The enzyme polygalacturonase (PGase) cleaves the α1-4 galactosidic bonds of polygalacturonic acid (PG), a major component of coffee mucilage, to produce galacturonic acid. PGase was found to be a very stable enzyme. It was stable to submersion in a boiling water bath for 1 hour or 10 minutes at 200 <sup>o</sup>C. When the PGase assay was conducted at a pH 3 or 14, no loss of PGase activity was noted. Guanidine HCl, a known protein denaturant, did not inhibit PGase at concentrations up to 162 mM. Addition of 0.3 M b-mercaptoethanol abolished PGase activity, so this concentration of b-mercaptoethanol was used as an assay control. Dithiothreitol was able to reduce PGase activity in a dose-dependent manner. A 38% reduction in enzyme activity was seen at 81 mM dithiothreitol and this increased to 95% inhibition at 162 mM dithiothreitol. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) inhibited PGase activity by 75% at 37.5 mM. b-Mercaptoethanol, dithiotheitol and TCEP reduced 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 at Seattle University, the Department of Chemistry at Universidad Centroamericana Managua, Nicaragua for sharing coffee mucilage samples and the Department of Chemistry and Biochemistry, University of Detroit Mercy for support of this project.)

Denaturation resistance of polygalacturonase obtained from coffee mucilage

249th National Meeting (2015)

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