Abstract
The anaerobic fermentation of glucose by Leuconostoc mesenteroides via the reductive pentose phosphate pathway leads to the accumulation of lactic acid and ethanol. The isotope redistribution coefficients (a(ij)) that characterize the specific derivation of each hydrogen atom in ethanol in relation to the non-exchangeable hydrogen atoms in glucose and the medium water have been determined using quantitative (2)H NMR. First, it is confirmed that the hydrogens of the methylene group are related only to the 1 and 3 positions of glucose via the NAD(P)H pool and not to the 4 position, in contrast to ethanol produced by Saccharomyces cerevisiae. Second, it is found that the conversion factors (C(f)) for the transfer of hydrogen to the pro-S and pro-R positions of the methylene group are not equivalent: the C(f)-1-R:C(f)-1-S ratio is 2.1, whereas the C(f)-3-R:C(f)-3-S ratio is 0.8. It is shown that this non-equivalence is not determined by the stereochemistry of the terminal NADH- and NADPH-dependent alcohol dehydrogenases, but is dependent on the cofactor selectivities of the reductive and oxidative steps of the reduced nucleotide cycle.
Highlights
Such studies mostly show only the qualitative relationships
J where (2H/1H)i is the isotopic ratio at position i of the product, (2H/1H)m is the isotopic ratio of the medium, and (2H/ 1H)j is the isotopic ratio at site j of the substrate
The technique of quantitatively describing the hydrogen isotope transfer coefficient (Cf) values using 2H NMR has been used to show that it is possible to distinguish the pathway used to ferment glucose to lactic acid by the lactic acid bacteria Lactococcus (Lc.) lactis and Leuconostoc (Ln.) mesenteroides (RPP) [5]
Summary
Enzyme Extracts—Bacterial cells were recovered from culture by centrifugation (4250 ϫ g for 10 min at 4 °C) and the pellet was resuspended in 20 ml of ice-cold sodium phosphate buffer (100 mM, pH 7.1). This suspension was transferred to a 30-ml centrifuge tube and repelleted (4250 ϫ g for 10 min at 4 °C). After 15 min at 30 °C, 1.0 ml of Ln. mesenteroides crude extract and 100 l of acetaldehyde (1.78 M, 100 l/ml H2O) were added, and the mixture was left to incubate for ϳ18 h at 30 °C. (2H/1H)i of ethanol produced by fermentation with Ln. mesenteroides using labeled water or glucose
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