From a screening of several Kluyveromyces strains, the yeast Kluyveromyces marxianus CBS 6556 was selected for a study of the parameters relevant to the commercial production of inulinase (EC 3.2.1.7). This yeast exhibited superior properties with respect to growth at elevated temperatures (40 to 45°C), substrate specificity, and inulinase production. In sucrose-limited chemostat cultures growing on mineral medium, the amount of enzyme decreased from 52 U mg of cell dry weight-' at D = 0.1 h-l to 2 U mg of cell dry weight1 l at D = 0.8 h-'. Experiments with nitrogen-limited cultures further confirmed that synthesis of the enzyme is negatively controlled by the residual sugar concentration in the culture. High enzyme activities were observed during growth on nonsugar substrates, indicating that synthesis of the enzyme is a result of a derepression/ repression mechanism. A substantial part of the inulinase produced by K. marxianus was associated with the cell wall. The enzyme could be released from the cell wall via a simple chemical treatment of cells. Results are presented on the effect of cultivation conditions on the distribution of the enzyme. Inulinase was active with sucrose, raffinose, stachyose, and inulin as substrates and exhibited an S/I ratio (relative activities with sucrose and inulin) of 15 under standard assay conditions. The enzyme activity decreased with increasing chain length of the substrate.

The possible physiological role of mitochondria in anaerobically grown Saccharomyces cerevisiae was investigated via enzyme localization and inhibitor studies. Almost all of the activity of citrate synthase (EC 4.1.3.7) was recovered in the mitochondrial fraction after differential centrifugation of spheroplast lysates. The enzyme exhibited a high degree of latency which was demonstrated by sonication of the mitochondrial fractions. Since citrate synthase is an important enzyme in anabolic reactions, a consequence of this localization is the requirement for transport of metabolites across the mitochondrial membranes. Such transport is likely to require energy which, as a result of anaerobiosis, cannot be supplied by respiration. It was therefore investigated whether ATP translocation into the mitochondria by an ADP/ATP translocase might be involved in anaerobic mitochondrial energy metabolism. It was shown that addition of the ADP/ATP translocase inhibitor bongkrekic acid to anaerobic cultures indeed inhibited growth, although only partially. It is concluded that mitochondria of S. cerevisiae fulfil a vital role in anaerobic sugar metabolism. Keywords INTRODUCTIONThe existence of mitochondria in anaerobically grown yeast cells has been disputed for a long period in the literature. Early reports (Wallace & Linnane, 1964;Linnane, 1965 ;Chapman & Bartley, 1968) claim that such cells are completely devoid of mitochondria and that adaptation to aerobic conditions involves de novo synthesis of these organelles. These reports were based on electron microscopic studies. Later it was shown that inadequate staining or fractionation procedures had led to these conclusions (Damsky e t al., 1969; Cartledge e t al., 1972;Cartledge & Lloyd, 1972; Jenkins e t a/., 1984). Since the membranous structures found under anaerobic conditions were quite different from the well-known aerobic mitochondria, the term ' pro-mitochondria ' was proposed to emphasize the relationship to aerobic organ- elles, as well as the differences between these organelles and fully functional mitochondria.In all reports on the disappearance of mitochondria under anaerobic conditions, a discussion of the physiological consequences was restricted to the respiratory system, i.e. cytochromes and oxidases and the F,-ATPase (Groot et a/., 1971). However, it is well-established that most enzymes of the citric acid cycle as well as some enzymes for sterol biosynthesis (Shimizu e t al., 1973) and amino acid synthesis (Ryan & Kohlhaw, 1974;Jauniaux et al., 1978) are localized inside the mitochondria and hence disappearance of the organelle could be lethal during growth under anaerobic conditions. Evidence for the indispensability of mitochondria is the fact that yeast proteins mediating protein import into mitochondria are essential for cell viability (Jensen & Yaffe, 1988;Baker & Schatz, 1991 ; Stuart e t al., 1994 On: Thu, 10 May 2018 13:33:32 W. V I S S E R a n d O T H E R S great attention has been given to the isolation of intact mitochondria. Citrate synthase...

In synchronized continuous cultures of Saccharomyces cerevisiae CBS 8066, the production of the extracellular invertase (EC 3.2.1.26) showed a cyclic behavior that coincided with the budding cycle. The invertase activity increased during bud development and ceased at bud maturation and cell scission. The cyclic changes in invertase production resulted in cyclic changes in amounts of invertase localized in the cell wall. However, the amount of enzyme invertase present in the culture liquid remained constant throughout the budding cycle. Also, in asynchronous continuous cultures of S. cerevisiae, the production and localization of invertase showed significant fluctuation. The overall invertase production in an asynchronous culture was two to three times higher than in synchronous cultures. This could be due to more-severe invertase-repressive conditions in a synchronous chemostat culture. Both the intracellular glucose-6-phosphate concentration and residual glucose concentration were significantly higher in synchronous chemostat cultures than in asynchronous chemostat cultures. In the asynchronous and synchronous continuous cultures of S. cerevisiae, about 40% of the invertase was released into the culture liquid; it has generally been believed that S. cerevisiae releases only about 5% of its invertase. In contrast to invertase production and localization in the chemostat cultures of S. cerevisiae, no significant changes in inulinase (EC 3.2.1.7) production and localization were observed in chemostat cultures of Kluyveromyces marxianus CBS 6556. In cultures of K. marxianus about 50% of the inulinase was present in the culture liquid.