Detection of Proteinases inSaccharomyces cerevisiaeby Flow Cytometry

Abstract

The physiological state of a yeast population used for inoculation determines how rapidly the cells adapt to new environmental conditions, begin proliferating and utilising extract. The decision as to whether a yeast culture is suitable for re-pitching should not be based only on viability determinations since this can be misleading. Increased proteolytic activity in a yeast population indicates the onset of senescence. A flow cytometric method has been developed for measuring a wide variety of proteinases in Saccharomyces cerevisiae employing a commercially available casein-dye conjugate. The detection of intracellular proteinase activity gives an early indication of apoptotic events and allows improved assessment of the physiological state of a yeast population. This knowledge will assist the industry to optimize the selection of yeast and its subsequent fermentation performance. Yeast cell autolysis with all its negative consequences for beer quality and stability will thus be minimised.

The Horace Brown Medal Lecture: Forty Years of Brewing Research

“…As previously described in this paper, the cell's sorting mechanism that targets proteinase A to the vacuole is partially failing under stress conditions (for example, high gravity wort fermentations) and proteinase A remains in the cytoplasm or is excreted into the environment. Laser scanning confocal microscopy as a qualitative tool, in conjunction with flow cytometry 61,177 -details later, is a powerful tool to gain insight into the response of yeast cells to the changing environmental conditions occurring during fermentation.…”

The Potential of Confocal Imaging for Measuring Physiological Changes in Brewer's Yeast

“…Although non-specific proteolysis of endocytosed proteins such as membrane receptors or extracellular proteins is largely confined to the lysosomal/ vacuolar system 22,55,56,61,66,67 proteolysis also takes place in other cell compartments. The staining procedure was conducted according to Hutter et al 46 with casein substrates labelled with quenched BODIPY ® FL dye, which is a substituted 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene derivative 35,53 . This casein-dye conjugate provides spectrally distinct substrates for detecting metallo, serine, acid and sulfhydryl proteinases.…”

“…The staining for intracellular proteinase was conducted according to the method of Hutter et al 46 . The EnzChek ® Proteinase Assay Kit E-6638, supplied by Molecular Probes (Molecular Probes, Eugene, OR) contains the lyophilized BODIPY FL substrate and the 20× digestion buffer.…”

“…Similar to FRAP, the latter technique is useful in the investigation of molecular mobility and dynamics in living cells. Laser scanning confocal microscopy as a qualitative tool, in conjunction with flow cytometry 46 as a quantitative measurement, is a powerful tool to gain insight into the response of yeast cells to the changing environmental conditions occurring during fermentation.…”

“…The present study builds on these two fundamental discoveries by using a confocal microscope to visualise cell compounds and compartments as well as physiological changes in brewer's yeast cells during fermentation. Most of the procedures and methods for staining and imaging yeast cells with the confocal microscope are based on those that have been developed over many years for use with the conventional wide field light microscope and with flow cytometry [41][42][43][44][45][46][47][48][49][50] . Some methods are inspired by fluorescence techniques applied to mammalian and human cells in biomedical and biomolecular sciences 1,[27][28][29]71,110 .…”