Selective and sensitive detection of pectin lyase activity using a colorimetric test: application to the screening of microorganisms possessing pectin lyase activity.
Several methods have been described for the detection and quantification of polygalacturonase (PG) and pectin lyase (PL) activities. The most frequently used tests are the Nelson method using copper(II) and an arsenomolybdate reagent to detect PG activity, and the colorimetric method using thiobarbituric acid (TBA) to detect PL activity. We observed that none of these methods are suitable to differentiate between these two enzymatic activities. Therefore, we optimized the test conditions of the TBA method. As a result, the detection of the enzymatic beta-elimination (PL activity) became sensitive and selective. A basic pretreatment at 80 degrees C for 5 min of the solution which contains the pectin fragments of the PL activity furnished aldehydes which were condensed with TBA or its derivatives. After acidification of the medium, a pink fluorescent dye was detected spectrophotochemically (lambda = 550 nm). The interference of galacturonic acid or oligomers resulting from PG activity was completely eliminated. The most sensitive reagent was N-(pyridin-2-yl)-thiobarbituric acid. The application of this method with the new reagent was extended to the screening of microorganisms possessing the PL activity. The obtained results confirm that Aspergillus niger strain and a Saccharomyces cerevisiae SCPP strain possess this activity.
Nedjma
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M
Pascal Biotech, 68 Bis, Boulevard Perreire, F-75017 Paris, France.
Hoffmann
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N
Belarbi
A
A
D001234:Aspergillus niger / Q000201:enzymology*; D003124:Colorimetry / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D010368:Pectins / Q000378:metabolism; D011096:Polygalacturonase / Q000378:metabolism; D011133:Polysaccharide-Lyases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012680:Sensitivity and Specificity; D013858:Thiobarbiturates / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors
D005456:Fluorescent Dyes; D013858:Thiobarbiturates; D010368:Pectins; D011096:Polygalacturonase; D011133:Polysaccharide-Lyases; C027349:pectin lyase; C029684:thiobarbituric acid