Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Record number 318528
Title Mode of action of pectin lyase A of Aspergillus niger on differently C6-substituted oligogalacturonides
Author(s) Alebeek, G.J.W.M. van; Christensen, T.M.I.E.; Schols, H.A.; Mikkelsen, J.D.; Voragen, A.G.J.
Source Journal of Biological Chemistry 277 (2002)29. - ISSN 0021-9258 - p. 25929 - 25936.
Department(s) Food Chemistry Group
Publication type Refereed Article in a scientific journal
Publication year 2002
Abstract A thorough investigation of the mode of action of Aspergillus niger (4M-147) pectin lyase A (PLA) on differently C6-substituted oligogalacturonides is described. PLA appeared to be very specific for fully methyl-esterified oligogalacturonides: removal of the methyl-ester or changing the type of ester (ethyl esterification) or transamidation resulted in (almost) complete loss of conversion. The PLA activity increased with increasing length of the substrate up to a degree of polymerization (DP) of 8 indicating the presence of at least eight subsites on the enzyme. Product analysis demonstrated the formation of several 4,5 unsaturated products and their saturated counterparts. The 4,5 unsaturated trimer was the main product up to DP 8. For DP 9 and 10 4,5 unsaturated tetramer was the major product. Based upon the bond cleavage frequencies, a provisional subsite map was calculated, which supports the presence of eight subsites. By limited alkaline de-esterification of fully methyl-esterified pentamer and hexamer two sets of partially methyl-esterified pentamers (x and y methyl groups) and hexamers (a and b methyl groups) were prepared. Matrix-assisted laser desorption/ionization time of flight mass spectroscopy (MALDI-TOF MS) analysis demonstrated that the methyl-ester distribution was fully random. Using these partially methyl-esterified oligogalacturonides as substrates for PLA a 10-fold decrease in reaction rate was recorded compared with the fully methyl-esterified counterparts. Analysis of the methyl-ester distribution of the products showed that PLA tolerates carboxyl groups in the substrate binding cleft. At either subsite 2, 4, or 1 to 4 a free carboxyl group could be tolerated, whereas methyl-esters were obligatory at subsite 1 and 3. So PLA is capable to cleave the bond between a methyl-esterified and a non-esterified galacturonic acid residue, where the newly formed 4,5 unsaturated non-reducing end residue always contains a methyl-ester.
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