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Alizarin Red S for Online Pyrophosphate Detection Identified by a Rapid Screening Method.

ABSTRACT: We identified Alizarin Red S and other well known fluorescent dyes useful for the online detection of pyrophosphate in enzymatic assays, including the loop mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) assays. An iterative screening was used for a selected set of compounds to first secure enzyme compatibility, evaluate inorganic pyrophosphate sensitivity in the presence of manganese as quencher and optimize conditions for an online detection. Of the selected dyes, the inexpensive alizarin red S was found to selectively detect pyrophosphate under LAMP and PCR conditions and is superior with respect to its defined red-shifted spectrum, long shelf life and low toxicity. In addition, the newly identified properties may also be useful in other enzymatic assays which do not generate nucleic acids but are based on inorganic pyrophosphate. Finally, we propose that our screening method may provide a blueprint for rapid screening of compounds for detecting inorganic pyrophosphate.

SUBMITTER: Fischbach J

PROVIDER: S-EPMC5364467 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Alizarin Red S for Online Pyrophosphate Detection Identified by a Rapid Screening Method.

Fischbach Jens J Loh Qiuting Q Bier Frank F FF Lim Theam Soon TS Frohme Marcus M Glökler Jörn J

Scientific reports 20170324

We identified Alizarin Red S and other well known fluorescent dyes useful for the online detection of pyrophosphate in enzymatic assays, including the loop mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) assays. An iterative screening was used for a selected set of compounds to first secure enzyme compatibility, evaluate inorganic pyrophosphate sensitivity in the presence of manganese as quencher and optimize conditions for an online detection. Of the selected dyes, ...[more]

PMID: 28338022

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Project description:BackgroundAlizarin red (AR) is a typical anthraquinone dye, and the resulting wastewater is toxic and difficult to remove. A study showed that the white rot fungus Trametes gibbosa (T. gibbosa) can degrade dye wastewater by decolorization and has its own enzyme-producing traits.MethodsIn this study, transcriptome sequencing was performed after alizarin red treatment for 0, 3, 7, 10, and 14 h. The key pathways and key enzymes involved in alizarin red degradation were found to be through the analysis of KEGG and GO. The Glutathione S-transferase (GST), manganese peroxidase (MnP) and laccase activities of T. gibbosa treated with alizarin red for 0-14 h were detected. LC-MS and GC-MS analyses of alizarin red decomposition products after 7 h and 14 h were performed.ResultsThe glutathione metabolic pathway ko00480, and the key enzymes GST, MnP, laccase and CYP450 were selected. Most of the genes encoding these enzymes were upregulated under alizarin red conditions. The GST activity increased 1.8 times from 117.55 U/mg prot at 0 h to 217.03 U/mg prot at 14 h. The MnP activity increased 2.9 times from 6.45 to 18.55 U/L. The laccase activity increased 3.7 times from 7.22 to 27.28 U/L. Analysis of the alizarin red decolourization rate showed that the decolourization rate at 14 h reached 20.21%. The main degradation intermediates were found to be 1,4-butene diacid, phthalic acid, 1,1-diphenylethylene, 9,10-dihydroanthracene, 1,2-naphthalene dicarboxylic acid, bisphenol, benzophenol-5,2-butene, acrylaldehyde, and 1-butylene, and the degradation process of AR was inferred. Overall, 1,4-butene diacid is the most important intermediate product produced by AR degradation.ConclusionsThe glutathione metabolic pathway was the key pathway for AR degradation. GST, MnP, laccase and CYP450 were the key enzymes for AR degradation. 1,4-butene diacid is the most important intermediate product. This study explored the process of AR biodegradation at the molecular and biochemical levels and provided a theoretical basis for its application in practical production.

Project description:Screening microbial cultures for specialised metabolites is essential for the discovery of new biologically active compounds. A novel, cost-effective and rapid screening method is described for extracting specialised metabolites from bacteria grown on agar plates, coupled with HPLC for basic identification of known and potentially novel metabolites. The method allows the screening of culture collections to identify optimal production strains and metabolite induction conditions. The protocol was optimised on two Burkholderia species known to produce the antibiotics, enacyloxin IIa (B. ambifaria) and gladiolin (B. gladioli), respectively; it was then applied to strains of each species to identify high antibiotic producers. B. ambifaria AMMD and B. gladioli BCC0238 produced the highest concentrations of the respective antibiotic under the conditions tested. To induce expression of silent biosynthetic gene clusters, the addition of low concentrations of antibiotics to growth media was evaluated as known elicitors of Burkholderia specialised metabolites. Subinhibitory concentrations of trimethoprim and other clinically therapeutic antibiotics were evaluated and screened against a panel of B. gladioli and B. ambifaria. To enhance rapid strain screening with more antibiotic elicitors, antimicrobial susceptibility testing discs were included within the induction medium. Low concentrations of trimethoprim suppressed the production of specialised metabolites in B. gladioli, including the toxins, toxoflavin and bongkrekic acid. However, the addition of trimethoprim significantly improved enacylocin IIa concentrations in B. ambifaria AMMD. Rifampicin and ceftazidime significantly improved the yield of gladiolin and caryoynencin by B. gladioli BCC0238, respectively, and cepacin increased 2-fold with tobramycin in B. ambifaria BCC0191. Potentially novel metabolites were also induced by subinhibitory concentrations of tobramycin and chloramphenicol in B. ambifaria. In contrast to previous findings that low concentrations of antibiotic elicit Burkholderia metabolite production, we found they acted as both inducers or suppressors dependent on the metabolite and the strains producing them. In conclusion, the screening protocol enabled rapid characterization of Burkholderia metabolites, the identification of suitable producer strains, potentially novel natural products and an understanding of metabolite regulation in the presence of inducing or suppressing conditions.

Dataset Information

Alizarin Red S for Online Pyrophosphate Detection Identified by a Rapid Screening Method.

Publications

Alizarin Red S for Online Pyrophosphate Detection Identified by a Rapid Screening Method.

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