Phosphorus-31 and carbon-13 nuclear magnetic resonance studies of glucose and xylose metabolism in Candida tropicalis cell suspensions

Abstract

The metabolism of glucose and xylose was studied as a function of oxygenation in suspensions of Candida tropicalis by 31 P and 13 C nuclear magnetic resonance spectroscopy. Both the rate of carbohydrate metabolism and the cytoplasmic pH were independent of the rate of oxygenation in cells metabolizing glucose. However, these two parameters were markedly dependent on the rate of oxygenation in C. tropicalis cells metabolizing xylose. For example, the cytoplasmic pH in fully oxygenated xylose-metabolizing cells was 7.8 but decreased to 6.3 in anoxic cells. In general, suspensions of cells consuming xylose had a lower rate of sugar uptake, a more acidic cytoplasmic pH, lower levels of sugarphosphomonoesters (SP) and ATP, higher levels of intracellular P i , a more alkaline vacuolar pH, and a lower rate of extracellular P i assimilation and polyphosphate synthesis than cells consuming glucose. These observations indicate that C. tropicalis metabolizing xylose is less energized than glucose-metabolizing cells. On both carbon sources, however, an inverse correlation between intracellular levels of SP and P i was observed. Also, uptake of extracellular P i correlated with the synthesis of polyphosphates within the cells. During anoxia, P i was not taken up, and polyphosphates were hydrolyzed instead to fulfill the cells' requirements for phosphate.

Phosphorus-31 and carbon-13 nuclear magnetic resonance studies of glucose and xylose metabolism in cell suspensions and agarose-immobilized cultures of Pichia stipitis and Saccharomyces cerevisiae

“…Figure 1 shows representative 13 C-NMR spectra obtained at the beginning and end of an experiment in which xylose was metabolized by an oxygenated P. stipitis cell suspension. Although this sample had been bubbled at a rate of 45 ml/min with pure oxygen, the signal-to-noise ratio is acceptable for data analysis (20). The time course of the fermentation was monitored by observing the decrease in the ␣ and ␤ anomeric C-1 resonances of xylose and the increase in the ethanol peaks as a function of time.…”

“…Organism and cultivation conditions. P. stipitis CBS 6054 and S. cerevisiae ATCC 28460 were maintained at 4ЊC on xylose-complex plates as previously described (20). Colonies of S. cerevisiae were much smaller when grown with xylose as the sole carbon source and took longer to establish than those of P. stipitis, suggesting that growth occurred mainly on the peptone or yeast extract components of the media.…”

“…The samples were bubbled with either oxygen or nitrogen at 45 ml/min. Under these conditions, cells are close to being fully oxygenated (20); more vigorous bubbling leads to deterioration of the NMR spectra. A capillary (inner diameter, 1.0 mm) containing 0.2 M methylene diphosphonic acid was used as an external chemical shift and intensity reference.…”

“…It may have even further beneficial effects; for example, the fermentation of D-glucose to ethanol is more rapid when immobilized Saccharomyces strains rather than cell suspensions of similar concentration are used (9,11,13,22,27,30,33). However, our recent attempts to improve the rate of D-glucose and D-xylose fermentation by using agarose-entrapped Candida tropicalis cells were not successful (19); in fact, the metabolism of both carbon sources was considerably slower for the immobilized cells than could be predicted on the basis of studies with cell suspensions (20). Potential reasons for the different behavior of these two yeast strains upon cell entrapment are threefold.…”

Inhibition kinetics of catabolic dehydrogenases by elevated moieties of ATP and ADP – implication for a new regulation mechanism in Lactococcus lactis

“…In stark contrast, there is a synergy between ATP and ADP (a < 1) at the active site of ADH and GAPDH, and, in addition, the dissociation constants were lower in the presence of the other inhibitor ( Table 2). In conclusion, when interpreted as the pool of ATP and ADP [using Eqn (6) and parameter values in Table 2], the two nucleotides affected ADH most strongly (> 95% inhibition), whereas most of the LDH activity was maintained (30-40% inhibition) (Fig. 3A).…”