For pectinmethylesterase, PME (EC 3.1.1.11) analysis, extraction
was performed on three sets of 15 g of berries after seed removal;
berries were manually ground in a mortar by adding liquid nitrogen
to obtain a fine powder and extracted with 0.2 M phosphate
buffer at pH 7.5 with the addition of 1 mM EDTA, 5% PVPP
(polyvinylpolypyrrolidone), and 2 M NaCl, up to a final volume of
10 ml. The homogenate was centrifuged at 39,800g for 1 h at
4 C. The supernatant was filtered through a Sephadex G25 column
to remove polyphenols. PME activity was assayed using Hagermanand Austin’s procedure (1986) with modifications for use in grapes.
0.4 ml of extract were incubated with 0.01% bromothymol blue,
0.5% w/v pectin in water at pH 7.5, 0.1 M NaOH, and 0.01 M phosphate
buffer. A spectrophotometric reading was taken at 620 nm
for 6 min. Enzyme activity is expressed as lmol/min gdw
For pectinmethylesterase, PME (EC 3.1.1.11) analysis, extractionwas performed on three sets of 15 g of berries after seed removal;berries were manually ground in a mortar by adding liquid nitrogento obtain a fine powder and extracted with 0.2 M phosphatebuffer at pH 7.5 with the addition of 1 mM EDTA, 5% PVPP(polyvinylpolypyrrolidone), and 2 M NaCl, up to a final volume of10 ml. The homogenate was centrifuged at 39,800g for 1 h at4 C. The supernatant was filtered through a Sephadex G25 columnto remove polyphenols. PME activity was assayed using Hagermanand Austin’s procedure (1986) with modifications for use in grapes.0.4 ml of extract were incubated with 0.01% bromothymol blue,0.5% w/v pectin in water at pH 7.5, 0.1 M NaOH, and 0.01 M phosphatebuffer. A spectrophotometric reading was taken at 620 nmfor 6 min. Enzyme activity is expressed as lmol/min gdw
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