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A malachite green-based assay to assess glucan phosphatase activity.

ABSTRACT: With the recent discovery of a unique class of dual-specificity phosphatases that dephosphorylate glucans, we report an in vitro assay tailored for the detection of phosphatase activity against phosphorylated glucans. We demonstrate that, in contrast to a general phosphatase assay using a synthetic substrate, only phosphatases that possess glucan phosphatase activity liberate phosphate from the phosphorylated glucan amylopectin using the described assay. This assay is simple and cost-effective, providing reproducible results that clearly establish the presence or absence of glucan phosphatase activity. The assay described will be a useful tool in characterizing emerging members of the glucan phosphatase family.

SUBMITTER: Sherwood AR

PROVIDER: S-EPMC3577954 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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A malachite green-based assay to assess glucan phosphatase activity.

Sherwood Amanda R AR Paasch Bradley C BC Worby Carolyn A CA Gentry Matthew S MS

Analytical biochemistry 20121129 1

With the recent discovery of a unique class of dual-specificity phosphatases that dephosphorylate glucans, we report an in vitro assay tailored for the detection of phosphatase activity against phosphorylated glucans. We demonstrate that, in contrast to a general phosphatase assay using a synthetic substrate, only phosphatases that possess glucan phosphatase activity liberate phosphate from the phosphorylated glucan amylopectin using the described assay. This assay is simple and cost-effective, ...[more]

PMID: 23201267

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Project description:Malachite green (MG) is an organic contaminant and the effluents with MG negatively influence the health and balance of the coastal and marine ecosystem. The diverse and abundant microbial communities inhabiting in mangroves participate actively in various ecological processes. Metagenomic sequencing from mangrove sediments was applied to excavate the resources MG-degradation genes (MDGs) and to assess the potential of their corresponding enzymes. A data set of 10 GB was assembled into 33,756 contigs and 44,743 ORFs were predicted. In the data set, 666 bacterial genera and 13 pollutant degradation pathways were found. Proteobacteria and Actinobacteria were the most dominate phyla in taxonomic assignment. A total of 44 putative MDGs were revealed and possibly derived from 30 bacterial genera, most of which belonged to the phyla of Proteobacteria and Bacteroidetes. The MDGs belonged to three gene families, including peroxidase genes (up to 93.54% of total MDGs), laccase (3.40%), and p450 (3.06%). Of the three gene families, three representatives (Mgv-rLACC, Mgv-rPOD, and Mgv-rCYP) which had lower similarities to the closest sequences in GenBank were prokaryotic expressed and their enzymes were characterized. Three recombinant proteins showed different MG-degrading activities. Mgv-rPOD had the strongest activity which decolorized 97.3% of MG (300 mg/L) within 40 min. In addition, Mgv-rPOD showed a more complete process of MG degradation compared with other two recombinant proteins according to the intermediates detected by LC-MS. Furthermore, the high MG-degrading activity was maintained at low temperature (20°C), wider pH range, and the existence of metal ions and chelating agent. Mgv-rLACC and Mgv-rCYP also removed 63.7% and 54.1% of MG (20 mg/L) within 24 h, respectively. The results could provide a broad insight into discovering abundant genetic resources and an effective strategy to access the eco-friendly way for preventing coastal pollution.

Dataset Information

A malachite green-based assay to assess glucan phosphatase activity.

Publications

A malachite green-based assay to assess glucan phosphatase activity.

Similar Datasets

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