Succinic Acid.
The origin of the succinic acid formed in fermentation has also been traced by Ehrlich [[1909]] to the alcoholic fermentation of the amino-acids. It was shown by Buchner and by Kunz [[1906]] that succinic acid like fusel oil is not formed during fermentation by yeast-juice or zymin, and, in the light of Ehrlich's work on fusel oil, several [p090] modes of formation appeared possible for this substance [Ehrlich, [1906, 3]]. The dibasic amino-acids might, for example, undergo simple reduction, the NH2 group being removed as ammonia and replaced by hydrogen. Aspartic acid would thus pass into succinic acid:—
COOH·CH2·CH(NH2)·COOH + 2 H = COOH·CH2·CH2·COOH + NH3.
This change can be effected in the laboratory only by heating with hydriodic acid. Biologically it has been observed [E. and H. Salkowski, [1879]] when aspartic acid is submitted to the action of putrefactive bacteria, and almost quantitatively when Bacillus coli communis is cultivated in a mixture of aspartic acid and glucose [Harden, [1901]]. In this case a well-defined source of hydrogen exists in the glucose, which when acted on by this bacillus yields a large volume of gaseous hydrogen, which is not evolved in the presence of aspartic acid. Some such source is also available in the case of yeast, although it cannot be chemically defined, for this organism is known to produce many reducing actions, which are usually ascribed to the presence of reducing ferments or reductases in the cell.
A similar action would convert glutamic acid,
COOH·CH2·CH2·CH(NH)2·COOH,
into glutaric acid,
COOH·CH2·CH2·CH2·COOH,
which also is found among the products of fermentation, whilst the monamino-acids would pass into the simple fatty acids.
On submitting these ideas to the test of experiment, however, Erhlich found that the addition of aspartic acid did not in any way increase the yield of succinic acid, and that of all the amino-acids which were tried only glutamic acid, COOH·CH2·CH2·CH(NH2)·COOH, produced a definite increase in the amount of this substance. Further experiments showed that glutamic acid was actually the source of the succinic acid, the relations being quite similar to those which exist for the production of fusel oil.
Succinic acid is formed whenever sugar is fermented by yeast, even in the absence of added nitrogenous matter, and amounts to 0·2 to 0·6 per cent. of the weight of the sugar decomposed, its origin in this case being the glutamic acid formed by the autolysis of the yeast protein. When some other source of nitrogen is present, such as asparagine or an ammonium salt, the amount falls to 0·05 to 0·1. If glutamic acid be added it rises to about 1 to 1·5 per cent. but falls again to about 0·05 to 0·1 when other sources of nitrogen, such as asparagine or ammonium salts, are simultaneously available, either in the presence or [p091] absence of added glutamic acid. As in the case of fusel oil, the production does not occur in the absence of sugar, and is not effected by yeast-juice or zymin.
The chemical reaction involved in the production of succinic acid differs to some extent from that by which fusel oil is formed, inasmuch as an oxidation is involved:—
COOH·CH2·CH·CH(NH2)·COOH + 2 O = COOH·CH2·CH2·COOH + NH3 + CO2.
From analogy with the production of amyl alcohol from leucine, glutamic acid would be expected to yield γ-hydroxybutyric acid:—
COOH·CH2·CH2·CH(NH2)·COOH + H2O = NH3 + CO2 + COOH·CH2·CH2·CH2·OH.
As a matter of fact this substance cannot be detected among the products of fermentation, but succinic acid as already explained is formed. This acid might, however, possibly be formed by the oxidation of the γ-hydroxybutyric acid:—
COOH·CH2·CH2·CH2·OH + 2 O = COOH·CH2·CH2·COOH + H2O,
although this change is on biological grounds improbable.
The conversion of the group —CH(NH2)— into the terminal CH2·OH in fusel oil, or COOH in succinic acid, may possibly be effected in several different ways, the most probable of which are the following:—
I. Direct elimination of carbon dioxide, followed by hydrolysis of the resulting amine:—
(1) R·CH(NH2)·COOH = R·CH2·NH2 + CO2.
(2) R·CH2·NH2 + H2O = R·CH2·OH + NH3.
The reaction (1) is actually effected by many bacteria and has been employed for the preparation of bases from amino-acids [cf. Barger, [1914], p. 7], although there is no direct evidence that it can be brought about by yeast. On the other hand reaction (2) has actually been observed with some yeasts. Thus it has been found [Ehrlich and Pistschimuka, [1912, 1]] that many "wild" yeasts produce this change with great readiness in presence of sugar, glycerol or ethyl alcohol as sources of carbon and grow well in media in which amines, such as p-hydroxyphenylethylamine or iso-amylamine, form the only source of nitrogen. Willia anomala (Hansen), a yeast which forms surface growths, succeeds admirably under these conditions, whereas culture yeasts are much less active in this way, although they produce a certain amount of change. It is therefore possible that this mode of decomposition plays some part in the production of fusel oil, but in the case of culture yeasts it is entirely subordinated to the mode next to be discussed. [p092]
II. Oxidative removal of the –NH2 group with formation of an α-ketonic acid:—
(1) R·CH(NH2)·COOH + O = R·CO·COOH + NH3
followed by the decomposition of the ketonic acid into carbon dioxide and an aldehyde and the subsequent reduction or oxidation of the aldehyde:—
(2) R·CO·COOH = R·CHO + CO2.
(3) (a) R·CHO + 2 H = R·CH2OH.
(b) R·CHO + O = R·COOH.
The evidence for the occurrence of reaction (1) is supplied by the experiments of Neubauer and Fromherz [[1911]]. Having previously found that amino-acids undergo a change of this kind in the animal body, Neubauer investigated their behaviour towards yeast. Taking dl-phenylaminoacetic acid, C6H5·CH(NH2)·COOH, it was found that the changes produced were essentially the same as in the animal body. The l-component of the acid was partly acetylated and partly unchanged, whereas the d-component of the acid yielded benzyl alcohol, C6H5·CH2·OH, phenylglyoxylic acid, C6H5·CO·COOH, and the hydroxy-acid C6H5·CH(OH)·COOH. Since however this hydroxy-acid was produced in the l-form it probably arose by the asymmetric reduction of phenylglyoxylic acid, a reaction which can be effected by yeast as was also found to be the case in the animal body [see Dakin, [1912], pp. 52, 78]. Moreover it was shown that when the effects of yeast on a ketonic acid and the corresponding hydroxy-acid were compared, the alcohol was formed in much better yield from the ketonic acid (70 per cent.) than from the hydroxy-acid (3–4 per cent.), the actual example being the production of tyrosol (p-hydroxyphenylethyl alcohol), OH·C6H4·CH2·CH2OH, from p-hydroxyphenylpyruvic acid, OH·C6H4·CH2·CO·COOH, and p-hydroxyphenyl-lactic acid, OH·C6H4·CH2·CH(OH)·COOH respectively.
Neubauer by these experiments established two extremely important points. 1. That the amino-acids actually yield the corresponding α-ketonic acids when treated with yeast and sugar solution. 2. That the a-ketonic acids under similar conditions give the alcohol containing one carbon atom less in good yield, whereas the corresponding hydroxy-acids only give an extremely small amount of these alcohols.
It is therefore probable that at an early stage in the decomposition of the amino-acids by yeast a ketonic acid is produced, which then undergoes further change.
The source of the oxygen required for this reaction and the mechanism of oxidation have not yet been definitely ascertained. It is possible [p093] that hydrated imino-acids of the type
| O | H | |
| │ | ||
| R· | C | —COOH |
| │ | ||
| N | H2 |
The spontaneous production of ketonic aldehydes from amino-acids and from hydroxy-acids in aqueous solution, which has been demonstrated by Dakin and Dudley [[1913]], points however to the possibility that the ketonic acid may be a secondary product derived from the corresponding ketonic aldehyde [see also Dakin, [1908]; Neuberg, [1908], [1909]]. This itself may either arise directly from the amino-acid or from a previously formed hydroxy-acid, the latter alternative being, however, improbable in view of the small yield of alcohol obtained from hydroxy-acids by the action of yeast in the experiments of Neubauer and Fromherz.
| R·CH(NH2)·COOH | → | R·CH(OH)·COOH |
| ⇅ | ⇅ | |
| R·CO·CHO | ||
| ↓ + Oxygen | ||
| R·CO·COOH | ||
(2) Whatever be the exact mode by which the ketonic acid is formed, it appears most probable that a compound of this nature forms the starting-point for the next stage in the production of the alcohols. The researches of Neuberg, which have already been discussed on p. 81, have revealed a mechanism in yeast—the enzyme carboxylase—by which these α-ketonic acids are rapidly broken up into an aldehyde and carbon dioxide:
R·CO·COOH = R·CHO + CO2
and it can scarcely be doubted that this is the actual course of the reaction.
(3) The final conversion of the aldehyde into the corresponding alcohol is also a change which it has been proved can be effected by yeast [Neuberg and Rosenthal, [1913]] probably by the aid of the reductase which is one of the weapons in its armoury of enzymes.
Yeast is capable of producing many vigorous reducing actions and rapidly reduces methylene blue and sodium selenite. It is in all probability due to a reaction of this kind that the iso-amylaldehyde and isovaleraldehyde were reduced to the alcohols in Neuberg and Steenbock's experiments [[1913], [1914]], and that considerable quantities of ethyl alcohol are formed in the sugar free fermentation of pyruvic acid [Neuberg and Kerb, [1913, 1]] (see later p. [110] for a discussion of this question).
A further possibility exists that in some cases the aldehyde may [p094] be simultaneously oxidised and reduced or the molecule of one aldehyde reduced and that of another oxidised with production of the corresponding acid and alcohol by an "aldehydo-mutase," similar to that which has been observed by Parnas [[1910]] in many animal tissues. Finally the aldehyde may simply be converted into the corresponding acid by oxidation as appears to take place in the formation of succinic acid.
The intermediate production of an aldehyde would thus be consistent both with the production of alcohols and acids from amino-acids.
Fusel oil would be formed by the reduction of the aldehydes arising from the simple monobasic amino-acids, succinic acid would be produced by oxidation of the aldehyde derived from the dibasic glutamic acid.
In favour of this view is to be adduced the fact that aldehydes such as isobutyraldehyde and valeraldehyde have been found in crude spirit, whilst acetaldehyde is a regular product of alcoholic fermentation [see Ashdown and Hewitt, [1910]]. Benzaldehyde, moreover, has been actually detected as a product of the alcoholic fermentation of phenylaminoacetic acid, C6H5·CH(NH2)·COOH [Ehrlich, [1907, 1]]. Further, the aldehydes so produced would readily pass by oxidation into the corresponding fatty acids, small quantities of which are invariably produced in fermentation.
This view of the nature of the alcoholic fermentation of the amino-acids is undoubtedly to be preferred to that previously suggested by Ehrlich [[1906, 3]] according to which a hydroxy-acid is first formed and then either directly decomposed into an alcohol and carbon dioxide or into an aldehyde and formic acid, the aldehyde being reduced and the formic acid destroyed (see p. [115]).
| R·CH(NH2)·COOH → | R·CH( | OH)·COOH | |
| ↓ | or | ↓ | |
| R·CH2OH + CO2 | R·CHO + H·CO2H | ||
| ↓ | |||
| R·CH2OH | |||
The most probable course of the decomposition by which isoamyl alcohol and succinic acid are produced from leucine and glutamic acid respectively is therefore the following:—
(a) Isoamyl Alcohol.
(1)
(CH3)2·CH·CH2·CH(NH2)·COOH
Leucine
(2)
(CH3)2·CH·CH2·CO·COOH
α-Ketoisovalerianic acid
(3)
(CH3)2CHCH2·CHO
Isovaleraldehyde
+ CO2
(4)
(CH3)2·CH·CH2·CH2OH
Isoamyl alcohol
(b) Succinic Acid.
(1)
COOH·CH2·CH2·CH(NH2)·COOH
Glutamic acid
(2)
COOH·CH2·CH2·CO·COOH
α-Keto-glutaric acid
(3)
COOH·CH2CH2·CHO
Succinic semialdehyde
+ CO2
(4)
COOH·CH2·CH2·COOH
Succinic acid