Integrated gene engineering synergistically improved substrate-product transport, cofactor generation and gene translation for cadaverine biosynthesis in E. coli.

Several approaches for efficient production of cadaverine, a bio-based diamine with broad industrial applications have been explored. Here, Serratia marcescens lysine decarboxylase (SmcadA) was expressed in E. coli; mild surfactants added in biotransformation reactions; the E. coli native lysine/cadaverine antiporter cadB, E. coli pyridoxal kinases pdxK and pdxY overexpressed and synthetic RBS libraries screened. Addition of mild surfactants and overexpression of antiporter cadB increased cadaverine biosynthesis of SmcadA. Moreover, expression of pdxY gene yielded 19.82 g/L in a reaction mixture containing added cofactor precursor pyridoxal (PL), without adding exogenous PLP. The screened synthetic RBS1, applied to fully exploit pdxY gene expression, ultimately resulted in PLP self-sufficiency, producing 27.02 g/L cadaverine using strain T7R1_PL. To boost SmcadA catalytic activity, the designed mutants Arg595Lys and Ser512Ala had significantly improved cumulative cadaverine production of 219.54 and 201.79 g/L respectively compared to the wild-type WT (181.62 g/L), after 20 h reaction. Finally, molecular dynamics simulations for WT and variants indicated that increased flexibility at the binding sites of the protein enhanced residue-ligand interactions, contributing to high cadaverine synthesis. This work demonstrates potential of harnessing different pull factors through integrated gene engineering of efficient biocatalysts and gaining insight into the mechanisms involved through MD simulations.

Indicator Layers Based on Ethylene-Vinyl Acetate Copolymer (EVA) and Dicyanovinyl Azobenzene Dyes for Fast and Selective Evaluation of Vaporous Biogenic Amines.

The article presents naked-eye methods for fast, sensitive, and selective detection of isopentylamine and cadaverine vapours based on 4-N,N-dioctylamino-4'-dicyanovinylazobenzene (CR-528) and 4-N,N-dioctylamino-2'-nitro-4'-dicyanovinylazobenzene (CR-555) dyes immobilized in ethylene-vinyl acetate copolymer (EVA). The reaction of CR-528/EVA and CR-555/EVA indicator layers with isopentylamine vapours caused a vivid colour change from pink/purple to yellow/orange-yellow. Additionally, CR-555/EVA showed colour changes upon exposure to cadaverine. The colour changes were analysed by ultraviolet⁻visible (UV/VIS) molecular absorption spectroscopy for amine quantification, and the method was partially validated for the detection limit, sensitivity, and linear concentration range. The lowest detection limits were reached with CR-555/EVA indicator layers (0.41 ppm for isopentylamine and 1.80 ppm for cadaverine). The indicator layers based on EVA and dicyanovinyl azobenzene dyes complement the existing library of colorimetric probes for the detection of biogenic amines and show great potential for food quality control.

Highly Sensitive, Printable Nanostructured Conductive Polymer Wireless Sensor for Food Spoilage Detection.

Near-field communication (NFC) labeling technology has been recently used to endow smartphones with nonline-of-sight sensing functions to improve the environment, human health, and quality of life. For applications in detecting food spoilage, the development of a sensor with high enough sensitivity to act as a switch for an NFC tag remains a challenge. In this Letter, we developed a nanostructured conductive polymer-based gas sensor with high sensitivity of Δ R/ R0 = 225% toward 5 ppm ammonia NH3 and unprecedented sensitivities of 46% and 17% toward 5 ppm putrescine and cadaverine, respectively. The gas sensor plays a critical role as a sensitive switch in the circuit of the NFC tag and enables a smartphone to readout meat spoilage when the concentration of biogenic amines is over a preset threshold. We envision the broad potential use of such intelligent sensing for food status monitoring applications in daily life, storage and supply chains.

Determination of Nonvolatile Amines in Foods by Improved Dansyl Derivatization Reaction.

An analytical method for the determination of nonvolatile amines (putrescine, cadaverine, histamine, tyramine, and spermidine) in foods was developed, using an improved dansyl derivatization technique. The five amines were extracted from food with 1% trichloroacetic acid. Three milliliter of extract was applied to a polymer-based strong cation exchange resin mini-column, which was washed with 5 mL of water, and eluted with 5 mL of 1 mol/L potassium carbonate solution. The eluate was dansylated, then 5 mL of toluene was added with shaking. The toluene layer was evaporated. The residue was taken up in 1 mL of acetonitrile and shaken with 1 mL of 5% proline in 1 mol/L potassium carbonate solution. The upper acetonitrile layer was collected, filtered, and subjected to HPLC. The limits of quantitation for putrescine and cadaverine in the samples were both 0.2 µg/g; those of spermidine, tyramine, and histamine were 0.8, 2.0, and 5.0 µg/g, respectively. The average recoveries of the five amines from nine foods exceeded 80%.

Cadaverine Production by Using Cross-Linked Enzyme Aggregate of Escherichia coli Lysine Decarboxylase.

Lysine decarboxylase (CadA) converts L-lysine into cadaverine (1,5-pentanediamine), which is an important platform chemical with many industrial applications. Although there have been many efforts to produce cadaverine through the soluble CadA enzyme or Escherichia coli whole cells overexpressing the CadA enzyme, there have been few reports concerning the immobilization of the CadA enzyme. Here, we have prepared a cross-linked enzyme aggregate (CLEA) of E. coli CadA and performed bioconversion using CadACLEA. CadAfree and CadACLEA were characterized for their enzymatic properties. The optimum temperatures of CadAfree and CadACLEA were 60°C and 55°C, respectively. The thermostability of CadACLEA was significantly higher than that of CadAfree. The optimum pH of both enzymes was 6.0. CadAfree could not be recovered after use, whereas CadACLEA was rapidly recovered and the residual activity was 53% after the 10th recycle. These results demonstrate that CadACLEA can be used as a potential catalyst for efficient production of cadaverine.

Free and Cell Wall-Bound Polyamines under Long-Term Water Stress Applied at Different Growth Stages of ×Triticosecale Wittm.

BACKGROUND: Long-stemmed and semi-dwarf cultivars of triticale were exposed to water stress at tillering, heading and anthesis stage. Quantitative determination of free and cell wall-bound polyamines, i.e. agmatine, cadaverine, putrescine, spermidine and spermine, was supplemented with an analysis of quantitative relationships between free and cell wall-bound polyamines. RESULTS: The content of free and cell wall-bound polyamines varied depending on the development stage, both under optimal and water stress conditions. Drought-induced increase in free agmatine content was observed at all developmental stages in long-stemmed cultivar. A depletion of spermidine and putrescine was also reported in this cultivar, and spermidine was less abundant in semi-dwarf cultivar exposed to drought stress at the three analyzed developmental stages. Changes in the content of the other free polyamines did not follow a steady pattern reflecting the developmental stages. On the contrary, the content of cell wall-bound polyamines gradually increased from tillering, through heading and until anthesis period. CONCLUSION: Water stress seemed to induce a progressive decrease in the content of free polyamines and an accumulation of cell wall-bound polyamines.

Electromembrane extraction of salivary polyamines followed by capillary zone electrophoresis with capacitively coupled contactless conductivity detection.

Electromembrane extraction (EME) as a novel sample preparation technique was firstly applied for the purification and enrichment of four polyamines mainly present in saliva samples. These four target analytes, putrescine, cadaverine, spermidine, and spermine, were directly determined by CZE with capacitively coupled contactless conductivity detection (CZE-C(4)D) after EME procedure. Several factors affecting extraction efficiency, electrophoretic separation, and detection were investigated. Under the optimum conditions, four polyamines were baseline separated within 22 min, exhibiting a linear calibration over three orders of magnitude (r>0.999); the highest enrichment factor could reach 106-fold (for spermidine), and the LODs were in the range of 1.4-7.0 ng mL(-1). The proposed EME/CZE-C(4)D method has been successfully applied to analyze human saliva samples with recoveries in the range of 78-97%.

Dual roles of cadaverine-producing Pseudomonas sp. on Microcystis spp. in hyper-eutrophic water.

A bacterium isolated from Lake Taihu was identified as Pseudomonas sp. A3CT, which performed different effects on Microcystis spp. Growth of Microcystis flos-aquae and Microcystis aeruginosa was assessed in co-culture with A3CT to determine the stimulatory or inhibitory effects on these toxic, bloom-forming Microcystis strains. Results demonstrated that the impacts of A3CT were species specific. A3CT promoted the growth of M. aeruginosa but inhibited growth of M. flos-aquae. To investigate the cause of this phenomenon, the chemical composition of A3CT exudates and the impact of exposure to A3CT exudates on the two Microcystis species were determined. Results suggested that the observed differential growth responses of the two microalgae to A3CT exposure might be related to two components in A3CT exudates NH4 (+) and cadaverine. Growth stimulation of M. aeruginosa by A3CT was significantly related to NH4 (+) concentration. Cadaverine possibly acted as a growth inhibitor of M. flos-aquae. The different effects of cadaverine on growth of the two Microcystis strains suggested that A3CT might play a role in intrageneric succession patterns observed during Microcystis blooms in Lake Taihu.

A bisamide and four diketopiperazines from a marine-derived Streptomyces sp.

A new bisamide N1-acetyl-N7-phenylacetyl cadaverine (1) and a series of diketopiperazines including a new diketopiperazine cyclo(2-hydroxy-Pro-R-Leu) (2), together with a new natural product cyclo(4-hydroxy-S-Pro-S-Trp) (3) and two known leucine-based diketopiperazines cyclo(4-hydroxy-R-Pro-S-Leu) (4) and cyclo (S-Pro-R-Leu) (5), were isolated from ethyl acetate extract of a fermentation broth of a marine-derived Streptomyces sp. Their structures were elucidated by the interpretation of spectroscopic analysis. The antitumor activities of compounds 1-3 against HL-60 cell lines were tested by MTT assay.

A sequential injection analysis/chemiluminescent plant tissue-based biosensor system for the determination of diamine.

In this paper, a new chemiluminescent plant tissue-based biosensor for diamine detection was presented by employing sequential injection analysis (SIA), which facilitates precise fluidic handling and lower consumption of sample and reagents. Pea-seedling tissue acted as the molecular recognition element and was packed in a mini-PTFE column and further incorporated in the SIA system. The analysis of diamines, such as putrescine and cadaverine, is based on an enzymatic conversion which takes place in the plant tissue column to produce hydrogen peroxide. The formed hydrogen peroxide was detected by a chemiluminescence reaction involving luminol and Co(2+). Under the optimal conditions, the linear calibration graphs were obtained within 0.2-80 microM (putrescine) and 0.5-100 microM (cadaverine). The detection limits of 0.03 and 0.06 microM were achieved for putrescine and cadaverine, respectively, along with the relative standard deviations of 2.14% and 3.08% (n=11) and a sampling frequency of 40 h(-1). The present biosensor has been used for the analysis of diamine in fish samples with an acceptable accuracy.

PCR detection of foodborne bacteria producing the biogenic amines histamine, tyramine, putrescine, and cadaverine.

This study describes an easy PCR method for the detection of foodborne bacteria that potentially produce histamine, tyramine, putrescine, and cadaverine. Synthetic oligonucleotide pairs for the specific detection of the gene coding for each group of bacterial histidine, tyrosine, ornithine, or lysine decarboxylases were designed. Under the conditions used in this study, the assay yielded fragments of 372 and 531 bp from histidine decarboxylase-encoding genes, a 825-bp fragment from tyrosine decarboxylases, fragments of 624 and 1,440 bp from ornithine decarboxylases, and 1,098- and 1,185-bp fragments from lysine decarboxylases. This is the first PCR method for detection of cadaverine-producing bacteria. The method was successfully applied to several biogenic amine-producing bacterial strains.

Separation of biogenic amines by micellar electrokinetic chromatography with on-line chemiluminescence detection.

A method of on-line chemiluminescence detection with capillary electrophoresis for biogenic amines (diaminopropane, putrescine, cadaverine and diaminohexane) labeled with N-(4-aminobutyl)-N-ethylisoluminol is reported for the first time. Two separation modes, capillary zone electrophoresis and micellar electrokinetic chromatography (MEKC), were studied. The results show that excellent resolution was achieved in MEKC. Parameters affecting separation process and chemiluminescence detection have been examined in detail. Under the optimum conditions, the baseline separation of four amines was obtained within 7.5 min. The detection limits (S/N=3) of diaminopropane, putrescine, cadaverine and diaminohexane are 3.5 x 10(-8), 3.5 x 10(-8), 3.9 x 10(-8) and 1.2 x 10(-7) M, respectively. The method was applied to the analysis of biogenic amines in lake water.

Separation of the enantiomers of substituted putrescine and cadaverine analogues by gas chromatography on chiral and achiral stationary phases.

Capillary gas chromatography (GC) on chiral stationary phases, i.e., Chirasil-Val [L-valine-tert.-(R)-alpha-butylamide] and XE-60-S-valine-(R)-alpha-phenylethylamide, has been applied to the resolution of various substituted analogues of putrescine as their N,N'-perfluoroacyl derivatives. The influence of the nature of the substituent on the retention behaviour and on the resolution of the enantiomers was studied. The results are discussed in terms of volatility and interaction with the chiral stationary phase. The 1,4-disubstituted putrescine analogues with two chiral centres were also clearly resolved into their corresponding stereoisomers. When the chain length between the two amino groups was increased, no clear resolution was obtained of the monosubstituted cadaverine analogues as their N,N'-perfluoroacyl derivatives. However, resolution was obtained after derivatization of the cadaverine analogues with (-)-alpha-methoxy-alpha-trifluoromethylphenylacetyl chloride, followed by GC analysis on an achiral phase.

A new rapid and simple assay for factor XIII activity using dansylcadaverine incorporation and gel filtration.

A new sensitive, reproducible and simple assay for factor XIII activity was developed, in which fluorescent dansylcadaverine (DC) is incorporated into casein and DC-casein is separated from free DC by gel filtration. A good correlation was found between our method and Lorand's. The main advantages of our method are: (1) our assay technique is simple with good reproducibility and takes only a couple of hours; (2) the detectable limit of factor XIII activity is about 1% of normal pool plasma, but, if necessary, can be lowered by increasing the sample amount and lengthening the reaction time.

[Method of determining low-molecular weight oligoamines in various biological materials]. / Metod opredeleniia nizkomolekuliarnykh oligoaminov v razlichnom biologicheskom materiale.

A modified procedure is described for thin-layer chromatography of dansyl-spermine, -spermidine, -cadaverine and -putrescine. Distinct separation of the dansyl derivatives studied from dansyl-ammonium, which usually causes difficulties, was achieved. This procedure enabled to estimate the pyckomolar quantities of biogenic oligoamines. The method does not require radioactive compounds, enables to carry out the measurements without the use of spectrofluorimeter or fluorescent densitometer. The densitometry of the chromatogram photo-prints was successfully used for quantitative estimation of the oligoamines.

An enzymatic fluorometric assay for L-lysine in plasma and tissue.

A simple, specific, and reliable method has been developed for the determination of L-lysine in blood plasma and tissue. The L-lysine in the sample is decarboxylated enzymatically, and fluorescamine is added to a pentan-1-ol extract of the cadaverine formed. This produces a stable product which is measured fluorometrically.

Improved analysis for urinary polyamines by use of high-voltage electrophoresis on paper.

This method for assay of polyamines (putrescine, spermidine, and spermine) in large numbers of urine samples is based on preliminary analyte isolation by use of a Dowex 50W column, separation by high-voltage paper electrophoresis, reaction with ninhydrin to detect the separated amines, and subsequent direct assay with a dual-wave-length thin-layer chromatography scanner by the zigzag scanning method. By optimizing the conditions for the Dowex 50W column and high-voltage paper electrophoresis, putrescine, cadaverine, spermidine, and spermine were completely separated. The major technical improvements are room-temperature collection of urine with toluene, shorter hydrolysis time, complete separation of putrescine from cadaverine and histamine, and elimination of interference with spermine measurement. Polyamine concentrations as measured by this method agreed with those obtained by use of an amino acid analyzer. Reference intervals by this method were similar to those reported by Russell (Clin. Chem. 23: 22, 1977) with an automated amino acid analyzer. Putrescine, spermidine, and spermine excretion in urine from patients with blood cancers and solid cancers was significantly increased; information on concentrations of putrescine and spermidine were especially useful for following the clinical efficacy of cancer treatments.

Partial purification and properties of a transamidinase from Lathyrus sativus seedlings. Involvement in homoarginine metabolism and amine interconversions.

A transamidinase was purified 463-fold from Lathyrus sativus seedlings by affinity chromatography on homoarginine--Sepharose. The enzyme exhibited a wide substrate specificity, and catalysed the reversible transfer of the amidino groups from donors such as arginine, homoarginine and canavanine to acceptors such as lysine, putrescine, agmatine, cadaverine and hydroxylamine. The enzyme could not be detected in the seeds, and attained the highest specific activity in the embryo axis on day 10 after seed germination. Its thiol nature was established by strong inhibition by several thiol blockers and thiol compounds in the presence of ferricyanide. In the absence of an exogenous acceptor, it exhibited weak hydrolytic activity towards arginine. It had apparent mol.wt. 210000, and exhibited Michaelis--Menten kinetics with Km 3.0 mM for arginine. Ornithine competitively inhibited the enzyme, with Ki 1.0 mM in the arginine--hydroxylamine amidino-transfer reaction. Conversion experiments with labelled compounds suggest that the enzyme is involved in homoarginine catabolism during the development of plant embryo to give rise to important amino acids and amine metabolites. Presumptive evidence is also provided for its involvement in the biosynthesis of the guanidino amino acid during seed development. The natural occurrence of arcain in L. sativus and mediation of its synthesis in vitro from agmatine by the transamidinase are demonstrated.