Localization of inulinase and invertase in Kluyveromyces species

Abstract

In vivo hydrolysis of inulin and sucrose was examined in selected yeasts of the genus Kluyveromyces. Cells, grown in sucrose-limited chemostat cultures, were subjected to treatments for the removal of inulinase, the enzyme responsible for the hydrolysis of both inulin and sucrose. The effects of these treatments were studied by measurement of inulin-dependent and sucrose-dependent oxygen consumption by cell suspensions. In Kluyveromyces marxianus var. marxianus, inulinase was partially secreted into the culture fluid. Removal of culture fluid inulinase by washing had no effect on sucrose-dependent oxygen consumption by this yeast. However, this treatment drastically reduced inulin-dependent oxygen consumption. Treatment of washed cells with sulfhydryls removed part of the cell wall-retained inulinase and reduced inulin-dependent oxygen consumption by another 80%. Sucrose-dependent oxygen consumption was less affected, decreasing by 40%. Cell suspensions of K. marxianus var. drosophilarum, K. marxianus var. vanudenii, and Saccharomyces kluyveri rapidly utilized sucrose but not inulin. This is in accordance with the classification of these yeasts as inulin negative. Supernatants of cultures grown at pH 5.5 did not catalyze the hydrolysis of inulin and sucrose. This suggested that these yeasts contained a strictly cell-bound invertase, an enzyme not capable of inulin hydrolysis. However, upon washing, cells became able to utilize inulin. The inulin-dependent oxygen consumption further increased after treatment of the cells with sulfhydryls. These treatments did not affect the sucrose-dependent oxygen consumption of the cells. Apparently, these treatments removed a permeability barrier for inulin that does not exist for sucrose. Nondenaturing polyacrylamide gel electrophoresis and determination of the S/I ratio (relative activity with sucrose and inulin) of enzyme preparations proved that in these yeasts, as in K. marxianus var. marxianus, hydrolysis of sucrose and inulin is catalyzed by the same enzyme, namely inulinase. This cryptic inulinase activity is not a physiological artifact. When cells were inoculated in media of pH 4.5 and incubated at 35°C instead of the standard cultivation conditions used in yeast taxonomy (pH 5.6, 25°C), rapid growth on inulin occurred. Both inulin-and sucrose-hydrolyzing activities could be detected in culture supernatants of these yeasts under these new conditions. Physiological, ecological, and taxonomic aspects of the occurrence and localization of inulinase in Kluyveromyces strains are discussed.

Purification and properties of an extracellular inulinase-like β-fructosidase from Bacillus stearothermophilus

“…ficuurn. On the other hand, the data are significantly different from those obtained by Rouwenhorst et al (1990a) and Workman and Day (1983) concerning inulinases isolated from Kluyveromyces species (50°C and p H 4-5) and invertase from S . cerevisiae (37°C and p H 4-5).…”

“…A considerable amount of information has been devoted to /?-fructosidases (invertase E.C.3.2.1.26) and inulinases (E.C.3.2.1.7) of yeasts (Parekh and Margarits 1986;Esmon et al 1987;Rouwenhorst et al 1990a) and molds (Ettalibi and Baratti 1987;Uhrn et al 1987) but only a few studies reporting /?-fructosidase activity in bacteria have been done (Efstathiou et al 1986;Allais et al 1987), except for Bacillus species (Uhm et al 1985;Allais et al 1987;Vullo et al 1991). These enzymes have potential use in the preparation of fructose syrups from inulin and invert sugar from sucrose in the food industry (Monsan and Combes 1984;Vullo et al 1990).…”

Structure and properties of the extracellular inulinase of Kluyveromyces marxianus CBS 6556

“…Remarkably, the S. cerevisiae SUC2 sequence hardly hybridized with the genome of the yeast S. kluyveri UCD 51-242 (6). Further research into the nature of the sucrose-hydrolyzing activity of S. kluyveri showed that this yeast possesses an inulinase, as defined by S/I ratio and behavior on native gel electrophoresis (31).…”

Morphological characterization of the dimorphic yeast Kluyveromyces marxianus var. marxianus NRRLy2415 by semi-automated image analysis

“…marxianus NRRLy2415, a strain which has previously been shown to be dimorphic in character (Walker and O'Neill, 1990). K. marxianus is extensively used for the fermentation of cheese whey permeate to ethanol (Vienne and von Stockar, 1985) and also possesses a wealth of hydrolytic enzymes, including galactosidase (Ku and Hang, 1992), inulinase (Rouwenhorst et al, 1990), and polygalacturonases (Schwan and Rose, 1994).…”