Determination Of L-lysine Research Articles

Perfluorosulfonic Acid Membranes with Short and Long Side Chains and Their Use in Sensors for the Determination of Markers of Viral Diseases in Saliva.

The development of accessible express methods to determine markers of viral diseases in saliva is currently an actual problem. Novel cross-sensitive sensors based on Donnan potential with bio-comparable perfluorosulfonic acid membranes for the determination of salivary viral markers (N-acetyl-L-methionine, L-carnitine, and L-lysine) were proposed. Membranes were formed by casting from dispersions of Nafion or Aquivion in N-methyl-2-pyrollidone or in a mixture of isopropyl alcohol and water. The influence of the polymer equivalent weight and the nature of dispersing liquid on water uptake, ion conductivity, and slope of Donnan potential for the membranes in H+ and Na+ form was investigated. The varying of the sorption and transport properties of perfluorosulfonic acid membranes provided a change in the distribution of the sensor sensitivity to N-acetyl-L-methionine, L-carnitine, and L-lysine ions, which was necessary for multisensory system development. The simultaneous determination of three analytes, and the group analysis of them in artificial saliva solutions, was performed. The errors of N-acetyl-L-methionine and L-carnitine determination were 4-12 and 3-11%, respectively. The determination of L-lysine was complicated by its interaction with Ca2+ ions. The error of the group analysis was no greater than 9%. The reverse character of the viral markers' sorption by the membranes provided long-term sensor operation.

Dye/metal ion-based chemosensing ensemble towards l-histidine and l-lysine determination in water via different optical responses.

In this study, a novel simple type of label-free, ultra-sensitive, and highly selective UV-Vis absorption and naked-eye detection of histidine (His) and lysine (Lys) using a dye/metal ion ensemble is developed. The outcoming high sensitivity and selectivity for histidine and lysine were attained by changing the metal ions. The indicator is released due to its displacement from the murexide (Mure)/Cu2+ complex by histidine and the change in absorbance may be due to the further complexation of lysine with the additional coordination sites present in the zinc atom of Mure/Zn2+ complex. The label-free chemosensor provided sensitive and selective detection of l-histidine and l-lysine with detection limits of 9.1 and 9.4nmolL-1, respectively. The protocol especially offers high selectivity for the determination of His and Lys among amino acids found in human urine samples. Furthermore, INHIBIT and NAND molecular logic gates were obtained using chemical inputs and UV-Vis absorbance signal output.

Amperometric l-lysine determination biosensor amplified with l-lysine oxidase nanoparticles and graphene oxide nanoparticles

We describe the amplification of amperometric l-lysine biosensor using l-lysine oxidase nanoparticles (LOxNPs) and graphene oxide nanoparticles (GrONPs) immobilized onto pencil graphite electrode (PGE). LOxNPs and GrONPs were characterized by UV spectroscopy and transmission electron microscopy (TEM). The working electrode (LOxNPs/GrONPs/PGE) was studied by scanning electron microscopy (SEM) and cyclic voltammetry at different stages of its construction. The biosensor showed optimum current at 0.7 V, pH 6.5, 35 °C, a detection limit of 0.01 μM, response time as 3.95 s and a wider linear range 0.01–1000 μM. The analytical recovery of added lysine in sera was 97 %. The within assay and between batch coefficients of variation for the biosensor were 0.068 and 0.074 % respectively. The biosensor measured l-lysine levels in sera of healthy adults and human immunodeficiency virus (HIV) patients. The biosensor exhibited good correlation with standard spectrophotometric method (R2 = 0.989). The biosensor lost 35 % of its original activity after its regular uses for a period of 180 days, while being stored dry at 4 °C.

Transcriptional study of the enhanced ε-poly-L-lysine productivity in culture using glucose and glycerol as a mixed carbon source.

A glucose-glycerol mixed carbon source (MCS) can substantially reduce batch fermentation time and improve ε-poly-L-lysine (ε-PL) productivity, which was of great significance in industrial microbial fermentation. This study aims to disclose the physiological mechanism by transcriptome analyses. In the MCS, the enhancements of gene transcription mainly emerged in central carbon metabolism, L-lysine synthesis as well as cell respiration, and these results were subsequently proved by quantitative real-time PCR assay. Intracellular L-lysine determination and exhaust gas analysis further confirmed the huge precursor L-lysine pool and active cell respiration in the MCS. Interestingly, in the MCS, pls was remarkably up-regulated than those in single carbon sources without transcriptional improvement of HrdD, which indicated that the improved ε-PL productivity was supported by other regulators rather than hrdD. This study exposed the physiological basis of the improved ε-PL productivity in the MCS, which provided references for studies on other biochemicals production using multiple substrates.

L-lysine determination in animal feed using microbiological microtiter plate assay

Chromatographic methods are most commonly used for the analysis of amino acids; however, there is growing need for faster, simpler and more price-effective assays. In this paper, the applicability of a rapid microbiological assay for quantification of the total content of L-lysine in feed samples was evaluated. The assay relies on the dependency of bacterial growth of Pediococcus acidilactici on the presence of L-lysine. Microbiological microtiter plate assay method for the quantitative determination of total (added and natural) L-lysine in feed samples has been verified, and parameters such as accuracy, precision, limit of detection, and limit of determination were evaluated. Results of total L-lysine determination in different feed samples have been compared with results of validated HPLC method. The microbiological microtiter plate assay method can be employed as a qualitative and quantification method for total L-lysine determination with detection and determination limit of 0.040 % and 0.085 %, respectively. However, further research on the influence of sample matrix on the determination of low lysine levels is required.

A Toolbox of Genetically Encoded FRET-Based Biosensors for Rapid l-Lysine Analysis.

Background: The fast development of microbial production strains for basic and fine chemicals is increasingly carried out in small scale cultivation systems to allow for higher throughput. Such parallelized systems create a need for new rapid online detection systems to quantify the respective target compound. In this regard, biosensors, especially genetically encoded Förster resonance energy transfer (FRET)-based biosensors, offer tremendous opportunities. As a proof-of-concept, we have created a toolbox of FRET-based biosensors for the ratiometric determination of l-lysine in fermentation broth. Methods: The sensor toolbox was constructed based on a sensor that consists of an optimized central lysine-/arginine-/ornithine-binding protein (LAO-BP) flanked by two fluorescent proteins (enhanced cyan fluorescent protein (ECFP), Citrine). Further sensor variants with altered affinity and sensitivity were obtained by circular permutation of the binding protein as well as the introduction of flexible and rigid linkers between the fluorescent proteins and the LAO-BP, respectively. Results: The sensor prototype was applied to monitor the extracellular l-lysine concentration of the l-lysine producing Corynebacterium glutamicum (C. glutamicum) strain DM1933 in a BioLector® microscale cultivation device. The results matched well with data obtained by HPLC analysis and the Ninhydrin assay, demonstrating the high potential of FRET-based biosensors for high-throughput microbial bioprocess optimization.

A novel potentiometric biosensor for determination of L-lysine in commercial pharmaceutical L-lysine tablet and capsule

The construction of an L-lysine biosensor on ammonium-selective poly(vinylchloride) (PVC) membrane electrode is described in this study. The construction procedure occurs in two stages: (I) the preparation of ammonium-selective poly(vinylchloride) (PVC) membrane electrode and (II) the chemical immobilization of lysine oxidase on this ammonium-selective electrode by using glutaraldehyde. The ammonium ions produced after enzymatic reaction were determined potentiometrically. The sensitivity of the lysine biosensor against ammonium ions and lysine were studied. The response time, linear working range, reproducibility and life time of the biosensor were also determined. The interfering effect of other amino acids on the biosensor performance was also studied and potentiometric selectivity coefficients were calculated. Although the biosensor responded mainly against tyrosine, a lot of amino acids and ascorbic acid that can be present in some real samples did not show any important interference. Additionally, lysine assay in commercial pharmaceutical lysine tablets and capsules was also successfully carried out. The results were in good agreement with previously reported values.

A novel amperometric biosensor based on a co-crosslinked l-lysine-α-oxidase/overoxidized polypyrrole bilayer for the highly selective determination of l-lysine

An amperometric biosensor for the determination of l-lysine based on l-lysine-α-oxidase immobilized by co-crosslinking on a platinum electrode previously modified by an overoxidized polypyrrole film is described. The optimization of experimental parameters, such as pH and flow rate, permitted to minimize significantly substrate interferences even using a low specific, commercial enzyme. The relevant biases introduced in the measurement of lysine were just about 1% for l-arginine, l-histidine and l-ornithine, roughly 4% for l-phenylalanine and l-tyrosine. The developed approach allowed linear lysine responses from 0.02mM up to 2mM with a sensitivity of 41nA/(mM×mm2) and a detection limit of 4μM (S/N=3). No appreciable loss in lysine sensitivity was observed up to about 40 days. Allowing polypyrrole layer to remove interference from electroactive compounds, the present method revealed suitable to detect l-lysine in a pharmaceutical and cheese sample, showing a good agreement with the expected values.

Amperometric biosensor based on diamond paste for the enantioanalysis of l-lysine

An amperometric biosensor was proposed for the enantioanalysis of l-lysine. The biosensor is based on the impregnation of l-lysine oxidase in diamond paste. The potential used for the determination of l-lysine was 650mV. The biosensor exhibited a linear concentration range between 1 and 100nmol/L with a limit of detection of 4pmol/L. The selectivity of the biosensor is high over other amino acids, such as l-serine, l-leucine, l-aspartic acid, l-glutamic acid, histamine, glycine. The proposed biosensor can be applied for the determination of l-lysine in serum samples and pharmaceutical compounds.

Flow injection electrochemiluminescence determination of l-lysine using tris(2,2′-bipyridyl) ruthenium(ii) (Ru(bpy)32+) on indium tin oxide (ITO) glass

Download options Please wait... Article information DOI https://doi.org/10.1039/C0AY00632G Article type Paper Submitted 20 Oct 2010 Accepted 02 Mar 2011 First published 12 Apr 2011 Download Citation Anal. Methods, 2011,3, 1163-1167 BibTex EndNote MEDLINE ProCite ReferenceManager RefWorks RIS Permissions Request permissions Flow injection electrochemiluminescence determination of L-lysine using tris(2,2′-bipyridyl) ruthenium(II) (Ru(bpy)32+) on indium tin oxide (ITO) glass S. Wang, J. Yu, F. Wan, S. Ge, M. Yan and M. Zhang, Anal. Methods, 2011, 3, 1163 DOI: 10.1039/C0AY00632G To request permission to reproduce material from this article, please go to the Copyright Clearance Center request page. If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given. If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page. Read more about how to correctly acknowledge RSC content. Social activity Tweet Share Search articles by author Shaowei Wang Jinghua Yu Fuwei Wan Shenguang Ge Mei Yan Meng Zhang

Determination of plasma and serum l-lysine using l-lysine ε-oxidase from Marinomonas mediterranea NBRC 103028 T

This article describes a successful application of l-lysine ε-oxidase (EC 1.4.3.20) for l-lysine determination. l-Lysine ε-oxidase was isolated from culture supernatant of Marinomonas mediterranea NBRC 103028 T and was used for l-lysine determination. Comparison of the characteristics of l-lysine ε-oxidase with l-lysine α-oxidase, a commercial enzyme used for l-lysine determination, suggests that the use of l-lysine ε-oxidase would be more valuable for the determination of l-lysine because of its selectivity and sensitivity, especially in samples with low l-lysine concentration. The enzyme acted only on l-lysine and l-ornithine, to which the relative activity was only 3.4% of that on l-lysine. The value obtained by the colorimetric assay using l-lysine ε-oxidase and horseradish peroxidase was not affected by l-ornithine. The enzyme also shows a higher affinity for l-lysine ( K m = 0.0018 mM). l-Lysine determination using l-lysine ε-oxidase in human plasma and serum was examined. The measured values were close to values determined by instrumental analyses using the precolumn AccQ·Tag Ultra Derivatization Kit. These results suggest that l-lysine ε-oxidase can be used for diagnosis based on plasma l-lysine concentration. This is the first report on the application of l-lysine ε-oxidase.

Partially disposable biosensors for the quick assessment of damage in foodstuff after thermal treatment

A novel approach towards a quick, easy and reliable solution to the quite complex problems connected with the Amadori's compounds and furosine formation in several foodstuff is here proposed and based on enzymatic reactions, either as far as L-lysine release after partial hydrolysis from proteic material of the food matrix (bioavailable L-lysine), or as far as the determination of total and free L-lysine is concerned. The contribution proposes the use of specific L-lysine sensitive bioelectrodes for the evaluation of the possible heat damage through the rapid measure of L-lysine consumption. The L-lysine biosensor has been realized by immobilizing the enzyme L-lysine-α-oxidase (LOx) on an amperometric H 2O 2 sensitive electrode. The bioelectrode-enzymatic reaction arrangement allows to perform determinations of L-lysine and bioavailable L-lysine in foodstuff from different sources and after different thermal treatments in a very rapid inexpensive and reliable way in the range 1.0–33 ppm, with a limit of detection LOD = 0.5 ppm, so that a considerable number of samples can be easily monitored quickly and with very low costs.

Sensitive determination of l-lysine with a new amperometric microbial biosensor based on Saccharomyces cerevisiae yeast cells

A new amperometric microbial biosensor based on Saccharomyces cerevisiae NRRL-12632 cells, which had been induced for lysine oxidase enzyme and immobilized in gelatin by a cross-linking agent was developed for the sensitive determination of L-lysine amino acid. To construct the microbial biosensor S. cerevisiae cells were activated and cultured in a suitable culture medium. By using gelatine (8.43 mg cm(-2)) and glutaraldehyde (0.25%), cells obtained in the logarithmic phase of the growth curve at the end of a 14 h period were immobilized and fixed on a pretreated oxygen sensitive Teflon membrane of a dissolved oxygen probe. The assay procedure of the microbial biosensor is based on the determination of the differences of the respiration activity of the cells on the oxygenmeter in the absence and the presence of L-lysine. According to the end point measurement technique used in the experiments it was determined that the microbial biosensor response depended linearly on L-lysine concentrations between 1.0 and 10.0 microM with a 1 min response time. In optimization studies of the microbial biosensor, the most suitable microorganism quantities were found to be 0.97x10(5)CFU cm(-2). In addition phosphate buffer (pH 7.5; 50 mM) and 30 degrees C were obtained as the optimum working conditions. In characterization studies of the microbial biosensor some parameters such as substrate specificity, interference effects of some substances on the microbial biosensor responses, reproducibility of the biosensor and operational and storage stability were investigated.

Flow Injection Analysis System for l-Lysine Estimation in Foodstuffs Using a Biosensor Based on Lysine Oxidase Immobilization on a Gold-Poly(m-Phenylenediamine) Electrode

The development and the evaluation of an amperometric FIA biosensing system for l-lysine determination in foodstuffs is described. The system employs a home-made flow-cell with an amperometric biosensor, prepared by immobilization of lysine oxidase on a gold-poly(m-phenylenediamine) electrode. A peristaltic pump is connected to the flow-cell via a 2-position 6-port sample injector/switching valve for the on-line transfer of the samples. The analytical procedure is based on the oxidation of l-lysine by l-lysine α-oxidase. The produced H2O2 is monitored amperometrically at 650 mV in FIA set-up. The lysine biosensor construction procedure comprises: a) the electropolymerization of m-phenylenediamine on a Si-gold strip electrode and b) the immobilization of l-lysine α-oxidase on the poly(m-phenylenediamine) membrane with glutaraldehyde in the presence of bovine serum albumin. Parameters, such as, flow rate, enzyme amount, monomer concentration, matrix effect, effect of interference's, etc., were studied. The flow-cell biosensor system provides a linear response from 1 × 10−3 to 5 × 10−5 M of lysine, with excellent characteristics of reproducibility, stability, and life-time. The system was further applied and evaluated for the estimation of lysine in various hydrolysate food samples (e.g., pasta, milk, wheat flour, etc.) without any pretreatment with an average recovery of 97.3%.

P-chip and P-chip bienzyme electrodes based on recombinant forms of horseradish peroxidase immobilized on gold electrodes.

Adsorption and bioelectrocatalytic activity of native horseradish peroxidase (HRP) and its recombinant forms on polycrystalline gold electrodes were studied. Recombinant forms of HRP were produced by a genetic engineering approach using an E. coli expression system. According to direct mass measurements with a quartz crystal microbalance, all the forms of HRP formed monolayer coverage of the enzyme on the gold surface. However, only gold electrodes modified with the recombinant HRP forms (non-glycosylated) exhibited high and stable current response to H2O2 due to its bioelectrocatalytic reduction based on direct electron transfer (ET) between gold and the active site of the enzyme. Introduction of a six-His tag either at the C-terminus or at the N-terminus of the enzyme molecule additionally increased the strength of the enzyme binding with the gold surface and the efficiency of direct ET. Immobilization of recombinant forms of HRP containing histidine functional groups on the surface of the gold electrode was used both for the development of a P-chip, a biosensor for hydrogen peroxide determination based on direct ET, and for the development of a bienzyme biosensor electrode for the determination of L-lysine based on co-immobilized recombinant forms of HRP and L-lysine-alpha-oxidase.

Development of an interferent free amperometric biosensor for determination of l-lysine in food

A highly selective, fast responding amperometric biosensor is described, useful for the determination of l-lysine in food. Common electrochemical interferences, like acetoaminophen and ascorbic acid have zero response at +100 mV applied onto a ruthenium/rhodium coated glassy carbon electrode covered with 1,2-diaminobenzene polymer. This novel transducer was coupled with l-lysine α-oxidase purified from Trichoderma viride and at the appropriate pH, classic substrate interferences from l-ornithine, l-arginine and l-phenylalanine are reduced to 3.4, 1.1 and 0.7% of the response to l-lysine (taken as 100%). No other amino acids respond. The sensor is inexpensive to produce, has excellent repeatability and very good reproducibility. Thus, the l-lysine (protein) content of foods can be almost specifically determined following rapid microwave digestion of the product.

Screen-printed enzyme sensors for l-lysine determination.

Sensors for the determination of L-lysine in samples of fermentation broth have been developed. Low-cost screen-printed sensors comprising a platinum working electrode, an Ag/AgCl pseudo reference and a carbon counter electrode were used as transducers for the enzyme sensors. L-lysine-(alpha)-oxidase from Trichoderma viride has been immobilized by entrapment into a polyurethane hydrogel. Sensors were characterized for L-lysine with respect to pH value, linear range, reproducibility, repeatability, storage and working stability. The sensitivities to other amino acids were also determined. A batch system with two working electrodes, one with immobilized enzyme and one without was adapted for the determination of L-lysine by differential measurements. Good agreement was found between L-lysine concentrations measured by the enzyme sensors and by a conventional amino acid analyzer.

Enantiomer Discrimination by Continuous Precipitation

A continuous turbidimetric method for the discrimination of enantiomers (L- and D-lysine) by the inhibitory action of L-lysine on the crystallization of L-glutamic acid is proposed. A multidetection flow system including an open-closed loop and a single detector permits the determination of kinetic parameters for the crystallization of L-glutamic acid in the presence of 2-propanol. L-Lysine can thus be determined in the presence of a 20 times higher D-lysine concentration. The proposed method was applied to the determination of L-lysine in pharmaceutical preparations with good results.

Flow-Injection Amperometric Biosensor Based on Immobilized L-Lysine-α-oxidase for L-Lysine Determination

Abstract Amperometric flow-injection analytical system for quantitation of low L-lysine concentration was described. L-lysine-α-oxidase from Trichoderme sp. was immobilized by covalent linking on silica gel. The shift in oxygen concentration during enzymatic reaction was detected with a Clark membrane electrode. The rate of oxygen consumption was linearly dependent on L-lysine concentration over the 0,20 - 5,5 mM. Response time was 14 s, the total assay time about 2 min. The influences of pH, ionic strength and nature of the buffer on enzyme catalytic activity were tested. The analysis conditions were optimized. The immobilized L-lysine-α-oxidase retained the catalytic activity for about 5 months.

Crystallization of organic crystals with “tailor-made” inhibitors. Determination of L-lysine using L-glutamic acid as substrate

A very simple turbidimetric procedure is used for determination of L-lysine, based on its inhibitory action on the crystallization of L-glutamic acid. The obtention of supersaturated solutions of L-glutamic acid was accomplished by means of the change in the solvent composition. Thus, the addition of ethanol to stable aqueous solutions of this amino acid allows the obtainment of unstable supersaturated solutions. The method suffers from very few interferences and permits the development of an analytical procedure to determine L-lysine in the presence of D-lysine. The method was also applied to the determination of L-lysine in pharmaceutical products.