title: Catabolite Inactivation in the Methylotrophic Yeast Pichia pastoris.
abstract: Inactivation of the alcohol oxidase enzyme system of Pichia pastoris, during the whole-cell bioconversion of ethanol to acetaldehyde, was due to catabolite inactivation. Electron microscopy showed that methanol-grown cells contained peroxisomes but were devoid of these microbodies after the bioconversion. Acetaldehyde in the presence of O(2) was the effector of catabolite inactivation. The process was initiated by the appearance of free acetaldehyde, and was characterized by an increase in the level of cyclic AMP, that coincided with a rapid 55% drop in alcohol oxidase activity. Further enzyme inactivation, believed to be due to proteolytic degradation, then proceeded at a constant but slower rate and was complete 21 h after acetaldehyde appearance. The rate of catabolite inactivation was dependent on acetaldehyde concentration up to 0.14 mM. It was temperature dependent and occurred within 24 h at 37 degrees C and by 6 days at 15 degrees C but not at 3 degrees C. Alcohol oxidase activity was psychrotolerant, with only a 17% decrease in initial specific activity over a temperature drop from 37 to 3 degrees C. In contrast, protease activity was inhibited at temperatures below 15 degrees C. When the bioconversion was run at 3 degrees C, catabolite inactivation was prevented. In the presence of 3 M Tris hydrochloride buffer, 123 g of acetaldehyde per liter was produced at 3 degrees C, compared with 58 g/liter at 30 degrees C. By using 0.5 M Tris in a cyclic-batch procedure, 140.6 g of acetaldehyde was produced.
authors.affiliation: Division of Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A 0R6, and Department of Microbiology, College of Biological Sciences, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
authors.forename: William D
authors.initials: WD
authors.lastname: Murray
authors.forename: Sheldon J B
authors.initials: SJ
authors.lastname: Duff
authors.forename: Terry J
authors.initials: TJ
authors.lastname: Beveridge