Tandem mass spectrometric analysis of Aspergillus niger pectin methylesterase: mode of action on fully methyl-esterified oligogalacturonates

The substrate specificity and the mode of action of Aspergillus niger pecti n methylesterase (PME) was determined using both fully methyl-esterified ol igogalacturonates with degrees of polymerization (DP) 2-6 and chemically sy nthesized monomethyl trigalacturonates. The enzymic activity on the differe nt substrates and a preliminary characterization of the reaction products w ere performed by using high-performance anion-exchange chromatography at ne utral pH. Electrospray ionization tandem MS (ESI-MS/MS) was used to localiz e the methyl esters on the O-18-labelled reaction products during the cours e of the enzymic reaction. A. niger PME is able to hydrolyse the methyl est ers of fully methyl-esterified oligogalacturonates with DP 2, and preferent ially hydrolyses the methyl esters located on the internal galacturonate re sidues, followed by hydrolysis of the methyl esters towards the reducing en d. This PME is unable to hydrolyse the methyl ester of the galacturonate mo iety at the non-reducing end.