In-Gel Determination of L-Amino Acid Oxidase Activity Based on the Visualization of Prussian Blue-Forming Reaction

Yu, Zhiliang; Zhou, Ning; Zhao, Changsong; Qiu, Juanping

doi:10.1371/journal.pone.0055548

Cited by 14 publications

(37 citation statements)

References 21 publications

(28 reference statements)

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“…Since Pox was one of the most enriched proteins in the inhibitory fractions after gel filtration, we determined whether we could increase hydrogen peroxide production in the STYM1 extracts by simply adding substrates for Pox. Prussian blue agar has been developed to detect oxidase activity in bacterial extracts (37). We adapted this system to be used with tryptic soy agar (TSA) plates so that we could detect hydrogen peroxide production in STYM1 extracts.…”

Section: Resultsmentioning

confidence: 99%

“…Prussian blue hydrogen peroxide detection. Prussian Blue TSA plates were prepared as described previously (37). To assay for hydrogen peroxide production from L. delbrueckii strains and Streptococcus sanguinis SK36, 5-l portions of broth cultures of each strain were spotted onto Prussian blue plates, allowed to dry, and incubated anaerobically at 37°C for 2 days.…”

Section: Methodsmentioning

confidence: 99%

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Interspecies Inhibition of Porphyromonas gingivalis by Yogurt-Derived Lactobacillus delbrueckii Requires Active Pyruvate Oxidase

Cornacchione

Klein

Duncan³

et al. 2019

Appl Environ Microbiol

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Despite a growing interest in using probiotic microorganisms to prevent disease, the mechanisms by which probiotics exert their action require further investigation. Porphyromonas gingivalis is an important pathogen implicated in the development of periodontitis. We isolated several strains of Lactobacillus delbrueckii from dairy products and examined their ability to inhibit P. gingivalis growth in vitro. We observed strain-specific inhibition of P. gingivalis growth in vitro. Whole-genome sequencing of inhibitory and noninhibitory strains of L. delbrueckii revealed significant genetic differences supporting the strain specificity of the interaction. Extracts of the L. delbrueckii STYM1 inhibitory strain contain inhibitory activity that is abolished by treatment with heat, proteinase K, catalase, and sodium sulfite. We purified the inhibitory protein(s) from L. delbrueckii STYM1 extracts using ammonium sulfate precipitation, anion-exchange chromatography, and gel filtration chromatography. Pyruvate oxidase was highly enriched in the purified samples. Lastly, we showed that purified, catalytically active, recombinant pyruvate oxidase is sufficient to inhibit P. gingivalis growth in vitro without the addition of cofactors. Further, using a saturated transposon library, we isolated transposon mutants of P. gingivalis in the feoB2 (PG_1294) gene that are resistant to killing by inhibitory L. delbrueckii, consistent with a mechanism of hydrogen peroxide production by pyruvate oxidase. Our results support the current understanding of the importance of strain selection, not simply species selection, in microbial interactions. Specific L. delbrueckii strains or their products may be effective in the treatment and prevention of P. gingivalis-associated periodontal disease. IMPORTANCE P. gingivalis is implicated in the onset and progression of periodontal disease and associated with some systemic diseases. Probiotic bacteria represent an attractive preventative therapy for periodontal disease. However, the efficacy of probiotic bacteria can be variable between studies. Our data support the known importance of selecting particular strains of bacteria for probiotic use, not simply a single species. Specifically, in the context of probiotic intervention of periodontitis, our data suggest that high-level expression of pyruvate oxidase with hydrogen peroxide production in L. delbrueckii could be an important characteristic for the design of a probiotic supplement or a microbial therapeutic.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Interspecies Inhibition of Porphyromonas gingivalis by Yogurt-Derived Lactobacillus delbrueckii Requires Active Pyruvate Oxidase

Cornacchione

Klein

Duncan³

et al. 2019

Appl Environ Microbiol

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“…Recently, a yellow-pigmented marine bacterium Pseudoalteromonas sp. B-3 was isolated to produce LAAO [ 21 , 22 ]. In this study, to explore the genes involved in regulating LAAO activity, a red-pigmented spontaneous mutant strain of Pseudoalteromonas sp.…”

Section: Introductionmentioning

confidence: 99%

Exploring Regulation Genes Involved in the Expression of L-Amino Acid Oxidase in Pseudoalteromonas sp. Rf-1

Lin

et al. 2015

PLoS ONE

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Bacterial L-amino acid oxidase (LAAO) is believed to play important biological and ecological roles in marine niches, thus attracting increasing attention to understand the regulation mechanisms underlying its production. In this study, we investigated genes involved in LAAO production in marine bacterium Pseudoalteromonas sp. Rf-1 using transposon mutagenesis. Of more than 4,000 mutants screened, 15 mutants showed significant changes in LAAO activity. Desired transposon insertion was confirmed in 12 mutants, in which disrupted genes and corresponding functionswere identified. Analysis of LAAO activity and lao gene expression revealed that GntR family transcriptional regulator, methylase, non-ribosomal peptide synthetase, TonB-dependent heme-receptor family, Na+/H+ antiporter and related arsenite permease, N-acetyltransferase GCN5, Ketol-acid reductoisomerase and SAM-dependent methytransferase, and their coding genes may be involved in either upregulation or downregulation pathway at transcriptional, posttranscriptional, translational and/or posttranslational level. The nhaD and sdmT genes were separately complemented into the corresponding mutants with abolished LAAO-activity. The complementation of either gene can restore LAAO activity and lao gene expression, demonstrating their regulatory role in LAAO biosynthesis. This study provides, for the first time, insights into the molecular mechanisms regulating LAAO production in Pseudoalteromonas sp. Rf-1, which is important to better understand biological and ecological roles of LAAO.

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“…As expected, the duplicate lanes 3 and 4 additionally showed CBB-stained protein bands in SDS-PAGE since R3-LAAO sample was precipitated from fermentation crude of Psudoalteromonas sp. R3 [17]. To determine which protein band bears the LAAO activity, all the CBB-stained protein bands near the purplish red band on Fe II XO agar were sliced out from a CBB-stained lane-4 replicate lane, and put on Fe II XO agar (lane 5).…”

Section: Resultsmentioning

confidence: 99%

“…R3 culture supernatant [17] or caLAAO solution (mixture of 1 µL caLAAO with 10 mL of 0.1 M PBS buffer with pH 7.5, Worthington Biochemical Corporation, USA). Unless otherwise described, the pH of reaction mixture was adjusted to about 7.5.…”

Section: Methodsmentioning

confidence: 99%

A Highly Sensitive Method for Quantitative Determination of L-Amino Acid Oxidase Activity Based on the Visualization of Ferric-Xylenol Orange Formation

Zhou

et al. 2013

PLoS ONE

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L-amino acid oxidase (LAAO) has important biological roles in many organisms, thus attracting great attention from researchers to establish its detection methods. In this study, a new quantitative in-gel determination of LAAO activity based on ferric-xylenol orange (FeIIIXO) formation was established. This method showed that due to the conversion of FeII to FeIII by H2O2 and subsequent formation of FeIIIXO complex halo in agar medium, the logarithm of H2O2 concentration from 5 to 160 µM was linearly correlated to the diameter of purplish red FeIIIXO halo. By extracting the LAAO-generated H2O2 concentration, the LAAO activity can be quantitatively determined. This FeIIIXO agar assay is highly sensitive to detect H2O2 down to micromolar range. More importantly, it is easy to handle, cheap, reproducible, convenient and accurate. Coupled with SDS-PAGE, it can directly be used to determine the number and approximate molecular weight of LAAO in one assay. All these features make this in-gel FeIIIXO assay useful and convenient as a general procedure for following enzyme purification, assaying fractions from a column, or observing changes in activity resulting from enzyme modifications, hence endowing this method with broad applications.

show abstract

In-Gel Determination of L-Amino Acid Oxidase Activity Based on the Visualization of Prussian Blue-Forming Reaction

Cited by 14 publications

References 21 publications

Interspecies Inhibition of Porphyromonas gingivalis by Yogurt-Derived Lactobacillus delbrueckii Requires Active Pyruvate Oxidase

Interspecies Inhibition of Porphyromonas gingivalis by Yogurt-Derived Lactobacillus delbrueckii Requires Active Pyruvate Oxidase

Exploring Regulation Genes Involved in the Expression of L-Amino Acid Oxidase in Pseudoalteromonas sp. Rf-1

A Highly Sensitive Method for Quantitative Determination of L-Amino Acid Oxidase Activity Based on the Visualization of Ferric-Xylenol Orange Formation

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