Summary. The kinetics and enzymology of o-xylose utilization were studied in aerobic and anaerobic batch cultures of the facultatively fermentative yeasts Candida utilis, Pachysolen tannophilus, and Pichia stipitis. These yeasts did not produce ethanol under aerobic conditions. When shifted to anaerobiosis cultures of C. utilis did not show fermentation of xylose; in Pa. tannophilus a very low rate of ethanol formation was apparent, whereas with Pi. stipitis rapid fermentation of xylose occurred. The different behaviour of these yeasts ist most probably explained by differences in the nature of the initial steps of xylose metabolism: in C. utilis xylose is metabolized via an NADPH-dependent xylose reductase and an NAD+-linked xylitol dehydrogenase. As a consequence, conversion of xylose to ethanol by C. utilis leads to an overproduction of NADH which blocks metabolic activity in the absence of oxygen. In Pa. tannophilus and Pi. stipitis, however, apart from an NADPH-linked xylose reductase also an NADH-linked xylose reductase was present. Apparently xylose metabolism via the NADH-dependent reductase circumvents the imbalance of the NAD+/NADH redox system, thus allowing fermentation of xylose to ethanol under anaerobic conditions. The finding that the rate of xylose fermentation in Pa. tannophilus and Pi. stipitis corresponds with the activity of the NADH-linked xylose reductase activity is in line with this hypothesis. Furthermore, a comparative study with various xylose-assimilating yeasts showed that significant alcoholic fermentation of xylose only occurred in those organisms which possessed NADH-linked aldose reductase.
Correlations for the prediction of biomass yields are valuable, and many proposals based on a number of parameters (Y(ATP), Y(Ave), eta(o), Y(c), Gibbs energy efficiencies, and enthalpy efficiencies) have been published. This article critically examines the properties of the proposed parameters with respect to the general applicability to chemotrophic growth systems, a clear relation to the Second Law of Thermodynamics, the absence of intrinsic problems, and a requirement of only black box information. It appears that none of the proposed parameters satisfies all these requirements. Particularly, the various energetic efficiency parameters suffer from major intrinsic problems. However, this article will show that the Gibbs energy dissipation per amount of produced biomass (kJ/C-mod) is a parameter which satisfies the requirements without having intrinsic problems. A simple correlation is found which provides the Gibbs energy dissipation/C-mol biomass as a function of the nature of the C-source (expressed as the carbon chain length and the degree of reduction). This dissipation appears to be nearly independent of the nature of the electron acceptor (e.g., O(2), No(3) (-), fermentation). Hence, a single correlation can describe a very wide range of microbial growth systems. In this respect, Gibbs energy dissipation is much more useful than heat production/C-mol biomass, which is strongly dependent on the electron acceptor used. Evidence is presented that even a net heat-uptake can occur in certain growth systems.The correlation of Gibbs energy dissipation thus obtained shows that dissipation/C-mol biomass increases for C-sources with smaller chain length (C(6) --> C(1)), and increases for both higher and lower degrees of reduction than 4. It appears that the dissipation/C-mol biomass can be regarded as a simple thermodynamic measure of the amount of biochemical "work" required to convert the carbon source into biomass by the proper irreversible carbon-carbon coupling and oxidation/reduction reactions. This is supported by the good correlation between the theoretical ATP requirement for biomass formation on different C-sources and the dissipation values (kJ/C-mol biomass) found. The established correlation for the Gibbs energy dissipation allows the prediction of the chemotrophic biomass yield on substrate with an error of 13% in the yield range 0.01 to 0.80 C-mol biomass/(C)-mol substrate for aerobic/anaerobic/denitrifying growth systems.
The coupling of membrane-bound glucose dehydrogenase (EC 1.1.99.17) to the respiratory chain has been studied in whole cells, cell-free extracts, and membrane vesicles of gram-negative bacteria. Several Escherichia coil strains synthesized glucose dehydrogenase apoenzyme which could be activated by the prosthetic group pyrrolo-quinoline quinone. The synthesis of the glucose dehydrogenase apoenzyme was independent of the presence of glucose in the growth medium. Membrane vesicles of E. coli, grown on glucose or succinate, oxidized glucose to gluconate in the presence of pyrrolo-quinoline quinone. This oxidation led to the generation of a proton motive force which supplied the driving force for uptake of lactose, alanine, and glutamate. Reconstitution of glucose dehydrogenase with limiting amounts of pyrrolo-quinoline quinone allowed manipulation of the rate of electron transfer in membrane vesicles and whole cells. At saturating levels of pyrrolo-quinoline quinone, glucose was the most effective electron donor in E. coli, and glucose oxidation supported secondary transport at even higher rates than oxidation of reduced phenazine methosulfate. Apoenzyme of pyrrolo-quinoline quinone-dependent glucose dehydrogenases with similar properties as the E. coli enzyme were found in Acinetobacter calcoaceticus (var. Iwoffi) grown aerobically on acetate and in Pseudomonas aeruginosa grown anaerobically on glucose and nitrate.on July 5, 2020 by guest http://jb.asm.org/ Downloaded from 494 van SCHIE ET AL.
867Acinetobacter lwofi, Azotobacter vinelandii, Agrobacterium and Rhizobium species contain quinoprotein glucose dehydrogenase apoenzyme (EC 1.1.99.17). Addition to whole cells of pyrrolo-quinoline quinone (PQQ), the prosthetic group of this enzyme, resulted in the production of gluconic acid from glucose. The in vivo reconstitution of apo-glucose dehydrogenase with PQQ was dependent on the presence of Ca2+ or Mg2+. Optimal conditions for reconstitution allowed maximal glucose dehydrogenase activity in the presence of 1-1 0 nmol PQQ 1-l. Synthesis of the apoenzyme of glucose dehydrogenase was not dependent on glucose in the growth media. The physiological significance of the synthesis of apo-glucose dehydrogenase, as found in a variety of bacteria, is discussed.
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