Combined assessment of lysine and N-acetyl cadaverine levels assist as a potential biomarker of the smoker periodontitis.

Periodontitis is an inflammatory condition of supporting structures of teeth leading to attachment and bone loss. Cigarette smoking is the single most important and modifiable risk factor with 5 to 20-fold susceptibility for periodontal diseases. Reverse smoking is a peculiar habit of smoking where the lit end is kept inside the mouth, which is predominant in the northern coastal districts of Andhra Pradesh. Polyamines are biologically active amines involved in tissue regeneration and modulation of inflammation. The study aimed to evaluate polyamines and check their utility as a marker in detection of periodontitis among different groups. Total polyamine levels showed significant increase in reverse smokers with periodontitis when compared to the other groups. Qualitative analysis by thin layer chromatography showed three polyamine bands with varying intensity among the different groups. Mass spectrometric and NMR analyses of the three bands identified them as N1, N8-diacetyl spermidine, N-acetyl cadaverine and lysine. Most significantly elevated levels of lysine was observed in the smoker and reverse smoker periodontitis groups when compared to healthy and non-smoker periodontitis groups. The significantly elevated levels of N-acetyl cadaverine could be responsible for the more destruction of periodontium in the reverse smoker group. Antioxidant potential decreased significantly in different smoker periodontitis groups. The present study suggests that the quantitative analysis of salivary polyamines, lysine and N-acetyl cadaverine can aid as an easy noninvasive diagnostic method for assessing the periodontal status, especially in smokers.

Cadaverine and putrescine exposure influence carbon and nitrogen cycling genes in water and sediment of the Yellow River.

The response patterns of microbial functional genes involved in biogeochemical cycles to cadaver decay is a central topic of recent environmental sciences. However, the response mechanisms and pathways of the functional genes associated with the carbon (C) and nitrogen (N) cycling to cadaveric substances such as cadaverine and putrescine remain unclear. This study explored the variation of functional genes associated with C fixation, C degradation and N cycling and their influencing factors under cadaverine, putrescine and mixed treatments. Our results showed only putrescine significantly increased the alpha diversity of C fixation genes, while reducing the alpha diversity of N cycling genes in sediment. For the C cycling, the mixed treatment significantly decreased the total abundance of reductive acetyl-CoA pathway genes (i.e., acsB and acsE) and lig gene linked to lignin degradation in water, while only significantly increasing the hydroxypropionate-hydroxybutylate cycle (i.e., accA) gene abundance in sediment. For the N cycling, mixed treatment significantly decreased the abundance of the nitrification (i.e., amoB), denitrification (i.e., nirS3) genes in water and the assimilation pathway gene (i.e., gdhA) in sediment. Environmental factors (i.e., total carbon and total nitrogen) were all negatively associated with the genes of C and N cycling. Therefore, cadaverine and putrescine exposure may inhibit the pathway in C fixation and N cycling, while promoting C degradation. These findings can offer some new insight for the management of amine pollution caused by animal cadavers.

A direct enzymatic evaluation platform (DEEP) to fine-tuning pyridoxal 5'-phosphate-dependent proteins for cadaverine production.

Pyridoxal 5'-phosphate (pyridoxal phosphate, PLP) is an essential cofactor for multiple enzymatic reactions in industry. However, cofactor engineering based on PLP regeneration and related to the performance of enzymes in chemical production has rarely been discussed. First, we found that MG1655 strain was sensitive to nitrogen source and relied on different amino acids, thus the biomass was significantly reduced when PLP excess in the medium. Then, the six KEIO collection strains were applied to find out the prominent gene in deoxyxylulose-5-phosphate (DXP) pathway, where pdxB was superior in controlling cell growth. Therefore, the clustered regularly interspaced short palindromic repeats interference (CRISPRi) targeted on pdxB in MG1655 was employed to establish a novel direct enzymatic evaluation platform (DEEP) as a high-throughput tool and obtained the optimal modules for incorporating of PLP to enhance the biomass and activity of PLP-dependent enzymes simultaneously. As a result, the biomass has increased by 55% using PlacI promoter driven pyridoxine 5'-phosphate oxidase (PdxH) with a trace amount of precursor. When the strains incorporated DEEP and lysine decarboxylase (CadA), the cadaverine productivity was increased 32% due to the higher expression of CadA. DEEP is not only feasible for high-throughput screening of the best chassis for PLP engineering but also practical in fine-tuning the quantity and quality of enzymes.

Multi-omics methods reveal that putrescine and cadaverine cause different degrees of enrichment of high-risk resistomes and opportunistic pathogens in the water and sediment of the Yellow River.

Contamination of antibiotic resistomes due to animal carcass decay has become a serious environmental concern. However, the relationship between main metabolite compounds of corpse decomposition (i.e., putrescine and cadaverine) and antibiotic resistomes remains unclear. To tackle this issue, the response of antibiotic resistance genes (ARGs) and microbiome in aquatic environment to excess putrescine, cadaverine and a mixture of both based on laboratory simulation experiment was investigated by high-throughput quantitative PCR and amplicon sequencing methods. Our results showed putrescine and cadaverine led to the increasing of TC (total carbon) and TN (total nitrogen) both in water and sediment. Under the exposure of putrescine and cadaverine, the total abundance of mobile genetic elements (MGEs) and most ARGs in water was higher than in sediment. In particular, putrescine and cadaverine caused significantly different decreases in alpha diversity of microbial community in water and sediment compared with the control group. Microbial community structures both in water and sediment were also significantly affected by cadaverine and putrescine. Furthermore, putrescine and cadaverine led to different degrees of increases of high-risk ARGs (like mecA) and opportunistic pathogens (like Delftia) in sediment, promoting the prevalence of antibiotic resistant bacteria. In conclusion, our findings revealed the influences of main metabolites of carcass decay on microbiome and resistomes, providing references for risk assessment and pollution management.

Functional Polymeric Membranes with Antioxidant Properties for the Colorimetric Detection of Amines.

Herein, the ability of highly porous colorimetric indicators to sense volatile and biogenic amine vapors in real time is presented. Curcumin-loaded polycaprolactone porous fiber mats are exposed to various concentrations of off-flavor compounds such as the volatile amine trimethylamine, and the biogenic amines cadaverine, putrescine, spermidine, and histamine, in order to investigate their colorimetric response. CIELAB color space analysis demonstrates that the porous fiber mats can detect the amine vapors, showing a distinct color change in the presence of down to 2.1 ppm of trimethylamine and ca. 11.0 ppm of biogenic amines, surpassing the limit of visual perception in just a few seconds. Moreover, the color changes are reversible either spontaneously, in the case of the volatile amines, or in an assisted way, through interactions with an acidic environment, in the case of the biogenic amines, enabling the use of the same indicator several times. Finally, yet importantly, the strong antioxidant activity of the curcumin-loaded fibers is successfully demonstrated through DPPH● and ABTS● radical scavenging assays. Through such a detailed study, we prove that the developed porous mats can be successfully established as a reusable smart system in applications where the rapid detection of alkaline vapors and/or the antioxidant activity are essential, such as food packaging, biomedicine, and environmental protection.

A Blue Light-Responsive Strong Synthetic Promoter Based on Rational Design in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii (C. reinhardtii) is a single-cell green alga that can be easily genetically manipulated. With its favorable characteristics of rapid growth, low cost, non-toxicity, and the ability for post-translational protein modification, C. reinhardtii has emerged as an attractive option for the biosynthesis of various valuable products. To enhance the expression level of exogenous genes and overcome the silencing of foreign genes by C. reinhardtii, synthetic promoters such as the chimeric promoter AR have been constructed and evaluated. In this study, a synthetic promoter GA was constructed by hybridizing core fragments from the natural promoters of the acyl carrier protein gene (ACP2) and the glutamate dehydrogenase gene (GDH2). The GA promoter exhibited a significant increase (7 times) in expressing GUS, over the AR promoter as positive control. The GA promoter also displayed a strong responsiveness to blue light (BL), where the GUS expression was doubled compared to the white light (WL) condition. The ability of the GA promoter was further tested in the expression of another exogenous cadA gene, responsible for catalyzing the decarboxylation of lysine to produce cadaverine. The cadaverine yield driven by the GA promoter was increased by 1-2 times under WL and 2-3 times under BL as compared to the AR promoter. This study obtained, for the first time, a blue light-responsive GDH2 minimal fragment in C. reinhardtii, which delivered a doubling effect under BL when used alone or in hybrid. Together with the strong GA synthetic promoter, this study offered useful tools of synthetic biology to the algal biotechnology field.

Evaluation of the ecotoxicological effects of biogenic amines derived from cadaverous putrefaction on springtails Folsomia candida.

Necro-leachate, a liquid released during cadaveric decomposition, is considered the main culprit for impacts on cemetery environments. The biogenic amines cadaverine and putrescine make up part of the composition of necro-leachate and have a certain toxicity to different organisms. Springtails are among the most used bioindicators to assess the impacts of soil contaminants. As there are no data on the acute and chronic toxicity of springtails exposed to cadaverine and putrescine, the objective of this study was to evaluate the toxic potential of both amines, under the behavioral effect of avoidance and reproduction in the species Folsomia candida. Springtails were exposed to soils contaminated with different concentrations of cadaverine and putrescine, and different mixtures of these amines. To evaluate the avoidance and reproduction test, the individuals were exposed for periods of 48 h and 28 days, respectively. The results obtained in the avoidance test showed that springtails exhibited a preference for the treated soil in both isolated and mixed treatments. The chronic evaluation assays showed that the reproduction was affected, particularly in the treatments with combined amines, resulting in a reduction in the total number of juveniles. From the results, it is possible to infer that the methods applied in this research have provided information that will contribute to a better understanding of the toxicity of putrefactive biogenic amines, since there exist few ecotoxicological studies carried out with these amines, and especially with those from cemetery environments.

Discovery of ancestral L-ornithine and L-lysine decarboxylases reveals parallel, pseudoconvergent evolution of polyamine biosynthesis.

Polyamines are fundamental molecules of life, and their deep evolutionary history is reflected in extensive biosynthetic diversification. The polyamines putrescine, agmatine, and cadaverine are produced by pyridoxal 5'-phosphate-dependent L-ornithine, L-arginine, and L-lysine decarboxylases (ODC, ADC, LDC), respectively, from both the alanine racemase (AR) and aspartate aminotransferase (AAT) folds. Two homologous forms of AAT-fold decarboxylase are present in bacteria: an ancestral form and a derived, acid-inducible extended form containing an N-terminal fusion to the receiver-like domain of a bacterial response regulator. Only ADC was known from the ancestral form and limited to the Firmicutes phylum, whereas extended forms of ADC, ODC, and LDC are present in Proteobacteria and Firmicutes. Here, we report the discovery of ancestral form ODC, LDC, and bifunctional O/LDC and extend the phylogenetic diversity of functionally characterized ancestral ADC, ODC, and LDC to include phyla Fusobacteria, Caldiserica, Nitrospirae, and Euryarchaeota. Using purified recombinant enzymes, we show that these ancestral forms have a nascent ability to decarboxylate kinetically less preferred amino acid substrates with low efficiency, and that product inhibition primarily affects preferred substrates. We also note a correlation between the presence of ancestral ODC and ornithine/arginine auxotrophy and link this with a known symbiotic dependence on exogenous ornithine produced by species using the arginine deiminase system. Finally, we show that ADC, ODC, and LDC activities emerged independently, in parallel, in the homologous AAT-fold ancestral and extended forms. The emergence of the same ODC, ADC, and LDC activities in the nonhomologous AR-fold suggests that polyamine biosynthesis may be inevitable.

Cadaverine regulates biotin synthesis to modulate primary root growth in Arabidopsis.

Cadaverine, a polyamine, has been linked to modification of root growth architecture and response to environmental stresses in plants. However, the molecular mechanisms that govern the regulation of root growth by cadaverine are largely unexplored. Here we conducted a forward genetic screen and isolated a mutation, cadaverine hypersensitive 3 (cdh3), which resulted in increased root-growth sensitivity to cadaverine, but not other polyamines. This mutation affects the BIO3-BIO1 biotin biosynthesis gene. Exogenous supply of biotin and a pathway intermediate downstream of BIO1, 7,8-diaminopelargonic acid, suppressed this cadaverine sensitivity phenotype. An in vitro enzyme assay showed cadaverine inhibits the BIO3-BIO1 activity. Furthermore, cadaverine-treated seedlings displayed reduced biotinylation of Biotin Carboxyl Carrier Protein 1 of the acetyl-coenzyme A carboxylase complex involved in de novo fatty acid biosynthesis, resulting in decreased accumulation of triacylglycerides. Taken together, these results revealed an unexpected role of cadaverine in the regulation of biotin biosynthesis, which leads to modulation of primary root growth of plants.

PaeA (YtfL) protects from cadaverine and putrescine stress in Salmonella Typhimurium and E. coli.

Salmonella and E. coli synthesize, import, and export cadaverine, putrescine, and spermidine to maintain physiological levels and provide pH homeostasis. Both low and high intracellular levels of polyamines confer pleiotropic phenotypes or lethality. Here, we demonstrate that the previously uncharacterized inner membrane protein PaeA (YtfL) is required for reducing cytoplasmic cadaverine and putrescine concentrations. We identified paeA as a gene involved in stationary phase survival when cells were initially grown in acidic medium, in which they produce cadaverine. The paeA mutant is also sensitive to putrescine, but not to spermidine or spermine. Sensitivity to external cadaverine in stationary phase is only observed at pH > 8, suggesting that the polyamines need to be deprotonated to passively diffuse into the cell cytoplasm. In the absence of PaeA, intracellular polyamine levels increase and the cells lose viability. Degradation or modification of the polyamines is not relevant. Ectopic expression of the known cadaverine exporter, CadB, in stationary phase partially suppresses the paeA phenotype, and overexpression of PaeA in exponential phase partially complements a cadB mutant grown in acidic medium. These data support the hypothesis that PaeA is a cadaverine/putrescine exporter, reducing potentially toxic levels under certain stress conditions.

Metabolic engineering of Escherichia coli for high production of 1,5-pentanediol via a cadaverine-derived pathway.

1,5-Pentanediol (1,5-PDO) is a high value-added chemical which is widely used as a monomer in the polymer industry. There are no natural organisms that could directly produce 1,5-PDO from renewable carbon sources. In this study, we report metabolic engineering of Escherichia coli for high-level production of 1,5-PDO from glucose via a cadaverine-derived pathway. In the newly proposed pathway, cadaverine can be converted to 1,5-PDO via 5-hydroxyvalerate (5-HV) by introducing only one heterologous enzyme in E. coli. Different endogenous genes of E. coli were screened and heterologous carboxylic acid reductase genes were tested to build a functional pathway. Compared to the previously reported pathways, the engineered cadaverine-based pathway has a higher theoretical yield (0.70 mol/mol glucose) and higher catalytic efficiency. By further combining strategies of pathway engineering and process engineering, we constructed an engineered E. coli strain that could produce 2.62 g/L 1,5-PDO in shake-flask and 9.25 g/L 1,5-PDO with a yield of 0.28 mol/mol glucose in fed-batch fermentation. The proposed new pathway and engineering strategies reported here should be useful for developing biological routes to produce 1,5-PDO for real application.

A novel co-production of cadaverine and succinic acid based on a thermal switch system in recombinant Escherichia coli.

BACKGROUND: Polyamide (nylon) is an important material, which has aroused plenty of attention from all aspects. PA 5.4 is one kind of nylon with excellent property, which consists of cadaverine and succinic acid. Due to the environmental pollution, bio-production of cadaverine and succinic acid has been more attractive due to the less pollution and environmental friendliness. Microbes, like Escherichia coli, has been employed as cell factory to produce cadaverine and succinic acid. However, the accumulation of cadaverine will cause severe damage on cells resulting in inhibition on cell growth and cadaverine production. Herein, a novel two stage co-production of succinic acid and cadaverine was designed based on an efficient thermos-regulated switch to avoid the inhibitory brought by cadaverine. RESULTS: The fermentation process was divided into two phase, one for cell growth and lysine production and the other for cadaverine and succinic acid synthesis. The genes of ldhA and ackA were deleted to construct succinic acid pathway in cadaverine producer strain. Then, a thermal switch system based on pR/pL promoter and CI857 was established and optimized. The fermentation conditions were investigated that the optimal temperature for the first stage was determined as 33 â„ƒ and the optimal temperature for the second stage was 39 â„ƒ. Additionally, the time to shifting temperature was identified as the fermentation anaphase. For further enhance cadaverine and succinic acid production, a scale-up fermentation in 5 L bioreactor was operated. As a result, the titer, yield and productivity of cadaverine was 55.58 g/L, 0.38 g/g glucose and 1.74 g/(L·h), respectively. 28.39 g/L of succinic acid was also obtained with yield of 0.19 g/g glucose. CONCLUSION: The succinic acid metabolic pathway was constructed into cadaverine producer strain to realize the co-production of succinic acid and cadaverine. This study provided a novel craft for industrial co-production of cadaverine and succinic acid.

Engineering synthetic microbial consortium for cadaverine biosynthesis from glycerol.

OBJECTIVES: 1,5-pentanediamine (cadaverine) is a C5 platform chemical, also an important raw material for bio-polyamide PA5X. With increasing concerns about the depletion of fossil resources and global environmental protection, cadaverine bio-production has attracted more attentions. RESULTS: Here, a microbial consortium consisting of Corynebacterium glutamicum cgl-FDK and Escherichia coli BL-ABST-Spy was constructed to de novo synthesize cadaverine utilizing glycerol as the sole carbon resource. The glycerol utilization pathway was initially constructed in C. glutamicum cgl-FDK to produce lysine from glycerol. Then, the pyridoxal 5'-phosphate (PLP) biosynthesis pathway and SpyTag/SpyCatcher protein-ligation system for lysine decarboxylase (CadA) and cadaverine-lysine antiporter protein (CadB) were introduced into E. coli BL-ABST-Spy to synthesize cadaverine from lysine. Furthermore, the fermentation conditions of microbial consortium were optimized and the cadaverine production reached 9.3 g/L with glycerol as the sole carbon source. CONCLUSIONS: This work provides a promising strategy for efficiently producing cadaverine from glycerol with an artificial microbial consortium.

Improvement of cadaverine production in whole cell system with baker's yeast for cofactor regeneration.

Cadaverine, 1,5-diaminopentane, is one of the most promising chemicals for biobased-polyamide production and it has been successfully produced up to molar concentration. Pyridoxal 5'-phosphate (PLP) is a critical cofactor for inducible lysine decarboxylase (CadA) and is required up to micromolar concentration level. Previously the regeneration of PLP in cadaverine bioconversion has been studied and salvage pathway pyridoxal kinase (PdxY) was successfully introduced; however, this system also required a continuous supply of adenosine 5'-triphosphate (ATP) for PLP regeneration from pyridoxal (PL) which add in cost. Herein, to improve the process further a method of ATP regeneration was established by applying baker's yeast with jhAY strain harboring CadA and PdxY, and demonstrated that providing a moderate amount of adenosine 5'-triphosphate (ATP) with the simple addition of baker's yeast could increase cadaverine production dramatically. After optimization of reaction conditions, such as PL, adenosine 5'-diphosphate, MgCl2, and phosphate buffer, we able to achieve high production (1740 mM, 87% yield) from 2 M L-lysine. Moreover, this approach could give averaged 80.4% of cadaverine yield after three times reactions with baker's yeast and jhAY strain. It is expected that baker's yeast could be applied to other reactions requiring an ATP regeneration system.

Surface Modification Enabling Reproducible Cantilever Functionalization for Industrial Gas Sensors.

Micro-cantilever sensors are a known reliable tool for gas sensing in industrial applications. We have demonstrated the application of cantilever sensors on the detection of a meat freshness volatile biomarker (cadaverine), for determination of meat and fish precise expiration dates. For achieving correct target selectivity, the cantilevers need to be functionalized with a cadaverine-selective binder, based on a cyclam-derivative. Cantilever surface properties such as surface energy strongly influence the binder morphology and material clustering and, therefore, target binding. In this paper, we explore how chemical and physical surface treatments influence cantilever surface, binder morphology/clustering and binding capabilities. Sensor measurements with non-controlled surface properties are presented, followed by investigations on the binder morphology versus surface energy and cadaverine capture. We demonstrated a method for hindering binder crystallization on functionalized surfaces, leading to reproducible target capture. The results show that cantilever surface treatment is a promising method for achieving a high degree of functionalization reproducibility for industrial cantilever sensors, by controlling binder morphology and uniformity.

Search for Structural Basis of Interactions of Biogenic Amines with Human TAAR1 and TAAR6 Receptors.

The identification and characterization of ligand-receptor binding sites are important for drug development. Trace amine-associated receptors (TAARs, members of the class A GPCR family) can interact with different biogenic amines and their metabolites, but the structural basis for their recognition by the TAARs is not well understood. In this work, we have revealed for the first time a group of conserved motifs (fingerprints) characterizing TAARs and studied the docking of aromatic (ß-phenylethylamine, tyramine) and aliphatic (putrescine and cadaverine) ligands, including gamma-aminobutyric acid, with human TAAR1 and TAAR6 receptors. We have identified orthosteric binding sites for TAAR1 (Asp68, Asp102, Asp284) and TAAR6 (Asp78, Asp112, Asp202). By analyzing the binding results of 7500 structures, we determined that putrescine and cadaverine bind to TAAR1 at one site, Asp68 + Asp102, and to TAAR6 at two sites, Asp78 + Asp112 and Asp112 + Asp202. Tyramine binds to TAAR6 at the same two sites as putrescine and cadaverine and does not bind to TAAR1 at the selected Asp residues. ß-Phenylethylamine and gamma-aminobutyric acid do not bind to the TAAR1 and TAAR6 receptors at the selected Asp residues. The search for ligands targeting allosteric and orthosteric sites of TAARs has excellent pharmaceutical potential.

Mapping of Transglutaminase-2 Sites of Human Salivary Small Basic Proline-Rich Proteins by HPLC-High-Resolution ESI-MS/MS.

Because of the distinctive features of the oral cavity, the determination of the proteins involved in the formation of the "oral protein pellicle" is demanding. The present study investigated the susceptibility of several human basic proline-rich peptides, named P-H, P-D, P-F, P-J, and II-2, as substrates of transglutaminase-2. The reactivity of the P-C peptide and statherin was also investigated. Peptides purified from human whole saliva were incubated with the enzyme in the presence or in the absence of monodansyl-cadaverine. Mass spectrometry analyses of the reaction products highlighted that P-H and P-D (P32 and A32 variants) were active substrates, II-2 was less reactive, and P-F and P-J showed very low reactivity. P-C and statherin were highly reactive. All of the peptides formed cyclo derivatives, and only specific glutamine residues were involved in the cycle formation and reacted with monodansyl-cadaverine: Q29 of P-H, Q37 of P-D, Q21 of II-2, Q41 of P-C, and Q37 of statherin were the principal reactive residues. One or two secondary glutamine residues of only P-H, P-D P32, P-C, and statherin were hierarchically susceptible to the reaction with monodansyl-cadaverine. MS and MS/MS data were deposited to the ProteomeXchange Consortium ( http://www.ebi.ac.uk/pride ) via the PRIDE partner repository with the data set identifier PXD014658.

Metabolic diversification of nitrogen-containing metabolites by the expression of a heterologous lysine decarboxylase gene in Arabidopsis.

Lysine decarboxylase converts l-lysine to cadaverine as a branching point for the biosynthesis of plant Lys-derived alkaloids. Although cadaverine contributes towards the biosynthesis of Lys-derived alkaloids, its catabolism, including metabolic intermediates and the enzymes involved, is not known. Here, we generated transgenic Arabidopsis lines by expressing an exogenous lysine/ornithine decarboxylase gene from Lupinus angustifolius (La-L/ODC) and identified cadaverine-derived metabolites as the products of the emerged biosynthetic pathway. Through untargeted metabolic profiling, we observed the upregulation of polyamine metabolism, phenylpropanoid biosynthesis and the biosynthesis of several Lys-derived alkaloids in the transgenic lines. Moreover, we found several cadaverine-derived metabolites specifically detected in the transgenic lines compared with the non-transformed control. Among these, three specific metabolites were identified and confirmed as 5-aminopentanal, 5-aminopentanoate and δ-valerolactam. Cadaverine catabolism in a representative transgenic line (DC29) was traced by feeding stable isotope-labeled [α-15 N]- or [ε-15 N]-l-lysine. Our results show similar 15 N incorporation ratios from both isotopomers for the specific metabolite features identified, indicating that these metabolites were synthesized via the symmetric structure of cadaverine. We propose biosynthetic pathways for the metabolites on the basis of metabolite chemistry and enzymes known or identified through catalyzing specific biochemical reactions in this study. Our study shows that this pool of enzymes with promiscuous activities is the driving force for metabolite diversification in plants. Thus, this study not only provides valuable information for understanding the catabolic mechanism of cadaverine but also demonstrates that cadaverine accumulation is one of the factors to expand plant chemodiversity, which may lead to the emergence of Lys-derived alkaloid biosynthesis.

Characteristic fragment ions associated with dansyl cadaverine and biotin cadaverine adducts on glutamine.

Glutamine residues susceptible to transglutaminase-catalyzed crosslinking can be identified by incorporation of dansyl cadaverine or biotin cadaverine. Bacterial transglutaminase and human transglutaminase 2 were used to modify residues in beta-casein with dansyl cadaverine. Bacterial transglutaminase was used to modify residues in human butyrylcholinesterase with biotin cadaverine. Tryptic peptides were analyzed by LC-MS/MS on an Orbitrap Fusion Lumos mass spectrometer. Modified residues were identified in Protein Prospector searches of mass spectrometry data. The MS/MS spectra from modified casein included intense peaks at 336.2, 402.2, and 447.2 for fragments of dansyl cadaverine adducts on glutamine. The MS/MS spectra from modified butyrylcholinesterase included intense peaks at 329.2, 395.2, and 440.2 for fragments of biotin cadaverine adducts on glutamine. No evidence for transglutaminase-catalyzed adducts on glutamic acid, aspartic acid, or asparagine was found. Consistent with expectation, it was concluded that bacterial transglutaminase and human transglutaminase 2 specifically modify glutamine. The characteristic ions associated with dansyl cadaverine and biotin cadaverine adducts on glutamine are useful markers for modified peptides.

Smartphone-interrogated test supports for the enzymatic determination of putrescine and cadaverine in food.

Diamino-oxidase (DAO), horseradish peroxidase (HRP), and tetramethylbenzidine (TMB) have been immobilized into cellulose to obtain circular cellulose test supports (CCTSs) for the determination of cadaverine (Cad) and putrescine (Put). During the enzymatic reaction, TMB is oxidized and a blue spot is obtained. This color (RGB coordinates) is measured with a smartphone and a commercial application. The highest sensitivity is provided by the component R and a linear response is observed for low biogenic amine (BA) concentrations, but a second-order polynomial response better fits the experimental results for a wider concentration range. This has been successfully explained with a model developed to explain the RGB values obtained in this type of analytical system. Optimization studies enable CCTSs to be obtained for Put and Cad determination, which could be used (kept at 4 °C) for at least 45 days if a stabilizer (StabilCoat™ or StabilGuard™) is added during its synthesis. In these conditions, the R coordinate follows the model up to at least 4 × 10-4 M Put and/or Cad (both analytes give the same response). The method permits the Put and Cad determination from 5 × 10-5 M up to 4 × 10-4 M (RSD = 3%, n = 3). The CCTSs have been applied to Put + Cad determination in a tuna sample without any interference by other biogenic amines. The concentration found statistically agrees with that obtained using a HPLC-MS-validated method. Graphical abstract.