Alcoholic Fermentation
by Arthur Harden · English · Gutenberg #44985 · 434,200 chars · public domain (US)
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Title: Alcoholic Fermentation
Author: Arthur Harden
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ALCOHOLIC FERMENTATION
2ND EDITION, 1914.
By ARTHUR HARDEN.
MONOGRAPHS ON BIOCHEMISTRY
EDITED BY
R. H. A. PLIMMER, D.Sc.
AND
F. G. HOPKINS, M.A., M.B., D.Sc., F.R.S.
GENERAL PREFACE.
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GILBERT T. MORGAN, D.Sc., F.I.C.
LONGMANS, GREEN AND CO.,
LONDON, NEW YORK, BOMBAY, CALCUTTA, AND MADRAS.
ALCOHOLIC FERMENTATION
BY
ARTHUR HARDEN, PH.D., D.SC., F.R.S.
PROFESSOR OF BIOCHEMISTRY, LONDON UNIVERSITY
HEAD OF THE BIOCHEMICAL DEPARTMENT, LISTER INSTITUTE, CHELSEA
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/SECOND EDITION/
LONGMANS, GREEN AND CO.
39 PATERNOSTER ROW, LONDON
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BOMBAY, CALCUTTA, AND MADRAS
1914
PREFACE.
The following chapters are based on courses of lectures delivered at
the London University and the Royal Institution during 1909-1910. In
them an account is given of the work done on alcoholic fermentation
since Buchner's epoch-making discovery of zymase, only in so far as
it appears to throw light on the nature of that phenomenon. Many
interesting subjects, therefore, have perforce been left untouched,
among them the problem of the formation of zymase in the cell, and
the vexed question of the relation of alcoholic fermentation to the
metabolic processes of the higher plants and animals.
My thanks are due to the Council of the Royal Society, and to the
Publishers of the "Journal of Physiology" for permission to make use
of blocks (Figs. 2, 4 and 7) which have appeared in their publications.
A. H.
PREFACE TO THE SECOND EDITION.
In the New Edition no change has been made in the scope of the work.
The rapid progress of the subject has, however, rendered necessary
many additions to the text and a considerable increase in the
bibliography.
A. H.
/May/,1914.
CONTENTS.
CHAPTER PAGE
I. HISTORICAL INTRODUCTION 1
II. ZYMASE AND ITS PROPERTIES 18
III. THE FUNCTION OF PHOSPHATES IN ALCOHOLIC FERMENTATION 41
IV. THE CO-ENZYME OF YEAST-JUICE 59
V. ACTION OF SOME INHIBITING AND ACCELERATING AGENTS ON
THE ENZYMES OF YEAST-JUICE 70
VI. CARBOXYLASE 81
VII. THE BY-PRODUCTS OF ALCOHOLIC FERMENTATION 85
VIII. THE CHEMICAL CHANGES INVOLVED IN FERMENTATION 96
IX. THE MECHANISM OF FERMENTATION 119
BIBLIOGRAPHY 136
INDEX 155
[p001]
CHAPTER I.
HISTORICAL INTRODUCTION.
The problem of alcoholic fermentation, of the origin and nature of
that mysterious and apparently spontaneous change which converted
the insipid juice of the grape into stimulating wine, seems to have
exerted a fascination over the minds of natural philosophers from the
very earliest times. No date can be assigned to the first observation
of the phenomena of the process. History finds man in the possession
of alcoholic liquors, and in the earliest chemical writings we find
fermentation, as a familiar natural process, invoked to explain and
illustrate the changes with which the science of those early days
was concerned. Throughout the period of alchemy fermentation plays
an important part; it is, in fact, scarcely too much to say that
the language of the alchemists and many of their ideas were founded
on the phenomena of fermentation. The subtle change in properties
permeating the whole mass of material, the frothing of the fermenting
liquid, rendering evident the vigour of the action, seemed to them
the very emblems of the mysterious process by which the long sought
for philosopher's stone was to convert the baser metals into gold. As
chemical science emerged from the mists of alchemy, definite ideas
about the nature of alcoholic fermentation and of putrefaction began
to be formed. Fermentation was distinguished from other chemical
changes in which gases were evolved, such as the action of acids
on alkali carbonates (Sylvius de le Boë, 1659); the gas evolved
was examined and termed gas vinorum, and was distinguished from
the alcohol with which it had at first been confused (van Helmont,
1648); afterwards it was found that like the gas from potashes it was
soluble in water (Wren, 1664). The gaseous product of fermentation
and putrefaction was identified by MacBride, in 1764, with the fixed
air of Black, whilst Cavendish in 1766 showed that fixed air alone
was evolved in alcoholic fermentation and that a mixture of this with
inflammable air was produced by putrefaction. In the meantime it had
been recognised that only sweet liquors could be fermented ("Ubi
notandum, nihil fermentare quod non sit dulce," Becher, 1682), and
finally Cavendish [p002] [1776] determined the proportion of fixed
air obtainable from sugar by fermentation and found it to be 57 per
cent. It gradually became recognised that fermentation might yield
either spirituous or acid liquors, whilst putrefaction was thought to
be an action of the same kind as fermentation, differing mainly in the
character of the products (Becher).
As regards the nature of the process very confused ideas at first
prevailed, but in the time of the phlogistic chemists a definite
theory of fermentation was proposed, first by Willis (1659) and
afterwards by Stahl [1697], the fundamental idea of which survived
the overthrow of the phlogistic system by Lavoisier and formed the
foundation of the views of Liebig. To explain the spontaneous origin
of fermentation and its propagation from one liquid to another, they
supposed that the process consisted in a violent internal motion
of the particles of the fermenting substance, set up by an aqueous
liquid, whereby the combination of the essential constituents of this
material was loosened and new particles formed, some of which were
thrust out of the liquid (the carbon dioxide) and others retained in
it (the alcohol).
Stahl specifically states that a body in such a state of internal
disquietude can very readily communicate the disturbance to another,
which is itself at rest but is capable of undergoing a similar change,
so that a putrefying or fermenting liquid can set another liquid in
putrefaction or fermentation.
Taking account of the gradual accumulation of fact and theory we
find at the time of Lavoisier, from which the modern aspect of the
problem dates, that Stahl's theoretical views were generally accepted.
Alcoholic fermentation was known to require the presence of sugar and
was thought to lead to the production of carbon dioxide, acetic acid,
and alcohol.
The composition of organic compounds was at that time not understood,
and it was Lavoisier who established the fact that they consisted
of carbon, hydrogen, and oxygen, and who made systematic analyses
of the substances concerned in fermentation (1784-1789). Lavoisier
[1789] applied the results of these analyses to the study of alcoholic
fermentation, and by employing the principle which he regarded as
the foundation of experimental chemistry, "that there is the same
quantity of matter before and after the operation," he drew up an
equation between the quantities of carbon, hydrogen, and oxygen in
the original sugar and in the resulting substances, alcohol, carbon
dioxide, and acetic acid, showing that the products contained the
whole matter of the sugar, and thus for the first time giving a clear
view of the chemical [p003] change which occurs in fermentation. The
conclusion to which he came was, we now know, very nearly accurate,
but the research must be regarded as one of those remarkable instances
in which the genius of the investigator triumphs over experimental
deficiencies, for the analytical numbers employed contained grave
errors, and it was only by a fortunate compensation of these that a
result so near the truth was attained.
Lavoisier's equation or balance sheet was as follows:--
Carbon. Hydrogen. Oxygen.
95·9 pounds of sugar (cane sugar) consist 26·8 7·7 61·4
of These yield:--
57·7 pounds of alcohol containing 16·7 9·6 31·4
35·3 " carbon dioxide containing 9·9 -- 25·4
2·5 " acetic acid containing 0·6 0·2 1·7
----- ---- -----
Total contained in products 27·2 9·8 58·5
The true composition of the sugar used was carbon 40·4, hydrogen 6·1,
oxygen 49·4.
Lavoisier expressed no view as to the agency by which fermentation
was brought about, but came to a very definite and characteristic
conclusion as to the chemical nature of the change. The sugar, which
he regarded in harmony with his general views as an oxide, was split
into two parts, one of which was oxidised at the expense of the other
to form carbonic acid, whilst the other was deoxygenised in favour
of the former to produce the combustible substance alcohol, "so that
if it were possible to recombine these two substances, alcohol and
carbonic acid, sugar would result".
From this point commences the modern study of the problem. Provided
by the genius of Lavoisier with the assurance that the hitherto
mysterious process of fermentation was to be ranked along with
familiar chemical changes, and that it proceeded in harmony with the
same quantitative laws as these simpler reactions, chemists were
stimulated in their desire to penetrate further into the mysteries
of the phenomenon, and the importance and interest of the problem
attracted many workers.
So important indeed did the matter appear to Lavoisier's countrymen
that in the year 8 of the French Republic (1800) a prize--consisting
of a gold medal, the value of which, expressed in terms of the newly
introduced metric system, was that of one kilogram of gold--was
offered by the Institute for the best answer to the question: "What
are the characteristics by which animal and vegetable substances which
act as ferments can be distinguished from those which they are capable
of fermenting?" [p004]
This valuable prize was again offered in 1802 but was never awarded,
as the fund from which it was to be drawn was sequestrated from the
Institute in 1804. The first response to this stimulating offer was
an important memoir by citizen Thenard [1803], which provided many
of the facts upon which Liebig subsequently based his views. Thenard
combats the prevailing idea, first expressed by Fabroni (1787-1799),
that fermentation is caused by the action of gluten derived from grain
on starch and sugar, but is himself uncertain as to the actual nature
of the ferment. He points out that all fermenting liquids deposit a
material resembling brewer's yeast, and he shows that this contains
nitrogen, much of which is evolved as ammonia on distillation. His
most important result is, however, that when yeast is used to ferment
pure sugar, it undergoes a gradual change and is finally left as a
white mass, much reduced in weight, which contains no nitrogen and is
without action on sugar. Thenard, moreover, it is interesting to note,
differs from Lavoisier, inasmuch as he ascribes the origin of some
of the carbonic acid to the carbon of the ferment, an opinion which
was still held in various degrees by many investigators (see Seguin,
quoted by Thenard).
Thenard's memoir was followed by a communication of fundamental
importance from Gay-Lussac [1810]. A process for preserving food had
been introduced by Appert, which consisted in placing the material
in bottles, closing these very carefully and exposing them to the
temperature of boiling water for some time. Gay-Lussac was struck
by the fact that when such a bottle was opened fermentation or
putrefaction set in rapidly. Analysis of the air left in such a sealed
bottle showed that all the oxygen had been absorbed, and these facts
led to the view that fermentation was set up by the action of oxygen
on the fermentable material. Experiment appeared to confirm this in
the most striking way. A bottle of preserved grape-juice was opened
over mercury and part of its contents passed through the mercury into
a bell-jar containing air, the remainder into a similar vessel free
from air. In the presence of air fermentation set in at once, in the
absence of air no fermentation whatever occurred. This connection
between fermentation and the presence of air was established by
numerous experiments and appeared incontestable. Fermentation, it
was found, could be checked by boiling even after the addition of
oxygen, and hence food could be preserved in free contact with the
air, provided only that it was raised to the temperature of boiling
water at short intervals of time. Gay-Lussac's opinion was that
the ferment was formed by the action of the oxygen on the [p005]
liquid, and that the product of this action was altered by heat and
rendered incapable of producing fermentation, as was also brewer's
yeast, which, however, he regarded, on account of its insolubility, as
different from the soluble ferment which initiated the change in the
limpid grape-juice. Colin, on the other hand [1825], recognised that
alcoholic fermentation by whatever substance it was started, resulted
in the formation of an insoluble deposit more active than the original
substance, and he suggested that this deposit might possibly in every
case be of the same nature.
So far no suspicion appears to have arisen in the minds of those who
had occupied themselves with the study of fermentation that this
change differed in any essential manner from many other reactions
familiar to chemists. The origin and properties of the ferment were
indeed remarkable and involved in obscurity, but the uncertainty
regarding this substance was no greater than that surrounding many, if
not all, compounds of animal and vegetable origin. Although, however,
the purely chemical view as to the nature of yeast was generally
recognised and adopted, isolated observations were not wanting which
tended to show that yeast might be something more than a mere chemical
reagent. As early as 1680 in letters to the Royal Society Leeuwenhoek
described the microscopic appearance of yeast of various origins as
that of small, round, or oval particles, but no further progress seems
to have been made in this direction for nearly a century and a half,
when we find that Desmazières [1826] examined the film formed on beer,
figured the elongated cells of which it was composed, and described it
under the name of Mycoderma Cerevisiæ. He, however, regarded it rather
as of animal than of vegetable origin, and does not appear to have
connected the presence of these cells with the process of fermentation.
Upon this long period during which yeast was regarded merely as a
chemical compound there followed, as has so frequently occurred in
similar cases, a sudden outburst of discovery. No less than three
observers hit almost simultaneously upon the secret of fermentation
and declared that yeast was a living organism.
First among these in strict order of time was Cagniard-Latour
[1838], who made a number of communications to the Academy and to
the Société Philomatique in 1835-6, the contents of which were
collected in a paper presented to the Academy of Sciences on 12
June, 1837, and published in 1838. The observations upon which this
memoir was based were almost exclusively microscopical. Yeast was
recognised as consisting of spherical particles, which were capable
of [p006] reproduction by budding but incapable of motion, and it
was therefore regarded as a living organism probably belonging to the
vegetable kingdom. Alcoholic fermentation was observed to depend on
the presence of living yeast cells, and was attributed to some effect
of their vegetative life (quelque effet de leur végétation). It was
also noticed that yeast was not deprived of its fermenting power by
exposure to the temperature of solid carbonic acid, a sample of which
was supplied to Cagniard-Latour by Thilorier, who had only recently
prepared it for the first time.
Theodor Schwann [1837], whose researches were quite independent of
those of Cagniard-Latour, approached the problem from an entirely
different point of view. During the year 1836 Franz Schulze [1836]
published a research on the subject of spontaneous generation, in
which he proved that when a solution containing animal or vegetable
matter was boiled, no putrefaction set in provided that all air which
was allowed to have access to the liquid was previously passed through
strong sulphuric acid. Schwann performed a very similar experiment by
which he showed that this same result, the absence of putrefaction,
was attained by heating all air which came into contact with the
boiled liquid. Wishing to show that other processes in which air took
part were not affected by the air being heated, he made experiments
with fermenting liquids and found, contrary to his expectation, that a
liquid capable of undergoing vinous fermentation and containing yeast
did not undergo this change after it had been boiled, provided that,
as in the case of his previous experiments, only air which had been
heated was allowed to come into contact with it.
Schwann's experiments on the prevention of putrefaction were
unexceptionable and quite decisive. The analogous experiments dealing
with alcoholic fermentation were not quite so satisfactory. Yeast was
added to a solution of cane sugar, the flask containing the mixture
placed in boiling water for ten minutes, and then inverted over
mercury. About one-third of the liquid was then displaced by air and
the flasks corked and kept inverted at air temperature. In two flasks
the air introduced was ordinary atmospheric air, and in these flasks
fermentation set in after about four to six weeks. Into the other two
flasks air which had been heated was led, and in these no fermentation
occurred. As described, the experiment is quite satisfactory, but
Schwann found on repetition that the results were irregular. Sometimes
all the flasks showed fermentation, sometimes none of them. This
was correctly ascribed to the experimental difficulties, but none
[p007] the less served as a point of attack for hostile and damaging
criticism at the hands of Berzelius (p. 8).
The origin of putrefaction was definitely attributed by Schwann to the
presence of living germs in the air, and the similarity of the result
obtained with yeast suggested the idea that alcoholic fermentation was
also brought about by a living organism, a conception which was at
once confirmed by a microscopical examination of a fermenting liquid.
The phenomena observed under the microscope were similar to those
noted by Cagniard-Latour, and in accordance with these observations
alcoholic fermentation was attributed to the development of a living
organism, the fermentative function of which was found to be destroyed
by potassium arsenite and not by extract of Nux vomica, so that the
organism was regarded rather as of vegetable than of animal nature.
This plant received the name of "Zuckerpilz" or sugar fungus (which
has been perpetuated in the generic term Saccharomyces). Alcoholic
fermentation was explained as "the decomposition brought about by
this sugar fungus removing from the sugar and a nitrogenous substance
the materials necessary for its growth and nourishment, whilst the
remaining elements of these compounds, which were not taken up by the
plant, combined chiefly to form alcohol".
Kützing's memoir, the third of the trio [1837], also dates from
1837, and his opinions, like those of Cagniard-Latour, are founded
on microscopical observations. He recognises yeast as a vegetable
organism and accurately describes its appearance. Alcoholic
fermentation depends on the formation of yeast, which is produced
when the necessary elements and the proper conditions are present
and then propagates itself. The action on the liquid thus increases
and the constituents not required to form the organism combine to
form unorganised substances, the carbonic acid and alcohol. "It is
obvious," says Kützing, in a passage which roused the sarcasm of
Berzelius, "that chemists must now strike yeast off the roll of
chemical compounds, since it is not a compound but an organised body,
an organism."
These three papers, which were published almost simultaneously,
were received at first with incredulity. Berzelius, at that time
the arbiter and dictator of the chemical world, reviewed them all
in his "Jahresbericht" for 1839 [1839] with impartial scorn. The
microscopical evidence was denied all value, and yeast was no more
to be regarded as an organism than was a precipitate of alumina.
Schwann's experiment (p. 6) was criticised on the ground that the
fermenting power of the added yeast had been only partially destroyed
in the [p008] flasks in which fermentation ensued, completely in
those which remained unchanged, the admission of heated or unheated
air being indifferent, a criticism to some extent justified by
Schwann's statement, already quoted, of the uncertain result of the
experiment.
Berzelius himself regarded fermentation as being brought about by the
yeast by virtue of that catalytic force, which he had supposed to
intervene in so many reactions, both between substances of mineral and
of animal and vegetable origin [1836], and which enabled "bodies, by
their mere presence, and not by their affinity, to arouse affinities
ordinarily quiescent at the temperature of the experiment, so that
the elements of a compound body arrange themselves in some different
way, by which a greater degree of electro-chemical neutralisation is
attained".
To the scorn of Berzelius was soon added the sarcasm of Wöhler and
Liebig [1839]. Stimulated in part by the publications of the three
authors already mentioned, and in part by the report of Turpin [1838],
who at the request of the Academy of Sciences had satisfied himself
by observation of the accuracy of Cagniard-Latour's conclusions,
Wöhler prepared an elaborate skit on the subject, which he sent to
Liebig, to whom it appealed so strongly that he added some touches
of his own and published it in the "Annalen," following immediately
upon a translation of Turpin's paper. Yeast was here described with a
considerable degree of anatomical realism as consisting of eggs which
developed into minute animals, shaped like a distilling apparatus, by
which the sugar was taken in as food and digested into carbonic acid
and alcohol, which were separately excreted, the whole process being
easily followed under the miscroscope.
Close upon this pleasantry followed a serious and important
communication from Liebig [1839], in which the nature of fermentation,
putrefaction, and decay was exhaustively discussed. Liebig did not
admit that these phenomena were caused by living organisms, nor did he
attribute them like Berzelius to the catalytic action of a substance
which itself survived the reaction unchanged. As regards alcoholic
fermentation, Liebig's chief arguments may be briefly summarised.
As the result of alcoholic fermentation, the whole of the carbon
of the sugar reappears in the alcohol and carbon dioxide formed.
This change is brought about by a body termed the ferment, which is
formed as the result of a change set up by the access of air to plant
juices containing sugar, and which contains all the nitrogen of the
nitrogenous constituents of the juice. This ferment is a substance
remarkably susceptible of change, which undergoes an uninterrupted and
progressive metamorphosis, of [p009] the nature of putrefaction or
decay, and produces the fermentation of the sugar as a consequence of
the transformation which it is itself undergoing.
The decomposition of the sugar is therefore due to a condition of
instability transferred to it from the unstable and changing ferment,
and only continues so long as the decomposition of the ferment
proceeds. This communication of instability from one substance
undergoing chemical change to another is the basis of Liebig's
conception, and is illustrated by a number of chemical analogies,
one of which will suffice to explain his meaning. Platinum is itself
incapable of decomposing nitric acid and dissolving in it; silver, on
the other hand, possesses this power. When platinum is alloyed with
silver, the whole mass dissolves in nitric acid, the power possessed
by the silver being transferred to the platinum. In like manner the
condition of active decomposition of the ferment is transferred to the
sugar, which by itself is quite stable. The central idea is that of
Stahl (p. 2) which was thus reintroduced into scientific thought.
In a pure sugar solution the decomposition of the ferment soon comes
to an end and fermentation then ceases. In beer wort or vegetable
juices, on the other hand, more ferment is continually formed in
the manner already described from the nitrogenous constituents of
the juice, and hence the sugar is completely fermented away and
unexhausted ferment left behind. Liebig's views were reiterated in
his celebrated "Chemische Briefe," and became the generally accepted
doctrine of chemists. There seems little doubt that both Berzelius and
Liebig in their scornful rejection of the results of Cagniard-Latour,
Schwann and Kützing, were influenced, perhaps almost unconsciously,
by a desire to avoid seeing an important chemical change relegated to
the domain of that vital force from beneath the sway of which a large
part of organic chemistry had just been rescued by Wöhler's brilliant
synthetical production of urea and by the less recognised synthesis of
alcohol by Hennell (see on this point Ahrens [1902]). A strong body of
evidence, however, gradually accumulated in favour of the vegetable
nature of yeast, so that it may be said that by 1848 a powerful
minority adhered to the views of Cagniard-Latour, Schwann, and Kützing
[see Schrohe, 1904, p. 218, and compare Buchner, 1904]. Among these
must be included Berzelius [1848], who had so forcibly repudiated the
idea only ten years before, whereas Liebig in the 1851 edition of his
letters does not mention the fact that yeast is a living organism
(Letter XV).
The recognition of the vegetable nature of yeast, however, by no
[p010] means disproved Liebig's view of the nature of the change
by which sugar was converted into carbon dioxide and alcohol, as
was carefully pointed out by Schlossberger [1844] in a research on
the nature of yeast, carried out in Liebig's laboratory but without
decisive results.
Mitscherlich was also convinced of the vegetable character of yeast,
and showed [1841] that when yeast was placed in a glass tube closed
by parchment and plunged into sugar solution, the sugar entered the
glass tube and was there fermented, but was not fermented outside the
tube. He regarded this as a proof that fermentation only occurred at
the surface of the yeast cells, and explained the process by contact
action in the sense of the catalytic action of Berzelius, rather than
by Liebig's transference of molecular instability. Similar results
were obtained with an animal membrane by Helmholtz [1843], who also
expressed his conviction that yeast was a vegetable organism.
In 1854 Schröder and von Dusch [1854, 1859, 1861] strongly reinforced
the evidence in favour of this view by succeeding in preventing the
putrefaction and fermentation of many boiled organic liquids by the
simple process of filtering all air which had access to them through
cotton-wool. These experiments, which were continued until 1861, led
to the conclusion that the spontaneous alcoholic fermentation of
liquids was due to living germs carried by the air, and that when the
air was passed through the cotton-wool these germs were held back.
At the middle of the nineteenth century opinions with regard to
alcoholic fermentation, notwithstanding all that had been done, were
still divided. On the one hand Liebig's theory of fermentation was
widely held and taught. Gerhardt, for example, as late as 1856 in the
article on fermentation in his treatise on organic chemistry [1856],
gives entire support to Liebig's views, and his treatment of the
matter affords an interesting glimpse of the arguments which were then
held to be decisive. The grounds on which he rejects the conclusions
of Schwann and the other investigators who shared the belief in the
vegetable nature of yeast are that, although in some cases animal and
vegetable matter and infusions can be preserved from change by the
methods described by these authors, in others they cannot, a striking
case being that of milk, which even after being boiled becomes sour
even in filtered air, and this without showing any trace of living
organisms. The action of heat, sulphuric acid, and filtration on the
air is to remove, or destroy, not living organisms but particles of
decomposing matter, that is to say, ferments which would add their
activity to that of the oxygen of the air. Moreover, many ferments,
as for example diastase, act without [p011] producing any insoluble
deposit whatever which can be regarded as an organism.
"Evidemment," he concludes, "la théorie de M. Liebig explique seule
tous les phénomènes de la manière la plus complète et la plus logique;
c'est à elle que tous les bons esprits ne peuvent manquer de se
rallier."
On the other hand it was held by many to have been shown that Liebig's
view of the origin of yeast by the action of the air on a vegetable
infusion was erroneous, and that fermentation only arose when the air
transferred to the liquid an active agent which could be removed from
it by sulphuric acid (Schulze), by heat (Schwann), and by cotton-wool
(Schröder and von Dusch). Accompanying alcoholic fermentation there
was a development of a living organism, the yeast, and fermentation
was believed, without any very strict proof, to be a phenomenon due to
the life and vegetation of this organism. This doctrine seems indeed
[Schrohe, 1904] to have been widely taught in Germany from 1840-56,
and to have established itself in the practice of the fermentation
industries.
In 1857 commenced the classical researches of Pasteur which finally
decided the question as to the origin and functions of yeast and
led him to the conclusion that "alcoholic fermentation is an act
correlated with the life and organisation of the yeast cells, not
with the death or putrefaction of the cells, any more than it is a
phenomenon of contact, in which case the transformation of sugar would
be accomplished in presence of the ferment without yielding up to it
or taking from it anything" [1860]. It is impossible here to enter
in detail into Pasteur's experiments on this subject, or indeed to
do more than indicate the general lines of his investigation. His
starting-point was the lactic acid fermentation.
The organism to which this change was due had hitherto escaped
detection, and as we have seen the spontaneous lactic fermentation
of milk was one of the phenomena adduced by Gerhardt (p. 10) in
favour of Liebig's views. Pasteur [1857] discovered the lactic acid
producing organism and convinced himself that it was in fact a living
organism and the active cause of the production of lactic acid. One
of the chief buttresses of Liebig's theory was thus removed, and
Pasteur next proceeded to apply the same method and reasoning to
alcoholic fermentation. Liebig's theory of the origin of yeast by
the action of the oxygen of the air on the nitrogenous matter of the
fermentable liquid was conclusively and strikingly disproved by the
brilliant device of producing a crop of yeast in a liquid medium
containing only comparatively [p012] simple substances of known
composition--sugar, ammonium tartrate and mineral phosphate. Here
there was obviously present in the original medium no matter which
could be put into a state of putrefaction by contact with oxygen and
extend its instability to the sugar. Any such material must first
be formed by the vital processes of the yeast. In the next place
Pasteur showed by careful analyses and estimations that, whenever
fermentation occurred, growth and multiplication of yeast accompanied
the phenomenon. The sugar, he proved, was not completely decomposed
into carbon dioxide and alcohol, as had been assumed by Liebig (p. 8).
A balance-sheet of materials and products was constructed which showed
that the alcohol and carbon dioxide formed amounted only to about 95
per cent. of the invert sugar fermented, the difference being made up
by glycerol, succinic acid, cellulose, and other substances [1860, p.
347]. In every case of fermentation, even when a paste of yeast was
added to a solution of pure cane sugar in water, the yeast was found
by quantitative measurements to have taken something from the sugar.
This "something" was indeterminate in character, but, including the
whole of the extractives which had passed from the yeast cells into
the surrounding liquid, it amounted to as much as 1·63 per cent. of
the weight of the sugar fermented [1860, p. 344].
Pasteur was therefore led to consider fermentation as a physiological
process accompanying the life of the yeast. His conclusions were
couched in unmistakable words: "The chemical act of fermentation is
essentially a phenomenon correlative with a vital act, commencing
and ceasing with the latter. I am of opinion that alcoholic
fermentation never occurs without simultaneous organisation,
development, multiplication of cells, or the continued life of cells
already formed. The results expressed in this memoir seem to me to
be completely opposed to the opinions of Liebig and Berzelius. If
I am asked in what consists the chemical act whereby the sugar is
decomposed and what is its real cause, I reply that I am completely
ignorant of it.
"Ought we to say that the yeast feeds on sugar and excretes alcohol
and carbonic acid? Or should we rather maintain that yeast in its
development produces some substance of the nature of a pepsin, which
acts upon the sugar and then disappears, for no such substance
is found in fermented liquids? I have nothing to reply to these
hypotheses. I neither admit them nor reject them, and wish only to
restrain myself from going beyond the facts. And the facts tell me
simply that all true fermentations are correlative with physiological
phenomena."
Liebig felt to the full the weight of Pasteur's criticisms; his reply
[p013] was long delayed [1870], and, according to his biographer,
Volhard [1909], caused him much anxiety. In it he admits the vegetable
nature of yeast, but does not regard Pasteur's conclusion as in any
way a solution of the problem of the nature of alcoholic fermentation.
Pasteur's "physiological act" is for Liebig the very phenomenon which
requires explanation, and which he still maintains can be explained by
his original theory of communicated instability. On some of Pasteur's
results, notably the very important one of the cultivation of yeast
in a synthetic medium, he casts grave doubt, whilst he explains
the production of glycerol and succinic acid as due to independent
reactions. The phenomenon of fermentation is still for him one which
accompanies the decomposition of the constituents of the cell, rather
than their building up by vegetative growth. "When the fungus ceases
to grow, the bond which holds together the constituents of the cell
contents is relaxed, and it is the motion which is thus set up in
them which is the means by which the yeast cells are enabled to bring
about a displacement or decomposition of the elements of sugar or
other organic molecules." Pasteur replied in a brief and unanswerable
note [1872]. All his attention was concentrated on the one question
of the production of yeast in a synthetic medium, which he recognised
as fundamental. The validity of this experiment he emphatically
reaffirmed, and finally undertook, from materials supplied by Liebig
himself, to produce as much yeast as could be reasonably desired. This
challenge was never taken up, and this communication formed the last
word of the controversy. Pasteur had at this time firmly established
his thesis, /no fermentation without life/, both for alcoholic
fermentation and for those other fermentations which are produced by
bacteria, and had put upon a sound and permanent basis the conclusions
drawn by Schulze, Cagniard-Latour, Schwann, and Kützing from their
early experiments. It became generally recognised that putrefaction
and other fermentative changes were due to specific organisms, which
produced them in the exercise of their vital functions.
Pasteur subsequently [1875] came to the conclusion that fermentation
was the result of life without oxygen, the cells being able, in the
absence of free oxygen, to avail themselves of the energy liberated by
the decomposition of substances containing combined oxygen. This view,
which did not involve any alteration of Pasteur's original thesis but
was an attempt to explain the physiological origin and function of
fermentation, gave rise to a prolonged controversy, which cannot be
further discussed in these pages. [p014]
Nevertheless, Liebig's desire to penetrate more deeply into the nature
of the process of fermentation remained in many minds, and numerous
endeavours were made to obtain further insight into the problem. In
spite of an entire lack of direct experimental proof, the conception
that alcoholic fermentation was due to the chemical action of some
substance elaborated by the cell and not directly to the vital
processes of the cell as a whole found strenuous supporters even
among those who were convinced of the vegetable character of yeast.
As early as 1833 diastase, discovered still earlier by Kirchhoff and
Dubrunfaut, had been extracted by means of water from germinating
barley and precipitated by alcohol as a white powder, the solution
of which was capable of converting starch into sugar, but lost this
power when heated [Payen and Persoz, 1833]. Basing his ideas in part
upon the behaviour of this substance, Moritz Traube [1858] enunciated
in the clearest possible manner the theory that all fermentations
produced by living organisms are caused by ferments, which are
definite chemical substances produced in the cells of the organism. He
regarded these substances as being closely related to the proteins and
considered that their function was to transfer the oxygen and hydrogen
of water to different parts of the molecule of the fermentable
substance and thus bring about that apparent intramolecular oxidation
and reduction which is so characteristic of fermentative change and
had arrested the attention of Lavoisier and, long after him, of Liebig.
Traube's main thesis, that fermentation is caused by definite ferments
or enzymes, attracted much attention, and received fresh support
from the separation of invertase in 1860 from an extract of yeast
by Berthelot, and from the advocacy and authority of this great
countryman of Pasteur, who definitely expressed his opinion that
insoluble ferments existed which could not be separated from the
tissues of the organism, and further, that the organism could not
itself be regarded as the ferment, but only as the producer of the
ferment [1857, 1860]. Hoppe-Seyler [1876] also supported the enzyme
theory of fermentation, but differed in some respects from Traube as
to the exact function of the ferment [see Traube, 1877; Hoppe-Seyler,
1877].
Direct experimental evidence was, however, still wanting, and
Pasteur's reiterated assertion [1875] that all fermentation
phenomena were manifestations of the life of the organism remained
uncontroverted by experience.
Numerous and repeated direct experimental attacks had been made
[p015] from time to time upon the problem of the existence of a
fermentation enzyme, but all had yielded negative or unreliable
results.
As early as 1846 a bold attempt had been made by Lüdersdorff [1846] to
ascertain whether fermentation was or was not bound up with the life
of the yeast by grinding yeast and examining the ground mass. A single
gram of yeast was thoroughly ground, the process lasting for an hour,
and the product was tested with sugar solution. Not a single bubble
of gas was evolved. A similar result was obtained in a repetition of
the experiment by Schmidt in Liebig's laboratory [1847], the grinding
being continued in this case for six hours, but the natural conclusion
that living yeast was essential for fermentation was not accepted, on
the ground that during the lengthy process of trituration in contact
with air the yeast had become altered and now no longer possessed the
power of producing alcoholic fermentation, but instead had acquired
that of changing sugar into lactic acid [see Gerhardt, 1856, p. 545].
Similar experiments made in 1871 by Marie von Manasseïn [1872, 1897],
in which yeast was ground for six to fifteen hours with powdered
rock crystal, yielded products which fermented sugar, but they
contained unbroken yeast cells, so that the results obtained could
not be considered decisive [Buchner and Rapp, 1898, 1], although
Frau von Manasseïn herself drew from them and from others in which
sugar solution was treated with heated yeast, but not under aseptic
conditions, the conclusion that living yeast cells were not necessary
for fermentation.
Quite unsuccessful were also the attempts made to accomplish the
separation of fermentation from the living cell by Adolf Mayer [1879,
p. 66], and, as we learn from Roux, by Pasteur himself, grinding,
freezing, and plasmolysing the cells, having in his hands proved alike
in vain. Extraction by glycerol or water, a method by which many
enzymes can be obtained in solution, gave no better results [Nägeli
and Loew, 1878], and the enzyme theory of alcoholic fermentation
appeared quite unjustified by experiment.
Having convinced himself of this, Nägeli [1879] suggested a new
explanation of the facts based on molecular-physical grounds.
According to this view, which unites in itself some of the conceptions
of Liebig, Pasteur, and Traube, fermentation is the transference
of a state of motion from the molecules, atomic groups, and atoms
of the compounds constituting the living plasma of the cell to
the fermentable material, whereby the equilibrium existing in the
molecules of the latter is disturbed and decomposition ensues [1879,
p. 29]. [p016]
This somewhat complex idea, whilst including, as did Liebig's theory,
Stahl's fundamental conception of a transmission of a state of
motion, satisfies Pasteur's contention that fermentation cannot occur
without life, and at the same time explains the specific action of
different organisms by differences in the constitution of their cell
contents. The really essential part of Nägeli's theory consisted in
the limitation of the power of transference of molecular motion to the
living plasma, by which the failure of all attempts to separate the
power of fermentation from the living cell was explained. This was the
special phenomenon which required explanation; to account for this the
theory was devised, and when this was experimentally disproved, the
theory lost all significance.
For nearly twenty years no further progress was made, and then in 1897
the question which had aroused so much discussion and conjecture, and
had given rise to so much experimental work, was finally answered
by Eduard Buchner, who succeeded in preparing from yeast a liquid
which, in the complete absence of cells, was capable of effecting the
resolution of sugar into carbon dioxide and alcohol [1897, 1].
In the light of this discovery the contribution to the truth made by
each of the great protagonists in the prolonged discussion on the
problem of alcoholic fermentation can be discerned with some degree of
clearness. Liebig's main contention that fermentation was essentially
a chemical act was correct, although his explanation of the nature
of this act was inaccurate. Pasteur, in so far as he considered the
act of fermentation as indissolubly connected with the life of the
organism, was shown to be in error, but the function of the organism
has only been restricted by a single stage, the active enzyme of
alcoholic fermentation has so far only been observed as the product
of the living cell. Nearest of all to the truth was Traube, who in
1858 enunciated the theorem, which was only proved for alcoholic
fermentation in 1897, that all fermentations produced by living
organisms are due to ferments secreted by the cells.
Buchner's discovery of zymase has introduced a new experimental
method by means of which the problem of alcoholic fermentation can
be attacked, and the result has been that since 1897 a considerable
amount of information has been gained with regard to the nature
and conditions of action of the enzymes of the yeast cell. It has
been found that the machinery of fermentation is much more complex
than had been surmised. The enzyme zymase, which is essential for
fermentation, cannot of itself bring about the alcoholic fermentation
of sugar, but is dependent on the presence of a second substance,
termed, for [p017] want of a more reasonable name, the co-enzyme.
The chemical nature and function of this mysterious coadjutor are
still unknown, but as it withstands the temperature of boiling water
and is dialysable, it is probably more simple in constitution than the
enzyme. This, however, is not all; for the decomposition of sugar a
phosphate is also indispensable. It appears that in yeast-juice, and
therefore also most probably in the yeast cell, the phosphorus present
takes an active part in fermentation and goes through a remarkable
cycle of changes. The breakdown of sugar into alcohol and carbon
dioxide is accompanied by the formation of a complex hexosephosphate,
and the phosphate is split off from this compound and thus again
rendered available for action by means of a special enzyme, termed
hexosephosphatase. In addition to this complex of ferments, the cell
also possesses special enzymes by which the zymase and the co-enzyme
can be destroyed, and, further, at least one substance, known as an
anti-enzyme, which directly checks this destructive action. It seems
probable, moreover, that the decomposition of the sugar molecule takes
place in stages, although much doubt yet exists as to the nature of
these.
At the present moment the subject remains one of the most interesting
in the whole field of biological chemistry, the limited degree of
insight which has already been gained into the marvellous complexity
of the cell lending additional zest to the attempt to penetrate the
darkness which shrouds the still hidden mysteries. [p018]
CHAPTER II.
ZYMASE AND ITS PROPERTIES.
DISCOVERY OF ZYMASE.
The history of Buchner's discovery is of great interest [Gruber, 1908;
Hahn, 1908]. As early as 1893 Hans and Eduard Buchner found that the
cells of even the smallest micro-organism could be broken by being
ground with sand [Buchner, E. and H., and Hahn, 1903, p. 20], and in
1896 the same process was applied by these two investigators to yeast,
with the object of obtaining a preparation for therapeutic purposes.
Difficulties arose in the separation of the cell contents from the
ground-up mixture of cell membranes, unbroken cells, and sand, but
these were overcome by carrying out the suggestion of Martin Hahn (at
that time assistant to Hans Buchner) that kieselguhr should be added
and the liquid squeezed out by means of a hydraulic press [Buchner,
E. and H., and Hahn, 1903, p. 58]. The yeast-juice thus obtained
was, in the first instance, employed for animal experiments, but
underwent change very rapidly. The ordinary antiseptics were found to
be unsuitable, and hence sugar was added as a preservative, and it
was the marked action of the juice upon this added cane sugar that
drew Eduard Buchner's attention to the fact that fermentation was
proceeding in the absence of yeast-cells.
As in the case of so many discoveries, the new phenomenon was brought
to light, apparently by chance, as the result of an investigation
directed to quite other ends, but fortunately fell under the eye of
an observer possessed of the genius which enabled him to realise its
importance and give to it the true interpretation.
In his first papers [1897, 1, 2; 1898], Buchner established the
following facts: (1) yeast-juice free from cells is capable of
producing the alcoholic fermentation of glucose, fructose, cane
sugar, and maltose; (2) the fermenting power of the juice is neither
destroyed by the addition of chloroform, benzene, or sodium arsenite
[Hans Buchner, 1897], by filtration through a Berkefeld filter, by
evaporation to dryness at 30° to 35°, nor by precipitation with
alcohol; (3) the fermenting power is completely destroyed when the
liquid is heated to 50°. [p019]
From these facts he drew the conclusion "that the production of
alcoholic fermentation does not require so complicated an apparatus as
the yeast cell, and that the fermentative power of yeast-juice is due
to the presence of a dissolved substance". To this active substance he
gave the name of zymase.
Buchner's discovery was not received without some hesitation. A
number of investigators prepared yeast-juice, but failed to obtain an
active product [Will, 1897; Delbrück, 1897; Martin and Chapman, 1898;
Reynolds Green, 1897; Lintner, 1899]. A more accurate knowledge of the
necessary conditions and of the properties of yeast-juice, however,
led to more successful results [Will, 1898; Reynolds Green, 1898;
Lange, 1898], and it was soon established that, given suitable yeast,
an active preparation could be readily procured by Buchner's method.
Criticism was then directed to the effect of the admitted presence
of a certain number of micro-organisms in yeast-juice [Stavenhagen,
1897], but Buchner [Buchner and Rapp, 1897] was able to show by
experiments in the presence of antiseptics and with juice filtered
through a Chamberland candle that the fermentation was not due to
living organisms of any kind.
The most weighty criticism of Buchner's conclusion consisted in an
attempt to show that the properties of yeast-juice might be due to the
presence, suspended in it, of fragments of living protoplasm, which,
although severed from their original surroundings in the cell, might
retain for some time the power of producing alcoholic fermentation.
This, it will be seen, was an endeavour to extend Nägeli's theory to
include in it the newly discovered fact.
In favour of this view were adduced the similarity between the effects
of many antiseptics on living yeast and on the juice, the ephemeral
nature of the fermenting agent present in the juice, the effect of
dilution with water, and the phenomenon of autofermentation which is
exhibited by the juice in the absence of added sugar [Abeles, 1898;
v. Kupffer, 1897; v. Voit, 1897; Wehmer, 1898; Neumeister, 1897;
Macfadyen, Morris, and Rowland, 1900; Bokorny, 1906; Fischer, 1903;
Beijerinck, 1897, 1900; Wroblewski, 1899, 1901].
A brief general description of the actual properties of yeast-juice
and of the phenomena of fermentation by its means is sufficient to
show the great improbability of this view.
The juice prepared by Buchner's method forms a somewhat viscous
opalescent brownish-yellow liquid, which is usually faintly acid in
reaction [compare Ahrens, 1900] and almost optically inactive. It has
a specific gravity of 1·03 to 1·06, contains 8·5 to 14 per cent.
[p020] of dissolved solids, and leaves an ash amounting to 1·4 to 2
per cent. About 0·7 to 1·7 per cent. of nitrogen is present, nearly
all in the form of protein, which coagulates to a thick white mass
when the juice is heated.
A powerful digestive enzyme of the type of trypsin is also present, so
that when the juice is preserved its albumin undergoes digestion at
a rate which depends on the temperature [Hahn, 1898; Geret and Hahn,
1898, 1, 2; 1900; Buchner, E. and H., and Hahn, 1903, pp. 287-340],
and is converted into a mixture of bases and amino-acids. After
about six days at 37°, or 10 to 14 days at the ordinary temperature,
the digestion is so complete that no coagulation occurs when the
juice is boiled. As this proteoclastic enzyme, like the alcoholic
enzyme, cannot be extracted from the living cells, it is termed
yeast endotrypsin or endotryptase. Fresh yeast-juice produces a slow
fermentation of sugar, which lasts for forty-eight to ninety-six
hours at 25° to 30°, about a week at the ordinary temperature, and
then ceases, owing, not to exhaustion of the sugar, but to the
disappearance of the fermenting agent. When the juice is preserved or
incubated in the absence of a fermentable sugar this disappearance
occurs considerably sooner, so that even after standing for a single
day at room temperature, or two days at 0°, no fermentation may
occur when sugar is added. The reason for this behaviour has not
been definitely ascertained. As will be seen later on (p. 64) the
phenomenon is a complex one, but the disappearance of the enzyme was
originally ascribed by Buchner to the digestive action upon it of
the endotrypsin of the juice [1897, 2], and no better explanation
has yet been found. Confirmation of this view is afforded by the
fact that the addition of a tryptic enzyme of animal origin greatly
hastens the disappearance of the alcoholic enzyme [Buchner, E. and
H., and Hahn, 1903, p. 126], and that some substances which hinder
the tryptic action favour fermentation [Harden, 1903]. The amount of
fermentation produced is almost unaffected by the presence of such
antiseptics as chloroform or toluene, although some others, such as
arsenites and fluorides, decrease it when added in comparatively high
concentrations, and it is only slightly diminished by dilution with
three or four volumes of sugar solution, somewhat more considerably
by dilution with water. When it is filtered through a Chamberland
filter the first portions of the filtrate are capable of bringing
about fermentation, but the fermenting power diminishes in the
succeeding portions and finally disappears. The juice can be spun
in a centrifugal machine without being in any way altered, and no
separation into more or less active layers takes place under these
conditions. [p021]
The amorphous powder obtained by drying the precipitate produced when
the juice is added to a mixture of alcohol and ether is also capable
of producing fermentation, and the process of precipitation may be
repeated without seriously diminishing the fermenting power of the
product.
These facts clearly show that the various phenomena adduced by
the supporters of the theory of protoplasmic fragments are quite
consistent with the presence of a dissolved enzyme as the active agent
of the juice, and at the same time that the properties demanded of
the living fragments of protoplasm to which fermentation is ascribed
are such as cannot be reconciled with our knowledge of living
matter. If living protoplasm is the cause of alcoholic fermentation
by yeast-juice, a new conception of life will be necessary; the
properties of the postulated fragments of protoplasm must be so
different from those which the protoplasm of the living cell possesses
as to deprive the theory of all real value [Buchner, 1900, 2; Buchner,
E. and H., and Hahn, 1903, p. 33].
Further and very convincing evidence against the protoplasm theory
is afforded by the behaviour of yeast towards various desiccating
agents. When yeast is dried at the ordinary temperature it retains
its vitality for a considerable period. If, however, the dried yeast
be heated for six hours at 100° it loses the power of growth and
reproduction but still retains that of fermenting sugar, and when
ground with sand, kieselguhr and 10 per cent. glycerol solution yields
an active juice [Buchner, 1897, 2; 1900, 1]. Preparations (known as
zymin) obtained by treating yeast with a mixture of alcohol and ether
[Albert, 1900, 1901, 1], or with acetone and ether [Albert, Buchner,
and Rapp, 1902], show precisely similar properties (p. 38). The
proof in this case has been carried a step further, for the active
juice obtained by grinding such acetone-yeast, when precipitated
with alcohol and ether, yields an amorphous powder, still capable of
fermenting sugar.
THE PREPARATION OF YEAST-JUICE.
Buchner's process for the preparation of active yeast-juice is
characterised by extreme simplicity. The yeast employed, which
should be fresh brewery yeast, is washed two or three times by being
suspended in a large amount of water and allowed to settle in deep
vessels. It is then collected on a filter cloth, wrapped in a press
cloth, and submitted to a pressure of about 50 kilos, per sq. cm. for
five minutes. The resulting friable mass contains about 70 per cent.
of water and is free from adhering wort. The washed yeast is then
[p022] mixed with an equal weight of silver sand and 0·2 to 0·3 parts
of kieselguhr, care being taken that this is free from acid. The
correct amount of kieselguhr to be added can only be ascertained by
experience, and varies with different samples of yeast. The dry powder
thus obtained is brought in portions of 300 to 400 grams into a large
porcelain mortar and ground by hand by means of a porcelain pestle
fastened to a long iron rod which passes through a ring fixed in the
wall (Fig. 1). The mortar used by Buchner has a diameter of 40 cm. and
the pestle and rod together weigh 8 kilos.
[Illustration: FIG. 1. (Mortar and Pestle--Ed.)]
As the grinding proceeds the light-coloured powder gradually darkens
and becomes brown, and the mass becomes moist and adheres to the
pestle, until finally, after two to three minutes' grinding, it takes
the consistency of dough, at which stage the process is stopped. The
mass is next enveloped in a press cloth and submitted to a pressure
of 90 kilos, per sq. cm. in a hydraulic hand press, the pressure
being very gradually raised in order to avoid rupture of the cloth.
The cloth required for 1000 grams of yeast measures 60 by 75 cm. and
is previously soaked in water and then submitted to a pressure of 50
kilos, per sq. cm., retaining about 35 to 40 c.c. of water.
The juice runs from the press on to a folded filter paper, to remove
kieselguhr and yeast cells, and passes into a vessel standing in ice
water.
The yield of juice obtained by Buchner in an operation of this kind
from 1 kilo. of yeast amounts to 320 to 460 c.c. It may be increased
by re-grinding the press cake and again submitting it to pressure, and
then amounts on the average to 450 to 500 c.c.
Since the cell membranes constitute about 20 per cent. of the weight
of the dry yeast, this yield corresponds to more than 60 per cent.
of the total cell contents of the yeast. It has been computed by
Will [quoted by Buchner, E. and H., and Hahn, 1903, p. 66] that
[p023] only about 20 per cent. of the cells are left unaltered by one
grinding and pressing, and only 4 per cent. after a repetition of the
process, at least 57 per cent. of the cells being actually ruptured by
the double process, and the remainder to some extent altered. It seems
probable from these figures that a certain amount of the juice may be
derived from the unbroken cells, and Will expressly states that many
unbroken cells have lost their vacuoles.
[Illustration: FIG. 2. (Hydraulic Hand Press--Ed.)]
If the yeast be submitted to a process of regeneration, which consists
in exposure to a well-aerated solution of sugar and mineral salts
until fermentation is complete, the juice subsequently obtained
[p024] is more active than that yielded by the original yeast
[Albert, 1899, 1].
A modified method of grinding yeast was introduced by Macfadyen,
Morris, and Rowland [1900], who placed a mixture of yeast and sand
in a jacketed and cooled vessel, in which a spindle carrying brass
flanges was rapidly rotated [Rowland, 1901]. One kilo. of yeast ground
in this way for 3·5 hours yielded 350 c.c. of juice.
This grinding process was at first adopted by Harden and Young in
their experiments but was afterwards abandoned in favour of Buchner's
hand-grinding process, as it was found liable to yield juices of low
fermenting power, probably on account of inefficient cooling during
the grinding process. A slight modification of Buchner's process has,
however, been introduced, the hand-ground mass being mixed with a
further quantity of kieselguhr until a nearly dry powder is formed,
and the mass packed between two layers of chain cloth in steel
filter plates and pressed out in a hydraulic press at about 2 tons
to the square inch (300 kilos. per sq. cm.). The press and plates
are shown in section in Fig. 2. It has also been found convenient to
remove yeast cells and kieselguhr from the freshly pressed juice by
centrifugalisation instead of by filtration through paper, and to wash
the yeast before grinding by means of a filter-press.
Working with English top yeasts Harden and Young have found the yield
of juice extremely variable, the general rule being that the amount
of juice obtainable from freshly skimmed yeast is smaller than that
yielded by the same yeast after standing for a day or two after being
skimmed. The yield for 1000 grams of pressed brewer's yeast varies
from 150 to 375 c.c., and is on the average about 250 c.c.
Very fresh yeast occasionally presents the peculiar phenomenon that
scarcely any juice can be expressed from the ground mass, although the
latter does not differ in appearance or consistency from a mass which
gives a good yield.
EXTRACTION OF ZYMASE FROM UNGROUND YEAST.
/1. Maceration of Dried Yeast./
A valuable addition to the methods of obtaining an active solution
of zymase was made in 1911 by Lebedeff [1911, 2; 1912, 2; see also
1911, 3, 7, and 1912, 1]. This investigator had been in the habit of
grinding dried yeast with water for preparing samples of yeast-juice
of uniform character and observed that when the dried yeast was
digested with sugar solution and the mixture heated, coagulation
[p025] took place throughout the whole liquid, the proteins of the
yeast having passed out of the cells. Further examination revealed the
interesting fact that dried yeast readily yielded an active extract
when macerated in water for some time. The quality of the resulting
"maceration extract" depends on a considerable number of factors, the
chief of which are: (1) the temperature of drying of the yeast; (2)
the temperature of maceration; (3) the duration of maceration; and (4)
the nature of the yeast, as well as, of course, the amount of water
added in maceration.
In general the yeast should be dried at 25°-30° and then macerated
with 3 parts of water for 2 hours at 35°.
The temperature of maceration may as a rule be varied, without
detriment to the product provided that the time of maceration is
also suitably altered; thus with dried Munich yeast, maceration for
4·5 hours at 25° is about as effective as 2 hours at 35°, whereas
treatment for a shorter time at 25° or a longer time at 35° produces
in general a less efficacious extract. Yeast dried at a lower
temperature than 25° tends to yield an extract poor in co-enzyme (p.
59) and hence of low fermenting power, this being especially marked at
air temperature.
The subsequent treatment of the yeast during maceration may, however,
be of great influence in such cases. Thus a yeast dried at 15° gave by
maceration at 25° for 4·5 hours a weak extract (yielding with excess
of sugar 0·33g. CO{2}), whereas when macerated at 35° for 2 hours it
yielded a normal extract (1·36g. CO{2}).
The nature of the yeast is of paramount importance. Thus while Munich
(bottom) yeast usually gives a good result, a top yeast from a Paris
brewery was found to yield extracts containing neither zymase nor its
co-enzyme in whatever way the preparation was conducted. The existence
of such yeasts is of great interest, and it was probably due to the
unfortunate selection of such a yeast for his experiments that Pasteur
was unable to prepare active fermenting extracts and therefore failed
to anticipate Buchner by more than 30 years (see p. 15). The English
top yeasts as a rule give poor results [see Dixon and Atkins, 1913]
and sometimes yield totally inactive maceration extract. It is not
understood why the enzyme passes out of the cell during the process of
maceration and the whole method gives rise to a number of extremely
interesting problems.
/Method./--A suitable yeast is washed by decantation, filtered through
a cloth, lightly pressed by means of a hand press, and then passed
through a sieve of 5mm. mesh, spread out in a layer 1-1·5cm. thick
and left at 25°-35° for two days. Fifty grams of the dried yeast is
[Pg 026] thoroughly and carefully mixed with 150 c.c. of water in
a basin by means of a spatula and the whole digested for two hours
at 35°. The mass often froths considerably. It is then filtered
through ordinary folded filter paper, preferably in two portions,
and collected in a vessel cooled by ice. The separation may also be
effected by centrifuging or pressing out the mass, and the maceration
may be conveniently conducted in a flask immersed in the water of a
thermostat. It is not advisable to macerate more than 50 grams in one
operation. Under these conditions 25-30 c.c. of extract are obtained
after 20 minutes' filtration, 70-80 c.c. in twelve hours. Dried Munich
yeast can be bought from Messrs. Schroder of Munich and serves as a
convenient source of the extract.[1]
[1] The material supplied is occasionally found to yield an inactive
extract and every sample should be tested.
This extract closely resembles in properties the juice obtained by
grinding the same yeast, but it is usually more active and contains
more inorganic phosphate (see p. 46).
/2. Other Methods./
Attempts to prepare active extracts from undried yeast in an analogous
manner have so far not been very successful. Thus Rinckleben [1911]
found that plasmolysis by glycerol (8 per cent.) or sodium phosphate
(5 per cent.) sometimes yielded an active juice and sometimes a juice
which contained enzyme but no co-enzyme, but more often an inactive
juice incapable of activation (p. 64) [see also Kayser, 1911].
Giglioli [1911] by the addition of chloroform also obtained an active
liquid. It appears in fact as though almost any method of plasmolysing
the yeast cell may yield a certain proportion of zymase in the exudate.
An ingenious process has been devised by Dixon and Atkins [1913] who
applied the method of freezing in liquid air which they had found
efficacious for obtaining the sap from various plant organs. They thus
succeeded in obtaining from yeast, derived from Guinness' brewery in
Dublin, liquids capable of fermenting sugar and of about the same
efficacy as the maceration extracts prepared by Lebedeff's method from
the same yeast. The results were, however, in both cases very low, the
maximum total production of CO{2} by 25 c.c. of liquid from excess
of sugar being 32·5 c.c. (air temperature) or about 0·06g. Munich
yeast on the other hand yields, either by maceration or grinding, a
liquid giving as much as 1·5-2g. of CO{2} per 25 c.c., whilst [p027]
English yeast-juice prepared by grinding often gives as much as
0·5-0·7g. of CO{2}.
No direct comparison with the juice prepared by grinding was made by
Dixon and Atkins, but it may be concluded from their results that the
best method of obtaining an active preparation from the top yeasts
used in this country is that of grinding. Maceration, freezing and
plasmolysis alike yield poor results. With Munich yeast on the other
hand the maceration process yields excellent results, whilst the
liquid air process has not so far been tried.
PRACTICAL METHODS FOR THE ESTIMATION OF THE FERMENTING POWER OF
YEAST-JUICE.
In order to estimate the amount of carbon dioxide evolved in a given
time and the total amount evolved by the action of yeast-juice on
sugar, Buchner adopted an extremely simple method, which consisted in
carrying out the fermentation in an Erlenmeyer flask provided with
a small wash-bottle, which contained sulphuric acid and was closed
by a Bunsen valve, and ascertaining the loss of weight during the
experiment. Corrections are necessary for the carbon dioxide present
in the original juice and retained in the liquid at the close of the
experiment and for that present in the air space of the apparatus,
but it was found that for most purposes these could be neglected.
In cases in which greater accuracy was desired, the carbon dioxide
was displaced by air before the weighings were made. A typical
experiment of this kind, without displacement of carbon dioxide, is
the following:--
March 22, 1899, Berlin bottom yeast V. 20 c.c. juice + 8 grams cane
sugar + 0·2 c.c. toluene as antiseptic at 16°. Grams of carbon dioxide
after
24 48 72 96 hours.
0·40 0·64 0·99 1·11
The total weight of carbon dioxide evolved under these conditions is
termed the fermenting power of the juice (Buchner).
A more accurate method [Macfadyen, Morris, and Rowland, 1900]
consists in passing the carbon dioxide into caustic soda solution and
estimating it by titration. The yeast-juice, sugar, and antiseptic are
placed in an Erlenmeyer flask provided with a straight glass tube,
through which air can be passed over the surface of the liquid, and a
conducting tube leading into a second flask which contains 50 c.c. of
10 per cent. caustic soda solution and is connected with the air by a
guard tube containing soda lime. The juice can be freed from carbon
dioxide by agitation in a current of air before the flask is connected
to [p028] that containing the caustic soda solution, and at the end
of the period of incubation air is passed through the apparatus, the
liquid being boiled out if great accuracy is required. The absorption
flask is then disconnected and the amount of absorbed carbon dioxide
estimated by titration. This is carried out by making up the contents
of the flask to 200 c.c., taking out an aliquot portion, rendering
this exactly neutral to phenophthalein by the addition first of normal
and finally of decinormal acid, adding methyl orange and titrating
with decinormal acid to exact neutrality. Each c.c. of decinormal acid
used in this last titration represents 0·0044 gram of carbon dioxide
in the quantity of solution titrated.
[Illustration: FIG. 3. (Schiff's Azotometer--Ed.)]
These methods are only suitable for observations at considerable
intervals of time. For the continuous observation of the course
of fermentation Harden, Thompson and Young [1910] connect the
fermentation flask with a Schiff's azotometer filled with mercury and
measure the volume of gas evolved, the liquid having been previously
saturated with carbon dioxide (Fig. 3). The level of the mercury in
the reservoir is kept constant by a syphon overflow, as shown in the
figure, or, according to a modification introduced by S. G. Paine,
by a specially constructed bottle provided with two tubulures near
the bottom. This ensures that no change in the pressure in the flask
occurs, and the volume of gas observed is reduced to normal pressure
by means of a table. Before making a reading it is necessary to shake
the fermenting mixture thoroughly, as the albuminous liquid very
readily becomes greatly supersaturated with carbon dioxide, so much
so in fact that very little gas is evolved in the intervals between
the shakings. The exact procedure in making an observation consists in
shaking the flask [p029] thoroughly, replacing in the thermostat,
allowing to remain for one minute, and then reading the level of the
mercury in the azotometer. After the required time, say five minutes,
has elapsed from the time at which the flask was first shaken, it is
again removed from the bath, shaken as before, replaced, allowed to
remain for one minute and the reading then taken. In this way readings
can be conveniently made at intervals of three or five minutes or even
less, and much more detailed information obtained about the course
of the reaction than is possible by means of observations made at
intervals of several hours.
Another form of volumetric apparatus, designed by Walton [1904], has
been used by Lebedeff [1909].
An apparatus on a different principle has been designed by Slator
[1906] for use with living yeast, but is equally applicable to
yeast-juice, and a very similar form has been more recently employed
by Iwanoff [1909, 2]. In this apparatus the change of pressure
produced by the evolution of carbon dioxide is measured at constant
volume, and comparative rates of evolution can be obtained with
considerable accuracy, although the method has the disadvantage that
the absolute volume of gas evolved is not measured. The apparatus
consists of a bottle or flask connected with a mercury manometer. The
fermenting mixture is placed in the bottle along with glass beads to
facilitate agitation, the pressure is reduced to a small amount by the
water-pump, and the rise of pressure is then observed at intervals,
this being proportional to the volume of gas produced. As in the
preceding case, the liquid must be well shaken before a reading is
made.
THE ALCOHOLIC FERMENTATION OF THE SUGARS BY YEAST-JUICE.
Yeast-juice brings about a slow fermentation of those sugars which
are fermented by the yeast from which it is prepared as well as of
dextrin, and of starch and glycogen, which are not fermented by living
yeast.
(/a/) /Relation to Fermentation by living Yeast./
Both in rate of fermentation and in the total fermentation produced,
yeast-juice stands far behind the equivalent amount of living yeast.
Taking 25 c.c. of yeast-juice to be equivalent to at least 36 grams of
pressed yeast containing 70 per cent. of moisture, it is found that
whereas the yeast-juice (from English top yeast) gives with glucose
a maximum rate of fermentation of about 3 c.c. in five minutes, the
living yeast ferments the sugar at the rate of about 126 c.c. in the
same time, or [p030] about forty times as quickly. The total carbon
dioxide obtainable from the yeast-juice, moreover, corresponds to the
fermentation of only 2 to 3 grams of sugar, whilst the living yeast
will readily ferment a much larger quantity, although the exact limit
in this respect has not been accurately determined. The reasons for
this great difference in behaviour will be discussed later on, after
the various factors concerned in fermentation have been considered (p.
123).
(/b/) /Relation of Alcohol to Carbon Dioxide./
In all cases of fermentation by yeast-juice and zymin, the relative
amounts of carbon dioxide and alcohol produced are substantially in
the ratio of the molecular weights of the compounds, that is as 44:
46, so that for 1 part of carbon dioxide 1·04 of alcohol are formed.
This has been shown for the juice and zymin from bottom yeasts by
Buchner [Buchner, E. and H., and Hahn, 1903, pp. 210, 211], who
obtained the ratios 1·01, 0·98, 1·01, and 0·99 from experiments in
which from 8 to 15 grams of alcohol were produced. Similar numbers,
0·90, 1·12, 0·95, 0·91 and 0·92, have been obtained for the juice from
top yeasts by Harden and Young [1904], who worked with much smaller
quantities. The variable results obtained with juice from top yeast by
Macfadyen, Morris and Rowland [1900], have not been confirmed.
(/c/) /Relation of Carbon Dioxide and Alcohol Produced to the Amount
of Sugar Fermented./
The construction of a balance-sheet between the sugar fermented and
the products formed is of special interest in the case of alcoholic
fermentation by yeast-juice, because, there being no cell growth as
in the case of living yeast, an opportunity appears to be afforded of
ascertaining whether the whole of the sugar is converted into alcohol
and carbon dioxide, or whether some fraction of the sugar passes into
any of the well-known subsidiary products of alcoholic fermentation by
yeast, such as glycerol, fusel oil, or succinic acid. Unfortunately
the question cannot be settled in this way. When the loss of sugar
during the fermentation is estimated directly, it is usually found
to be considerably greater than the sum of the alcohol and carbon
dioxide produced from it. This fact was first observed by Macfadyen,
Morris and Rowland [1900], and was then confirmed by Buchner [Buchner,
E. and H., and Hahn, 1903, p. 212], in one instance, the excess of
sugar lost over products being in this case about 15 per cent. of the
total sugar which had disappeared. The matter was then more thoroughly
investigated by Harden and Young [1904]. [p031]
The conditions under which the experiment must be carried out are not
very favourable to the attainment of extreme accuracy. Yeast-juice
contains glycogen and a diastatic enzyme which converts this into
dextrins and finally into sugar. This process goes on throughout
fermentation, tending to increase the sugar present and to make the
apparent loss of sugar less than the sum of the products. In spite
of this it was found that a certain amount of sugar invariably
disappeared without being accounted for as alcohol or carbon dioxide,
and this whether the fermentation lasted sixty or a hundred and
eight hours, and independently of the dilution of the juice. This
disappearing sugar amounted in some cases to 44 per cent. of the
total loss of sugar, and on the average of twenty-five experiments
was 38 per cent. Further information was sought by converting all the
sugar-yielding constituents of the juice into sugar by hydrolysis
before and after the fermentation. This process revealed the fact that
when the glucose equivalent of the juice before and after fermentation
was determined after hydrolysis with three times normal acid for
three hours (and a correction made for the loss of reducing power
experienced by glucose itself when submitted to this treatment),
the difference was almost exactly equal to the alcohol and carbon
dioxide produced. In other words, accompanying fermentation, a change
proceeds by which sugar is converted into a less reducing substance,
reconvertible into sugar by hydrolysis with acids. Similar results
were subsequently obtained by Buchner and Meisenheimer [1906], who
employed 1·5 normal acid and observed a small nett loss of sugar.
Still more recently Lebedeff [1909, 1910, see also 1913, 2] has
carried out similar estimations with the same result. It is doubtful
whether the experiments which have so far been made on this point are
sufficiently accurate to decide with certainty whether or not the
loss of sugar is exactly equal to the sum of the carbon dioxide and
alcohol produced. It has been shown by Buchner and Meisenheimer [1906]
that glycerol is a constant product of alcoholic fermentation by
yeast-juice (p. 95), and no other source for this than the sugar has
yet been found, so that it is not improbable that a small amount of
sugar is converted into non-carbohydrate substances other than carbon
dioxide and alcohol.
It has also been shown [Harden and Young, 1913] that the deficit of
sugar is not due to the formation of hexosephosphate (p. 47), which
has a lower reduction than glucose, and that the solution from which
the sugar (either glucose or fructose) has disappeared actually
contains some substance of relatively high dextrorotation and of low
reducing power. [p032]
However this may be, it may be considered as established that
during alcoholic fermentation sugar is converted by an enzyme into
some compound of less reducing power, which again yields sugar on
hydrolysis with acids. The exact nature of this substance has not been
ascertained, but it appears likely that the process is a synthetical
one resulting in the formation of some polysaccharide, possibly
intermediate between the hexoses and glycogen.
A similar phenomenon has been observed with living yeast by Euler
and Johansson [1912, 1], and Euler and Berggren [1912], whose
interpretation of the observation is discussed later on (p. 57).
(/d/) /Fermentation of Different Carbohydrates. Autofermentation./
Yeast-juice and zymin ferment all the sugars which are fermented by
the yeast from which they are prepared, and, in addition, a number
of colloidal substances which cannot pass through the membrane of
the living yeast cell, but which are hydrolysed by enzymes in the
juice and thus converted into simpler sugars capable of fermentation
[Buchner and Rapp, 1898, 3; 1899, 2]. Of the simple sugars which have
been examined, glucose, fructose, and mannose are freely fermented,
l-arabinose not at all, whilst the case of galactose is doubtful.
Galactose is, however, fermented by juice prepared from a yeast which
has been "trained" to ferment galactose [Harden and Norris, 1910]. As
regards both the rate of fermentation and the total amount of carbon
dioxide evolved from glucose and fructose by the action of a definite
amount of yeast-juice, Buchner and Rapp obtained practically identical
numbers. Harden and Young [1909], using juice from top yeast, found
that fructose was slightly more rapidly fermented and gave a somewhat
larger total than glucose, whilst mannose was initially fermented at
almost the same rate as glucose, but gave a decidedly lower total, the
following being the average result:--
Sugar. Relative Rates. Relative Totals.
Glucose 1 1
Fructose 1·29 1·15
Mannose 1·04 0·67
Among the disaccharides, cane sugar and maltose are freely fermented,
and the juice can be shown like living yeast to contain invertase and
maltase. The extent of fermentation does not differ materially from
that attained with glucose. Lactose is not fermented.
Of the higher sugars raffinose is fermented by juice from bottom
yeast, but more slowly than cane sugar or maltose. No experiments seem
to have been made with juice from top yeast. [p033]
As regards the fermentation of the higher carbohydrates, very little
experimental work has been carried out. Buchner and Rapp found that
the fermentation of starch paste was doubtful, but that soluble starch
and commercial dextrin were fermented with some freedom. No special
study has been made of the diastatic enzymes which bring about the
hydrolysis of these substances.
The fermentation of glycogen by yeast-juice is of considerable
interest, since it is known that the characteristic reserve
carbohydrate of the yeast cell is glycogen [see Harden and Young,
1902, where the literature is cited], and moreover that in living
yeast the intracellular fermentation of glycogen proceeds readily,
whereas glycogen added to a solution in which yeast is suspended is
not affected. Yeast-juice contains a diastatic enzyme which hydrolyses
glycogen to a reducing and fermentable sugar, so that in a juice poor
in zymase to which glycogen has been added, the amount of sugar is
found to increase, the hydrolysis of the glycogen proceeding more
quickly than the fermentation of the resulting sugar [Harden and
Young, 1904], but the course of this enzymic hydrolysis of glycogen by
yeast-juice has not yet been studied. As a rule, it is found both with
juices from top and bottom yeast that the evolution of carbon dioxide
from glycogen proceeds less rapidly and reaches a lower total than
from an equivalent amount of glucose.
Since nearly all samples of yeast contain glycogen, yeast-juice and
also zymin usually contain this substance as well as the products of
its hydrolysis. These provide a source of sugar which enters into
alcoholic fermentation, so that a slow spontaneous production of
carbon dioxide and alcohol proceeds when yeast-juice is preserved
without any addition of sugar. The extent of this autofermentation
varies considerably, as might be expected, with the nature of the
yeast employed or the preparation of the material, but is generally
confined within the limits of 0·06 to 0·5 gram of carbon dioxide for
25 c.c. of juice.
In juice from bottom yeast it amounts to about 5 to 10 per cent.
of the total fermentation obtainable with glucose [Buchner, 1900,
2], whereas in juice from top yeasts, which gives a smaller total
fermentation with glucose, it may occasionally equal, or even exceed,
the glucose fermentation, and frequently amounts to 30 to 50 per cent.
of it. It is therefore generally advisable in studying the effect of
yeast-juice on any particular substance to ascertain the extent of
autofermentation by means of a parallel experiment.
The maceration extract of Lebedeff (p. 24) is usually, but not
invariably [Oppenheimer, 1914, 2], free from glycogen, which is
hydrolysed [p034] and fermented during the processes of drying
and macerating, and therefore as a rule shows no appreciable
autofermentation.
(/e/) /Effect of Concentration of Sugar on the Total Amount of
Fermentation./
The kinetics of fermentation by zymase will be considered later on
(p. 120), but the effect on the total fermentation of different
concentrations of sugar, this substance being present throughout in
considerable excess, may be advantageously discussed at this stage.
The subject has been investigated by Buchner [Buchner, E. and H., and
Hahn, 1903, pp. 150-8; Buchner and Rapp, 1897] using cane sugar, and
he has found both for yeast-juice and for dried yeast-juice dissolved
in water that (/a/) the total amount of fermentation increases with
the concentration of the sugar; (/b/) the initial rate of fermentation
decreases with the concentration of the sugar. The following are the
results of a typical experiment, 20 c.c. of yeast-juice being employed
in presence of toluene at 22°:--
Cane Sugar. │ CO{2} in grams after
─────────┬───────────┼──────────┬──────────┬──────────
Weight. │ Per cent. │ 6 hours. │24 hours. │ 96 hours.
─────────┼───────────┼──────────┼──────────┼──────────
2·2 │ 10 │ 0·17 │ 0·50 │ 0·55
3·52 │ 15 │ 0·14 │ 0·53 │ 0·64
5 │ 20 │ 0·13 │ 0·54 │ 0·73
6·66 │ 25 │ 0·13 │ 0·52 │ 0·80
8·56 │ 30 │ 0·12 │ 0·46 │ 0·81
10·76 │ 35 │ 0·12 │ 0·40 │ 0·82
13·33 │ 40 │ 0·11 │ 0·36 │ 0·82
10·76 │ 35 │ 0·12 │ 0·40 │ 0·82
13·33 │ 40 │ 0·11 │ 0·36 │ 0·82
The results as to the total fermentations in experiments of this kind
are liable to be vitiated by the circumstance that when a low initial
concentration of sugar is employed, the supply of sugar may be so
greatly exhausted before the close of the experiment as to cause a
marked diminution in the rate of fermentation and hence an unduly
low total. Even allowing, however, for any effect of this kind, the
foregoing table clearly shows the increase in total fermentation
and the decrease in initial rate accompanying the increase of sugar
concentration from 10 to 40 per cent. Working with a greater range
of concentrations (3·3-53·3 grm. per 100 c.c.) Lebedeff has obtained
similar results with maceration extract [1911, 4], but has found
that the total amount fermented diminishes after a certain optimum
concentration (about 33·3 grm. per 100 c.c.) is reached.
A practical conclusion from these experiments is that a high [p035]
concentration of sugar tends to preserve the enzyme in an active state
for a longer time. Simultaneously it prevents the development of
bacteria and yeast cells.
(/f/) /Effect of Varying Concentration of Yeast-Juice./
This subject, which is of considerable importance with reference to
the question of the protoplasmic or enzymic nature of the active agent
in yeast-juice, has been examined in some detail by Buchner [Buchner,
E. and H., and Hahn, 1903, pp. 158-65] and by Meisenheimer [1903]
for juices from bottom yeast, by Harden and Young [1904] for those
from top yeast, and by Lebedeff [1911, 4] for maceration extract, the
results obtained being in substantial agreement.
Dilution of yeast-juice with sugar solution, so that the concentration
of the sugar remains constant, produces a small progressive diminution
in the total fermentation, which only becomes marked when more than 2
volumes are added, and this independently of the actual concentration
of the sugar. Dilution with water produces a somewhat more decided
diminution, which, however, does not exceed 50 per cent. of the total
for the addition of 3 volumes of water. The effect on maceration
extract is somewhat greater but of the same kind. The autofermentation
of juice from top yeast is scarcely affected by dilution with 4
volumes of water.
┌───────────────────────────────────────────────────────────────────┐
│ Nature of Per cent. Volumes of Volumes of Total │
│ Juice of Sugar Sugar Water Added. Fermentation │
│ (1,2,3) Employed Solution in g. │
│ by Weight Added. of CO{2}. │
│ Bottom Yeast──────────────────────────────────────────────────────┤
│ 1 29 0 -- 0·99 │
│ 1 -- 1·13 │
│ 2 -- 0·92 │
│ 4 -- 0·79 │
│ 2 9 0 -- 0·43 │
│ 1 -- 0·60 │
│ 2 -- 0·53 │
│ 4 -- 0·41 │
│ 3 9 -- 0 0·46 │
│ -- 1 0·32 │
│ -- 2 0·33 │
│ -- 3 0·36 │
│ Top Yeast─────────────────────────────────────────────────────────┤
│ 1 0 -- 0 0·29 │
│ (Autofermentation) -- 2 0·29 │
│ -- 3 0·28 │
│ 2 29 0 -- 0·31 │
│ 1 -- 0·34 │
│ 2 -- 0·31 │
│ 4 -- 0·35 │
│ 6 -- 0·28 │
│ 3 7·4 -- 0 0·44 │
│ -- 1 0·35 │
│ -- 2 0·30 │
│ -- 3 0·28 │
└───────────────────────────────────────────────────────────────────┘
[p036]
On the whole, therefore, yeast-juice may be said to be only slightly
affected by dilution even with pure water, and the effect of the
latter can in no way be regarded as comparable with the poisonous
effect which it exerts on living protoplasm, as suggested by
Macfadyen, Morris, and Rowland [1900].
(/g/) /The Effect of Antiseptics on the Fermentation of Sugars by
Yeast-Juice./
Buchner has paid special attention to the effect of antiseptics
on the course of fermentation by yeast-juice [Buchner and Rapp,
1897; 1898, 2, 3; 1899, 1; Buchner and Antoni, 1905, 1; Buchner and
Hoffmann, 1907; Buchner, E. and H., and Hahn, 1903, pp. 169-205; see
also Albert, 1899, 2; Gromoff and Grigorieff, 1904; Duchaček, 1909]
in order (1) to obtain evidence as to the possibility of the active
agent in yeast-juice consisting of fragments of protoplasm and not of
a soluble enzyme, and (2) also to provide a safe method of avoiding
contamination, by the growth of bacteria or yeasts, of the liquids
used which were often kept at 25° for several days. The results of
these experiments are briefly summarised in the following table, in
which the effect of each substance on the total fermentation produced
is noted:--
Substance. Effect on Total Fermentation.
Concentrated solution of glycerol Slight diminution
" " " sugar " increase
Toluene (to saturation or excess) Less than 10 per cent.
diminution
Chloroform 0·5 per cent. Slight increase
" 0·8 per cent. (saturation) No change
" Large excess (17 per cent.) 64 per cent. diminution
Chloral hydrate 0·7 per cent. Increase up to 27 per cent.
" 3·5-5·4 per cent. Completely destroyed
Phenol 0·1 per cent. No change
" 0·5 " 40 per cent. diminution
" 1·2 " Completely destroyed
Thymol 1 " Slight diminution
" 5 " Marked "
Benzoic acid 0·1 " 7 per cent. diminution
" 0·25 " 26 " "
Salicylic acid 0·1 " 10 " "
" 0·27 " 35 " "
Formaldehyde 0·12 " 20 " "
" 0·24 " 30-60 " "
Acetone 6 " 20 " "
" 14 " 80 " "
Alcohol 6 " 0-20 " "
" 14 " 75 " "
Sodium fluoride 0·5 " 90 " "
" 2 " Almost completely destroyed
Ammonium fluoride 0·55 per cent. Completely destroyed
Sodium azoimide, NaN{3}, 0·36 per cent. Slight diminution
" " 0·71 " Marked "
Quinine hydrochloride 1 " Slight increase
Ozone 10·4-34·8 milligrams per 20 c.c. Marked diminution
Hydrocyanic acid 1·2 per cent. Completely destroyed
[p037]
The general result of these experiments is to show that quantities
of antiseptics which are sufficient to inhibit the characteristic
action of living cells have only a slight effect on the fermentative
activity of yeast-juice. A large excess of the antiseptic in many
cases produces a very decided diminution or total destruction of
the fermenting power, and accompanying this a precipitation of the
constituents of the juice. The decided increase of activity produced
by small quantities of chloral hydrate, and to a less marked extent by
chloroform and a few other substances, is of considerable interest.
It is ascribed by Duchaček to a selective action on the proteoclastic
enzyme, but without satisfactory evidence.
Hydrocyanic acid, even in dilute solution, completely suspends the
fermenting power of the juice, without, however, producing any
permanent change in the fermenting complex, as is shown by the fact
that when the hydrocyanic acid is removed by a current of air, the
juice regains its fermenting power. In this respect hydrocyanic acid
behaves precisely as with many other enzymes and with colloidal
platinum [Bredig, 1901]. Sodium arsenite is a pronounced protoplasmic
poison, which rapidly destroys the power of growth and reproduction
in living cells, and was therefore applied to yeast-juice to
differentiate between protoplasmic and enzymic action. It was,
however, found that the action of this substance was complicated by
some unknown factor and very irregular results were obtained [Buchner,
E. and H., and Hahn, 1903, pp. 193 ff.]. These phenomena appear to be
of the same order as those produced by the addition of arsenates to
yeast-juice [Harden and Young, 1906, 3], and will be discussed along
with the latter (p. 77).
PERMANENT PREPARATIONS CONTAINING ACTIVE ZYMASE.
A considerable number of preparations have been obtained in the dry
state which retain some proportion of the fermenting power of yeast or
yeast-juice.
Starting with yeast-juice, it is possible to arrive at this result
either by evaporation or precipitation. When the juice is very rapidly
evaporated to a syrup at 20° to 25° and then further dried at 35°,
either in the air or in a vacuum and finally exposed over sulphuric
acid in a vacuum desiccator, a dry brittle mass is obtained which is
soluble in water and retains practically the whole of the fermenting
power of the juice. The success of the preparation depends on the
nature of the yeast from which the juice is derived, Berlin yeasts V
and S yielding much less satisfactory results than Munich yeast. The
powder when [p038] thoroughly dry is found to retain its properties
almost unimpaired for at least a year, and can be heated to 85° for
eight hours without undergoing any serious loss of fermenting power
[Buchner and Rapp, 1898, 4; 1901; Buchner, E. and H., and Hahn, 1903,
pp. 132-9].
Active powders can also be obtained by precipitating yeast-juice
with alcohol, alcohol and ether, or acetone. The preparation is
best effected by bringing the juice into 10 volumes of acetone,
centrifuging at once and as rapidly as possible, washing, first with
acetone and then with ether, and finally drying over sulphuric acid.
The white powder thus obtained is not completely soluble in water
but is almost entirely dissolved by aqueous glycerol (2·5 to 20 per
cent.), forming a solution which has practically the same fermenting
power as the original juice. The precipitation can be repeated without
any serious loss of fermenting power. Prolonged contact of the
precipitate with the supernatant liquid, especially when alcohol or
alcohol and ether are used, causes a rapid loss of the characteristic
property [Albert and Buchner, 1900, 1, 2; Buchner, E. and H., and
Hahn, 1903, pp. 228-246; Buchner and Duchaček, 1909].
Dry preparations capable of fermenting sugar can also be readily
obtained from yeast without any preliminary rupture of the cells.
Heat alone (yielding a product known as hefanol) or treatment with
dehydrating agents may be used for this purpose, and a brief allusion
has already been made (p. 21) to the different varieties of permanent
yeast (Dauerhefe) obtainable in these ways. The most important of
these products are the dried Munich yeast (Lebedeff, see p. 25), and
the material known as zymin, which is now made under patent rights for
medicinal purposes by Schroder of Munich. The latter has proved of
value in the investigation of the production of zymase in the yeast
cell [Buchner and Spitta, 1902], and of many other problems concerned
with alcoholic fermentation. In order to prepare it 500 grams of
finely divided pressed brewer's yeast, containing about 70 per cent.
of water, are brought into 3 litres of acetone, stirred for ten
minutes, and filtered and drained at the pump. The mass is then well
mixed with 1 litre of acetone for two minutes and again filtered and
drained. The residue is roughly powdered, well kneaded with 250 c.c.
of ether for three minutes, filtered, drained, and spread on filter
paper or porous plates. After standing for an hour in the air it is
dried at 45° for twenty-four hours. About 150 grams of an almost white
powder containing only 5·5 to 6·5 per cent. of water are obtained.
This is quite incapable of growth or reproduction but produces a very
considerable amount of alcoholic fermentation, far greater indeed than
a corresponding [p039] quantity of yeast-juice. Two grams of the
powder corresponding to 6 grams of yeast and about 3·5 to 4 c.c. of
yeast-juice, are capable of fermenting about 2 grams of sugar, whereas
the 4 c.c. of yeast-juice would on the average only ferment from
one-quarter to one-sixth of this amount of sugar. The rate produced
by this amount of zymin is about one-eighth of that given by the
corresponding amount of living yeast [Albert, 1900; Albert, Buchner,
and Rapp, 1902]. The proteoclastic ferment is still present in zymin,
which undergoes autolysis in presence of water in a similar manner to
yeast-juice [Albert, 1901, 2].
As already mentioned an active juice can be prepared by grinding
acetone-yeast with water, sand, and kieselguhr, and this process
presents the advantage that samples of yeast-juice of approximately
constant composition can be prepared at intervals from successive
portions of a uniform supply of acetone-yeast.
Preparations of acetone-yeast, made from yeast freed from glycogen
by exposure in a thin layer to the air for three or four hours at
35° to 45°, or eight hours at the ordinary temperature [Buchner and
Mitscherlich, 1904], show practically no autofermentation and may be
used analytically for the estimation of fermentable sugars.
All the foregoing preparations exhibit the same general properties as
yeast-juice, as regards their behaviour towards the various sugars,
antiseptics, etc.
When zymin is mixed with sugar solution without being previously
ground, it exhibits a peculiarity which is of some practical interest.
The time which elapses before the normal rate of fermentation is
attained and the total fermentation obtainable vary with the amount of
sugar solution added, the time increasing and the total diminishing
as the quantity of this increases. This phenomenon appears to have
been noticed by Trommsdorff [1902], and a single experiment of Buchner
shows the influence of the same conditions [Buchner, E. and H., and
Hahn, 1903, p. 265, Nos. 700-1]. Harden and Young have found that when
2 grams of zymin are mixed with varying quantities of 10 per cent.
sugar solution the following results are obtained:--
───────────┬──────────────────────────────────────────
Volumes of │ Total Gas Evolved in
Sugar │ 1 2 3 4 22·5 hours.
Solution │
───────────┼──────────────────────────────────────────
5 c.c. │ 15·7 31·6 44·8 56·5 233·3
10 " │ 2·2 10·5 23 31·8 202·3
20 " │ 0·9 2·4 13·6 23·7 125·5
40 " │ 1·4 1·7 2·3 2·9 56·3
───────────┴──────────────────────────────────────────
[p040]
This behaviour appears to be due to the removal of soluble matter
essential for fermentation from the cell, which is discussed later
on. It follows that when zymin is being tested for fermenting power,
a uniform method should be adopted, and all comparative tests should
be made with the same volumes of added sugar solution. Ground zymin
appears to begin to ferment somewhat more slowly than unground (2 grm.
to 12·4 c.c. of sugar solution in each case), but eventually produces
the same total volume of gas [Buchner and Antoni, 1905, 1]. [p041]
CHAPTER III.
THE FUNCTION OF PHOSPHATES IN ALCOHOLIC FERMENTATION.
In the course of some preliminary experiments (commenced by the late
Allan Macfadyen, but subsequently abandoned) on the production of
anti-ferments by the injection of yeast-juice into animals, the serum
of the treated animals was tested for the presence of such antibodies
both for the alcoholic and proteoclastic enzymes of yeast-juice, and
it was then observed that the serum of normal and of treated animals
alike greatly diminished the autolysis of yeast-juice.
As the explanation of the comparatively rapid disappearance of
the fermenting power from yeast-juice had been sought, as already
mentioned (p. 20), in the hydrolytic action of the tryptic enzyme
which always accompanies zymase, the experiment was made of carrying
out the fermentation in the presence of serum, with the result that
about 60 to 80 per cent. more sugar was fermented than in the absence
of the serum [Harden, 1903].
This fact was the starting-point of a series of attempts to obtain a
similar effect by different means, in the course of which a boiled
and filtered solution of autolysed yeast-juice was used, in the hope
that the products formed by the action of the tryptic enzyme on the
proteins of the juice would, in accordance with the general rule,
prove to be an effective inhibitant of that enzyme. This solution
was, in fact, found to produce a very marked increase in the total
fermentation effected by yeast-juice, the addition of a volume of
boiled juice equal to that of the yeast-juice doubling the amount of
carbon dioxide evolved [Harden and Young, 1905, 1]. This effect was
found to be common to the filtrates from boiled fresh yeast-juice and
from boiled autolysed yeast-juice, and was ultimately traced in the
main, not to the antitryptic effect which had been surmised, but to
two independent factors, either of which was capable in some degree of
bringing about the observed result.
Boiled yeast-juice was indeed found to possess a decided
anti-autolytic effect, as determined by a comparison of the amounts
of nitrogen rendered non-precipitable by tannic acid in yeast-juice
alone [p042] and in a mixture of yeast-juice and boiled juice on
preservation [Harden, 1905]. The anti-autolytic effect, however,
appeared to vary independently of the effect on the fermentation, and
the conclusion was drawn, as stated above, that the increase in the
alcoholic fermentation was not directly dependent on the decrease in
the action of the proteoclastic enzyme but was due to some independent
cause. The property possessed by boiled yeast-juice of diminishing the
autolysis of yeast-juice has now been carefully examined by Buchner
and Haehn [1910, 2] and ascribed by them to a soluble antiprotease (p.
65).
The two factors to which the increase in fermentation produced by the
addition of boiled juice were ultimately traced were (1) the presence
of phosphates in the liquid, and (2) the existence in boiled fresh
yeast-juice of a co-ferment or co-enzyme, the presence of which is
indispensable for fermentation [Harden and Young, 1905, 1, 2].
The former of these factors will be here discussed and the co-enzyme
will form the subject of the following chapter.
The general fact that sodium phosphate increases the total
fermentation produced by a given volume of yeast juice was observed on
several occasions by Wroblewski [1901] and also by Buchner [Buchner,
E. and H., and Hahn, 1903, pp. 141-2], who ascribed the action of this
salt to its alkalinity, comparing it in this respect with potassium
carbonate and remarking that the increase in both cases took place
chiefly in the first twenty hours of fermentation. The increased
amount of fermentation following the addition of boiled yeast-juice
was also noted by Buchner and Rapp [1899, 2, No. 265, p. 2093] in a
single experiment.
Observations made at intervals of a few minutes instead of twenty
hours have, however, revealed the fact that phosphates play a part
of fundamental importance in alcoholic fermentation and that their
presence is absolutely essential for the production of the phenomenon.
EFFECT OF THE ADDITION OF PHOSPHATE TO A FERMENTING MIXTURE OF
YEAST-JUICE AND SUGAR.
When a suitable quantity[2] of a soluble phosphate is added to a
fermenting mixture of glucose, fructose, or mannose with yeast-juice,
the rate of fermentation rapidly rises, sometimes increasing as
much as twenty-fold, continues at this high value for a certain
period and then falls again to a value approximately equal to, but
generally [p043] somewhat higher than, that which it originally had.
Careful experiments have shown that during this period of enhanced
fermentation the amounts of carbon dioxide and alcohol produced exceed
those which would have been formed in the absence of added phosphate
by a quantity exactly equivalent to the phosphate added in the ratio
CO{2} or C{2}H{6}O:R′{2}HPO{4} [Harden and Young, 1906, 1].
[2] The effect of an excess of phosphate is discussed later on, p. 71.
This result is of fundamental importance, and the evidence on which it
rests deserves some consideration. Quantitative experiments on this
subject require certain preliminary precautions. The acid phosphates
are too acid to permit of any extended fermentation and the phosphates
of the formula R′{2}HPO{4} absorb a considerable volume of carbon
dioxide with production of a bicarbonate, according to the reaction:--
R{2}HPO{4} + H{2}CO{3} ⇌ RHCO{3} + RH{2}PO{4}.
The method which has been adopted, therefore, is to employ either a
secondary phosphate saturated with carbon dioxide at the temperature
of the experiment, or a mixture of five molecular proportions of
the secondary phosphate with one molecular proportion of a primary
phosphate, in which the amount of bicarbonate formed is negligible. In
the former case it is necessary to ascertain whether any of the carbon
dioxide evolved is derived from the bicarbonate by the action of acid
originally present or produced in the yeast-juice or by a disturbance
of the original equilibrium owing to the chemical change which occurs.
This is done by acidifying duplicate samples with hydrochloric acid
before and after the fermentation and measuring the gas evolved in
each case. Any necessary correction can then be made. The calculation
of the extra amount of carbon dioxide evolved from yeast-juice
containing sugar when a phosphate is added involves an estimation
of the amount which would have been evolved in the absence of added
phosphate, and this is a matter of some difficulty. Since the final
steady rate of fermentation attained is often slightly different from
the initial rate, the practice has been adopted of ascertaining this
final rate and then calculating the total evolution corresponding to
it for the whole period from the time of the addition of the phosphate
to the end of the observations. This amount deducted from the observed
total leaves the extra amount of carbon dioxide formed, and it is
this quantity which is equivalent to the phosphate added. Alcohol is
simultaneously produced in the normal ratio. The justification for
this method of calculation will be found later (p. 54).
The following table, containing the results of experiments with
[Pg 044] glucose, fructose, and mannose, indicates very clearly the
nature of the method of calculation and also of the agreement between
observation and theory.
Three quantities of 25 c.c. of yeast-juice + 5 c.c. of a solution
containing 1 gram of the sugar to be examined (a large excess) were
incubated with toluene at 25° for one hour, in order to remove all
free phosphate, and to each were then added 5 c.c. of a solution of
sodium phosphate corresponding to 0·1632 gram of Mg{2}P{2}O{7} and
equivalent to 32·6 c.c. of carbon dioxide at N.T.P. The rates of
fermentation were then observed until they had passed through the
period of acceleration and had fallen and attained a steady value, the
gases being measured moist at 19·3° and 760·15 mm.
─────────────────────────────────────────────────┬─────────────────────
│ Glucose.
│ ┌──────────────
│ │ Mannose.
│ │ ┌─────────
│ │ │Fructose.
─────────────────────────────────────────────────┼──────┼────┴─────────
Maximum rate attained, c.cs. per five minutes │ 9·6 │ 7 11·3
Final rate of fermentation │ 1·1 │ 0·96 1·08
Total carbon dioxide produced by fermentation in │ │
fifty-five minutes after addition of phosphate│ 49·7 │ 47·8 47·6
Correction for evolution in absence of phosphate │ │
in fifty-five minutes │ 12·1 │ 10·6 11·9
Extra carbon dioxide equivalent to phosphate │ 37·6 │ 37·2 35·7
" " " " " " at N.T.P.│ 34·4 │ 34 32·6
─────────────────────────────────────────────────┴──────┴──────────────
These numbers agree well with the value calculated from the phosphate
added, viz. 32·6 [Harden and Young, 1909].
Another experiment is illustrated graphically in Fig. 4, in which
the volume of carbon dioxide evolved is plotted against time.
The determination was in this case made by adding 25 c.c. of an
aqueous solution containing 5 grams of glucose to one quantity of
25 c.c. of yeast-juice (curve A) and 5 c.c. of 0·3 molar solution
of the mixed primary and secondary sodium phosphates, and 20 c.c.
of a solution containing 5 grams of glucose to a second equal
quantity of yeast-juice (curve B). Curve A shows the normal course
of fermentation of yeast-juice with glucose. There is a slight
preliminary acceleration during the first twenty minutes, due to free
phosphate in the juice, and the rate then becomes steady at about
1·4 c.c. in five minutes. During this preliminary acceleration 10
c.c. of extra carbon dioxide are evolved, this number being obtained
graphically by continuing the line of steady rate back to the axis of
zero time. Curve B shows the effect of the added phosphate. The rate
rises to about 9·5 c.c. in five minutes, i.e. to more than six times
the normal rate, and then gradually falls until after an hour it is
again steady and almost exactly equal to 1·4 c.c. per five minutes.
Continuing the line of steady rate back to the axis of zero [p045]
time it is found that the extra amount of carbon dioxide is 48 c.c.
Subtracting from this the 10 c.c. shown in curve A as due to the juice
alone, a difference of 38 c.c. is obtained due to the added phosphate.
The amount calculated from the phosphate added in this case is, at
atmospheric temperature and pressure, 38·9 c.c.
[Illustration: FIG. 4 (Graph of Carbon Dioxide Evolution v Time--Ed.)]
After the expiration of seventy minutes from the commencement of the
experiment, a second addition is made of an equal amount of phosphate.
The whole phenomenon then recurs, as shown in curve C, the maximum
rate being slightly lower than before, about 6 c.c. per five minutes,
and the rate again becoming finally steady at 1·4 c.c. as before. The
extra amount of carbon dioxide evolved in this second period obtained
graphically as in the former case, is 107-68 = 39 c.c.
It may be noted that in this case the observations after each addition
last fifty to seventy minutes, so that an error of 0·1 c.c. per five
minutes in the estimated final rate would make an error of 1 to 1·4
c.c. in the extra amount of carbon dioxide, i.e. about 3 to 4 per
cent. of the total, and this is approximately the limit of accuracy of
the method. [p046] The results are more precise when the yeast-juice
employed is an active one, since, when the fermenting power of the
juice is low, the initial period of accelerated fermentation is unduly
prolonged and the calculation of the extra amount of carbon dioxide is
rendered uncertain.
Zymin (p. 38) yields precisely similar results to yeast-juice, but
in this case the rate of fermentation is not so largely increased.
This has the effect that the extra amount of carbon dioxide cannot
be quite so accurately estimated for zymin, because a slight error
in the determination of the final rate of fermentation has a greater
influence on the result. The equivalence between the extra amount
of carbon dioxide evolved and the phosphate added is, however,
unmistakable, as is shown by the following results of an experiment
with zymin, in which 6 grams of zymin (Schroder) + 3 grams of fructose
(Schering) + 25 c.c. of water were incubated at 25° in presence of
toluene until a steady rate had been attained. Five c.c. of a solution
of sodium phosphate equivalent to 32·2 c.c. carbon dioxide at N.T.P.
were then added.
Maximum rate attained,
c.c. per five minutes 14·1
Final rate of fermentation 6·2
Total evolved by fermentation in eighty
minutes after addition of phosphate 131
Correction for evolution in absence of
phosphate in eighty minutes 99·2
Extra carbon dioxide at 16° and 767·1 mm 31·8
" " " " N.T.P 29·8
Considering the small proportional rise in rate and the long period of
accelerated fermentation, the agreement between the volume observed,
29·8 c.c., and that calculated from the phosphate, 32·2, is quite
satisfactory [Harden and Young, 1910, 1.] Precisely the same relations
hold for maceration extract, but in this case it must be remembered
that a large amount of free phosphate is present in the extract, as
much as 0·3129 grm. Mg{2}P{2}O{7} being obtained from 20 c.c. in one
preparation, so that the original extract had the concentration of a
0·14 molar solution of sodium phosphate. It is in fact not improbable
that the delay in the onset of fermentation sometimes observed with
maceration extract (see Lebedeff, 1912, 2; Neuberg and Rosenthal,
1913) may be due to the presence of phosphate in so great an excess
of the amount which can be rapidly esterified by the enzymes that the
rate of fermentation is at first greatly lowered (see p. 71). When
this phosphate is removed by incubation with glucose or fructose,
the subsequent addition of phosphate produces the characteristic
action and the extra carbon dioxide evolved is, as with other yeast
preparations, equivalent to the phosphate added. An actual estimation
carried out in this way gave 35 c.c. of CO{2} for an addition of
phosphate equivalent to 32·9 c.c. [Harden and Young, 1912]. [p047]
Within the limits imposed by the experimental conditions, then, the
fact is well established that the addition of a soluble phosphate to a
fermenting mixture of a hexose with yeast-juice, maceration extract,
dried yeast, or zymin causes the production of an equivalent amount of
carbon dioxide and alcohol.
This fact indicates that a definite chemical reaction occurs in
which sugar and phosphate are concerned, and this conclusion is
confirmed when the fate of the added phosphate is investigated. If
an experiment, such as one of those described above, be interrupted
as soon as the rate of fermentation has again become normal, and the
liquid be boiled and filtered, it is found that nearly the whole of
the phosphorus present passes into the filtrate, but that only a small
proportion of this exists as mineral phosphate, whilst the remainder,
including that added in the form of a soluble phosphate, is no longer
precipitable by magnesium citrate mixture [Harden and Young, 1905, 2].
A similar observation was made at a later date by Iwanoff [1907],
who had previously observed [1905] that living yeast, like many
other vegetable organisms, converted mineral phosphates into organic
derivatives. Iwanoff employed zymin and hefanol (p. 38) instead
of yeast-juice, and found that phosphates were thereby rendered
non-precipitable by uranium acetate solution, but did not observe the
accelerated fermentation caused by their addition.
The foregoing conclusions have been strikingly confirmed by
experiments with maceration extract carried out by Euler and Johansson
[1913], in which both the carbon dioxide evolved and the phosphate
rendered non-precipitable by magnesia were determined at intervals.
When dried yeast is employed as the fermenting agent, the amount of
phosphate esterified in the earlier stages is greater than would be
expected, but ultimately becomes exactly equivalent to the carbon
dioxide evolved.
NATURE OF THE PHOSPHO-ORGANIC COMPOUND FORMED BY YEAST-JUICE AND ZYMIN
FROM THE HEXOSES AND PHOSPHATE.
The formation and properties of the compound produced from phosphates
in the manner just described have been investigated by Harden and
Young [1905, 2; 1908, 1; 1909; 1911, 2], Young [1909; 1911], Iwanoff
[1907; 1909, 1], Lebedeff [1909; 1910; 1911, 5, 6; 1912, 3; 1913, 1];
and Euler [1912, 1; Euler and Fodor, 1911; Euler and Kullberg, 1911,
3; Euler and Ohlsén, 1911; 1912; Euler and Johansson, 1912, 4; Euler
and Bäckström, 1912], but its exact constitution cannot as yet be
regarded as definitely known. [p048]
Phosphates undergo this characteristic change when the sugar
undergoing fermentation is glucose, mannose, or fructose, and it
may be said at once that no distinction can be established between
the products formed from these various hexoses; they all appear
to be identical. The compound produced is, as already mentioned,
not precipitated by ammoniacal magnesium citrate mixture, nor by
uranium acetate solution. It can, however, be precipitated by copper
acetate (Iwanoff) and by lead acetate (Young). The preparation of the
pure lead salt from the liquid obtained by fermenting a sugar with
yeast-juice or zymin in presence of phosphate is commenced by boiling
and filtering the liquid. Magnesium nitrate solution and a small
quantity of caustic soda solution are then added to precipitate any
free phosphate, and the liquid well stirred and allowed to stand over
night. To the neutralised filtrate lead acetate is then added together
with sufficient caustic soda solution to maintain the reaction neutral
to litmus, until no further precipitate is formed. The liquid is then
filtered or, better, centrifugalised, and the precipitate repeatedly
washed with water until a portion of the clear filtrate gives no
reduction when boiled with Fehling's solution. It is essential that
this washing should be thorough as evidence has recently been obtained
of the formation under certain conditions of a hexosephosphate,
the lead salt of which is not so sparingly soluble as that of the
hexosediphosphate [Harden and Robison, 1914]. The lead precipitate
is then suspended in water, decomposed by a current of sulphuretted
hydrogen, the clear filtrate freed from sulphuretted hydrogen by a
current of air, and finally neutralised with caustic soda. The removal
of phosphate and conversion into lead salt are repeated twice, and the
resulting lead salt is then found to be free from nitrogen and to have
a composition represented by the formula C{6}H{10}O{4}(PO{4}Pb){2}.
Lebedeff carries out the preparation in a somewhat different manner.
The fermentation is effected by means of air-dried yeast (150 grams
to 1 litre of water, 210 grams cane-sugar and 105 grams of a mixture
of 2 parts Na{2}HPO{4} and 1 part NaH{2}PO{4}) and the liquid (about
700 c.c.) after boiling and filtering, is treated with an equal volume
of acetone. About 300 c.c. of a thick liquid is precipitated and
this is redissolved in water and precipitated by an equal volume of
acetone two or three times. The final liquid is then precipitated with
warm lead acetate solution and filtered and washed with dilute lead
acetate solution until the filtrate is clear and no longer reduces
Fehling's solution after removal of the lead [1910]. Euler and Fodor
[1911] on the other hand precipitate the free phosphate with magnesia
mixture and then add acetone, dissolve the syrup thus precipitated in
water and add copper [p049] acetate solution. A blue copper salt
is precipitated which is thoroughly washed with water and used for
the preparation of solutions of the acid. A solution of the free acid
can readily be prepared by the action of sulphuretted hydrogen on the
lead salt suspended in water. It forms a strongly acid liquid, which
requires exactly two equivalents of base for each atom of phosphorus
present to render it neutral to phenolphthalein. It decomposes when
evaporated, leaving a charred mass containing free phosphoric acid.
The acid is slightly optically active, and has [/a/{D}] = + 3·4°. A
number of amorphous salts have been prepared by precipitation from
a solution of the sodium salt, and of these the silver, barium, and
calcium salts have been analysed with results agreeing with the
general formula C{6}H{10}O{4}(PO{4}R′{2}){2}. The magnesium, calcium,
barium, and manganese salts, which are only sparingly soluble, are
all precipitated when their solutions are boiled but re-dissolve on
cooling, and this property can be utilised for their purification. The
alkali salts have only been obtained as viscid residues.
A difference of opinion exists as to the molecular weight and
constitution of this substance. Iwanoff [1909, 1] regards it as a
triosephosphoric acid, C{3}H{5}O{2}(PO{4}H{2}), basing this view
on the preparation of an osazone which melted at 142°, but when
recrystallised from benzene gave a product melting at 127°-8°,
which had the same appearance, melting-point, and nitrogen content
as the triosazone formed by the action of phenylhydrazine on the
oxidation products of glycerol. Neither Lebedeff [1909] nor Young
could obtain Iwanoff's osazone, and all attempts to reduce the acid
with formation of glycerol either by sodium amalgam or hydriodic acid
were unsuccessful (Young). There is therefore practically no serious
experimental evidence in favour of Iwanoff's view.
On the other hand, Harden and Young regard the acid as a diphosphoric
ester of a hexose. This view is based on the fact that when the
acid is boiled with water, or an acid, free phosphoric acid is
produced along with a levo-rotatory solution containing fructose and
possibly a small proportion of some other sugar or sugars. (Euler
and Fodor however did not obtain a hexose in this way [1911].) The
acid itself only reduces Fehling's solution after some hours in
the cold, rapidly when boiled, whereas when its solution is first
boiled, and then treated with Fehling's solution in the cold, the
products of decomposition bring about reduction in a few minutes.
The reduction brought about when the acid is boiled with Fehling's
solution is considerably less (33 per cent.) than that produced by an
equivalent amount of glucose. The behaviour of the compound towards
phenylhydrazine is also in complete agreement [p050] with this
view. Lebedeff found [1909, 1910] that the acid or its salts heated
with phenylhydrazine in presence of acetic acid gave an insoluble
compound which was ultimately found to be the /phenylhydrazine salt of
hexosemonophosphoric acid osazone/
C{6}H{5}NH·NH{2}·H{2}PO{4}·C{4}H{5}(OH){3}·
C(N{2}HC{6}H{5})CH(N{2}HC{6}H{5})
[Lebedeff, 1910; 1911, 6; Young, 1911]. After recrystallisation from
alcohol this compound forms yellow needles, melting at 151°-152°. It
is decomposed by caustic soda yielding a /sodium salt/
Na{2}PO{4}·C{4}H{5}(OH){3}·(CN{2}HC{6}H{5})·CH(N{2}HC{6}H{5})
and on boiling with caustic soda decomposes giving a hexosazone (free
from phosphorus) which is probably glucosazone, and in addition
glyoxalosazone, probably as the result of a secondary decomposition.
Towards acids it is remarkably stable yielding with hydrochloric acid
a /hexosonephosphoric ester/ from which the original osazone can be
regenerated (Lebedeff). Lebedeff at first [1910] argued from the
formation of this osazone that the original hexosephosphate contained
only one phosphoric acid group per molecule of hexose. It was however
shown by Young [1911] and subsequently confirmed by Lebedeff [1911,
6] that one molecule of phosphoric acid is split off during the
formation of the osazone, even in neutral solution. Moreover it has
been found that in the cold hexosediphosphoric acid reacts with 3
molecules of phenylhydrazine forming the /diphenylhydrazine salt of
hexosediphosphoric acid phenylhydrazone/
(C{6}H{5}NH·NH{2}·H{2}PO{4}){2}·C{6}H{7}(OH){3}·N{2}HC{6}H{5}.
This compound crystallises out when 1 volume of alcohol is added to
a solution of 3 molecules of phenylhydrazine in one of the acid and
forms colourless needles melting at 115°-117°. p-Bromophenylhydrazine
yields an analogous compound melting at 127°-128°.
Precisely the same products are given with phenylhydrazine by the
hexosephosphoric acid prepared from glucose, mannose, and fructose,
proving that all these sugars yield the same hexosediphosphoric acid,
a point of fundamental importance.
Direct measurements of the molecular weight of the acid by the
freezing-point method, combined with the determination of the degree
of dissociation by the rate of cane-sugar inversion, are indecisive,
but indicate that the acid has a molecular weight considerably higher
than that required for a triosephosphoric acid.
A similar uncertainty attaches to the determination of the molecular
weight from the freezing-point depression and conductivity of the
acid potassium salt [Euler and Fodor, 1911]. Euler however concludes
[p051] that both a hexosediphosphoric acid and a triosemonophosphoric
acid are formed, but has not prepared any derivatives of the latter.
As regards the constitution of the hexosephosphoric ester several
suggestions have been made by Young, but no decisive evidence at
present exists. The identity of the products from glucose, mannose,
and fructose may be explained by regarding the acid as a derivative
of the enolic form common to these three sugars (p. 97), or by
supposing that portions of two sugar molecules may be concerned in its
production. The formation and composition of the hydrazone and osazone
are of great importance as they indicate that in all probability
one of the phosphoric acid residues is united with the carbon atom
adjacent to the carbonyl group of the hexose. They moreover render it
certain that the original phosphoric ester is a hexosediphosphoric
ester and not a triosemonophosphoric ester.
Hexosediphosphoric acid has not as yet been discovered in the animal
body. The action of a number of enzymes upon it has been examined
[Euler, 1912, 2; Euler and Funke, 1912; Harding, 1912; Plimmer, 1913]
with the following results.
The lipase of castor oil seeds, a glycerol extract of the intestinal
mucous membrane of the rabbit and pig, and an aqueous extract of bran
have a slow hydrolytic action, whereas pepsin and trypsin are without
effect. Feeding experiments with rabbits and dogs indicate that the
ester is capable of hydrolysis in the animal body, a large proportion
of the phosphorus being excreted as inorganic phosphate. The ester is
also decomposed by /Bacillus coli communis/.
It is remarkable that the hexosephosphate is not fermented nor
hydrolysed by living yeast, a fact observed by Iwanoff, Harden and
Young, and Euler, although, according to the experiments of Paine
[1911], the yeast cell is at all events partially permeable to the
sodium salt.
THE EQUATION OF ALCOHOLIC FERMENTATION.
An equation can readily be constructed for the reaction in which
hexosephosphate is formed, the data available being the formula of the
product and the relation between the phosphate added and the carbon
dioxide and alcohol produced:--
(1) 2C{6}H{12}O{6} + 2PO{4}HR{3} =
2CO{2} + 2C{2}H{6}O + 2H{2}O + C{6}H{10}O{4}(PO{4}R{2}){2}.
According to this, two molecules of sugar are concerned in the change,
the carbon dioxide and alcohol being equal in weight to one [p052]
half of the sugar used, and the hexosephosphate and water representing
the other half.
Additional confirmation of this equation is afforded by the
determination of the ratio between sugar used and carbon dioxide
evolved when a known weight of sugar together with an excess of
phosphate is added to yeast-juice at 25°. The phenomena then observed
are precisely similar to those which occur when a phosphate is
added to a fermenting mixture of yeast-juice and excess of sugar as
described above. The rate of fermentation rapidly rises and then
gradually falls until a rate is attained approximately equal to that
of the autofermentation of the juice in presence of phosphate. At this
point it is found that the extra amount of carbon dioxide evolved,
beyond that which would have been given off in the absence of added
sugar, bears the ratio expressed in equation (1) to the sugar added
[Harden and Young, 1910, 2]. The results of four estimations made in
this way were (/a/) 0·2 grams of glucose gave 26·5 and 27·9 c.c. of
carbon dioxide at N.T.P.; (/b/) 0·2 grams of fructose gave 27·9 and
28·9 c.c. The carbon dioxide calculated from the sugar added in each
of the four cases is 26·96 c.c.
It has also been shown by Euler and Johansson [1913] that in the
fermentation of a mixture of equivalent amounts of phosphate and
glucose, the whole of the glucose had disappeared when the whole of
the phosphate had become esterified.
CYCLE OF CHANGES UNDERGONE BY PHOSPHATE IN ALCOHOLIC FERMENTATION.
According to equation (1) the free phosphate present is used up in the
reaction, and the question at once arises whether there is any source
from which a constant supply of free phosphate can be elaborated
in the juice, or whether some other change occurs which results in
the formation of carbon dioxide and alcohol in the absence of free
phosphate. The experimental evidence points in the direction of the
former of these alternatives, but the question is a very difficult one
to decide with absolute certainty.
When a mixture of a phosphate with yeast-juice and sugar is examined
at intervals and the amount of free phosphate estimated, the following
stages are observed:--
1. During the initial period of accelerated fermentation following the
addition of the phosphate, the concentration of free phosphate rapidly
diminishes.
2. At the close of this period, the amount of free phosphate [p053]
present is very low, and, as long as active fermentation continues, no
marked increase in it occurs.
3. As alcoholic fermentation slackens and finally ceases, a marked
and rapid rise in the amount of free phosphate occurs at the expense
of the hexosephosphate, which steadily diminishes in amount, and this
change is brought about by an enzyme in the juice and ceases if the
liquid be boiled.
This last reaction may be represented by the equation
(2) C{6}H{10}O{4}(PO{4}R{2}){2} + 2H{2}O =
C{6}H{12}O{6} + 2PO{4}HR{2}.
In the light of this equation, together with equation No. 1, given
above, all these facts can be simply and easily understood.
The rapid diminution in the amount of free phosphate during stage 1
corresponds with the occurrence of reaction (1). During the whole
period of fermentation the enzymic hydrolysis of the hexosephosphate
is proceeding according to equation (2). Up to the end of stage 2 the
phosphate thus produced enters into reaction, according to equation
(1), with the sugar which is present in excess and is thus reconverted
into hexosephosphate, so that as long as alcoholic fermentation is
proceeding freely, no accumulation of free phosphate can occur.
As soon as alcoholic fermentation ceases, however, it is no longer
possible for the phosphate to pass back into hexosephosphate, and
hence it accumulates in the free state.
A similar hydrolysis of hexosephosphate and accumulation of phosphate
occur when a solution of hexosephosphate is treated with yeast-juice
which has been deprived of the power of fermentation by dialysis, or
with zymin freed from co-enzyme by washing (p. 63).
The actual rate of fermentation observed in any particular case in
presence of excess of sugar, enzyme, and co-enzyme must on this view
depend on the supply of phosphate which is available.
In presence of an adequate amount of phosphate, as well as of sugar,
the highest rate attained represents the maximum velocity at which
reaction (1) can proceed in that sample of yeast-juice or zymin, and
this high rate is characteristic of the initial period of accelerated
fermentation which follows the addition of a suitable quantity of
phosphate. By the simple expedient of renewing the supply of phosphate
as rapidly as it is converted into hexosephosphate, this high rate can
be maintained for a considerable time [Harden and Young, 1908, 1]. In
this way, for example, an average rate of evolution of carbon dioxide
of 15 c.c. in five minutes was maintained for an hour and a [p054]
quarter, whereas the normal rate in the absence of added phosphate was
3 c.c.
As soon as all the free phosphate has entered into the reaction,
however, the supply of phosphate depends in the main on the rate
at which the resulting hexosephosphate is decomposed, and the rate
of fermentation now attained is conditioned by the rate at which
reaction (2) proceeds, and this evidently depends on the existing
concentration of the hydrolytic enzyme, which may be provisionally
termed /hexosephosphatase/.
The rates attained during the initial period of rapid fermentation and
the subsequent period of slow fermentation are thus seen to represent
the velocities of two entirely different chemical reactions.
These considerations also explain why it is the /extra/ carbon dioxide
evolved during the initial period, and not the total carbon dioxide,
which is equivalent to the added phosphate. As the production of
phosphate is proceeding throughout the whole period at a rate which
is equivalent to the normal rate of fermentation, it is obviously
necessary to deduct the corresponding amount of carbon dioxide from
the total evolved in order to ascertain the amount equivalent to the
added phosphate.
An explanation is also afforded of the fact that a considerable
increase in the concentration of hexosephosphate does not materially
increase the normal rate of fermentation. This is probably due to
the circumstance that, in accordance with the general behaviour
of enzymes in presence of excess of the fermentable substance,
the hexosephosphatase hydrolyses approximately equal amounts of
hexosephosphate in equal times whatever the concentration of the
latter may be, above a certain limit.
According to the experiments of Euler and Johansson [1913] the
hydrolytic activity of the hexosephosphatase is greatly diminished by
the presence of toluene.
EFFECT OF PHOSPHATE ON THE TOTAL FERMENTATION PRODUCED BY YEAST-JUICE.
The addition of a phosphate to yeast-juice not only produces the
effect already described, but also enables a given volume of
yeast-juice to effect a larger total fermentation, even after
allowance is made for the carbon dioxide equivalent to the quantity of
phosphate added. The increase in the case of ordinary yeast-juice has
been found to amount to from 10 to 150 per cent. of the original total
fermentation [p055] produced by the juice in the absence of added
phosphate. The numbers contained in columns 1 and 2 of the table on
p. 56 illustrate this effect, the ratio of the total in the presence
of phosphate to that obtained in its absence being given, as well as
that of the total in presence of phosphate less the equivalent of
the phosphate added, to the original fermentation. The cause of this
increase in the total fermentation is probably to be sought mainly in
a protective action of the excess of hexosephosphate on the various
enzymes, for, as has been stated above, the rate of fermentation after
the termination of the initial period, is practically the same as in
the absence of added phosphate (see p. 43).
Now it follows from equation (1) (p. 51) that in the total absence
of phosphate no fermentation should occur, and the experimental
realisation of this result would afford very strong evidence in favour
of this interpretation of the phenomenon.
Hitherto, however, it has not been found possible to free the
materials employed completely from phosphorus compounds which
yield phosphates by enzymic hydrolysis during the experiment, but
it has been found that when the phosphate contents are reduced to
as low a limit as possible, the amount of sugar fermented becomes
correspondingly small, and, further, that in these circumstances the
addition of a small amount of phosphate or hexosephosphate produces a
relatively large increase in the fermenting power of the enzyme.
When the total phosphorus present is thus largely reduced, the
increase produced by the addition of a small amount of phosphate may
amount to as much as eighty-eight times the original, in addition to
the quantity equivalent to the phosphate, whilst the actual total
evolved, including this equivalent, may be as much as twenty times the
original fermentation. This result must be regarded as strong evidence
in favour of the view that phosphates are indispensable for alcoholic
fermentation.
The results indicated above were experimentally obtained in three
different ways and are exhibited in the following table. In the first
place (cols. 3 and 4), advantage was taken of the fact that the
residues obtained by filtering yeast-juice through a Martin gelatin
filter (p. 59) are sometimes found to be almost free from mineral
phosphates, whilst they still contain a small amount of co-enzyme.
The experiment then consists in comparing the fermentation produced
by such a residue poor in phosphate with that observed when a small
amount of phosphate is added. The second method (col. 5) consisted in
carrying out two parallel fermentations by means of a residue rendered
inactive by filtration [p056] and a solution of co-enzyme free from
phosphate and hexosephosphate (p. 67) [Harden and Young, 1910, 2].
The third method (col. 6) consisted in washing zymin with water,
to remove soluble phosphates, and then adding to it a solution
of co-enzyme containing only a small amount of phosphate, and
ascertaining the effect of the addition of a small known amount of
hexosephosphate upon the fermentation produced by this mixture [Harden
and Young, 1911, 1].
───────────────────────────┬──────┬──────┬──────┬──────┬──────┬───────
│ 1 │ 2 │ 3 │ 4 │ 5 │ 6
───────────────────────────┼──────┼──────┼──────┼──────┼──────┼───────
│ c.c. │ c.c. │ c.c. │ c.c. │ c.c │ c.c.
│ │ │ │ │ │
Gas evolved in absence of │ │ │ │ │ │
added phosphate │ 369 │ 220 │ 1·4 │ 1·2 │ 20·3 │ 1·5
In the presence of │ 629 │ 629 │ 25·8 │ 26·8 │ 92·3 │ 132·7
───────────────────────────┼──────┼──────┼──────┼──────┼──────┼───────
Increase due to phosphate │ 260 │ 409 │ 24·4 │ 25·6 │ 72·0 │ 131·2
Carbonic acid equivalent │ │ │ │ │ │
to phosphate │ 63 │ 61 │ 16·9 │ 16·8 │ 16·8 │ --
───────────────────────────┼──────┼──────┼──────┼──────┼──────┼───────
Increase after initial │ │ │ │ │ │
period │ 197 │ 348 │ 7·5 │ 8·8 │ 55·2 │ --
───────────────────────────┼──────┼──────┼──────┼──────┼──────┼───────
Ratio of totals │ 1·7 │ 2·9 │ 18·4 │ 21·3 │ 4·5 │ 88
Ratio of increase after │ │ │ │ │ │
initial period to │ │ │ │ │ │
original fermentation │ 0·5 │ 1·6 │ 5·3 │ 7·3 │ 2·7 │ --
───────────────────────────┴──────┴──────┴──────┴──────┴──────┴───────
PRODUCTION OF A FERMENTABLE SUGAR FROM HEXOSEPHOSPHATE BY THE ACTION OF
AN ENZYME CONTAINED IN YEAST-JUICE.
The sugar which, according to equation (2) accompanies the phosphate
formed by the enzymic hydrolysis of hexosephosphate is under ordinary
circumstances fermented by the alcoholic enzyme of the juice and thus
escapes detection.
When, however, a solution of a hexosephosphate is exposed to the
action of either yeast-juice or zymin, entirely or partially freed
from co-enzyme, this sugar, being no longer fermented, accumulates and
can be examined. It has thus been found [Harden and Young, 1910, 2]
that a sugar is in fact produced in this way which can be fermented
by living yeast and exhibits the reactions of fructose, although the
presence of other hexoses is not excluded. The products of the enzymic
hydrolysis of the hexosephosphates therefore appear to be the same as,
or similar to, those formed by the action of acids [Young, 1909].
A further consequence of these facts is that a hexosephosphate will
yield carbon dioxide and alcohol when it is added to yeast-juice or
zymin, and this has also been found to be the case [Harden and Young,
1910, 2; Iwanoff, 1909, 1]. [p057]
MECHANISM OF THE FORMATION OF HEXOSEDIPHOSPHORIC ACID.
On this subject little is yet known, but a number of extremely
interesting results, the interpretation of which is still doubtful,
have been obtained by Euler and his colleagues. Euler has obtained
a yeast [Yeast H of the St. Erik's brewery in Stockholm] which
differs from Munich yeast in several respects. A maceration extract
prepared from the yeast dried at 40° in a vacuum produces no effect
on a glucose solution containing phosphate. If, however, the glucose
solution be previously partially fermented with living yeast and
then boiled and filtered, the addition of the extract prepared from
Yeast H brings about the esterification of phosphoric acid without
any accompanying evolution of carbon dioxide [Euler and Ohlsén, 1911,
1912].
Euler interprets this as follows: (/a/) Glucose itself is not directly
esterified, but must first undergo some preliminary change, which is
brought about by the action of living yeast. No proof of the existence
of a new modification of glucose in this solution has however been
advanced, other than its behaviour to extract of Yeast H, so that
Euler's conclusion cannot be unreservedly accepted. It is moreover
possible and even more probable that some thermostable catalytic
substance (perhaps a co-enzyme) passes from the yeast into the
glucose solution and enables the yeast extract to attack the glucose
and phosphoric acid. A very small degree of esterification was also
produced when an extract having no action on glucose and phosphate
was added to glucose which had been treated with 2 per cent. caustic
soda for forty hours, but the nature of the compound formed was not
ascertained [Euler and Johansson, 1912, 4]. (/b/) The esterification
of phosphoric acid without the evolution of carbon dioxide implies
that the enzyme by which this process is effected is distinct from
that which causes the actual decomposition of the sugar. Euler goes
further than this and regards the enzyme as a purely synthetic one,
giving it the name of hexosephosphatese to distinguish it from the
hexosephosphatase which hydrolyses the hexosephosphate.
The evidence on which this conclusion is based cannot be regarded as
satisfactory, inasmuch as it consists in the observation that /in
presence of sugar/ yeast extract does not hydrolyse the phosphoric
ester. This, however, could not be expected since hydrolysis and
synthesis under these conditions would ultimately proceed at equal
rates.
In any case the adoption of this nomenclature is inconsistent with the
conception of an enzyme as a catalyst and is therefore inadvisable
until the reaction has been much more thoroughly studied. [p058]
It may further be pointed out that no proof has yet been advanced that
the phosphoric ester produced without evolution of carbon dioxide is
identical with hexosediphosphoric acid produced with evolution of
carbon dioxide. It is by no means improbable that it represents some
intermediate stage in the production of the latter (see p. 117).
Euler's other results on this subject may be briefly summarised as
follows:--
(1) In presence of excess of sugar the esterification of the
phosphoric acid proceeds by a monomolecular reaction and is most rapid
in faintly alkaline reaction [Euler and Kullberg, 1911, 3].
(2) When yeast extract has been heated for 30 minutes to 40° it
effects the esterification of phosphoric acid at a much greater
rate than the unheated extract (2-10 times). Heating at 50° for 30
minutes however completely inactivates the extract. The cause of the
activation is as yet unknown. The temperature coefficient for the
unheated extract (17·5°-30°) is 1·4-1·5 for 10° rise of temperature
[Euler and Ohlsén, 1911].
(3) Yeasts which in the dried state all produce rapid esterification
of phosphoric acid, yield extracts of very unequal powers in this
respect [Euler, 1912, 1]. [p059]
CHAPTER IV.
THE CO-ENZYME OF YEAST-JUICE.
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.
[Illustration FIG. 5. (Filter Apparatus, Tube/Flange Detail--Ed.)]
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.
[Illustration: FIG. 6. (Filter Apparatus--Ed.)]
/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:--
──────────────────────┬────────┬──────────┬────────┬────────┬──────────
│ │ Filtrate │ Boiled │ Water │ CO{2}
No. Material. │ Volume.│ added. │ Juice │ added. │ evolved.
│ │ │ added. │ │
│ │ c.c. │ c.c. │ c.c. │
──────────────────────┼────────┼──────────┼────────┼────────┼──────────
1 Undried and │ │ │ │ │
unwashed residue │ 15 c.c.│ 0 │ 0 │ 15 │ 0 g.
│ 15 " │ 15 │ 0 │ 0 │ 0·035 "
2 " " │ 15 " │ 0 │ 0 │ 15 │ 0·024 "
│ 15 " │ 0 │ 15 │ 0 │ 0·282 "
3 Undried and washed│ │ │ │ │
residue │ 25 " │ 0 │ 0 │ 0 │ 0·4 c.c.
│ 25 " │ 0 │ 25 │ 0 │268 "
4 " " │ 20 " │ 0 │ 0 │ 0 │ 8·3 "
│ 20 " │ 20 │ 0 │ 0 │ 90·3 "
5 Washed and dried │ │ │ │ │
residue │ 1 gram │ │ │ │
│ in │ │ │ │
│ 15 c.c.│ 0 │ 0 │ 0 │ 0 "
│ " │ 0 │ 12 │ 0 │108 "
6 " " │ 1 gram │ │ │ │
│ in │ │ │ │
│ 25 c.c.│ 0 │ 0 │ 0 │ 0 "
│ " │ 0 │ 25 │ 0 │364 "
──────────────────────┴────────┴──────────┴────────┴────────┴──────────
[p062]
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 CO{2}.
(2) 20 c.c. of dialysed juice + 10 c.c. of evaporated dialysate gave
0·52 gram CO{2}.
(3) 20 c.c. of dialysed juice + 10 c.c. of boiled juice gave
0·89 gram CO{2}.
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 Duchaček, 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
[Pg 067] 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. [Pg 070]
CHAPTER V.
ACTION OF SOME INHIBITING AND ACCELERATING AGENTS ON THE ENZYMES OF
YEAST-JUICE.
One of the most interesting and at the same time most difficult
problems concerning enzyme action in general is the nature of the
inhibiting or accelerating effect produced by many substances upon
the rate or total result of the chemical process set up in presence
of the enzyme. Inhibition, it is usually supposed, involves either
the decomposition of the enzyme, in which case it is irreversible,
its removal from the sphere of action by some change in its mode of
solution, or the formation of an inactive or less active compound
between the enzyme and the inhibiting agent. This compound it may
sometimes be possible to decompose, with the result that the activity
of the enzyme is restored. A striking example of this, to which
allusion has already been made, is the effect of hydrocyanic acid on
alcoholic fermentation (p. 37).
Acceleration of enzyme action can in some cases be ascribed to the
fact that the accelerating substance possesses an assignable chemical
function in the reaction, so that an increase in the concentration of
this substance causes an increase in the rate of the reaction. As we
have seen in Chapter III, this is the explanation of the accelerating
effect of phosphates on fermentation by yeast-juice. In many other
cases, however, no such chemical function can be traced, as, for
example, in the effect of neutral salts on the hydrolytic action of
invertase, or the effect of the addition of the co-enzyme to zymase,
and it is necessary to fall back on some assumption, such as that the
accelerating agent acts by increasing the effective concentration of
the enzyme or by combining either with the enzyme or the substrate,
forming a compound which undergoes the reaction more readily.
The interest in the following examples of inhibition and acceleration
of fermentation by yeast-juice lies not only in their relation to
these general problems but also, and perhaps chiefly, in their
bearing on the specific problem of the nature and mode of action of
the various agents concerned in the production of alcohol and carbon
dioxide from sugar in the yeast-cell. [p071]
I. INFLUENCE OF CONCENTRATION OF PHOSPHATE ON THE COURSE OF
FERMENTATION.
Prominent among these instances of inhibition and acceleration are
the phenomena attendant on the addition of excess of phosphate to
yeast-juice.
When a phosphate is added to a fermenting mixture of a sugar and
yeast-juice, the effect varies with the concentration of the phosphate
and the sugar and with the particular specimen of yeast-juice
employed. With low concentrations of phosphate in presence of excess
of glucose the acceleration produced is so transient that no accurate
measurements of rate can be made. As soon as the amount of phosphate
added is sufficiently large, it is found that the rate of evolution of
carbon dioxide very rapidly increases from five to ten times, and then
quickly falls approximately to its original value.
As the concentration of phosphate is still further increased, it is
first observed that the maximum velocity, which is still attained
almost immediately after the addition of the phosphate, is maintained
for a certain period before the fall commences, and then, as the
increase in concentration of phosphate proceeds, that the maximum is
only gradually attained after the addition, the period required for
this increasing with the concentration of the phosphate. Moreover,
with still higher concentrations, the maximum rate attained is less
than that reached with lower concentrations, and further, the rate
falls off more slowly. The concentration of phosphate which produces
the highest rate, which may be termed the optimum concentration,
varies very considerably with different specimens of yeast-juice
[Harden and Young, 1908, 1].
All these points are illustrated by the accompanying curves (Fig. 7)
which show the rate of evolution per five minutes plotted against
the time for four solutions in which the initial concentrations of
phosphate were (A) 0·033, (B) 0·067, (C) 0·1, and (D) 0·133 molar,
the volumes of 0·3 molar phosphate being 5, 10, 15, and 20 c.c. in
each case added to 25 c.c. of yeast-juice, and made up to 45 c.c, each
solution containing 4·5 grams of glucose. The time of addition is
taken as zero, the rate before addition being constant, as shown in
the curves.
It will be observed that 5 and 10 c.c. (A and B) give the same
maximum, whilst 15 c.c. (C) produce a much lower maximum, and 20 c.c.
(D) a still lower one, the rate at which the velocity diminishes
after the attainment of the maximum being correspondingly slow in
these last two cases. By calculating the amount of phosphate which
has disappeared as such from the amount of carbon dioxide evolved,
[p072] it is found that the maximum does not occur at the same
concentration of free phosphate in each case.
[Illustration: FIG. 7. (Graph CO{2} rate vs time--Ed.)]
These results suggest that the phosphate is capable of forming two
or more different unstable associations with the fermenting complex.
One of these, formed with low concentrations of the phosphate, has
the composition most favourable for the decomposition of sugar,
whilst the others, formed with higher concentrations of phosphate,
contain more of the latter, probably associated in such a way with
the fermenting complex as to render the latter partially or wholly
incapable of effecting the decomposition of the sugar molecule. As the
fermentation proceeds slowly in the presence of excess of phosphate,
the concentration of the latter is reduced by conversion into
hexosephosphate, and a re-distribution of phosphate occurs, resulting
in the gradual change of the less active into the more active
association of phosphate with fermenting complex, and a consequent
rise in the rate of fermentation.
In those cases in which the maximum rate corresponding to the optimum
concentration of phosphate is never attained, some secondary cause may
be supposed to intervene, such as a permanent change in a portion of
the fermenting complex, accumulation of the products of the reaction,
etc.
It is also possible as suggested by Buchner for the analogous case of
arsenite (p. 78) that the addition of increasing amounts of phosphate
causes a progressive but reversible change in the mode of dispersion
[p073] of the colloidal enzyme, and that this has the secondary
effect of altering the rate of fermentation. No decisive evidence is
as yet available upon the subject.
The results obtained by Euler and Johansson [1913] to which reference
has already been made indicate that in presence of a moderate excess
of phosphate esterification is more rapid than production of carbon
dioxide. No explanation of this phenomenon has yet been given, but it
might obviously be due either to the production of some phosphorus
compound which subsequently takes part in the production both of
hexosediphosphate and of carbon dioxide, or, less probably, to the
entire independence of the two changes--esterification of phosphate
and production of carbon dioxide--which might then be differently
affected by the presence of excess of phosphate and therefore take
place at different rates.
II. REACTION OF FRUCTOSE WITH PHOSPHATES IN PRESENCE OF YEAST-JUICE.
Although, as has been pointed out (p. 42), glucose, mannose, and
fructose all react with phosphate in a similar manner in presence of
yeast-juice, there are nevertheless certain quantitative differences
between the behaviour of glucose and mannose on the one hand, and
fructose on the other, which appear to be of considerable importance.
Fructose differs from the other two fermentable hexoses in two
particulars: (1) the optimum concentration of phosphate is much
greater; (2) the maximum rate of fermentation attainable is much
higher [Harden and Young, 1908, 2; 1909].
These points are clearly illustrated by the following results, which
all refer to 10 c.c. of yeast-juice, and show that the optimum
concentration of phosphate for the fermentation of fructose is
from 1·5 to 10 times that of glucose, and that the maximum rate of
fermentation for fructose in presence of phosphate is 2 to 6 times
that of glucose.
───────┬─────────┬──────────────────────┬──────────────────────
Sugar │ Total │ Optimum Volume of │ Maximum Rate in Cubic
in │ Volume. │ 0·6 Molar Phosphate │ Centimetres of CO{2}
Grams.│ │ in c.c. │ per Five Minutes
│ │ Glucose. │ Fructose. │ Glucose. │ Fructose.
───────┼─────────┼──────────┼───────────┼──────────┼───────────
2 │ 35 │ 2 │ 5 │ 7·5 │ 32·2
4 50 1 10 5·4 28·4
1·6 23 2 5 8 17
1 25 1·75 5 5·2 25·9
2 25 5 7·5 16·2 31·2
2 20 2 3·5 7·9 22·6
2 22·5 0·75 2 3·4 22·2
───────────────────────────────────────────────────────────────
[p074]
It is interesting to note that the two high rates, 32·2 and 31·2 c.c.
per five minutes, are equal to about half the rate obtainable with
an amount of living yeast corresponding to 10 c.c. of yeast-juice,
assuming that about 16 to 20 grams of yeast are required to yield this
volume of juice, and that this amount of yeast would give about 56
to 70 c.c. of carbon dioxide per five minutes at 25°, which has been
found experimentally to be about the rate obtainable with the top
yeast employed for these experiments.
III. EFFECT OF THE ADDITION OF FRUCTOSE ON THE FERMENTATION OF GLUCOSE
OR MANNOSE IN PRESENCE OF A LARGE EXCESS OF PHOSPHATE.
When the maximum rate of fermentation of glucose or mannose by
yeast-juice in presence of phosphate is greatly lowered by the
addition of a large excess of phosphate, the addition of a relatively
small amount of fructose (as little as 2·5 per cent. of the weight of
the glucose) causes rapid fermentation to occur. This induced activity
is not due solely to the selective fermentation of the added fructose,
since the amount of gas evolved may be greatly in excess of that
obtainable from the quantity added.
Another way of expressing the same thing is to say that the optimum
concentration of phosphate (p. 71) is greatly raised when 2·5 per
cent. of fructose is added to glucose, and that consequently the
rate of fermentation rises. The effect is extremely striking, since
a mixture of glucose and yeast-juice fermenting in the presence of a
large excess of phosphate at the rate of less than 1 c.c. of carbon
dioxide in five minutes may be made to ferment at six to eight times
this rate by the addition of only 0·05 gram of fructose (2·5 per cent.
of the glucose present), and to continue until the total gas evolved
is at least five to six times as great as that obtainable from the
added fructose, the concentration of the phosphate being the whole
time at such a height as would limit the fermentation of glucose alone
to its original value.
The effect is not produced when the concentration of the phosphate is
so high that the rate of fermentation of fructose is itself greatly
lowered.
This remarkable inductive effect is specific to fructose and is not
produced when glucose is added to mannose or fructose, or by mannose
when added to glucose or fructose, under the proper conditions of
concentration of phosphate in each case.
This interesting property of fructose, taken in connection with the
[p075] facts that this sugar in presence of phosphate is much more
rapidly fermented than glucose or mannose, and that the optimum
concentration of phosphate for fructose is much higher than for
glucose or mannose, appears to indicate that fructose when added to
yeast-juice does not merely act as a substance to be fermented, but in
addition, bears some specific relation to the fermenting complex.
All the phenomena observed are, indeed, consistent with the
supposition that fructose actually forms a permanent part of the
fermenting complex, and that, when the concentration of this sugar in
the yeast-juice is increased, a greater quantity of the complex is
formed. As the result of this increase in the concentration of the
active catalytic agent, the yeast-juice would be capable of bringing
about the reaction with sugar in presence of phosphate at a higher
rate, and at the same time the optimum concentration of phosphate
would become greater, exactly as is observed. The question whether,
as suggested above, fructose actually forms part of the fermenting
complex, and the further questions, whether, if so, it is an essential
constituent, or whether it can be replaced by glucose or mannose with
formation of a less active complex, remain at present undecided, and
cannot profitably be more fully discussed until further information is
available.
It must, moreover, be remembered that different samples of yeast-juice
vary to a considerable extent in their relative behaviour to glucose
and fructose, so that the phenomena under discussion may be expected
to vary with the nature and past history of the yeast employed.
IV. EFFECT OF ARSENATES ON THE FERMENTATION OF SUGARS BY YEAST-JUICE
AND ZYMIN.
The close analogy which exists between the chemical functions of
phosphorus and arsenic lends some interest to the examination of the
action of sodium arsenate upon a mixture of yeast-juice and sugar, and
experiments reveal the fact that arsenates produce a very considerable
acceleration in the rate of fermentation of such a mixture [Harden
and Young, 1906, 3; 1911, 1]. The phenomena observed, however, differ
markedly from those which accompany the action of phosphate.
The acceleration produced is of the same order of magnitude as that
obtained with phosphate, but it is maintained without alteration
for a considerable period, so that there is no equivalence between
the amount of arsenate added and the extra amount of fermentation
effected. Further, no organic arsenic compound corresponding in
composition with the hexosephosphates appears to be formed.
Increase of concentration of arsenate produces a rapid inhibition of
[p076] fermentation, probably due to some secondary effect on the
fermenting complex, possibly to be interpreted as the formation of
compounds incapable of combining with sugar and hence unable to carry
on the process of fermentation. An optimum concentration of arsenate
therefore exists just as of phosphate, at which the maximum rate is
observed, and this optimum concentration and the corresponding rate
vary with different samples of juice and are less for glucose than
for fructose. The rate of fermentation by zymin is relatively less
increased than that by yeast-juice.
Owing to the fact that the rate is permanently maintained the addition
of a suitable amount of arsenate increases the total fermentation
produced to a much greater extent than phosphate.
The nature of these effects may be gathered from the result of a few
typical experiments. In one case the rate of fermentation of glucose
by yeast-juice was raised by the presence of 0·03 molar arsenate from
2 to 23 c.c. per five minutes, and the total evolved in ninety-five
minutes from 51 to 459 c.c. The accelerating effect on 20 c.c. of
juice, of as little as 0·005 c.c. of 0·3 molar arsenate, containing
0·11 mgrm. of arsenic, can be distinctly observed, but the maximum
effect is usually produced by about 1 to 3 c.c., the concentration
being therefore 0·015 to 0·045 molar. Greater concentrations than
this produce a less degree of acceleration accompanied by a shorter
duration of fermentation, as shown by the following numbers which
refer to 20 c.c. of yeast-juice in a total volume of 40 c.c.
containing 10 per cent. of glucose:--
─────────────┬──────────────────┬────────────────
C.cs. of │ Molar │ Maximum
0·3 Molar │ Concentration of │ Rate of
Arsenate │ Arsenate. │ Fermentation.
in 40 c.c. │ │
─────────────┴──────────────────┴────────────────
0 0 3·5
0·005 0·0000375 6·3
0·01 0·000075 8
0·02 0·00015 14·2
0·04 0·0003 19·9
0·1 0·00075 29·7
0·2 0·0015 35
─────────────────────────────────────────────────
0·5 0·00375 34·9
1·0 0·0075 29·5
2·0 0·015 23·2
5·0 0·0375 14·5
10·0 0·075 8·7
15·0 0·1125 5·3
20 0·15 3·2
─────────────────────────────────────────────────
The contrast between glucose and fructose in their relations to [p077]
arsenate are well exhibited in the following table, in which the
rates of fermentation produced by arsenate in presence of excess of
glucose and fructose respectively are given:--
────────────────────────────────────────────────────
Rate.
Concentration of Arsenate. ───────────────────────
Glucose. Fructose.
────────────────────────────────────────────────────
0·0075 molar 12·1 26·6
0·0225 (opt. for glucose) 13·4 --
0·0525 (opt. for fructose) -- 45·8
0·1125 5·1 39
────────────────────────────────────────────────────
Here the optimum concentration for fructose is more than twice that
for glucose, whilst the maximum rate of fermentation obtainable with
fructose is between three and four times the maximum given by glucose.
V. EFFECT OF ARSENITES ON THE FERMENTATION PRODUCED BY YEAST-JUICE.
Effects somewhat similar to those produced by arsenates were observed
by Buchner [Buchner and Rapp, 1897; 1898, 1, 2, 3; 1899, 2; Buchner,
E. and H., and Hahn, 1903, pp. 184-205] when potassium arsenite was
added to yeast-juice. This substance, the action of which on yeast had
been adduced by Schwann as a proof of the vegetable nature of this
organism, was employed by Buchner on account of its poisonous effect
on vegetable cells as an antiseptic and as a means of testing for the
protoplasmic nature of the agent present in yeast-juice. Its effect on
the fermentation was, however, found to be irregular, and at the same
time it did not act as an efficient antiseptic in the concentrations
which could be employed. Even 2 per cent. of arsenious oxide, added as
the potassium salt, had in many cases a decided effect in diminishing
the total fermentation obtained with cane sugar, and this effect
increased with the concentration. A number of irregularities were also
observed which cannot here be discussed. It was further found that in
some cases 2 per cent. of arsenious oxide inhibited the fermentation
of glucose but not of saccharose, or of a mixture of glucose and
fructose, whilst its effect on fructose alone was of an intermediate
character.
The important observation was also made by Buchner that the
addition of a suitable quantity of arsenite as a rule caused a
greatly increased fermentation during the first sixteen hours even
in experiments in which the total fermentation was diminished. By
examining the effect of arsenite on fermentation in a similar manner
to that of arsenate, Harden and Young [1911, 1] have found that a
close analogy exists [Pg 078] between the effects and modes of
action of these substances, but that arsenite produces a much smaller
acceleration than arsenate. An optimum concentration of arsenite
exists, just as in the case of arsenate, which produces a maximum
rate of fermentation. Further increase in concentration leads to
inhibition, and in no case is there any indication of the production
of an exactly equivalent amount of fermentation as in the case of
phosphate. In various experiments with dialysed, evaporated, and
diluted yeast-juice in which 2 per cent. of arsenious oxide was found
by Buchner to inhibit fermentation, it is probable that, owing to the
small amount of fermenting complex left, this amount of arsenious
oxide was considerably in excess of the optimum concentration,
although Buchner ascribes the effect to the removal of some of the
protective colloids of the juice, owing to the prolonged treatment to
which it had been subjected.
The extent of the action of arsenite appears from the following
results. In one case a rate of 1·7 c.c. was increased to 7 c.c. by
0·06 molar arsenite. In another experiment it was found that th
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