APPLICATION OF ANALYTICAL SIZE-EXCLUSION CHROMATOGRAPHY TO THE INVESTIGATION OF SUBSTRATE DEGRADATION BY A COMMERCIAL PECTATE LYASE FROM ERWINIA-CAROTOVORA

Within the scope of the characterization of a commercial pectate lyase (PAL) the pattern of substrate degradation by that enzyme was investi gated with oligogalacturonic acids as well as pectins with various deg rees of esterification as substrates. For this purpose an analytical s ize exclusion chromatographic method (HPSEC) was elaborated in order t o evaluate the reaction products of the degraded substrates over the w hole range of molecular weight with the main interest lying in the eme rging oligogalacturonic acids. The degradation of polygalacturonic aci d by PAL was additionally investigated by means of thin-layer chromato graphy (TLC) and viscosimetry. Furthermore spontaneous depolymerizatio n and deesterification of the substrates occurring under the chosen co nditions were examined. The PAL showed to be an endo-enzyme both by me ans of TLC and HPSEC. Polygalacturonic acid was broken down, mainly ge nerating unsaturated tri- and digalacturonic acid; the corresponding m onomer was not found. The mode of cleavage is discussed.