system, comprising invertase immobilized within a nylon tube acting in series with glucose oxidase immobilized in a similar way, was used for the automated determination of sucrose. 3. The linked enzyme system, comprising beta-galactosidase immobilized within a nylon tube acting in series with glucose oxidase immobilized in a similar way, was used for the automated determination of lactose. 4. The linked enzyme system, comprising amyloglucosidase immobilized within a nylon tube acting in series with glucose oxidase immobilized in a similar way, was used for the automated determination of maltose. 5. Mixtures of glucose oxidase and amyloglucosidase were immobilized within the same piece of nylon tube and used for the automated determination of maltose. 6. Mixtures of glucose oxidase and invertase were immobilized within the same piece of nylon tube and used for the automated determination of sucrose.     

Immobilization of invertase on modified nylon tubes

Invertase was immobilized on O-alkylated nylon tubes modified with amine arms and activated with glutaraldehyde. The performance of the immobilized invertase derivative was dependent on the nylon tube modification that preceded the enzyme coupling procedure. Optimal experimental conditions for the tube modification and the enzyme coupling step are described and discussed.     

The preparation of nylon-tube-supported hexokinase and glucose 6-phosphate dehydrogenase and the use of the co-immobilized enzymes in the automated determination of glucose.

Triethyloxonium tetrafluoroborate was used to O-alkylate nylon-tube thus producing the imidate salt of the nylon which was further made to react with 1,6-diaminohexane. 2. Hexokinase (EC 2.7.1.1) and glucose 6-phosphate dehydrogenase (EC 1.1.1.49) were immobilized on the amino-substituted nylon tube through glutaraldeyde and bisimidates. 3. The effect of varying the conditions of O-alkylation and the amount of enzyme immobilized on the activity of nylon tube-hexokinase derivatives was determined. 4. The effect of varying the amount of enzyme immobilized on the activity of nylon-tube-glucose 6-phosphate dehydrogenase derivatives was determined. 5. The thermal stability of nylon-tube-hexokinase and nylon-tube-glucose 6-phosphate dehydrogenase derivatives was studied. 6. Different ratios of hexokinase and glucose 6-phosphate dehydrogenase were co-immobilized on nylon tube, and the rate of conversion of glucose into 6-phosphogluconolactone was compared with the individual activities of the immobilized enzymes. 7. Hexokinase and glucose 6-phosphate dehydrogenase co-immobilized on nylon tube were used in the automated analysis of glucose.     

Polysaccharide derivatives as coats for nylon tube urease

Urease was immobilized on O-alkylated nylon tubes coated with polyaminated derivatives of starch or dextran. The specific activity of the enzyme coil and the relative stability of the immobilized enzyme, compared with immobilized urease derived from other nylon tube modifications, were enhanced. Also, the nonspecific binding of urease to O-alkylated nylon tubes was virtually eliminated by the coating process.     

An Affinity Chromatographic Reactor for Highly Efficient Turnover of Dissociable Cofactors

A conjugated enzyme system of alcool dehydrogenase and lactate dehydrogenase was immobilized in an ultrafiltration hollow fiber tube, which was inserted in a fine nylon tube to form a hollow-fiber-capillary reactor. To this reactor, the substrates, pyruvate and ethanol, were supplied continuously. The necessary cofactor, NAD, was supplied as a pulse for a short time. The retention time of NAD in the reactor, estimated from the response curve of lactate produced, was much longer than those of the other substrates and products because of the strong adsorption of NAD to the immobilized enzymes through affinity. Therefore, the reactor could produce lactate from pyruvate for a long time without any more NAD. As a typical case, when the enzyme concentration is sufficiently high, the estimated retention time of NAD was 50 times as long as those of other materials so that the NAD turnover obtained was 412,000. The effects of NAD pulse concentration and the immobilized enzyme concentration on the retention time of NAD and NAD turnover were investigated experimentally and theoretically.     

The immobilization of enzymes on nylon structures and their use in automated analysis

1. Glucose oxidase (EC 1.1.3.4) and urease (EC 3.5.1.5) were covalently attached through glutaraldehyde to low-molecular-weight nylon powder. 2. Immobilized derivatives of glucose oxidase and urease were prepared by cross-linking the respective enzymes within the matrix of a nylon membrane. 3. An improved process is described for the immobilization of glucose oxidase and urease on the inside surface of partially hydrolysed nylon tube. 4. Automated analytical procedures are described for the determination of glucose with each of the three immobilized glucose oxidase derivatives and for the determination of urea with each of the three immobilized urease derivatives. 5. The efficiencies of the three immobilized enzyme structures as reagents for the automated determination of their substrates were compared.     

Active protein and substrate flow effects in a tubular immobilized invertase reactor

Investigations of invertase (EC 3.2.1.26) immobilized inside modified nylon tubes showed that between 4% and 20% (w/w) of the protein exposed to binding sites on the tube was immobilized. An enhanced activity consistent with enzyme purification during immobilization was also evident, suggesting that, in scaled-up commercial applications, nylon tube invertase would be a more economical converter of sucrose than the free enzyme. The quantity and specific activity of the immobilized protein were not stochiometrical with the amount used in the coupling solution and, in the system studied, a concentration of 2 mg ml^?1 was optimal. K_m and V_max values confirmed higher rates of immobilized invertase catalysis when the rates of substrate flow through the reactor were higher. Higher rates of substrate flow imply a shortened residence time in the reactor and would lower the fractional conversion per pass of the substrate, reducing the efficiency of the reactor in flow-through situations. Thus, these higher catalysis rates, attributable at the higher flow rates to a reduction of the diffusion barrier between enzyme and substrate, would not translate into improved economy in the commercial flow-through processes at which the reactor is aimed.     

Polymethylglutamate as a new matrix for covalently immobilized enzymes: Preparation and properties of urease and uricase

Polymethylglutamate (PMG), a synthetic polypeptide, was used as a new carrier to immobilize urease (EC 3.5.1.5) and uricase (EC 1.7.3.3) by the azide method. The enzymes could be immobilized onto PMG in various forms, such as film, fiber, coating on various beads, and a silicon tube. The retained activities of the immobilized enzymes were excellent (more than 95%), therefore it was possible to immobilized almost all activities of the enzymes added in the coupling mixtures. Heat stabilities of the resulting immobilized enzymes were markedly improved, while the optimal pH and K_m values remained almost unchanged. The urease immobilized on the PMG-coated glass beads packed in a column, was found to retain its activity more than 80% of the initial value, even after the occasional use for a year. In view of the improved retained activities and stabilities of the immobilized enzymes, PMG may therefore be a very versatile matrix for the immobilized enzymes.     

Mathematical modeling of flow-tube extracorporeal shunts with immobilized enzymes

A complex of computer programs "IF-reactor" for model calculations and processing of experimental data related to flow tube extracorporeal shunts with immobilized enzymes has been developed. Qualitative and quantitative comparison of theory with experiment has been made.     

Short Communication: Membrane-impregnated probe for simultaneous PCR amplification and detection

We report a rapid and highly versatile one-step single tube method for gene amplification and detection using a non-radiolabelled probe. The technique requires nylon on which a third primer has been immobilized that acts as a probe and digoxigenin-labelled-dUTP added to the conventional PCR mixture. After PCR amplification the membrane-impregnated probe is labelled with dig-11-dUTP that serves to confirm the identity of the PCR product.     

Invertase embedded-PVC tubing as a flow-through reactor aimed at conversion of sucrose into inverted sugar

A simple flow-through reactor system is prepared by covalent linking of a biomolecule on the inner surface of a polyvinyl chloride (PVC) tube. This is achieved by introducing an active functional group on the surface of an inert PVC tube through 1-fluoro-2-nitro-4-azidobenzene (FNAB), a precursor of highly reactive nitrene, which can insert to any C–H bond. CCl₄ lacking C–H bond is taken as a solvent for loading FNAB solution into the tube. FNAB loaded tube is then allowed to expose to sunlight for 20 min during which azido group of FNAB generates nitrene and attaches itself to PVC tube through insertion reaction. Invertase is immobilized in the activated PVC tube at 50 °C in 45 min. Invertase embedded-PVC tube is used as a flow-through reactor to convert sucrose to invert sugar. The flow-through reactor converted sucrose into invert sugar with 97% yield as shown by HPLC analysis. The reactor is reused for eight cycles with 17% loss of its initial activity. The reactor system is cheap as PVC tube is working both as a carrier of biomolecule and a reaction vessel. This reactor system overcomes the problem of back pressure and can be used for any enzymatic conversion in a flow-through system.     

Enzyme-gas chromatography: Theoretical and experimental aspects

A method based on the production of gas by enzymes was developed to determine the concentration of amino acids. The enzyme was immobilized by coreticulation on the external surface of a capillary silicone tube. The gas produced diffused through the silicone membrane to the lumen of the tube and was carried by a vector gas to a gas chromatograph. The amount of measured gas has been shown to be a function of the amino acid concentration. A model of the system that gave good agreement between experimental and calculated values was developed.     

Invertase embedded-PVC tubing as a flow-through reactor aimed at conversion of sucrose into inverted sugar

A simple flow-through reactor system is prepared by covalent linking of a biomolecule on the inner surface of a polyvinyl chloride (PVC) tube. This is achieved by introducing an active functional group on the surface of an inert PVC tube through 1-fluoro-2-nitro-4-azidobenzene (FNAB), a precursor of highly reactive nitrene, which can insert to any C–H bond. CCl₄ lacking C–H bond is taken as a solvent for loading FNAB solution into the tube. FNAB loaded tube is then allowed to expose to sunlight for 20 min during which azido group of FNAB generates nitrene and attaches itself to PVC tube through insertion reaction. Invertase is immobilized in the activated PVC tube at 50 °C in 45 min. Invertase embedded-PVC tube is used as a flow-through reactor to convert sucrose to invert sugar. The flow-through reactor converted sucrose into invert sugar with 97% yield as shown by HPLC analysis. The reactor is reused for eight cycles with 17% loss of its initial activity. The reactor system is cheap as PVC tube is working both as a carrier of biomolecule and a reaction vessel. This reactor system overcomes the problem of back pressure and can be used for any enzymatic conversion in a flow-through system.     

Freeze-thaw immobilization of liposomes in chromatographic gel beads: evaluation by confocal microscopy and effects of freezing rate

Biological membranes immobilized in chromatographic gel beads constitute a multifunctional affinity matrix. Membrane protein-solute interactions and drug partitioning into the lipid bilayers can conveniently be studied. By the use of confocal laser-scanning microscopy (CLSM) the distribution of immobilized model membranes in the beads has been visualized for the first time. Freeze-thaw-immobilized liposomes in Superdex 200 gel beads were situated in a thick shell surrounding a liposome-free core. The amount of phospholipids immobilized by freeze-thawing was dependent on the temperature in the cooling bath and the type of test tube used. A bath temperature of -25 degrees C gave higher immobilization yield than freezing at -75 or -8 degrees C did. Freeze-thawing in the presence of liposomes did not affect the gel bead shape or the refractive index homogeneity of the agarose network of the beads, as shown by confocal microscopy.     

The Application of Chemiluminescence of a Cypridina Luciferin Analog to Immobilized Enzyme Sensors

Chemiluminescence of a Cypridina luciferin analog, 2-methyl-6-phenyl-3,7-dihydro-imidazo[l,2-a]pyrazin-3-one, was applied to immobilized enzyme sensors. Xanthine oxidase, peroxidase, glucose oxidase, uricase and cholesterol oxidase were immobilized by using photo-crosslinkable resin prepolymer or ion-exchangeable cellulose beads. The immobilized enzyme sensor system was composed of a photoncounter and a test tube in which the immobilized enzyme membrane or particles were placed. A linear relation between the concentration of substrates and luminescence rate was obtained on a logarithmic scale. This immobilized enzyme sensor system could be used repeatedly. Hydrogen peroxide, xanthine and hypoxanthine were measured sensitively and rapidly within 100 sec. Glucose, cholesterol and uric acid were measured sensitively within 10 min but could be measured within 100 sec, although less sensitive. The detection limits for xanthine, hypoxanthine, hydrogen peroxide, glucose, cholesterol and uric acid were 0.02, 0.02, 0.2, 0.4, 2 and 2 μM, respectively. Concentrations of hypoxanthine in tuna muscle, and glucose and cholesterol in serum measured using this sensor system were comparable with those measured by the standard methods.     

Kinetics of yeast alcohol dehydrogenase and its coenzyme coimmobilized in a tubular flow reactor

Yeast alcohol dehydrogenase and nicotinamide adenine dinucleotide (NAD) were coimmobilized, with covalent attachment, to the interior surface of a nylon tube. The NAD was attahed at the N(6) group of the adenine moiety; an NAD derivative was prepared and attached to free carboxyl groups at a partially hydrolyzed nylon surface. The enzyme was attached, through glutaraldehyde residues, to free amino groups on the surface. Kinetic studies were carried out in which the reduced NAD was recycled by means of phenazine ethosulfate and 2,6-dichlorophenol indophenol. The reaction was studied over a range of flow rates and ethanol concentrations. The variation of rate with flow rate suggested that there was little diffusion control with respect to ethanol and that there was no observable inhibition by the reaction products. These conclusions were confirmed by evidence based on dimensionless parameters for the reaction and by direct inhibition experiments. The apparent Michaelis constant was lower than when only the enzyme was immobilized, suggesting that the immobilized enzyme-coenzyme system is of high efficiency. Overall rates of reaction were lower than when there was saturation with NAD. The tube showed no measurable loss of catalytic activity over a period of one month.     

Glucose oxidation in a dual hollow fiber bioreactor with a silicone tube oxygenator

A dual hollow fiber bioreactor, consisting of an outer silicone membrane for oxygen supply and an inner polyamide membrane for substrate permeation, was used as an immobilized enzyme reactor to carry out enzymatic glucose oxidation. Attaching a silicone tube oxygenator to provide an additional oxygen supply improved the conversion in glucose oxidation when the oxygen supply was rate-limiting. The reactor was operated in both diffusion and ultrafiltration modes. In the latter case, the conversion was much higher, but the stability of the immobilized enzyme was better maintained in the diffusion mode. As the inlet glucose concentration increased from 10mM to 500mM, the conversion decreased from 70 to 20%.     

Immobilized-enzyme pipette. Scope and limitations of a simple device.

Disposable pipette tips made of polymeric nylon tube with enzymes bound covalently to their inside surface and fixed to the stem of an automatic, adjustable-volume pipette holder together constitutes an immobilized enzyme pipette or 'Impette'. The present paper describes the application in research laboratories and clinics of this new development, with urease as an example in the determination of blood urea.     

Effects of fluid shear on immobilized enzyme kinetics.

There have been a number of reports concerning the damaging effects of shear on globular proteins in solution. Some recent work has indicated, however, that globular proteins in solution are relatively stable, but may be inactivated at air-liquid interfaces during shearing. This study investigated the effects of fluid shear on immobilized enzyme activity. Immobilized enzyme reactors were built to operate with the enzyme immobilized at the boundary of a fluid flow field. Two different enzymes, penicillinase and lactate dehydrogenase, were covalently bound to the interior surface of nylon tubes. Fluid shear rate was changed by varying the flow rate of substrate (reactant) solution through the tube, and fluid shear stresses were increased by increasing the viscosity of the recirculating solution. There were no observed effects of fluid shear on immobilized penicillinase or lactate dehydrogenase activity at shear rates of up to 10,350 s-1 or at shear stresses of up to 73 Pa.     

Affinity chromatography on immobilized anhydrotrypsin: general utility for selective isolation of C-terminal peptides from protease digests of proteins

Recently we have succeeded in the efficient isolation of the C-terminal peptides from tryptic digests of the tail sheath protein (with C-terminal Gly) and the tube protein (with C-terminal Glu) of bacteriophage T4, by taking advantage of a unique property of immobilized anhydrotrypsin, that is, a strong specific affinity for peptides containing Arg or Lys residues at their C-termini. In this study, the utility of affinity chromatography on immobilized anhydrotrypsin was further demonstrated in the cases of Streptomyces subtilisin inhibitor (as a reduced and S-carboxymethylated form, with C-terminal Phe) and alpha 1-antitrypsin (with C-terminal Lys). By subjecting a tryptic digest of the former protein and a chymotryptic digest of the latter protein to the affinity chromatography, the C-terminal peptides were specifically recovered in the breakthrough fraction and in the adsorbed fraction, respectively. It was further shown that immobilized anhydrotrypsin can also adsorb peptides with C-terminal S-aminoethyl-Cys residues and exerts adsorptive ability even toward the peptides in solution containing urea at a high concentration if appropriate precautions are taken. These findings suggest the general utility of this simple method for C-terminal peptide isolation, which is extremely helpful for studies to confirm amino acid sequences deduced from nucleotide sequences of the cDNA (or genomic DNA) of proteins.