Zuteck, Melissa L., Edwin A. Gay, Joshua M. Henning, and Mary Lou Caspers

The enzyme polygalacturonase (PGase) cleaves the α1-4 galactosidic bonds of polygalacturonic acid (PG), a major component of coffee mucilage, to produce galacturonic acid.  A unit of PGase activity is defined as the number of mmol of galacturonic acid produced per min at pH 5.0.  PGase is found to be a very stable enzyme.  It is stable to submersion in a boiling water bath for 1 hour or 10 minutes at 200 oC.  When the PGase assay is conducted at a pH 3 or 14, no loss of PGase activity is noted.  Addition of 0.3 M b-mercaptoethanol abolishes PGase activity and so this concentration of b-mercaptoethanol is used as an assay control.  Dithiothreitol, a less noxious compound than b-mercaptoethanol, is able to reduce PGase activity in a dose-dependent manner.  A 38 % reduction in enzyme activity is seen at 81 mM dithiothreitol and this increases to 95 % inhibition at 162 mM dithiothreitol. Both b-mercaptoethanol and dithiotheitol reduce the disulfide bonds formed between the side chains of cysteine residues.  This suggests that the resistance of PGase to denaturation by high temperature and acid or base is due to the stabilization of its tertiary structure by disulfide bonds. (We gratefully acknowledge Susan Jackels, Seattle University, for sharing coffee mucilage samples and the Department of Chemistry and Biochemistry, University of Detroit Mercy for support of this project.)