# Popular Science Monthly/Volume 59/September 1901/The Soluble Ferments or Enzymes

 THE SOLUBLE FERMENTS OR ENZYMES.[1]
By Professor EDWIN O. JORDAN,

UNIVERSITY OF CHICAGO.

IT has been said somewhat sententiously that the advance of science consists simply in a change of problems; we achieve progress when we substitute for one problem another at once more delicate and more precise. The recent history of the theory of alcoholic fermentation furnishes a conspicuous illustration of this aphorism. Liebig's ingenious conception concerning the breaking-down of the sugar molecule by the decomposing albuminous compounds in dead and dying yeast cells—his notion being that the sugar is toppled over, so to speak, by the mechanical shock of other falling molecules—was forced to yield to Pasteur's apparently clear demonstration of the part played under natural conditions by the living yeast cell. More recently the conception of the living cell as the essential feature of the process has been dethroned in its turn, and alcoholic fermentation is now shown to rest on the action of an 'unorganized,' 'lifeless,' or 'soluble' ferment or enzyme, secreted by the yeast plant. Further, the action of this enzyme and of other and more familiar 'unorganized' ferments has been brought into line with some of the most characteristic activities of the living cell, and many general life phenomena have been shown to be in reality phenomena of fermentation.

The consequent focusing of attention upon the enzymes, their nature, mode of action and chemical relations, has already been prolific in results of great biological interest. The science of experimental medicine, in particular, is discovering that many of the problems relating to immunity, to toxins, antitoxins and agglutinating substances are closely connected with the problems of fermentation and the enzymes.

So far as is known, the peculiar substances called enzymes are produced only by the living cells of animals and plants, although there are certain inorganic substances that so closely reproduce the essential qualities of enzyme action that they might almost be termed 'inorganic enzymes.' The enzymes obtained from the animal or plant cell have never been isolated in a pure condition, and hence no definite knowledge of their chemical composition and constitution has yet been secured. In general, enzymes are precipitated by alcohol from the watery extract of plant or animal tissue, but such a precipitate contains the enzyme inextricably mingled with other chemical compounds, and all attempts to separate out a pure enzyme have thus far signally failed. The whole field of enzyme study is therefore at present beset with the same sort of difficulties that the science of bacteriology encountered before the days of 'pure cultures,' and it is doubtless true that effects that are at the present time ascribed to individual enzymes are in reality caused by mixtures of distinct varieties.

The different kinds of enzymes are at present chiefly distinguished through the differences in the changes that they produce in other substances. The enzymes that act upon starchy substances, for instance, may be conveniently grouped together; those that disintegrate albuminous compounds may be similarly treated, and so on.

Enzymes converting starchy substances into sugar. The longest known and perhaps most thoroughly studied enzyme is a representative of the group that transforms insoluble carbohydrate substances into soluble ones. Amylase, or diastase, is the well-known enzyme that accomplishes the conversion of the starch of the barley-grain into sugar in the process of malting. The action of the enzyme in this process appears to be quite elaborate since the complex starch molecule passes through several stages during its conversion into sugar, the hardly less complex substances known as 'erythrodextrins' and 'achroödextrins' being formed on the way. The theory has been advanced that the starch molecule breaks down by the taking on of successive molecules of water and by subsequent decompositions, sugar (maltose) being formed at each splitting, together with a dextrin of lower molecular weight. Duclaux, however, maintains the existence of two enzymes in malt, one, a liquefying or decoagulating enzyme to which he would restrict the name amylase, and which converts the insoluble starch into the soluble dextrins, and a second (dextrinase), which has a saccharifying power and converts the dextrins into sugar. Other enzymes that may be placed in the same group with amylase are inulase and cytase. Inulase converts into fruit sugar a reserve food-substance found in many plants and known as inulin. Inulin is allied to starch in its chemical composition, and probably breaks down by successive stages under the action of inulase just as starch does under the influence of amylase. Another form of reserve food-substance stored up by many plants is the familiar substance comprising the cell-wall of most plants and known as cellulose. This substance, like inulin and starch, can be changed into a more directly utilizable substance by the action of cytase, an enzyme found in many plant cells. The products of the activity of cytase have as yet been imperfectly studied, and it is possible that several different enzymes, corresponding to the different kinds of cellulose, are at present included under this name. Cytase may be useful in various ways to the organisms secreting it. Some parasitic fungi are able to attack and penetrate the cell-walls of the host-plant by virtue of the dissolving power of the cytase secreted in the tip of the growing hyphæ.

Enzymes acting upon sugars. Another group of enzymes is concerned with the conversion of the higher sugars or polysaccharides into the lower. The classic instance is the so-called 'inversion' of cane-sugar or sucrose into equal parts of grape-sugar and fruit-sugar, according to the equation:

 C12H22O11 ${\displaystyle +}$ H2O ${\displaystyle =}$ C6H12O6 ${\displaystyle +}$ C6H12O6 sucrose or dextrose or levulose or cane-sugar. grape-sugar. fruit-sugar.

A solution of cane-sugar turns a ray of polarized light to the right, but the mixture of dextrose and levulose, owing to the superior lævo-rotatory power of levulose, turns the ray to the left, whence the term 'inversion' as applied to this process. The enzyme that is able to produce the inversion of cane-sugar was discovered by Berthelot in 1860. Sucrase, or invertase, is found in many yeasts and other fungi, in pollen-grains, in the beet-root, and to a slight extent in some animal secretions. Cane-sugar is not directly assimilable by the animal body, and if injected into a vein is excreted almost unchanged. The inversion which occurs in the intestine when the cane-sugar is taken into the body by way of the alimentary tract seems to be a necessary preliminary to the utilization of this sugar as a food substance.

The same is true to a certain extent of maltose, which is a sugar of the same percentage composition as sucrose, but with a different arrangement of the atoms within the molecule. Maltose is split up by the action of the enzyme maltase into two molecules of dextrose. Maltose, like sucrose, is only with difficulty assimilable, and its conversion into dextrose constitutes an important phase of carbohydrate nutrition. Maltase, like sucrase, is a widely distributed enzyme and is found in many animal and plant tissues.

The action of maltase upon maltose presents a significant example of what chemists call a 'balanced' action. When a certain proportion of maltose has been converted into dextrose, the action of the maltase ceases, and if now an excess of dextrose be added the action of the enz}Tne is reversed, and a certain proportion of the dextrose is converted back into maltose until a new equilibrium be reached. This reversibility of action has been thought to indicate that the action of maltase falls in line with other chemical reactions and is not essentially different from that evinced by many well-studied inorganic substances.

Together with sucrase and maltase may be grouped other enzymes that split up the higher sugars into those of lower molecular weight, such as lactase which converts lactose or milk-sugar into dextrose and galactose, trehalase which splits up trehalose, a sugar obtained from Syrian manna, into two molecules of dextrose, and raffinase and melizitase which act upon certain of the higher polysaccharides.

Coagulating enzymes. The group of clotting or coagulating enzymes includes two comparatively well-known enzymes, rennet and plasmase or fibrin ferment. The use of rennet in setting curd for cheese and in preparing the delicate dessert known as junket is generally familiar. The source of most of the commercial rennet is the extract of the mucous membrane of the stomach of the calf; this enzyme is found also in many other young mammals during the period of lactation. Rennet has been obtained likewise from several vegetable sources; parts of the plant Galium are used in the country districts of England to aid in the formation of curd in cheese-making, and the peasants of the Italian Alps use the leaves of the butterwort (Pinguicula) for a similar purpose. The curdling or precipitation of the casein by rennet is singularly dependent upon the presence of salts of lime. A very minute quantity of rennet in the presence of calcium salts will curdle a prodigious quantity of casein. It is in fact uncertain whether rennet can act at all in the entire absence of calcium. In the presence of calcium the potency of rennet ranks higher than that of any other enzyme yet studied, one part of rennet being able to coagulate more than 250,000 times its own weight of casein.

The phenomenon of the clotting of blood is dependent upon a variety of factors as yet imperfectly understood. That the fibrin or solid portion of the clot is separated out from the blood plasma by the action of an enzyme is, however, solidly established. The character and mode of action of this enzyme—termed plasmase, or fibrin ferment—are still quite obscure, although the fact that in mammalian blood the enzyme originates from the leucocytes, or white blood corpuscles, seems to be generally admitted. In birds the enzyme exists in the cells of the tissues and not in the blood corpuscles. The blood of all vertebrates with nucleated red corpuscles presents a marked resistance to spontaneous coagulation; clotting, on the contrary, is almost immediate among the mammals, which possess enucleated red corpuscles. As is the case with rennet, calcium salts favor coagulation; their presence seems, however, not to be necessary.

Other clotting phenomena have been shown to be due to enzyme action. The formation of jelly from the juices of various fruits and berries is due to the gelatinizing or coagulating effect of an enzyme, pectase, which acts upon pectose, a carbohydrate allied to cellulose and occurring in many fruits and vegetables.

The various phenomena of clotting and gelatinizing belong in the debatable border land between physics and chemistry. Duclaux has given in his treatise a clear exposition of his reasons for believing that the changes involved in the various processes of coagulation are due to a disturbance of the physical equilibrium of the substances in solution rather than to any chemical reaction.

Enzymes acting upon proteid substances. One of the most important groups of enzymes is that of the proteolytic enzymes, characterized by their property of breaking down albuminous or proteid compounds into simpler ones. Owing to the prominent rôle they play in the human body in connection with digestive processes they have been subjected to exhaustive study. Two chief groups are recognized, the peptic enzymes, of which pepsin, the enzyme of the gastric juice, is the type, and the tryptic enzymes, the best known of which is the trypsin secreted by the mammalian pancreas. The peptic enzymes are almost unique among known enzymes, inasmuch as they can act only in an acid medium; they are further characterized by their inability to carry the decomposition of proteid substances beyond the 'peptone' stage. The tryptic enzymes, on the other hand, are most potent in a slightly alkaline medium, and they are able to push proteid decomposition to a point beyond that reached by the pentic enzymes. Two of the most characteristic end-products of tryptic digestion are the substances leucin and tryosin which, like urea, are not assimilable by the tissues and are eliminated from the body. Several tryptic enzymes of vegetable origin are known, among which bromelin from the juice of the pineapple and papain from the fruit of the papaw-tree have been thoroughly studied. It is probable that the digestive enzyme secreted by insectivorous plants belongs to the tryptic class.

An interesting tryptic enzyme has been recently discovered in fresh milk by Professors Babcock and Russell. This enzyme, called galactase by its discoverers, acts upon the proteids in milk and plays a most important part in the manufacture of cheese; it is probably responsible for many of the phenomena of cheese-ripening that were formerly ascribed to bacteria.

Owing to the great chemical complexity of proteid substances and to the fact that little is known about their chemical constitution, the study of proteolytic enzymes is hampered by difficulties of an especially serious nature. Since neither the initial composition of the proteid compound nor the substances to which it gives rise on decomposition can be accurately determined, the distinction of different kinds of proteolytic enzymes is attended with greater difficulty than is experienced in the case of enzymes that attack chemical compounds so comparatively well understood as the sugars.

The alcoholic enzyme. The enzyme that converts sugar into alcohol and carbon dioxide has been only recently discovered, although it has been diligently sought for since the time of Pasteur. The discovery of Buchner in 1896 that, by applying great pressure to a mass of yeast cells, an enzyme, which he named zymase, could be extracted gave in fact a new impulse to all enzyme study. Zymase appears to dislocate the sugar molecule according to the classic formula:

 C6H12O6 ${\displaystyle =}$ 2C2H5OH ${\displaystyle +}$ 2CO2 dextrose alcohol carbon dioxide 180 gr. 92 gr. 88 gr.

The explanation of the prolonged failure of investigators to discover zymase lies in the fact that this enzyme is closely associated with the substance of the living yeast cell and does not diffuse out into the surrounding medium as does another common yeast enzyme already mentioned under the name of invert-ferment or sucrase. In solution, zymase quickly loses its strength, probably partly because of oxidation, partly because of the destructive action of the tryptic enzymes of yeast. Zymase is able to convert a number of different sugars into alcohol and carbon dioxide: maltose and sucrose are readily fermentable, galactose much less readily and lactose not at all. Glycogen can be slowly fermented by zymase, but is not fermented by the living yeast cell because it can not pass through the cell-membrane into the cell and zymase can not pass out. The brilliant researches of Emil Fischer upon the relation of the configuration of the sugar molecule to its fermentability have demonstrated how delicate is the relation obtaining between the structure of the sugar molecule and the enzyme that attacks it. A slight rearrangement in the position of the atoms within the molecule, the actual number of atoms remaining all the while the same, is sufficient to determine whether a sugar can be fermented or not. Only in those cases where the geometrical build of the enzyme conforms to that of the sugar molecule can fermentation occur. To use Fischer's metaphor, the enzyme must fit the substance it attacks as closely as the right key fits the wards of the lock that it opens.

Other enzymes. A few other important enzymes can be but briefly mentioned, since the limits of this review do not permit of a fuller consideration. A group of enzymes of which emulsin is the type may be classed as the glucoside-splitting enzymes. These ferments are able to split up glucosides—which may be described as compounds of glucose (or some other sugar) with an alcohol, ether, aldehyde, or similar body—into glucose and the aldehyde or other associated compound. Emulsin is found in many plant tissues, but it is doubtful if it occurs in any animal body. The physiological role of emulsin is not wholly understood; it is possible that the glucose formed by the enzyme action is useful in the nutrition of the plant, or it may be true that the toxic or bitter principle also split off has a protective value and prevents injury to the plant by animals. Besides emulsin, a few other glucoside-splitting enzymes are known.

Lipase, a fat-splitting enzyme; laccase, an oxidizing enzyme concerned in the production of the famous black varnish used in lacquer work; and urease, an enzyme that converts urea into ammonium carbonate, are among the other better-known enzymes.

There is reason to believe also that the various anti-microbic substances found in the bodies of some artificially immunized and some naturally immune animals are to be regarded as enzymes, as is likewise the substance that is found in the blood of typhoid patients and that has a 'clumping' or 'agglutinating' action upon the typhoid bacilli. Eesearch in this direction has not, however, proceeded far enough to enable us to offer anything more than a conjecture as to the real character of the 'agglutinines' and lysines.'

The precise mode of action of enzymes has been the theme of much speculation. Perhaps the simplest and most natural view of some cases is to suppose that the enzyme combines first, for example, with a molecule of water, and then attaches itself to the body upon which it acts. This new compound, meeting with another molecule of the same substance, is then decomposed into the body which the enzyme produces and the enzyme itself. The enzyme thus acts as a simple intermediary, bringing the molecule of water or oxygen in closer contact with the fermentable substance. This view has certain arguments in its favor, as for instance the fact that the enzyme does not exhaust itself in the course of the changes that it produces. If it is unceasingly decomposed and reconstituted the reason for this is clear.

Such an explanation, however, is hardly valid for the action of zymase upon sugar and for the reversible action of maltase. Now there are certain facts regarding the action of mineral acids upon sugars and proteids, of various salts upon the phenomena of clotting and oxidation and of other changes brought about by inorganic substances which render it difficult to set enzyme action apart as a' thing by itself. The action of an enzyme is essentially 'catalytic,' that is, it is able to exert an influence wholly out of proportion to its quantity, and itself remain unaltered at the end of the process. It has been pointed out that the influence of an enzyme or indeed any catalytic agent is simply to retard or accelerate changes which ordinarily take place more slowly or more rapidly. In other words, an enzyme simply influences the rate of change, not the final condition of the substance upon which it acts. The nature of the change, the final state of chemical equilibrium, is determined by the chemical forces within the substance itself, the speed at which the change occurs is determined by the enzyme.

1. The Soluble Ferments and Fermentation. J. Reynolds Green. Cambridge University Press, 1899.
Masson, Paris, 1899.
Traité de microbiologie, II., diastases, toxines et venins. E. Duclaux.
Les enzymes et leurs applications. J. Effront. Carre et Naud, Paris, 1899.