Effect of ethanol, carbon tetrachloride, and methyl ethyl ketone on butanol oxidase activity in rat lung and liver.

The ability of the rat liver to oxidize 2-butanol via a cytochrome P-450-mediated mixed-function oxidase reaction is well known. The purpose of this study was to examine this microsomal alcohol oxidizing system in rat lung and determine if it could be altered by treatments that inhibit or induce this activity. 2-Butanol was incubated with microsomal preparations from male rats, and methyl ethyl ketone production was measured by gas chromatography. The rate was six to eight times lower in lung than in liver. Administration of low doses of ethanol (0.5 ml/kg and 1.0 ml/kg) ip for 7 d did not alter activity in the liver but was inhibitory in the lung, as was a high dose of 3.0 ml/kg in the liver. Carbon tetrachloride (1.0 ml/kg, ip) decreased activity in both tissues, especially the lung. The effects of the two inhibitors were not additive. Methyl ethyl ketone induced 2-butanol oxidation in both tissues. The lung possesses butanol oxidase activity that is alterable by both inhibitors and inducers.

Acetoin production by wild-type strains and a lactate dehydrogenase-deficient mutant of Streptococcus mutans.

Eleven different laboratory strains of Streptococcus mutans representing the various serogroups were found to produce an average of 6.0 +/- 4.8 mM acetoin when grown in glucose-containing medium under aerobic conditions. None of the strains produced detectable acetoin when grown anaerobically. A lactate dehydrogenase-deficient mutant produced acetoin both aerobically and anaerobically and in substantially greater amounts than the wild-type strains did. Substitution of mannitol for glucose resulted in decreased acetoin production by wild-type strains and the lactate dehydrogenase-deficient mutant, indicating a role for NADH2 in the regulation of the acetoin pathway. Pyruvate incorporated into the growth medium of a wild-type strain caused acetoin to be produced anaerobically and stimulated acetoin production aerobically. Cell extracts of a wild-type S. mutans strain were capable of producing acetoin from pyruvate and were (partly) dependent on thiamine PPi. Extracts prepared from aerobically grown cells had approximately twice the acetoin-producing activity as did extracts prepared from anaerobically grown cells. The results indicate that acetoin production by S. mutans may represent an auxiliary reaction of pyruvate dehydrogenase in this organism.

Brewers' yeast pyruvate decarboxylase produces acetoin from acetaldehyde: a novel tool to study the mechanism of steps subsequent to carbon dioxide loss.

A gas-liquid chromatographic technique was developed for the determination of both acetaldehyde and the 3-4% acetoin side product that results from the brewers' yeast pyruvate decarboxylase (EC 4.1.1.1) catalyzed reaction of pyruvic acid. Employing this method enabled the demonstration of the catalysis of acetaldehyde condensation to acetoin by the enzyme. It was found that the acetoin produced enzymatically from pyruvic acid or from acetaldehyde was optically active, thus providing stereochemical information about the reaction. Deuterium kinetic isotope effects (employing CH3CHO and CH3CDO) were determined on the steady-state kinetic parameters to be 4.5 (Vmax) and 3.2 (Vmax/Kappm), respectively. This enabled, for the first time, the estimation of relative kinetic barriers for steps past decarboxylation. It could be concluded that (a) C-H bond scission was part of rate limitation in the enzyme-catalyzed condensation of acetaldehyde to acetoin and that (b) among the steps leading to the release of acetaldehyde, protonation of the key enamine intermediate was part of rate limitation. This latter finding is also directly applicable to the mechanism of pyruvate decarboxylation.

[The intensification of the Voges-Proskauer-reaction by fumarate]. / Die Verstärkung der Voges-Proskauer-Reaktion durch Fumarat.

The influence of different substances and growth temperatures on the Voges-Proskauer-reaction of Vibrio spec. (HIM 627-1) was investigated by gas chromatographic analysis of acetoin produced and the Voges-Proskauer-reaction itself. The most efficacious conditions for a maximum production of acetoin and the result of the Voges-Proskauer-reaction, respectively, were cultivation of the bacteria in complex Voges-Proskauer-medium in the presence of 0.03 M glucose and 0.05 M fumarate at 37 degrees C. The examination of various so-called Voges-Proskauer positive bacterial strains showed that the production of acetoin and the Voges-Proskauer-reaction, respectively, was increased by cultivation of the bacteria in the presence of fumarate. Fumarate did not influence the reaction of the so-called Voges-Proskauer negative strains, so that false-positive reactions did not occur.

Laboratory-scale production of acetoin plus diacetyl by Enterobacter cloacae ATCC 27613.

Conditions for the laboratory-scale production of acetoin plus diacetyl by Enterobacter cloacae ATCC 27613 were studied. Thirty-five g acetoin plus diacetyl/50 g sucrose were obtained when fermentation was carried out in 2.5 liter medium containing 12.5 g peptone and 12.5 g yeast extract, at pH 7.0, in a 5 liter conical flask on a shaker (240 rpm) at 28-30 degrees C for 48 hr. Recovery of pure diacetyl was 85% of the total acetoin plus diacetyl.

Rapid test for acetyl-methyl-carbinol formation by Enterobacteriaceae.

A modified Voges-Proskauer test is described which distinguishes within 4 to 8 hours between organisms that can produce acetyl-methyl-carbinol (acetoin) from glucose fermentation and those that cannot.

Studies on the formation of acetoin from acetaldehyde by the pyruvate dehydrogenase complex and its regulation.

1. Pyruvate dehydrogenase complex was isolated from pigeon breast muscle involving steps of isoelectric precipitation, poly(ethyleneglycol) fractionation and separation on a glycerine gradient in the ultracentrifuge. 2. Arsenite, a potent inhibitor of the dihydrolipoyl transcetylase, did not affect the formation of acetoin from acetaldehyde, indicating that the pyruvate dehydrogenase component was operative in this reaction. 3. Production of acetoin by the pyruvate dehydrogenase complex is subject to regulation by phosphorylation and dephosphorylation, the dephosphorylated form only being active. 4. The inhibition by acetaldehyde of the pyruvate dehydrogenase complex could be partly explained by the formation of acetoin as an alternative reaction.

Electron capture gas chromatographic detection of acethylmethylcarbinol produced by neisseria gonorrhoeae.

Acetylmethylcarbinol (acetoin) production by Neisseria gonorrhoeae and other Neisseria species was established by gas-liquid chromatography and by mass spectrometric data. Sixty-nine isolates of Neisseria were tested by incubating them in a chemically defined fluid medium. The medium was extracted with organic solvents and derivatized with heptafluorobutryic anhydride for gas chromatography and mass spectrometry. Cultures of 58 of the same strains were tested with the conventional Voges-Proskauer reagents, and results were compared with those of gas-liquid chromatography. When glucose was used as an energy source, N. gonorrhoeae, some N. meningitidis, and N. lactamica produced enough acetoin in 16 h to be detectable by either method, whereas other Neisseria species produce amounts detectable only by gas chromatography. The conventional acetylmethylcarbinol test with the chemically defined medium and maltose as an energy source might be used to develop methods that would differentiate certain members of the genus, including the pathogenic species.

Effect of substrates on acetoin production by Torulopsis colliculosa and Enterobacter species.

Under optimal conditions, Torulopsis colliculosa NRRL 172 and Enterobacter B-87 (ATCC 27613) produced 50 to 500 mg of acetoin per g of substrate. Whereas cane molasses, gur, glucose, and sucrose were suitable substrates for acetoin production, lactose and mannitol supported very good growth but yielded little or no acetoin. Production of acetoin increased with increases in the concentration of glucose, yeast extract, and peptone. Combination of substrates and intermittent feeding of substrate failed to increase the yields.

[Formation of acetoin by Actinomyces olivaceus cultures]. / Obrazovanie atsetoina kul'turoi Actinomyces olivaceus

Actinomyces olivaceus and some other actinomycetes accumulate acetoin and 2,3-butanediol in the cultural broth. Addition of cobalt to the medium favours the accumulation of acetoin.