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As a result of the cloning and overexpression of individual Aspergillus niger pectinase genes the corresponding enzymes are now readily available for detailed characterisation using defmed substrates, including derivatised oligogalacturonides. In this chapter we give an overview of the results of these studies for a pectate and pectine lyase and a polygalacturonase. Detailed kinetic studies on pectate lyase have revealed that the substrate binds to the enzyme as a Ca 2 +-substrate complex, thus explaining the absolute requirement of pectate lyases for Ca 2 +-ions. Using derivatised oligogalacturonides it was shown that pectin lyase A cannot cleave substrates that are: 1) fully de-esterified, 2) fully ethyl-esterified, 3) fully amidated, and 4) fully methylamidated. Furthermore, partial de-esterification of fully methyl esterified oligogalacturonides, one methyl group on average, resulted in a strong reduction of the catalytic efficiency and a change in the preferred binding mode. From these studies it can be concluded that pectin lyase A is highly specific for pectin with a high degree of methylesterification. Endopolygalacturonases (endoPGs) were thouroughly characterised kinetically which allowed to ca1culate subsite affinities. Site directed mutagenesis of endoPGII in combination with the 3D-structure allowed to pin-point amino acids involved in catalysis as well as amino acids that are important for binding of the substrate. At subsite -5 the presence of an arginine appeared critical for displaying multiple attack attack on a single chain (processive) behavior.

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