Quantification of the amount of galacturonic acid residues in blocksequences in pectin homogalacturonan by enzymatic fingerprinting with exo- and endo-polygalacturonase II from Aspergillus niger

Carbohydr Res. 2000 Jul 24;327(3):321-32. doi: 10.1016/s0008-6215(00)00068-9.

Abstract

A method to determine the amount of galacturonic acid in blocksequence (BS) in pectin homogalacturonan (HG) is described. The method is based on a combination of endopolygalacturonase II (endo-PG II) and exopolygalacturonase (exo-PG) digestion followed by quantification of the liberated galacturonic acid monomer. The amount of monomers released is directly related to the amount of non-esterified galacturonic acid units located between two other non-esterified galacturonic acids units on the HG chain. The amount released for exo-PG digestion only corresponds to the BS located at the non-reducing end of the polymer. The difference between total- and exo-BS was calculated to be the amount of endo-BS located either within or on the reducing end of the HG. Three series of model pectins obtained by de-esterification of a high-ester pectin with either plant pectin methyl-esterase (p-PME, P-series), fungal pectin methyl-esterase (f-PME, F-series) and chemical de-esterification using base (B-series) were analysed and compared with a fully de-esterified pectic acid sample obtained from the same raw material. Clear differences for the increase of the amounts of blocksequence could be seen between de-esterification of the P- and F-series samples supporting a blockwise and a homogenous de-esterification mechanism, respectively. f-PME and base treatment showed only minor differences in the increase of galacturonic acid units in BS, despite differences seen in their methyl-esterification pattern. Differences between the amounts of galacturonic acid located in exo- and endo-BS, provided evidence for the need of a certain start side or blocklength for p-PME to de-esterify blockwise.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aspergillus niger / enzymology*
  • Carbohydrate Conformation
  • Carbohydrate Sequence
  • Hexuronic Acids / analysis*
  • Models, Molecular
  • Molecular Sequence Data
  • Pectins / chemistry*
  • Pectins / metabolism*
  • Polygalacturonase / metabolism*

Substances

  • Hexuronic Acids
  • galacturonic acid
  • Pectins
  • endopolygalacturonase II
  • Polygalacturonase
  • polygalacturonic acid