Relation of Carboxylase to Alcoholic Fermentation.
With regard to the relation of carboxylase to the process of alcoholic fermentation, nothing definite is yet known. As Neuberg points out [see Neuberg and Kerb, [1913, 1]] the universal presence of the enzyme in yeasts capable of producing alcoholic fermentation, and the extreme readiness with which the fermentation of pyruvic acid takes place create a [p083] strong presumption that the decomposition of pyruvic acid actually forms a stage in the process of the alcoholic fermentation of the sugars. On the other hand Ehrlich's alcoholic fermentation of the amino-acids (p. [87]) provides another function for carboxylase—that of decomposing the α-ketonic acids produced by the deaminisation of the amino-acids. It must be remembered in this connection that carboxylase is not specific in its action, but catalyses the decomposition not only of pyruvic acid but also of a large number of other α-ketonic acids, including many of those which correspond to the amino-acids of proteins and are doubtless formed in the characteristic decomposition of these amino-acids by yeast. Carboxylase undoubtedly effects one stage in the production of alcohols from amino-acids, whether it is also the agent by which one stage in the alcoholic fermentation of sugar is brought about still remains to be proved.
A comparison of the conditions of action of carboxylase and zymase has revealed several interesting points of difference. Neuberg and Rosenthal [[1913]] have observed that the fermentation of pyruvic acid by maceration extract commences much more rapidly than that of glucose and interpret this to mean that in the fermentation of glucose a long preliminary process occurs before sufficient pyruvic acid has been produced to yield a perceptible amount of carbon dioxide. The long delay (3 hours) which they sometimes observed in the action of maceration juice on glucose is however by no means invariable (see p. [46]), but in any case indicates that the sugar fermentation can be affected by conditions which are without influence on the pyruvic fermentation. A similar conclusion is to be drawn from the fact that the pyruvic acid fermentation is less affected by antiseptics than the glucose fermentation [Neuberg and Karczag, [1911, 4]; Neuberg and Rosenthal, [1913]], chloroform sufficient to stop the glucose fermentation brought about by yeast or dried yeast being usually without effect on the fermentation of the pyruvates either alone or in presence of boric or arsenious acid. A more important difference is that carboxylase decomposes pyruvic acid in the absence of the co-enzyme which is necessary for the fermentation of glucose [Harden, [1913]; Neuberg and Rosenthal, [1913]]. This can be demonstrated experimentally by washing dried yeast or zymin with water (see p. [63]) until it is no longer capable of decomposing glucose (Harden), or by allowing maceration extract to autolyse or dialyse until it is free from co-enzyme (Neuberg and Rosenthal). The zymase of maceration extract is moreover inactivated in 10 minutes at 50–51°, whereas after this treatment the carboxylase is still active. [p084]
The only conclusion that can be legitimately drawn from these highly interesting facts is that if the decomposition of pyruvic acid actually be a stage in the alcoholic fermentation of glucose the soluble co-enzyme is required for some change precedent to this, so that in its absence the production of pyruvic acid cannot be effected.
CHAPTER VII. THE BY-PRODUCTS OF ALCOHOLIC FERMENTATION.
When pure yeast is allowed to develop in a solution of sugar containing a suitable nitrogenous diet and the proper mineral salts, the liquid at the close of the fermentation contains not only alcohol and some carbon dioxide but also a considerable number of other substances, some arising from the carbonaceous and others from the nitrogenous metabolism of the cell. Prominent among the non-nitrogenous substances which are thus found in fermented sugar solutions are fusel oil, succinic acid, glycerol, acetic acid, aldehyde, formic acid, esters, and traces of many other aldehydes and acids. In addition to these substances which are found in the liquid, there are also the carbonaceous constituents of the newly formed cells of the organism, comprising the material of the cell walls, yeast gum, glycogen, complex organic phosphates, as well as other substances.
The attention of chemists has been directed to these compounds since Pasteur first emphasised their importance as essential products of the alcoholic fermentation of sugar, and his example was generally followed in attributing their origin to the sugar.
The study of cell-free fermentation by means of yeast-juice or zymin has, however, revealed the facts that certain of these substances are not formed in the absence of living cells, and that their origin is to be sought in the metabolic processes which accompany the life of the cell. Their source, moreover, has been traced not to the sugar but to the amino-acids, formed by the hydrolysis of the proteins, which occur in all such liquids as beer wort, grape juice, etc., which are usually submitted to alcoholic fermentation. This has so far been proved with certainty for the fusel oil and succinic acid, and rendered highly probable for all the various aldehydes and acids of which traces have been detected.