In the previous chapter reference was made to the fact that the addition of boiled yeast-juice greatly increases the amounts of carbon dioxide and alcohol formed from sugar by the action of a given volume of yeast-juice.

When the boiled juice is dialysed the substance or substances to which this effect is due pass into the dialysate, the residue being quite inactive. In order to ascertain the effect on the process of alcoholic fermentation of the complete removal of these unknown substances from yeast-juice itself, dialysis experiments were instituted with fresh yeast-juice, capable of bringing about an active production of carbon dioxide and alcohol from sugar. It was already known from the experiments of Buchner and Rapp [1898, 1] that dialysis in parchment paper for seventeen hours at 0° against water or physiological salt solution only produced a diminution of about 20 per cent. in the total amount of fermentation obtainable, and in view of the less permanent character of the juice from top yeasts a more rapid method of dialysis was sought. This was found in the process of filtration under pressure through a film of gelatin, supported in the pores of a Chamberland filter candle, which had been introduced by Martin [1896].

In this way it was found possible to divide the juice into a residue and a filtrate, each of which was itself incapable of setting up the alcoholic fermentation of glucose, whereas, when they were reunited, the mixture produced almost as active a fermentation as the original juice [Harden and Young, 1905, 1; 1906, 2].

The apparatus employed for this purpose consists of a brass tube provided with a flange in which the gelatinised candle is held by a compressed india-rubber ring, and is shown in section in Fig. 5. Two such apparatus are used, each capable of holding about 70 c.c. of the liquid to be filtered. The tubes, after being filled with the yeast-juice, are connected by means of a screw joint with a cylinder of compressed air and the filtration carried out under a pressure of 50 atmospheres, [p060] the arrangement employed being shown in Fig. 6. In the earlier experiments 25 to 50 c.c. of yeast-juice were placed in each tube and the filtration continued until no more liquid passed through. The residue was then washed several times in situ by adding water and forcing it through the candle. The time occupied in this process varied from six to twelve hours with different preparations of yeast-juice. The candle was then removed from the brass casing and the thick, brown-coloured residue scraped off, dissolved in water, and at once examined. It was subsequently found to be possible to dry this residue in vacuo over sulphuric acid without seriously altering the fermenting power, and this led to a slight modification of the method, which is now conducted as follows. Two quantities of 50 c.c. each of yeast-juice are filtered, without washing, and the residues spread on watch-glasses and dried in vacuo. Two fresh quantities of 50 c.c. are then filtered through the same candles and the residues also dried. The 200 c.c. of juice treated in this way give a dry residue of 17 to 24 grams. The residue is then dissolved in 100 c.c. of water and filtered in quantities of 50 c.c. through two fresh gelatinised candles and the residue again dried. A considerable diminution in weight occurs, partly owing to incomplete removal from the candle and brass casing, and the final residue only amounts to about 9 to 12 grams. Occasionally it is necessary to repeat the processes of dissolving in water, filtering, and drying, but a considerable loss both of material and fermenting power attends each such operation.

The sticky residue dries up very rapidly in vacuo to a brittle, scaly mass, which is converted by grinding into a light yellow powder.

The filtrate was invariably found to be quite devoid of fermenting power, none of the enzyme passing through the gelatin.

Properties of the Filtered and Washed Residue.—The residue prepared as described above consists mainly of the protein, glycogen, and dextrins of the yeast-juice, and is almost free from mineral phosphates, but contains a certain amount of combined phosphorus. It also contains the enzymes of the juice, including the proteoclastic enzyme, and the hexosephosphatase (p. 54). Its solution in water is usually quite inactive to glucose or fructose, but in some cases produces a small and evanescent fermentation. When the original filtrate or a corresponding [p061] quantity of the filtrate from boiled fresh yeast-juice is added, the mixture ferments glucose or fructose quite readily. The following table shows the quantitative relations observed, the sugar being in all cases present in excess:—

These experiments lead to the conclusion that the fermentation of glucose and fructose by yeast-juice is dependent upon the presence, not only of the enzyme, but also of another substance which is dialysable and thermostable.

Precisely similar results were subsequently obtained by Buchner and Antoni [1905, 2] by the dialysis of yeast-juice. One hundred c.c. of juice were dialysed for twenty-four hours at 0° against 1300 c.c. of distilled water, and the dialysate was then evaporated at 40° to 50° to 20 c.c. The fermenting power of 20 c.c. of the dialysed juice was then determined with the following additions:—

• (1) 20 c.c. of dialysed juice+ 10 c.c. of water gave 0·02 gram CO₂.
• (2) 20 c.c. of dialysed juice+ 10 c.c. of evaporated dialysate gave 0·52 gram CO₂.
• (3) 20 c.c. of dialysed juice+ 10 c.c. of boiled juice gave 0·89 gram CO₂.

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.

This result is probably to be explained, at all events in the main, by the presence in the co-enzyme solution of the antiprotease to which reference has already been made [Buchner and Haehn, 1910, 2]. This agent, the constitution of which is still unknown, protects proteins in general from the action of digestive enzymes, and on the assumption that the alcoholic enzyme of yeast-juice belongs to the class of proteins, may be supposed to lessen the rate at which this enzyme is destroyed by the endotryptase of the juice. This antiprotease is, like the co-enzyme (p. 68), destroyed by lipase but is more stable than the co-enzyme towards hydrolytic agents, and can be obtained free from co-enzyme by boiling the solution for some hours alone or by heating with dilute sulphuric acid. Such a solution possesses no regenerative power, but still retains its power of protecting proteins against digestion and of preserving the fermenting power of yeast-juice. [p066]

It must, however, be remembered that the addition of a phosphate alone may greatly prolong the period of fermentation of yeast-juice (p. 55), and sugar is well known to exert a similar action. It appears, therefore, that the existence of the enzyme is prolonged not only by the presence of the antiprotease but also by that of sugar and hexosephosphate, into which phosphate passes in presence of sugar. Similar effects are exerted on the co-enzyme by sugar and probably also by hexosephosphate.

The fermenting complex, therefore, in the presence of these substances, either separately or together, falls off more slowly in activity and is present for a longer time, and for both of these reasons produces an increased amount of fermentation. It seems probable also that the hexosephosphatase is similarly affected, so that the supply of free phosphate is at the same time better maintained, and the rate of fermentation for this reason decreases more slowly than would otherwise be the case.

It is in this way that an explanation may be found of the remarkable increase in total fermentation, which is produced by the addition to yeast-juice and sugar of boiled yeast-juice, containing free phosphate (which passes into hexosephosphate) as well as co-enzyme, of boiled autolysed yeast-juice, containing free phosphate but no co-enzyme, or of phosphate solution alone.

In no case is the original rate of fermentation greatly increased after the initial acceleration has disappeared, but in every case the total fermentation is considerably augmented, and this is no doubt mainly to be attributed, as just explained, to the diminished rate of decomposition of the fermenting complex and probably of the hexosephosphatase.

Although both enzyme and co-enzyme are completely precipitated from yeast-juice, as already described (p. 38), by 10 volumes of acetone, the co-enzyme is less easily precipitated than the enzyme, and a certain degree of separation can therefore be attained by fractional precipitation [Buchner and Duchacek, 1909]. The enzyme cannot, however, be completely freed from co-enzyme in this manner, and the process is attended by a very considerable loss of enzyme. This is probably due to the fact that only small quantities of acetone can be added (1·5 to 3 volumes), in order to avoid precipitation of co-enzyme, and that the precipitates thus formed contain a large proportion of water, a condition which appears to be fatal to the preservation of the enzyme.

It is, however, not quite certain whether it is the zymase or the hexosephosphatase which is destroyed in these cases, as no attempt [p067] was made to distinguish between them. In any case the precipitates obtained by fractional treatment with acetone, even when reunited, produce a much smaller fermentation than the original juice or the powder prepared by bringing it into 10 volumes of acetone.

Attempts to isolate the co-enzyme from boiled yeast-juice have also been hitherto unsuccessful. It has, however, been found possible to remove a considerable amount of material from the solution without affecting the co-enzyme. When 1 volume of alcohol is added to boiled yeast-juice, a bulky precipitate, consisting largely of carbohydrates, is produced, and the filtrate from this is found to contain the co-enzyme and can be freed from alcohol by evaporation. Further precipitation with alcohol has not led to useful results.

When a solution which has been treated in this way is precipitated with lead acetate and kept neutral to litmus, the free phosphate and hexosephosphate are thrown down and the co-enzyme remains in solution. The filtrate can be freed from lead by means of sulphuretted hydrogen and neutralised, and then forms a solution of co-enzyme free from phosphate and hexosephosphate but still containing combined phosphorus. More complete purification than this has not yet been accomplished. Occasionally the precipitate of lead salts retains some of the co-enzyme, apparently by adsorption, but usually the greater part remains in the solution (Harden and Young).

The co-enzyme is partially removed from yeast-juice by means of a colloidal solution of ferric hydroxide (Resenscheck). A precipitate is thus obtained which contains phosphorus and resembles boiled yeast-juice in its regenerative action on yeast-juice rendered inactive by fermentation. It has not, however, so far been found possible to isolate any definite compound from this precipitate. There are also indications that when yeast-juice, either fresh or boiled, is electrolysed, the co-enzyme tends to accumulate at the cathode [Resenscheck, 1908, 1, 2].

Buchner and Klatte [1908] made use of yeast-juice rendered free from co-enzyme by incubation with sugar solution to examine the nature of the agent by which the co-enzyme is destroyed. This agent is certainly an enzyme, since boiled yeast-juice can be preserved with unimpaired powers for a considerable length of time, and suspicion fell naturally, in the first instance, on the endotryptase of the yeast cell. Direct experiment showed, however, that yeast-juice, which, when fresh, rapidly destroyed the co-enzyme of boiled juice, lost this power on preservation, but retained its proteoclastic properties without diminution, so that the tryptic enzyme could not be the one concerned. The direct action of commercial trypsin on boiled yeast-juice also yielded [p068] a negative result, although this cannot strictly be regarded as an indication of the effect of the specific proteoclastic enzymes of yeast-juice. On the other hand, it was found that when boiled juice was treated for some time with an emulsion containing the lipase of castor oil seeds, the co-enzyme was completely destroyed. This is a result of great importance, inasmuch as it probably indicates that the co-enzyme is chemically allied to the class of substances hydrolysable by lipase, i.e. to the fats and other esters.

Further, observations by Buchner and Haehn [1909] have shown that digestion with potassium carbonate solution containing 2·5 grams per 100 c.c. also brings about the destruction of the co-enzyme, and that this is also slowly accomplished by the repeated boiling of the juice. The co-enzyme is also destroyed both by acid and alkaline hydrolysis, and when the solution is evaporated to dryness and the residue ignited.

Beyond this general indication nothing is known of the chemical nature of the co-enzyme. The intimate relation of phosphoric acid to the process of fermentation renders it not impossible that the co-enzyme may contain this group, but there is no definite evidence for such a belief. Purely negative results have been obtained with all the substances of known composition which have yet been tested, among these being soluble phosphates, hexosephosphates and a number of oxidisable and reducible substances, such as quinol, p-phenylenediamine, methylene blue, peptone beef broth, etc. (Harden and Young; Harden and Norris [1914]; see also Euler and BÄckstrÖm [1912]), glycero-phosphates (Buchner and Klatte).

The precise function of the co-enzyme is even more obscure than its chemical nature. The system of reacting substances consisting of fermentable material, enzyme and co-enzyme, bears, however, an obvious superficial resemblance to many of the systems required for the accomplishment of chemical changes in the animal or vegetable organism. Such a triad of substances is, for example, requisite for the process by which the red blood corpuscles of an animal are broken up by the serum of a different animal into the blood of which the red corpuscles of the first animal have been injected. This effect is only produced when two substances are present, the amboceptor or immune body and the complement. The analogy may be carried to a further stage since the amboceptor is, like the co-enzyme, more thermostable than the complement, which therefore corresponds with the enzyme. Immune serum can, in fact, be freed from complement by being heated at 57–60° for half an hour, whereas the amboceptor is unaffected by this treatment. On the other hand, the complement and amboceptor do not [p069] appear to act like enzymes but rather like ordinary chemical reagents, remaining in combination even after the blood corpuscle has been broken up, whereas the enzyme and co-enzyme of yeast-juice are again liberated when the reaction between sugar and phosphate has been completed.