The development of an immobilized lactate oxidase system for lactic acid analysis

An enzyme designated as lactate oxidase was purified from Acetobacter peroxydans by using the partition methods of separation. A DE-52 cellulose column was used for the primary purification of lactate oxidase, and the purified enzyme was covalently bound to a porous cellulose bead matrix in which benzoquinone was used as the coupling reagent. The physicochemical properties of the native and immobilized enzymes were determined including molecular weight, cofactor requirements, and optimal reaction conditions. Lactate oxidase was shown not to be subject to product inhibition, and to require Mg(2+) as a metal cofactor. Analysis of an immobilized lactate oxidase packed-bed reactor indicated that this system may not be subject to internal diffusional limitations. Molecular oxygen appeared to be a cosubstrate of the enzyme, and a reaction mechanism was postulated to predict the kinetic behavior of the immobilized reactor system. Applications of the immobilized lactate oxidase reactor for the pulse-flow analysis of lactic acid in whole milk and in a yeast fermentation system were considered.     

Kinetic studies on the reaction mechanism of sarcosine oxidase

A sarcosine oxidase (sarcosine: oxygen oxidoreductase (demethylating), EC 1.5.3.1) isolated from Corynebacterium sp. U-96 contains both covalently bound FAD and noncovalently bound FAD. The noncovalent FAD reacts with sarcosine, the covalent FAD with molecular oxygen (Jorns, M.S. (1985) Biochemistry 24, 3189-3194). To clarify the reaction mechanism of the enzyme, kinetic investigations were performed by the stopped-flow method as well as by analysis of the overall reaction. The absorption spectrum of the enzyme in the steady state was very similar to that of the oxidized enzyme, and no intermediate enzyme species, such as a semiquinoid flavin, was detected. The rate for anaerobic reduction of the noncovalently bound FAD and the covalently bound FAD by sarcosine were 31 and 6.7 s-1, respectively. The latter value was smaller than the value of respective Vmax/e0 obtained by the overall reaction kinetics (Vmax/e0: the maximum velocity per enzyme concentration). Both rate constants for oxidation of the two FADs by molecular oxygen were 100 s-1. A reaction scheme of sarcosine oxidase is proposed to account for the data obtained; 70% of the enzyme functions via a fully reduced enzyme, and 30% of the enzyme goes along a side-path, without forming the fully reduced enzyme. In addition, it is suggested that the reactivity of noncovalently bound FAD with sarcosine is affected by the oxidation-reduction state of the covalently bound FAD, in contrast to the reactivity of the covalently bound FAD with molecular oxygen, which is independent of the oxidation-reduction state of the noncovalently bound FAD.     

Deactivation studies of immobilized glucose oxidase

Studies have been performed in a tubular flow reactor to characterize the deactivation of immobilized glucose oxidase. The effects of oxygen concentration in the range of 0.09 to 0.467mM and hydrogen peroxide concentrations in the range of 0.1 to 10mM were studied. A simple mathematical model assuming first-order reaction and deactivation was found to describe the deactivation behavior adequately. The deactivation rate constant was found to increase with increasing levels of feed oxygen. Hydrogen peroxide was found to deactivate the enzyme severely and the deactivation rate constants were higher than those for oxygen deactivation. The influence of external and internal diffusion effects on the deactivation rate constant were examined. Although diffusional restrictions were negligible for oxygen transfer to the pellet, they were significant for transfer of hydrogen peroxide to the bulk stream. Increasing deactivation rates. Severe internal diffusion limitations were observed for the glucose oxidase system. However, for particle sizes in the range of 500 to 2000 μm, no effect on the rate of deactivation of the enzyme was observed.     

Development of post-column enzymic reactors with immobilized alcohol oxidase for use in the high-performance liquid chromatographic assay of alcohols with electrochemical detection

The development of a very sensitive, direct injection high-performance liquid chromatographic method, using a post-column reactor with immobilized alcohol oxidase, was undertaken with the aim of determining methanol and ethanol levels in microlitre volumes of biological samples. After reversed-phase chromatography to separate methanol and ethanol, the analytes were enzymically converted into the respective aldehydes with formation of stoichiometric amounts of hydrogen peroxide, which could be measured via electrochemical oxidation at a platinum electrode. Some problems were encountered in the development of solid-phase enzymic reactors, using a delicate enzyme, that is prone to lose activity, such as alcohol oxidase. Owing to the slightly alkaline pH required for the optimum activity of alcohol oxidase, polymeric columns seemed to be preferable for the chromatography. HEMA copolymer was chosen as the stationary phase, but the methanol and ethanol peaks eluted close together and posed severe problems of limiting post-column band spreading. Reactors based on coarse supports for enzyme immobilization gave unacceptable band spreading, causing the methanol and ethanol peaks to overlap. On the other hand high-performance liquid chromatographic packings maintained the efficiency of the chromatographic separation, quite independently of the reactor volume. Polymeric supports proved superior to silicas in maintaining the enzyme activity. However, relevant changes in the enzyme substrate specificity were observed after immobilization.     

Sulphydryl oxidase: Properties and applications

Sulphydryl oxidase has been isolated and purified from bovine milk and partially characterized. The enzyme is obtained from the whey fraction produced by chymosin (rennin) treatment of skim milk. Several procedures have been developed for its isolation, all of which use to advantage its concentration-dependent aggregate size. This enzyme exhibits a much broader substrate specificity than that shown by other ‘aerobic oxidases’ presently characterized which catalyse the formation of disulphides from thiols. Using molecular oxygen as an electron acceptor in a twoelectron reduction to hydrogen peroxide, the enzyme catalyses oxidation of cysteine and its analogues, some volatile thiols, peptides, and also thiols in proteins.Vis-a-vis protein-disulphide oxidoreductase and protein-disulphide isomerase, this enzyme does not catalyse thiol-disulphide interchange and thus functions only in de novo formation of disulphides. Enzyme-bound iron and most likely a free sulphydryl group are essential for catalytic activity; however, the carbohydrate moiety of this glycoprotein is probably not required. The broad substrate specificity suggests several potential industrial uses for the enzyme, including flavour modification in the food industry or synthesis of disulphides in pharmaceuticals. Consequently, immobilized forms were developed and characterized. Reactors containing sulphydryl oxidase covalently immobilized on porous glass and silica have been examined for their efficacy in eliminating the cooked flavour in ultra-high temperature sterilized milk. Both the degree of oxidation and cooked flavour were correlated with a normalized residence time in the reactor.     

Hydrogen peroxide generation with immobilized glucose oxidase for textile bleaching.

Glucose oxidase was covalently immobilized on commercially available alumina and glass supports, with a high level of protein recovery. The operational stability of the alumina carrier was an advantage over the glass support, though the rate of generation of hydrogen peroxide in the case of the latter was higher. The immobilization technique provided repeated application of the enzyme even in low concentration, and the hydrogen peroxide generated in the enzymatic reaction was successively used for textile bleaching.     

A hydrogen peroxide-forming NADH oxidase that functions as an alkyl hydroperoxide reductase in Amphibacillus xylanus

The Amphibacillus xylanus NADH oxidase, which catalyzes the reduction of oxygen to hydrogen peroxide with beta-NADH, can also reduce hydrogen peroxide to water in the presence of free flavin adenine dinucleotide (FAD) or the small disulfide-containing Salmonella enterica AhpC protein. The enzyme has two disulfide bonds, Cys128-Cys131 and Cys337-Cys340, which can act as redox centers in addition to the enzyme-bound FAD (K. Ohnishi, Y. Niimura, M. Hidaka, H. Masaki, H. Suzuki, T. Uozumi, and T. Nishino, J. Biol. Chem. 270:5812-5817, 1995). The NADH-FAD reductase activity was directly dependent on the FAD concentration, with a second-order rate constant of approximately 2.0 x 10(6) M(-1) s(-1). Rapid-reaction studies showed that the reduction of free flavin occurred through enzyme-bound FAD, which was reduced by NADH. The peroxidase activity of NADH oxidase in the presence of FAD resulted from reduction of peroxide by free FADH(2) reduced via enzyme-bound FAD. This peroxidase activity was markedly decreased in the presence of oxygen, since the free FADH(2) is easily oxidized by oxygen, indicating that this enzyme system is unlikely to be functional in aerobic growing cells. The A. xylanus ahpC gene was cloned and overexpressed in Escherichia coli. When the NADH oxidase was coupled with A. xylanus AhpC, the peroxidase activity was not inhibited by oxygen. The V(max) values for hydrogen peroxide and cumene hydroperoxide reduction were both approximately 150 s(-1). The K(m) values for hydrogen peroxide and cumene hydroperoxide were too low to allow accurate determination of their values. Both AhpC and NADH oxidase were induced under aerobic conditions, a clear indication that these proteins are involved in the removal of peroxides under aerobic growing conditions.     

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.     

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.     

Direct injection high-performance liquid chromatographic method with electrochemical detection for the determination of ethanol and methanol in plasma using an alcohol oxidase reactor

A highly sensitive reversed-phase high-performance liquid chromatographic assay for ethanol and methanol in plasma, using a post-column enzymic reactor with electrochemical detection, has been developed. The alcohols, separated on the column, were converted by immobilized alcohol oxidase into their respective aldehydes with formation of stoichiometric amounts of hydrogen peroxide, detected via oxidation at a platinum electrode. As the chromatographic column, two glass cartridges (150 mm × 3 mm I.D.) in series, packed with 10-μm HEMA-S 1000^® packing, were used. Alcohol oxidase from Candida boidinii was immobilized onto HEMA-BIO 1000 VS-L (10 μm), packed in a 30 mm × 3 mm I.D. glass cartridge. The reaction product, hydrogen peroxide, was detected with an amperometric detector with a platinum electrode, operated at +500 mV vs. an Ag/AgCl reference electrode. A 20-μl volume of ten-fold diluted plasma was injected without any pre-treatment. Under the described conditions, methanol and ethanol were well resolved from each other and from the “front” of the chromatogram. The limit of detection was ca. 2.5 nmol for ethanol and 0.6 nmol for methanol in plasma, at a signal-to-noise ratio of 3. Excellent linearity was observed for ethanol, in the range 0.125–4 μg injected (r = 0.9999). In contrast, the response for methanol was markedly non-linear above 500 μg injected, presumably owing to progressive saturation of the reactor. The precision and accuracy of the assay were satisfactory, as was the reactor life (one month).     

Immobilized glucose oxidase–catalase and their deactivation in a differential-bed loop reactor

Glucose oxidase containing catalase was immobilized with a copolymer of phenylenediamine and glutaraldehyde on pumice and titania carrier to study the enzymatic oxidation of glucose in a differential-bed loop reactor. The reaction rate was found to be first order with respect to the concentration of limiting oxygen substrate, suggesting a strong external mass-transfer resistance for all the flow rates used. The partial pressure of oxygen was varied from 21.3 up to 202.6 kPa. The use of a differential-bed loop reactor for the determination of the active enzyme concentration in the catalyst with negligible internal pore diffusion resistance is shown. Catalyst deactivation was studied, especially with respect to the presence of catalase. It is believed that the hydrogen peroxide formed in the oxidation reaction deactivates catalase first; if an excess of catalase is present, the deactivation of glucose oxidase remains small. The mathematical model subsequently developed adequately describes the experimental results.     

Determination of inorganic phosphorus using immobilized pyruvate oxidase

A new method for the automated analysis of inorganic phosphorus using immobilized enzyme was established. The method was based on the determination of hydrogen peroxide formed by the action of pyruvate oxidase on inorganic phosphate and pyruvate. Since pyruvate oxidase required inorganic phosphate for its stability and therefore had to be kept in a buffer containing inorganic phosphate, it could hardly be used as a reagent in the form of aqueous solution for the determination of inorganic phosphorus. This difficulty was overcome by using immobilized pyruvate oxidase in column form. When the present method was applied to the determination of inorganic phosphorus in serum, it gave perfect linearity of the data up to 0.20 g inorganic phosphorus/L with satisfactory precision, reproducibility, high sensitivity, and accurate recoveries. The immobilized enzyme reactor unit showed enhanced heat stability and good operational stability for a one-month period, during which time it was used over 900 times for analyses. The enzyme column was not affected by organic phosphorus compounds. The results correlated satisfactorily with those obtained by another well-established method.     

A chemiluminescence automatic analyser for the measurement of biological compounds

A compact automated analyser which could analyse constituents in biological fluids with a small sample volume and in a short time has been developed. The instrument was composed of a flow injection analysis system equipped with chemiluminometric detection and an immobilized enzyme column reactor used in combination. Chemiluminescence has high sensitivity, and its reaction proceeds very quickly. Furthermore, an immobilized enzyme column reactor can produce a sufficient amount of hydrogen peroxide from compounds in serum in a short time. When enzymes are used as reagents for the analysis of substances in blood or blood serum, the final signals emitted by different enzyme reactions are usually not only hydrogen peroxide but also ammonia, NAD(P)H and so on. However, the practical chemiluminescence method for ammonia and NAD(P)H has not been established. We have discovered a new practical method for ammonia and NAD(P)H using an enzyme column reactor consisting of both immobilized L -glutamate dehydrogenase and L -glutamate oxidase. The determinations of glucose and uric acid in serum by chemiluminometry after production of hydrogen peroxide by the respective oxidases are presented. A newly chemiluminometric determination of ammonia, NAD(P)H and its applications to other enzymatic analyses that give ammonia and NAD(P)H as a final signal are also described.     

An immobilized enzyme reactor for the detoxification of bilirubin

An immobilized enzyme reactor has been developed for the degradation of bilirubin as a potential treatment for neonatal jaundice. It utilizes the enzyme bilirubin oxidase from Myrothecium verrucaria, which in the presence of molecular oxygen converts bilirubin to biliverdin and other products that are much less toxic than bilirubin. Bilirubin oxidase was covalently attached to agarose beads using cyano transfer activation. Forty percent of the specific activity of bilirubin oxidase was retained after immmobilization, and preparations with 20 units of enzymatic activity per gram of drained wet weight of gel were obtained. The stability of bilirubin oxidase at pH 7.4 and 37 degrees C was improved fivefold by immobilization. A 15-mL column containing immobilized bilirubin oxidase, through which a 37 degrees C solution of 332muM bilirubin and 450muM human serum albumin in 0.05M phosphate buffer (pH 7.4) was passed at 1 mL/min, converted more than 60 percent of the bilirubin per pass. The substrate specificity of the enzyme and the small volume of the reactor are important characteristics for this clinical application where it is desirable to remove only one compound from the blood and to minimize the volume of blood in the extracorporeal circuit. This reactor, by detoxifying the jaundiced infant's blood of bilirubin, would eliminate the risks associated with the use of donor blood as is done currently in treating severe neonatal jaundice.     

Steady-state generation of hydrogen peroxide: kinetics and stability of alcohol oxidase immobilized on nanoporous alumina

Alcohol oxidase from Pichia pastoris was immobilized on nanoporous aluminium oxide membranes by silanization and activation by carbonyldiimidazole to create a flow-through enzyme reactor. Kinetic analysis of the hydrogen peroxide generation was carried out for a number of alcohols using a subsequent reaction with horseradish peroxidase and ABTS. The activity data for the immobilized enzyme showed a general similarity with literature data in solution, and the reactor could generate 80 mmol H₂O₂/h per litre reactor volume. Horseradish peroxidase was immobilized by the same technique to construct bienzymatic modular reactors. These were used in both single pass mode and circulating mode. Pulsed injections of methanol resulted in a linear relation between response and concentration, allowing quantitative concentration measurement. The immobilized alcohol oxidase retained 58 % of initial activity after 3 weeks of storage and repeated use.     

Immobilization of manganese peroxidase from Lentinula edodes and its biocatalytic generation of MnIII-chelate as a chemical oxidant of chlorophenols

Manganese peroxidase (MnP) purified from commercial cultures of Lentinula edodes was covalently immobilized through its carboxyl groups using an azlactone-functional copolymer derivatized with ethylenediamine and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) as a coupling reagent. The tethered enzyme was employed in a two-stage immobilized MnP bioreactor for catalytic generation of chelated MnIII and subsequent oxidation of chlorophenols. Manganese peroxidase immobilized in the enzyme reactor (reactor 1) produced MnIII-chelate, which was pumped into another chemical reaction vessel (reactor 2) containing the organopollutant. Reactor 1-generated MnIII-chelates oxidized 2,4-dichlorophenol and 2,4, 6-trichlorophenol in reactor 2, demonstrating a two-stage enzyme and chemical system. H2O2 and oxalate chelator concentrations were varied to optimize the immobilized MnP's oxidation of MnII to MnIII. Oxidation of 1.0 mM MnII to MnIII was initially measured at 78% efficiency under optimized conditions. After 24 h of continuous operation under optimized reaction conditions, the reactor still oxidized 1.0 mM MnII to MnIII with approximately 69% efficiency, corresponding to 88% of the initial MnP activity.     

A flow injection analysis system involving immobilized NADH oxidase in column form for clinical analysis.

A highly sensitive FIA system for chemiluminometric determination of reduced coenzyme, NADH, was developed, using immobilized NADH oxidase from Brevibacterium ammoniagenes. The enzyme catalyzed the oxidation of NADH generating hydrogen peroxide which emitted chemiluminescence when mixed with luminol and potassium ferricyanide. The immobilized enzyme reactor was a mini-column, measuring 1 or 2 mm in inner diameter and 20 mm in length, and the sample volume was only 1 microliter per assay, with a feeding speed of one sample per min and a lowest detection limit of 10 pmol NADH. A FIA system was also developed for the determination of magnesium in human serum, using an enzyme column reactor with simultaneously coimmobilized hexokinase, D-glucose-6-phosphate dehydrogenase, and NADH oxidase. The performance of the system was as satisfactory as a routine colorimetric assay, but with much higher sensitivity.     

Immobilized enzymes: a prototype apparatus for oxidase enzymes in chemical analysis utilizing covalently bound glucose oxidase

A prototype analytical device incorporating an immobilized oxidase enzyme reactor is described. The monitor assembly involves the continuous measurement of dissolved oxygen in the sample stream and uses a commercially available Clark electrode mounted into a miniature vortex mixer. Utilizing glucose oxidase covalently coupled to porous glass particles a reagentless analytical procedure is presented for glucose in both simple and complex biological fluids. Operational limitations are described and characterized for both kinetic and endopoint methods of analysis.     

A comparative study of free and immobilized soybean and horseradish peroxidases for 4-chlorophenol removal: protective effects of immobilization

Horseradish peroxidase (HRP) and soybean peroxidase (SBP) were covalently immobilized onto aldehyde glass through their amine groups. The activity yield and the protein content for the immobilized SBP were higher than for the immobilized HRP. When free and immobilized peroxidases were tested for their ability to remove 4-chlorophenol from aqueous solutions, the removal percentages were higher with immobilized HRP than with free HRP, whereas immobilized SBP needs more enzyme to reach the same conversion than free enzyme. In the present paper the two immobilized derivatives are compared. It was found that at an immobilized enzyme concentration in the reactor of 15 mg l(-1), SBP removed 5% more of 4-chlorophenol than HRP, and that a shorter treatment was necessary. Since immobilized SBP was less susceptible to inactivation than HRP and provided higher 4-chlorophenol elimination, this derivative was chosen for further inactivation studies. The protective effect of the immobilization against the enzyme inactivation by hydrogen peroxide was demonstrated.     

Immobilization of epoxide hydrolase from Aspergillus niger onto DEAE-cellulose: enzymatic properties and application for the enantioselective resolution of a racemic epoxide

Recombinant epoxide hydrolase (EH) from Aspergillus niger can be a very promising tool for the resolution of various racemic epoxides by enantioselective hydrolysis. The enzyme was successfully immobilized by ionic adsorption onto DEAE-cellulose (99% yield, 70% of retention activity). The temperature for maximal activity (40 °C) and the activation energy (38.8 kJ/mol) were similar for both the immobilized and free EHs, whereas the optimal pH was about one unit less for the immobilized enzyme. Thermal stability was also affected by immobilization; the immobilized enzyme appeared to be slightly less stable than the free one. However, a gram-scale resolution of racemic para-chlorostyrene oxide (pCSO) was successfully carried out in a repeated batch reactor, operated for seven cycles. Furthermore, using a very high substrate concentration of 2 M (306 g/L), i.e. biphasic conditions, the resolution of 3 g of pCSO was also achieved in a repeated batch reactor using approximately 300 mg of immobilized EH, corresponding to less than 3 mg of the enzymatic powder.