Lysine Oxidase Research Articles

Flow injection chemiluminescence determination of ornithine and sequential determination of ornithine and lysine by using immobilized lysine oxidase

The flow injection chemiluminescence (CL) determination of ornithine and the sequential determination of lysine and ornithine by using immobilized lysine oxidase are described. Hydrogen peroxide produced in the enzyme-catalysed reaction is determined by luminol CL. The calibration graph obtained by injecting ornithine solutions into the carrier stream (phosphate buffer, pH 8.0), is linear over the range 1.0×10 −6–2.4×10 −5 M. The detection limit (3σ) for ornithine is 4×10 −7 M. The sample throughput is 60 h −1. Sequential determination of lysine and ornithine is based on chemiluminescence measurements on a lysine/ornithine mixture at both pH 6.0 (lysine response only) and 8.0 (lysine and ornithine). Lysine and ornithine can be determined in the range 1.2×10 −7–6×10 −5 M. Relative errors are in the range 3–6%. The interference effects of some amino acids and metal ions are investigated.

Amperometric determination of lysine using a lysine oxidase biosensor based on rigid-conducting composites

In this study, amperometric biosensors based on rigid conducting composites are developed for the determination of lysine. These lysine biosensors consist of chemically immobilized lysine oxidase membranes attached to either graphite-methacrylate or peroxidase-modified graphite-methacrylate electrodes. The enzymatic degradation of lysine releases hydrogen peroxide, which is the basis of the amperometric detection. The direct oxidation of hydrogen peroxide is monitored at +1000 mV with a graphite-methacrylate electrode, while with the peroxidase-modified electrode reductive detection is performed. In addition, for the peroxidase-modified biocomposite electrode, both direct electron transfer and hydroquinone-mediated detection are studied. For the lysine biosensor based on the hydroquinone-mediated peroxidase biocomposite, the linear range is up to 1.6×10 −4 M, the sensitivity 11300 μA/M, the repeatability 1.8%, the detection limit 8.2×10 −7 M and the response time t 95% is 42 s. The proposed biosensors are used to determine lysine in pharmaceutical samples. Results are consistent with those obtained with the standard method.

Determination of lysine in pharmaceutical samples containing endogenous ammonium ions by using a lysine oxidase biosensor based on an all-solid-state potentiometric ammonium electrode

A new potentiometric method is proposed to determine lysine in pharmaceutical samples. This method is based on a lysine biosensor consisting of a chemically immobilized lysine oxidase membrane attached to an all-solid-state ammonium electrode. Lysine is degraded in the sensor to release ammonium, which is detected by means of the ammonium electrode. The presence of endogenous ammonium in the samples interferes with these determinations, since the response measured corresponds to the sum of the ammonium generated enzymatically and that present in the sample. This is a general drawback for all biosensors based on the detection of ammonium. Study of samples containing both lysine and ammonium showed that concentration ranges exist in which a near-logarithmic relationship between potentials measured and lysine concentrations is found. Therefore, within these ranges, lysine can be determined by using the standard addition method, with the subsequent data treatment involving an iterative linearization procedure. Results obtained with the proposed potentiometric method are consistent with those given by the standard method for amino acid analysis.

Potentiometric biosensor for lysine analysis based on a chemically immobilized lysine oxidase membrane

An enzymatic lysine electrode is developed for the potentiometric analysis of lysine (Lys). This lysine biosensor consists of a chemically immobilized lysine oxidase membrane attached to an all-solid-state ammonium electrode. In this system, the enzymatic degradation of lysine produces ammonia, which is then detected with the ammonium electrode. The influence of immobilization conditions on the potentiometric response and the lifetime of the electrode was studied. Under optimal conditions, the relationship between the potentiometric response and the log [Lys] was linear between 3×10 −5 and 10 −3 M, the sensitivity was 50 mV/decade, the repeatability 1.4%, and the response time t 95% was 15 s.

Flow injection amperometric and chemiluminescence individual and simultaneous determination of lysine and glucose with immobilized lysine oxidase and glucose oxidase

Flow injection amperometric and chemiluminescence methods for the individual and simultaneous determination of lysine and glucose by using lysine oxidase and glucose oxidase are described in this paper. The calibration graphs for lysine are linear over the range 1 × 10 −5 − 1.6 × 10 −4 and 2.05 × 10 −7 − 2.75 × 10 −5 M for amperometric and chemiluminescence detection respectively. The detection limits (3σ) for lysine are 5 × 10 −6 M with amperometric and 4.0 × 10 −8 M with chemiluminescence detection. The sample throughput rates are 80 and 100 h −1, respectively. Lysine and glucose concentrations can be monitored simultaneously in the range 1.5 − 9 × 10 −5 M and 7 × 10 −7−8 × 10 −6 M with amperometric and chemiluminescence detection respectively. The interference effects of some amino acids and metal ions are examined. An on-line cation-exchange minicolumn is inserted into the chemiluminescence manifold to remove the interference effect of Fe 2+ and Cu 2+.

Fast Analysis of Lysine in Food Using Protein Microwave Hydrolysis and an Electrochemical Biosensor

Abstract A fast procedure for lysine analysis in food was developed by coupling in sequence a microwave protein hydrolysis technique with a lysine enzyme electrode. Protein hydrolysis was carried out in 6N HCl using sealed vessels located in a microwave digestion system. Parameters such as irradiation power, pressure, time and temperature were varied to select the best conditions for the hydrolysis. The hydrolysate was then analyzed for lysine using an electrochemical biosensor based on an amperometric H2O2 transducer and the enzyme lysine oxidase covalently immobilized on a preactivated polymer support. Analytical parameters, such as pH, buffer and measurement system were studied in order to eliminate or minimize the enzymatic interferences. Bovine serum albumin and two farro (T. dicoccum) meals (whole and sieved) were processed for lysine content. The analysis was carried out with this new procedure (microwave hydrolysis + biosensor) and by traditional hydrolysis coupled with ion-exchange chromatography (IEC). Results obtained with the two procedures correlated well.

Selective Biosensing of l-Lysine by a Low-Temperature Flow-Injection Technique Using an Immobilized Lysine Oxidase Reactor

An automated flow-injection analytical system, equipped with an immobilized L-lysine-α-oxidase reactor (stable for over 6 months) and having a hydrogen peroxide electrode in the manifold, was developed for biosensing L-lysine. A substantial improvement in the selectivity for lysine was achieved by lowering the system temperature to 10°C; the sensor response was linearly dependent on the L-lysine concentration (from tens of μM to mM order). The analytical results were verified by HPLC using protein hydrolyzates as test solutions comprising various interfering amino acids. By the aid of an in-line filter, the leaching of lysine from L-lysine-enriched fish feed into an aqueous buffer was automatically monitored.

Production of fructosyl lysine oxidase from Fusarium oxysporum S-1F4 on autoclave-browned medium.

In Fusarium oxysporum S-1F4, fructosyl lysine oxidase (FLOD) was induced with N epsilon-fructosyl N alpha-Z-lysine (epsilon-FL), which is a model compound of a glycated protein, and the induction was inhibited by the addition of cycloheximide in the growing cells. FLOD formation was greatly enhanced in an autoclave-browned medium containing glucose and L-lysine. Some Amadori compounds formed from glucose and L-lysine during autoclaving were assumed to induce the enzyme. After optimization of the culture conditions, FLOD produced in the browned medium was comparable to that in the medium with epsilon-FL.

Dual Functionalities of 4-Aminodiphenylamine in Enzymatic Assay and Mediated Biosensor Construction

4-Aminodiphenylamine (N-phenyl-1,4-phenylenedi-amine, CAS 101-54-2) and its water-soluble HCl salt (CAS 2198-59-6) were demonstrated to be efficient mediators for glucose oxidase, lactate oxidase, xanthine oxidase, and lysine oxidase. Using cyclic voltammetry, single oxidative peak potentials were observed for scans ranging from 0 to 0.5 V vs Ag/AgCl. The half-wave potential for both preparations was 0.11 V vs Ag/AgCl at pH 7 and decreased 59 mV per unit pH increase. Peak current data were analyzed to estimate diffusivities of 0.8 × 10−5cm2/s for soluble 4-ADPA HCl, and 2.36 × 10−5cm2/s for 4-ADPA solubilized in 2.5 mM2-hydroxypropyl-β-cyclodextrin. The overall second-order kinetic constants (k) for the reaction of reduced glucose oxidase with oxidized 4-ADPA HCl and 4-ADPA in cyclodextrin were estimated to be 1.8 × 105and 1.7 × 105M−1s−1, respectively, using cyclic voltammetry measurements at varied scan rates and enzyme concentrations. Both preparations proved to be suitable electron acceptors for horseradish peroxidase, as indicated by changes in absorbance spectra upon oxidation or reduction. The electrochemical and spectral behavior of the preparations were applied in conjunction with glucose oxidase to devise mediated amperometric and hydrogen peroxide-coupled spectrophotometric assays for glucose. The results of both assays compared favorably with the hexokinase reference method.

Purification and properties of fructosyl lysine oxidase from Fusarium oxysporum S-1F4.

Fructosyl lysine oxidase (FLOD) was examined for its use in the enzymatic measurement of the level of glycated albumin in blood serum. To isolate microorganisms having such an enzyme activity, we used N epsilon-fructosyl N alpha-Z-lysine (epsilon-FL) as a sole nitrogen source in the enrichment culture medium. The isolated fungus, strain S-1F4, showed a high FLOD activity in the cell-free extract and was identified as Fusarium oxysporum. FLOD was purified to an apparent homogeneity on SDS-PAGE. The molecular mass of the subunit was 50 kDa on SDS-PAGE and seemed to exist in a monomeric form. The enzyme had an absorption spectrum characteristic of a flavoprotein and the flavin was found to be covalently bound to the enzyme. The enzyme acted against N epsilon-fructosyl N alpha-Z-lysine and N alpha-fructosyl N epsilon-Z-lysine and showed specificity for fructosyl lysine residues.

Photodiode-based chemiluminometric biosensors for hydrogen peroxide and L-lysine

Photodiode-based chemiluminometric biosensors for hydrogen peroxide and L-lysine were developed. The sensors were used in a flow injection set-up to determine hydrogen peroxide and lysine. Hydrogen peroxide could be measured with a detection limit of 10 −6 M. Lysine determinations were performed with a lysine oxidase / microbial peroxidase membrane in a range between 5 μM and 10 mM lysine.

Chemiluminometric l-lysine determination with immobilized lysine oxidase by flow-injection analysis

An enzymatic flow-injection procedure for the determination of l-lysine was developed. A lysine oxidase reactor is combined with a fibre-optic hydrogen peroxide detector. Hydrogen peroxide detection is based on the peroxidase-catalysed luminol reaction. The chemiluminescent light is detected by a photomultiplier. l-Lysine can be determined in the range 10–1000 μM. A sampling rate of up to 90 h − can be achieved. The whole sensing assay works for more than 1 month. The double logarithmic graph of the peak signal height vs. the lysine concentration is linear, the slope being larger than unity ( r 2 = 0.991, n = 4).

Amperometric lysine bioprobes analysis in feeds

Amperometric enzyme electrode probes have been constructed for the specific determination of L-lysine and used in batch and flow analysis. The enzyme lysine oxidase was immobilized on a preactivated polymer support which was placed on a platinum electrode. Additional blocking membranes conferred high stability, reproducibility and avoided electrochemical and enzyme interferences. Parameters including pH, temperature, storage and operational times were optimized. Lysine was determined in the range 10 −6–2.10 −3 M with a detection limit of 5 × 10 −7 M. The Michaelis constant was 2 × 10 −3 M. This value was approximately two order of magnitudes higher than that reported in literature for the free enzyme. The response time of the probe was about 2 min in batch and flow analysis and 30 sec in flow injection analysis (FIA). The resulting probes were stable for more than three months with more than 300 analyses performed. The determination of lysine was carried out by both flow-through analysis and FIA. Analysis in feeds was carried out by acid hydrolysis to liberate lysine; then the solution was analyzed by the bioprobe and HPLC procedures. Results by the two methods correlated well.

Oxidation of peptidyl lysine by copper complexes of pyrroloquinoline quinone and other quinones. A model for oxidative pathochemistry

Various o- and p-quinones were assessed as oxidants of peptidyl lysine in elastin and collagen substrates in the presence and absence of divalent copper as paradigms of protein-lysine 6-oxidase (lysyl oxidase) which contains both quinone and copper cofactors. Pyrroloquinoline quinone was among the most active in the absence and the most active of the o- and p-quinones tested in the presence of copper. The optimal rate of elastin oxidation occured at a 2:1 PQQ/CU(II) ratio while Cu(II) itself oxidized elastin relatively slightly. Elastin oxidation by 2:1 PQQ/Cu(II) required aerobic conditions consistent with oxygen-dependent turnover of this catalytic pair. Dimethylsulfoxide and catalase individually or in combination inhibited elastin oxidation by PQQ/CuA(II) by approx. 50%, suggesting that oxygen free radical species participate in the reaction. Amino-acid analysis of elastin and collagen substrates oxidized by 2:1 PQQ/Cu and then reduced with borohydride revealed that α- maminoadipic-δ-semialdehyde and lesser amounts of covalent cross-linkages were generated by this oxidant. In contrast, lysine oxidase produced aldehydes and significantly greater quantities of cross-linkage products, consistent with the known specificity of the enzyme. These data, thus, indicate the potential for free quinones, such as PQQ, particularly when stimulated by appropriate metal ions, to act as adventitious oxidants of lysine side-chains in proteins.

Deficiency of copper can cause neuronal degeneration

The aim of this article is to emphasize the important role that copper plays in the function of nerve cells. We are reporting preliminary data which suggest that the swelling of axons which we produce in rats by iminodipropionitrile, IDPN, is due to its chelating action on copper, and how conversely supplementation with copper abolishes both symptoms and lesions. The copper values we obtained by atomic absorption spectrophotometry of the spinal cord and brain from the animals fully support this contention. In comparing these results with the diseases that are known to be due to copper deficiency, namely Menkes disease in man, swayback in lambs and several neurological mutant mice, we find not only similar axonal swellings, but also amelioration of symptoms and lesions by early administration of copper. Considering the main forms in which copper is present, we discuss the cuproproteins, i.e. ceruloplasmin and metallothionein, and their role in transport and delivery of copper to various organs. Further, the many cuproenzymes i.e. superoxide dismutase, tryptophan-2,3-dioxygenase, lysine oxidase, cytochrome oxidase, monoamine oxidases, tyrosinase, dopamine-β-hydroxylase and d-amino levulinate dehydratase are noted for their roles in the nervous system. Finally, we suggest that neuronal copper deficiency should be more fully investigated as a possible etiological factor in the more common neurodegenerative diseases, such as Alzheimer's disease and amyotrophic lateral sclerosis, ALS.

Anti-invasive and antimetastatic action of lysine oxidase fromTrichoderma sp. in vitro and in vivo

A new fungal strain, Trichoderma sp., discovered in Moscow, produces the antitumor enzyme, lysine-oxidase, which demonstrates an anti-invasive effect in vitro and anti-metastatic activity in vivo. Maximal inhibition of the in vitro invasion of MM1 clone cells was obtained when the tumor cells were pretreated with 2.5 mU/ml of lysine-oxidase; the pretreatment caused a 1.9-times reduction in cell growth and a 1.6-times reduction in the invasive capacity. We studied its anti-metastatic effect on the spreading Lewis lung carcinoma (3LL) in mice from which the primary tumor had been removed. The administration of the enzyme (50 U/kg, i.v.) significantly decreased not only the extent but the number of lung metastases, as compared with the untreated mice. In addition to that, the lysine-oxidase treatment considerably increases the life-span of mice from which the primary tumor had been removed (200 days after 3LL implantation, lysine-oxidase treatment caused surviving of 50% mice in experimental group).

Colorimetric detection of microbially excreted lysine directly on agar plates

We have developed a procedure for colorimetrically detecting microbially excreted lysine directly on agar plates. The methodology described here, using lysine as the target metabolite, is meant to serve as a model system that could be generalized to any metabolite if suitably specific detection chemistry is available. The plate procedure that we have developed utilizes a lysine oxidase [4]/peroxidase-coupled chemical methodology to generate a dye having an absorption maximym at 460 nm. A two-layer plate format was adopted, which consists of a nutrient layer to support microbial growth deposited over a detection layer containing the enzymes and the precursor chromagen system.

The inhibition of protein-lysine 6-oxidase by various lathyrogens. Evidence for two different mechanisms.

Lathyrogens decrease collagen and elastin cross-linking by inhibiting lysine oxidase. The lathyrogens isoniazid and semicarbazide decrease liver pyridoxal phosphate and are teratogenic; all their effects are reversed by pyridoxal. beta-Aminopropionitrile, another lathyrogen, does not affect liver pyridoxal phosphate, and its lathyrogenic and teratogenic effects are not reversed by pyridoxal. Time courses of these effects differ greatly, suggesting enzyme inhibition by different mechanisms.

The effect of hypoxia on the synthesis of collagen and glycosaminoglycans by cultured pig aortic endothelium

Porcine aortic endothelium cultured at 20% oxygen concentration synthesizes collagen and three of its marker enzymes — proline and lysine hydroxylases and lysine oxidase, the cross-link enzyme. It also synthesizes and secretes hyaluronic acid, dermatan sulphate, possibly heparan sulphate, large amounts of chondroitin 4-sulphate and smaller amounts of chondroitin 6-sulphate. Growth of these cells for 24 h in 0%, or 2% oxygen results in little change in cell numbers or cell protein but a fall in collagen synthesis and in proline and lysine hydroxylases, but a rise in lysine oxidase. There is a considerable increase in synthesis and secretion of all the glycosaminoglycans found. The cell lipids appear qualitatively unchanged. Apart from increased lysosomes seen at 0% oxygen, no ultrastructural changes appear to occur. These findings illustrate the lability of the endothelial response to oxygen lack.