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Rapid enzymatic method for pectin methyl esters determination.

ABSTRACT: Pectin is a natural polysaccharide used in food and pharma industries. Pectin degree of methylation is an important parameter having significant influence on pectin applications. A rapid, fully automated, kinetic flow method for determination of pectin methyl esters has been developed. The method is based on a lab-made analyzer using the reverse flow-injection/stopped flow principle. Methanol is released from pectin by pectin methylesterase in the first mixing coil. Enzyme working solution is injected further downstream and it is mixed with pectin/pectin methylesterase stream in the second mixing coil. Methanol is oxidized by alcohol oxidase releasing formaldehyde and hydrogen peroxide. This reaction is coupled to horse radish peroxidase catalyzed reaction, which gives the colored product 4-N-(p-benzoquinoneimine)-antipyrine. Reaction rate is proportional to methanol concentration and it is followed using Ocean Optics USB 2000+ spectrophotometer. The analyzer is fully regulated by a lab written LabVIEW program. The detection limit was 1.47?mM with an analysis rate of 7 samples h(-1). A paired t-test with results from manual method showed that the automated method results are equivalent to the manual method at the 95% confidence interval. The developed method is rapid and sustainable and it is the first application of flow analysis in pectin analysis.

SUBMITTER: Lekawska-Andrinopoulou L

PROVIDER: S-EPMC3888753 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Rapid enzymatic method for pectin methyl esters determination.

Lękawska-Andrinopoulou Lucyna L Vasiliou Efstathios G EG Georgakopoulos Dimitrios G DG Yialouris Constantinos P CP Georgiou Constantinos A CA

Journal of analytical methods in chemistry 20131226

Pectin is a natural polysaccharide used in food and pharma industries. Pectin degree of methylation is an important parameter having significant influence on pectin applications. A rapid, fully automated, kinetic flow method for determination of pectin methyl esters has been developed. The method is based on a lab-made analyzer using the reverse flow-injection/stopped flow principle. Methanol is released from pectin by pectin methylesterase in the first mixing coil. Enzyme working solution is in ...[more]

PMID: 24455426

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Project description:Background5' methylation of cytosines in DNA molecules is an important epigenetic mark in eukaryotes. Bisulfite sequencing is the gold standard of DNA methylation detection, and whole-genome bisulfite sequencing (WGBS) has been widely used to detect methylation at single-nucleotide resolution on a genome-wide scale. However, sodium bisulfite is known to severely degrade DNA, which, in combination with biases introduced during PCR amplification, leads to unbalanced base representation in the final sequencing libraries. Enzymatic conversion of unmethylated cytosines to uracils can achieve the same end product for sequencing as does bisulfite treatment and does not affect the integrity of the DNA; enzymatic methylation sequencing may, thus, provide advantages over bisulfite sequencing.ResultsUsing an enzymatic methyl-seq (EM-seq) technique to selectively deaminate unmethylated cytosines to uracils, we generated and sequenced libraries based on different amounts of Arabidopsis input DNA and different numbers of PCR cycles, and compared these data to results from traditional whole-genome bisulfite sequencing. We found that EM-seq libraries were more consistent between replicates and had higher mapping and lower duplication rates, lower background noise, higher average coverage, and higher coverage of total cytosines. Differential methylation region (DMR) analysis showed that WGBS tended to over-estimate methylation levels especially in CHG and CHH contexts, whereas EM-seq detected higher CG methylation levels in certain highly methylated areas. These phenomena can be mostly explained by a correlation of WGBS methylation estimation with GC content and methylated cytosine density. We used EM-seq to compare methylation between leaves and flowers, and found that CHG methylation level is greatly elevated in flowers, especially in pericentromeric regions.ConclusionWe suggest that EM-seq is a more accurate and reliable approach than WGBS to detect methylation. Compared to WGBS, the results of EM-seq are less affected by differences in library preparation conditions or by the skewed base composition in the converted DNA. It may therefore be more desirable to use EM-seq in methylation studies.

Dataset Information

Rapid enzymatic method for pectin methyl esters determination.

Publications

Rapid enzymatic method for pectin methyl esters determination.

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