ABSTRACT
The effect of inositol supplementation on glucose derepression, invertase secretion and SUC2 gene expression in Saccharomyces sp. W4 was studied. Invertase secretion was repressed, when the yeast cells, grown the synthetic medium without inositol (I(-) medium) contained more than 0.2% (w/v) initial concentration of glucose. However, in the same medium plus inositol (I(+) medium, inositol conc. 100 microg/100 ml), invertase secretion was repressed only at glucose concentrations higher than 2.0% (w/v). Results showed that secreted invertase activity increased only in the I+ medium, whereas intracellular invertase activity remained constant in both media during the cell, growth. The mRNA encoding secreted invertase was higher in the glucose-derepressed cells grown in the I(+) medium than in the glucose-repressed cells grown in the I(-) medium. Similarly, phosphatidylinositol (PI) content was significantly higher in the cells grown in the I(+) medium than in the I(-) medium. These results indicated that PI might be involved in the glucose derepression, invertase secretion and SUC2 gene expression at the transcriptional level in the yeast.
Subject(s)
Cell Culture Techniques , Culture Media , Dose-Response Relationship, Drug , Gene Expression Regulation, Fungal , Glucose/metabolism , Inositol/metabolism , Phospholipids/metabolism , RNA/metabolism , RNA, Fungal/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Saccharomyces/metabolism , Saccharomyces cerevisiae Proteins/biosynthesis , Time Factors , beta-Fructofuranosidase/biosynthesisABSTRACT
Mutant A11, a mutant of Saccharomycopsis fibuligera Sdu with low acid and neutral trehalase was found to accumulate over 18% (w/w) trehalose from starch in its cells. In this study, trehalose-6-phosphate synthase (Tps1) was purified to homogeneity from this mutant, with a 30-fold increase in the specific enzyme activity, as compared to the concentrated cell-free extract, from initial cells. The molecular mass of the purified enzyme as determined by SDS-PAGE was 66 kDa. The optimum pH and temperature of the purified enzyme were 6.6 and 37 degrees C, respectively. The enzyme was activated by Ca2+, K+ and Mg2+, with K+ showing the highest activation at 35 mM. On the other hand, Mn2+, Cu2+, Fe3+, Hg2+ and Co2+ inhibited the enzyme. The enzyme was also strongly inhibited by protease inhibitors such as iodoacetic acid, EDTA and PMSF.