Yeast Polygalacturonase Activity is Resistant to Extreme Temperature and pH

Polygalacturonase cleaves the α1–4 galactosidic bonds of polygalacturonic acid (PG), a major component of fruit mucilage, to produce galacturonic acid. A unit of polygalacturonase activity is defined as the number of mmol of galacturonic acid produced per min at pH 5.0. Polygalacturonase is stable to submersion in a boiling water bath for 1 hour or 10 minutes at 200 °C. When the polygalacturonase assay is conducted at a pH 3 or 14, no loss of activity is noted. Addition of 0.3 M β‐mercaptoethanol abolishes polygalacturonase activity and so this concentration of β‐mercaptoethanol is used as an assay control. Dithiothreitol is able to reduce polygalacturonase 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. Tris(2‐carboxyethyl)phosphine at 50 mM completely inhibits polygalacturonase activity. β‐Mercaptoethanol, dithiotheitol and tris(2‐carboxyethyl)phosphine all reduce the disulfide bonds formed between the side chains of cysteine residues. This suggests that the resistance of polygalacturonase to denaturation by high temperature and acid or base is due to the stabilization of its tertiary structure by disulfide bonds. (I gratefully acknowledge the work of Melissa L. Zuteck, Edwin A. Gay, Joshua Henning, Ayah Bakri, Bianca Yaldo and Glenn R. Oppenlander who assisted in this work while undergraduates.) Support or Funding Information I thank the Department of Chemistry and Biochemistry, University of Detroit Mercy for support of this project This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .