Expression of Cryptic β-Fructofuranosidase in Saccharomyces rouxii

Abstract

Raffinose hydrolysis was studied in Saccharomyces rouxii. The responsible enzyme was identified as a fB-fructofuranosidase (EC 3.2.1.26), which has a pH optimum of 5.5 and a Km of 83 mM for raffinose. This enzyme was cryptic in cells from a 3-day culture. A 2-min treatment with 0.1 volume of ethyl acetate in sodium acetate buffer (pH 6) gave complete expression of the enzyme, which was still retained by the cell. Ghosts were prepared by modifying membrane structure with small basic proteins in distilled water, and after washing they showed the full complement of enzymatic activity. The enzyme remained cryptic in osmotically protected spheroplasts; however, after lysis (by dilution) release, as well as expression, was effected. Mechanical disruption of fresh cells revealed and released all of the enzyme. Cells in early stationary phase had all of their ,8-fructofuranosidase in a cryptic state. Aging yielded fractional expression of activity; initially this was proportional to cell death, but later the degree of expression exceeded the death rate. Media from aged cultures or cell-free extracts of aged cells were not effective in revealing the cryptic enzyme of younger cells. S. rouxii ,8-fructofuranosidase has a different autolytic-release pattern from its counterpart in S. cerevisiae. Also, high concentrations of glucose do not repress the S. rouxii enzyme. The f-fructofuranosidase in young cells of S. rouxii must be enclosed by the protoplasmic membrane or a special vesicular structure. This system was compared with other Saccharomyces species in connection with the translocation of enzymes across the protoplasmic membrane.

Isolation and Characterization of Protoplasts from Saccharomyces rouxii

“…This is in contrast with the ,B-fructofuranosidase of S. cerevisiae, which is typical for the genus in that over 95% of the enzyme activity is expressed at all ages (1). "Expression" here refers to the enzyme being fully assayable in intact cells without recourse to prior membrane disruption (which is necessary in the assay of young cells of S. rouxii [2]).…”

“…Stationary-phase cells of S. rouxii exhibit an unusual propensity for vesicles in the periplasmic space. These organelles have been called periplasmic bodies (3), and their presence or absence (older cells) has been circumstantially linked with this species' /8-fructofuranosidase, which is "cryptic" in young cells and "expressed" in aged cultures (2). This is in contrast with the ,B-fructofuranosidase of S. cerevisiae, which is typical for the genus in that over 95% of the enzyme activity is expressed at all ages (1).…”

Effects of Polyenes, Detergents, and Other Potential Membrane Perturbants on an Osmotolerant Yeast, Saccharomyces rouxii

“…,B-Fructofuranosidase assays were conducted at 30°C with sucrose as substrate. Specific activities are expressed as International Units (IU) per gram (dry weight) of yeast (2). A 15-min pretreatment of a given yeast sample with 0.1 volume of ethylacetate (2) was taken as revealing the total enzyme concentration; degrees of expression in companion treatments are given as percentages of the total activity.…”