Alcoholic Fermentation / Second Edition, 1914

by Arthur Harden

Simultaneously with this, the yeast acquires the property of decomposing and fermenting hexosephosphate and of responding to the addition of phosphate. This last property is only acquired to a small degree in this way but it becomes much more strongly developed if the pressed yeast be washed with toluene on the filter pump. Thus 10 grams of yeast after this treatment fermented fructose at 1·2 c.c. per three minutes; after the addition of phosphate (5 c.c. of 0·6 molar phosphate) the rate rose to 6·9 and then gradually fell in the typical manner [Harden, [1910]; see also Euler and Johansson, [1912, 3]].

The current explanation of the great decrease in rate of fermentation which attends the action of toluene and other antiseptics on living yeast, and also follows upon the disintegration of the cell, appears to be that in living yeast the high rate of fermentation is maintained by the continued production of relatively large fresh supplies of fermenting complex, and that when the power of producing this catalytic agent is destroyed by the poison, the rate of fermentation falls to a low value, corresponding to the store of zymase still present in the cell (cf. Buchner, E. and H., and Hahn, [1903], pp. 176, 180).

This explanation implies that the rate of fermentation after the action of the toluene represents the amount of fermenting complex present, a supposition which has been shown (p. [53]) to be highly improbable. It further necessitates, as also pointed out independently by Euler and Ugglas [[1911]], a rapid destruction of the fermenting complex both in the process of fermentation and by the action of the antiseptic, as otherwise the store of zymase remaining in the dead cell would be practically the same as that contained in the living cell at the moment when it was subjected to the antiseptic, and this store would therefore suffice to carry out fermentation at the same rate in the dead as in the living cell. No such rapid destruction, however, occurs in yeast-juice, as judged by the rate of fermentation, which falls off [p126] slowly and to about the same extent in the presence or absence of toluene. Moreover, as shown above, it is highly probable that the actual amount of fermenting complex in yeast-juice is a large fraction of that present at any moment in the cell, and is capable under suitable conditions of producing fermentation at a rate comparable with that of the living cell.

This last criticism also applies to the view expressed by Euler [Euler and Ugglas, [1911]; Euler and Kullberg, [1911, 1], [2]] that in the living cell the zymase is partly free and partly combined with the protoplasm; when the vital activity of the cell is interfered with, the combined portion of the zymase is thrown out of action and only that which was free remains active.

The suggestion made by Rubner [[1913]] that the action of yeast on sugar is in reality chiefly a vital act, but that a small proportion of the change is due to enzyme action, is similar in its consequences to that of Euler and may be met by the same arguments. Buchner and Skraup [[1914]] have moreover shown that the effects of sodium chloride and toluene on the fermenting power of yeast which were observed by Rubner, can be explained in other ways.

Some other explanation must therefore be sought for this phenomenon. Great significance must be attached in this connection to the relation noted above between the degree of disintegration and disorganisation of the cell and the fall in the normal rate of fermentation. It seems not impossible that fermentation may be associated in the living cell with some special structure, or carried on in some special portion of the cell, perhaps the nuclear vacuole described by Janssens and Leblanc [[1898]], Wager [[1898], [1911]; Wager and Peniston, [1910]] and others which undergoes remarkable changes both during fermentation and autofermentation [Harden and Rowland, [1901]]. The disorganisation of the cell might lead to many modifications of the conditions, among others to the dilution of the various catalytic agents by diffusion throughout the whole volume of the cell. As a matter of observation the dilution of yeast-juice leads to a considerable diminution of the rate of fermentation of sugar, and it is possible that this is one of the chief factors concerned. That phenomena of this kind may be involved is shown by the remarkable effect of toluene on the autofermentation of yeast. Whereas the fermentation of sugar is greatly diminished by the action of toluene, the rate of autofermentation, which is carried on at the expense of the glycogen of the cell, is greatly increased. In a typical case, for example, the autofermentation of 10 grams of yeast suspended in 20 c.c. of water amounted to 28 c.c. in 4·8 hours [p127] at 25°, whereas the same amount of yeast in presence of 2 c.c. of toluene gave 97·6 c.c. in the same time.

Many salts produce a similar effect on English top yeasts (in which the autofermentation is large) [Harden and Paine, [1912]], whereas Neuberg and Karczag in Berlin [[1911, 2]] were unable to observe this phenomenon.

A necessary preliminary of the fermentation of glycogen is its conversion by a diastatic enzyme into a fermentable sugar, and it is probable that the effect of the disorganisation of the cell by toluene is that this enzyme finds more ready access to the glycogen, which is stored in the plasma of the cell. No such acceleration of autofermentation is effected by the addition of toluene to yeast-juice, and hence the result is not due to an acceleration of the action of the diastatic enzyme on the glycogen.

This effect of toluene is similar in character to the action of anæsthetics on the leaves of many plants containing glucosides and enzymes, whereby an immediate decomposition of the glucoside is initiated [see H. E. and E. F. Armstrong, [1910]].

Although as indicated above Euler's theory cannot apply to zymase itself, if applied to the hexosephosphatase it would afford a consistent explanation of the facts. According to this modified view it would be the hexosephosphatase of yeast which existed largely in the combined form, so that in extracts, in dried yeast and in presence of toluene only the small fraction which was free would remain active. The zymase on the other hand would have to be regarded as existing to a large extent in the free state so that it would pass into extracts comparatively unimpaired in amount and capable under proper conditions (i.e. when supplied with sufficient phosphate) of bringing about a very vigorous fermentation. The theory of combined and free enzymes is undoubtedly of considerable value, although it cannot be considered as fully established.