Determination of Polygalacturonase Activity in Coffee Mucilage

Sakowicz, Stephanie E., Brian M. Roy, 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.  The long range goal of this project is to determine if a relationship exists between the amount of PGase activity and the quality of the coffee produced in small coffee plantations in Nicaragua. These smaller plantations generally use traditional methods of coffee bean processing and the coffee receives low quality ratings, therefore selling at a lower rate on international coffee markets.  If it is found that the quality does depend on PGase activity, the smaller plantations can be assisted in developing an improved method for coffee bean fermentation.  A unit of PGase activity is defined as the number of mmol of galacturonic acid produced per min.  PGase is found to be a very stable enzyme.  It is not possible to denature it by submersion in a boiling water bath for 10 minutes.  Addition of  25 mM urea reduces PGase activity by 15%; however, PGase is denatured by 97.4% in the presence of 2.5% b-mercaptoethanol.  Therefore, this concentration of b-mercaptoethanol is used as an assay control.  The specific activities (U/mg total protein) of PGase in three different samples of coffee mucilage are 4.13+ 1.16, 1.59 + 0.91 and 1.68 + 0.99 U/mg. The mean of the first sample is significantly different (P<0.0002) than the means of the other two samples.  Additional analyses of more mucilage samples are needed to determine if statistically significant differences in PGase activity exist among greater numbers of different coffee mucilage samples. (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.)