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.     

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.     

The enzyme coupling process in urease immobilization on O-alkylated nylon tubes

Coupling of Jack bean urease (EC 3.5.1.5) to the inside surface of type 6 nylon tubes, activated by high-temperature O-alkylation with dimethyl sulphate and modified subsequently with lysine and glutaraldehyde, was investigated to establish optimal experimental conditions for the coupling process. For the system described, the most active immobilized urease derivatives were prepared with 2 mg/ml of the solubilized urease solution and use of higher enzyme concentrations proved wasteful. Although urease coupling without thermal denaturation of the solubilized enzyme was achieved at 20 degrees C, derivatives prepared at 37 degrees C yielded maximal activity over the 3 h coupling period. Also, longer incubations of the enzyme solution in the tube were unnecessary under these conditions. Optimal pH for the coupling process was 6.5, one at which the solubilized enzyme was most stable.     

Determination of heat changes in the proximity of immobilised enzymes with an enzyme thermistor and its use for the assay of metabolites.

A device, the enzyme thermistor, is described which is capable of measuring changes in heat due to enzymic reactions. The sensor, a thermistor, is in direct contact with the site of reaction through its placement in a microcolumn filled with an immobilised enzyme preparation. The substrate solution flows past the thermistor tip, and as much as approx. one half of the total heat evolved can be registered as temperature change, deltat. Glass-bound glucose oxidase (EC 1.1.3.4), penicillinase (EC 3.5.2.6), trypsin (EC 3.4.21.4) and urease (EC 3.5.1.5) were used for the determination of glucose, penicillin G, benzoyl-L-arginine ethyl ester and urea respectively. Linear relationships between the deltat recorded and the concentration of substrate were obtained in all cases.     

Nylon shavings enzyme reactor for batch determination of urea

The design and performance of an enzyme reactor (enzyme electrode) which features (i) incorporating nylon shavings onto which an enzyme is covalently bonded, (ii) a flat-surface combination pH electrode for proton monitoring, and (iii) a body providing an injection port for sample injection and washing and stirring capabilities is described. The reactor configuration described here offers good diffusional and partition characteristics which result in relatively fast response, good stability, simplicity of operation, low sample and reagent consumption, and adaptability to flow systems. Application to the determination of urea in standards and physiological salt solutions is demonstrated by use of immobilized urease (EC 3.5.1.5).     

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.     

Enhanced oxidation of bilirubin by an immobilized tri-enzyme system of glucose oxidase, bilirubin oxidase and horseradish peroxidase

Bilirubin oxidase was immobilized to nylon fibres. A tri-enzyme system composed of glucose oxidase, bilirubin oxidase and horseradish peroxidase was also immobilized to the fibres. Both immobilized systems were tested and it was found that the latter gave enhanced oxidation rates for bilirubin.     

Immobilization of enzymes on chitin and chitosan

During the last few years, d-glucose isomerase, glucoamylase, β-d-galactosidase (lactase), β-d-glucosidase, d-glucose oxidase, AMP deaminase, urease, pronase, subtilisin, trypsin, papain, alkaline phosphatase, acid phosphatase, pepsin, chymotrypsin and lysozyme have been immobilized on chitin and on some of its derivatives, mainly with glutaraldehyde. The preparation and performances of the immobilized enzymes are described.     

Urease immobilized on nylon: preparation and properties

Urease (EC 3.5.1.5) was covalently attached through glutaraldehyde to partially hydrolysed nylon 6/6 tubes. The highest activity of immobilized enzyme was obtained at 65?°C and pH 6.5, while the optimum temperature for free urease was found to be 25?°C. Immobilized urease showed an improved thermal stability in comparison to free urease. It retained 76% of the original activity after 60 days when stored at 4?°C and 78% of the activity after 5 repeated uses.     

Flow kinetics of nylon open-tubular and beaded immobilized glucose oxidase reactors

Equations describing the flow kinetics of immobilized glucose oxidase in open nylon tubes and in tubes filled with solid glass spheres were experimentally determined. Reactors of three different tube (dt) and bead (db) diameters were tested using various linear flow rates (vf) and glucose concentrations ([S]). The kinetics of the open-tubular reactors were described by [P] = 1.5.10(-3) [S] L0.86 vf-0.78 dt-0.9 and the kinetics of the beaded-tubular reactors by [P] = 5.10(-3) [S] L0.98 vf-0.75 dt-1.0 (db/dt)2.7, where [P] equals the concentration of H2O2 formed. The aspect ratio, db/dt, is the critical design factor for beaded reactors.     

Immobilization of glucose oxidase and peroxidase to a urea derivative of granulated microcrystallized cellulose

Glucose oxidase and peroxidase were immobilized individually to a urea derivative of granulated microcrystallized cellulose activated by formaldehyde. The catalytic properties of the immobilized enzymes were studied and compared to those of the soluble enzymes. The immobilized glucose oxidase and peroxidase were used for the manual determination of the concentration of glucose in sera. The developed method is characterized by high analytical reliability and comparatively low cost. The results correlated well with those obtained by using a BECKMAN glucoanalyzer, utilizing soluble glucose oxidase.     

Immobilization of β-Glucuronidase on Pellicular Nylon

β-Glucuronidase (EC 3.2.1.31), From Patella vulgala, was immobilized on a pellicular, polyethyleneimine-derivatized nylon net. Several types of nylon nets were used in order to ensure the best relationship between activity of the immobilized enzyme and net characteristics. No differences were observed between the two most used reagents for nylon activation by alkylation under standard working conditions. The influence of attachment by several protein aminoacidic side chains was determined, and enzyme immobilization involving lysyl and tyrosyl protein residues showed better activity. Coupling conditions for these derivatives have been optimized by studying the influence of p^H, temperature and reaction time on derivative activity. The saturation profiles obtained for these derivatives showed a high protein content, 30mg/g, for a non-porous support. Characterization of these derivatives against p^H, ionic strength and temperature was also studied, and no differences were found between soluble and immobilized activity-profiles apart from those found when thermal stability studies were performed. High stability towards intermittent use was found when assayed against p-nitrophenylglucuronide as substrate. After 18 months of use a loss of activity of only 22 and 33% for derivatives through lysyl and tyrosyl residues, respectively, was observed.     

Immobilization of glucose oxidase on silica-based supports

Glucose oxidase (beta-D-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4) was covalently coupled to silica-based supports containing aldehyde functional groups. The activity of the immobilized enzyme was about 1000 U/g support. The optimum pH of the catalytic activity was 5.5 for the soluble enzyme and 6.0 for the immobilized enzyme. With glucose as a substrate the Km value of the immobilized enzyme was higher than in case of the soluble enzyme. The immobilized enzyme was found to be more thermostable than the soluble one. The immobilization did not affect the stability of glucose oxidase against the denaturing effect of urea.     

Effect of enzyme-matrix composition on potentiometric response to glucose using glucose oxidase immobilized on platinum

Glucose oxidase (beta-D-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) was immobilized in a crosslinked matrix of bovine serum albumin, catalase, glucose oxidase and glutaraldehyde on platinum foil. When placed in glucose solution, this enzyme-electrode elicited a potentiometric response that varied with the changes in glucose concentration. The immobilized glucose oxidase was present at 7.4-10.1 micrograms enzyme protein/ml of matrix, as determined with 125I-labelled enzyme. The coupled enzyme activity was stable over 120 h; however, the apparent activity of the immobilized glucose oxidase was markedly less than that for the same amount of enzyme free in solution. This indicated a significant level of diffusional resistance within the enzyme-matrix. The potentiometric response to glucose increased significantly as either the thickness of the enzyme-matrix or the glutaraldehyde content was reduced; this also was attributed to diffusional effects. Several enzyme-electrodes, constructed without exogenous catalase and with different amounts of glucose oxidase, showed greater sensitivity in potentiometric response at low glucose oxidase loadings. These results are consistent with the hypothesis that the potentiometric response arises from an interfacial reaction involving a hydrogen peroxide redox couple at a platinum surface. The data also suggest that an optimum range of hydrogen peroxide concentration exists for maximum electrode sensitivity.     

Various applications of immobilized glucose oxidase and polyphenol oxidase in a conducting polymer matrix

In this study, glucose oxidase and polyphenol oxidase were immobilized in conducting polymer matrices; polypyrrole and poly(N-(4-(3-thienyl methylene)-oxycarbonyl phenyl) maleimide-co-pyrrole) via electrochemical method. Fourier transform infrared and scanning electron microscope were employed to characterize the copolymer of (N-(4-(3-thienyl methylene)-oxycarbonyl phenyl) maleimide) with pyrrole. Kinetic parameters, maximum reaction rate and Michealis-Menten constant, were determined. Effects of temperature and pH were examined for immobilized enzymes. Also, storage and operational stabilities of enzyme electrodes were investigated. Glucose and polyphenol oxidase enzyme electrodes were used for determination of the glucose amount in orange juices and human serum and phenolic amount in red wines, respectively.     

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.     

Synthesis of a highly substituted N(6)-linked immobilized NAD(+) derivative using a rapid solid-phase modular approach: suitability for use with the kinetic locking-on tactic for bioaffinity purification of NAD(+)-dependent dehydrogenases

This study is concerned with further development of the kinetic locking-on strategy for bioaffinity purification of NAD(+)-dependent dehydrogenases. Specifically, the synthesis of highly substituted N(6)-linked immobilized NAD(+) derivatives is described using a rapid solid-phase modular approach. Other modifications of the N(6)-linked immobilized NAD(+) derivative include substitution of the hydrophobic diaminohexane spacer arm with polar spacer arms (9 and 19.5 A) in an attempt to minimize nonbiospecific interactions. Analysis of the N(6)-linked NAD(+) derivatives confirm (i) retention of cofactor activity upon immobilization (up to 97%); (ii) high total substitution levels and high percentage accessibility levels when compared to S(6)-linked immobilized NAD(+) derivatives (also synthesized with polar spacer arms); (iii) short production times when compared to the preassembly approach to synthesis. Model locking-on bioaffinity chromatographic studies were carried out with bovine heart l-lactate dehydrogenase (l-LDH, EC 1.1.1.27), bakers yeast alcohol dehydrogenase (YADH, EC 1.1.1.1) and Sporosarcinia sp. l-phenylalanine dehydrogenase (l-PheDH, EC 1.4.1.20), using oxalate, hydroxylamine, and d-phenylalanine, respectively, as locking-on ligands. Surprisingly, two of these test NAD(+)-dependent dehydrogenases (lactate and alcohol dehydrogenase) were found to have a greater affinity for the more lowly substituted S(6)-linked immobilized cofactor derivatives than for the new N(6)-linked derivatives. In contrast, the NAD(+)-dependent phenylalanine dehydrogenase showed no affinity for the S(6)-linked immobilized NAD(+) derivative, but was locked-on strongly to the N(6)-linked immobilized derivative. That this locking-on is biospecific is confirmed by the observation that the enzyme failed to lock-on to an analogous N(6)-linked immobilized NADP(+) derivative in the presence of d-phenylalanine. This differential locking-on of NAD(+)-dependent dehydrogenases to N(6)-linked and S(6)-linked immobilized NAD(+) derivatives cannot be explained in terms of final accessible substitutions levels, but suggests fundamental differences in affinity of the three test enzymes for NAD(+) immobilized via N(6)-linkage as compared to thiol-linkage.     

On-line monitoring of glucose in mammalian cell culture using a flow injection analysis (FIA) mediated biosensor

A flow injection analysis (FIA) biosensor system has been developed for on-line determination of glucose during mammalian cell cultivation. The culture sample was peristaltically withdrawn from the bioreactor and after cell separation by a steam sterilizable ceramic microfilter, the filtrate was continuously fed to the FIA mediated-biosensor system at 4 mLh(-1), whereas the cell-containing retentate was recirculated to the bioreactor. In the amperometric biosensor system, glucose oxidase was covalently immobilized onto a preactivated nylon membrane and attached to the sensing area of a platinum working electrode. The enzyme reaction was coupled with the mediator 1,1'-dimethylferricinium (DMFe(+))-cyclodextrin inclusion complex to recycle the reduced glucose oxidase to its original active state. 1,1'-Dimethylferrocene (DMFe) was then reoxidized to DMFe(+) at the surface of the platinum electrode poised at + 0.15 V vs silver/silver chloride. The FIA mediated-biosensor was linear up to 6 mM glucose, with a detection limit of 0.1 mM, and possessed excellent reproducibility (+/- 0.4 %, 95 % confidence interval) over 123 repeated analyses during a 62 h continuous operation. The immobilized glucose oxidase was stable for up to 7 days when applied to glucose measurement during 5-10 day fed-batch cultivation of 293S mammalian cells. The results obtained from the mediated-biosensor system compared well with the hexokinase and HPLC data. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 497-504, 1997.     

A chemiluminometric method for the determination of urea in serum using a three-enzyme bioreactor

A flow injection chemiluminometric assay for urea has been developed based on a minicolumn bioreactor packed with immobilized enzyme-bearing glass beads. The reactor contains immobilized urease, L -glutamate dehydrogenase and L -glutamate oxidase, aligned in this order (upstream to the downstream). When the sample is introduced into the bioreactor, urea is first hydrolysed by urease to produce ammonia, which is then converted into L -glutamate by L -glutamate dehydrogenase. L -Glutamate is finally oxidized by L -glutamate oxidase to produce hydrogen peroxide, which is quantified by measuring chemiluminescence emitted upon admixing with luminol and potassium ferricyanide. One assay cycle is completed within 1 minute. The method is sensitive (detection limit 0.5 nmol) and is linear in the range 0–30 mmol/l. It can be readily applied to the determination of urea in human serum, and requires no blank corrections for ammonia and/or L -glutamate present in serum samples.     

尿素氮-葡萄糖双功能分析仪的研究

由固定化脲酶、谷氨酸脱氢酶、谷氨酸氧化酶、葡萄糖氧化酶的复合酶膜组成的双电极系统,可以同时测定尿素氮和葡萄糖的含量,每次进样量为25μl,20s即可测定出尿素氮和葡萄糖的含量。在0～60mg/dl尿素氮、0～500mg/dl葡萄糖范围内具有良好的线性关系。连续测定20次的变异系数分别为1.02％和1.05％。酶膜使用寿命为两星期以上。此仪器可广泛应用于临床检验和体育训练中。