It was shown in the previous chapter that phosphates are essential to fermentation, and hence it becomes necessary to inquire whether the effect of dialysis is simply to remove these. Experiment shows that this is not the case. Soluble phosphates do not confer the power of producing fermentation on the inactive residue obtained by filtration. Moreover, when yeast-juice is digested for some time before being boiled, it is found, as will be subsequently described, that the boiled autolysed juice is quite incapable of setting up fermentation in the inactive residue, although free phosphates are abundantly present [Harden and Young, [1906, 2]].
The filtration residue is never obtained quite free from combined phosphorus, but the production from this of the phosphate necessary for fermentation to proceed, may be so slow as to render the test for co-enzyme uncertain, owing to the absence of sufficient phosphate. When a filtration residue is being tested it is therefore necessary to secure the presence of sufficient phosphate to enable the characteristic reaction to proceed, and at the same time to avoid adding phosphate in too great concentration, as this may, in the presence of only small amounts of enzyme or co-enzyme, inhibit the fermentation (p. [71]). The proof that a filtration residue or dialysed juice is quite free from co-enzyme is therefore a somewhat complicated matter, and not only involves the experimental demonstration that the material will not ferment sugar, but also that this power is not imparted to it by the addition of a small concentration of phosphate. As it has been found (p. [73]) that the fermentation of fructose is less affected than that of glucose by the presence of excess of phosphate, the practical method of examining a filtration residue for co-enzyme is to test its action on a solution of fructose (1) alone and (2) in presence of a small concentration of phosphate. If the residue produces no action [p063] in either case, but produces fermentation when a solution of co-enzyme is added in the presence of the same concentration of phosphate as was previously employed, it may be concluded that this sample was free from co-enzyme but contained enzyme; such an experiment also affords a definite proof that the co-enzyme does not consist of phosphate.
This dialysable, thermostable substance, without which alcoholic fermentation cannot proceed, has been provisionally termed the co-ferment or co-enzyme of alcoholic fermentation. This expression was first introduced by Bertrand [[1897]], to denote substances of this kind, and he applied it in two instances—to the calcium salt which he considered was necessary for the action of pectase on pecten substances, and to the manganese which he supposed to be essential for the activity of laccase. Without inquiring whether these substances are precisely comparable in function with that contained in yeast-juice, the term may be very well applied to signify the substance of unknown constitution without the co-operation of which the thermolabile enzyme of yeast-juice is unable to set up the process of alcoholic fermentation. The active agent of yeast-juice consisting of both enzyme and co-enzyme may be conveniently spoken of as the fermenting complex, and this term will occasionally be employed in the sequel.
The co-enzyme is present alike in the filtrates from fresh yeast-juice and from boiled yeast-juice, and is also contained in the liquids obtained by boiling yeast with water and by washing zymin or dried yeast with water.
Practically the only chemical property of the co-enzyme, other than that of rendering possible the process of alcoholic fermentation, which has so far been observed, is that it is capable of being decomposed, probably by hydrolysis, by a variety of reagents, prominent among which is yeast-juice. This was observed by Harden and Young in the course of their attempts to prepare a completely inactive residue by filtration. In many cases a residue was obtained which still possessed a very limited power of fermentation, only a small amount of carbon dioxide being formed and the action ceasing entirely after the expiration of a short period; on the subsequent addition of boiled juice, however, a very considerable evolution of carbon dioxide was produced. This was interpreted to mean that the residue in question contained an ample supply of enzyme but only a small proportion of co-enzyme, and that the latter was rapidly destroyed, so that the fermentation soon ceased. The boiled juice then added provided a further proportion of co-enzyme by the aid of which the surplus enzyme was [p064] enabled to carry on the fermentation. This view was confirmed by adding to a solution of a completely inactive filtration residue and glucose successive small quantities of boiled juice and observing the volumes of carbon dioxide evolved after each such addition. Thus in one case successive additions of volumes of 3 c.c. of boiled juice produced evolutions of 8·2, 6, and 6 c.c. of carbon dioxide. In another case two successive additions of 15 c.c. of boiled juice produced evolutions of 54 and 41·2 c.c. On the other hand, the enzyme itself also gradually disappears from yeast-juice when the latter is incubated either alone or with sugar (p. [20]).
The cessation of fermentation in any particular mixture of enzyme and co-enzyme may, therefore, be due to the disappearance of either of these factors from the liquid. If the amount of co-enzyme present be relatively small it is the first to disappear, and fermentation can then only be renewed by the addition of a further quantity, whilst the addition of more enzyme produces no effect. If, on the other hand, the amount of co-enzyme be relatively large, the inverse is true; the enzyme is the first to disappear, and fermentation can only be renewed by the addition of more enzyme, a further quantity of co-enzyme producing no effect. It has, moreover, been found that the co-enzyme, like the enzyme, disappears more rapidly in the absence of glucose than in its presence, incubation at 25° for two days being as a rule sufficient to remove all the co-enzyme from yeast-juice from top yeasts in the absence of sugar, whilst in the presence of fermentable sugar co-enzyme may still be detected at the end of four days.
In all the experiments carried out by Harden and Young with juice from English top yeast it was found that when a mixture of the juice with glucose was incubated until fermentation had ceased, the further addition of co-enzyme in the form of boiled juice did not cause any renewal of the action; in other words, the whole of the enzyme had disappeared.
On the other hand, Buchner and Klatte [[1908]], working with juice and zymin prepared from bottom yeast, observed the extremely interesting fact that after the cessation of fermentation the addition of an equal volume of boiled juice caused a renewed decomposition of sugar, and that the processes of incubation until no further evolution of gas occurred and re-excitation of fermentation by the boiled juice could be repeated as many as six times. Thus in one experiment the duration of the fermentation was extended from three to a total of twenty-four days, and the total gas evolved from 0·73 gram to 2·19 grams. The phenomenon has been found to be common to yeast from Munich and [p065] from Berlin as well as to zymin and maceration extract, and it was further observed that the boiled juice from one yeast could regenerate the juice from another, although the quantitative relations were different.
In these samples of yeast-juice, therefore, there is present a natural condition of affairs precisely similar to that obtaining in the artificial mixtures of inactive filtration residue and co-enzyme solution made by Harden and Young. The balance of quantities is such that the co-enzyme disappears before the enzyme, leaving a certain amount of enzyme capable of exercising its usual function as soon as sufficient co-enzyme is added. This establishes an interesting point of contrast with the juice prepared from top yeast in England, in which the enzyme does not outlast the co-enzyme [Harden and Young, [1907]]. The difference may be due to some variation in the relative proportions of enzyme and co-enzyme or of the enzymes to which the disappearance of each of these is presumptively due, or to a combination of these two causes. It was, however, found, even in the juice from bottom yeast, that incubation for three days at 22° without the addition of sugar caused the disappearance of the enzyme as well as of the co-enzyme, and left a residue alike incapable of being regenerated by the addition of co-enzyme or of restoring the power of producing fermentation to an inactive mixture containing enzyme and sugar.
If the fermenting power of the juice is to be preserved by repeated regeneration for a long period, it is absolutely necessary to add the co-enzyme solution each time as soon as fermentation has ceased, since the enzyme in the absence of this addition rapidly disappears, even in the presence of sugar.