Yeast emulsin

The use of the enzyme system then known as invertin, which was extracted from beer yeast with water and precipitated from the aqueous solution, was available to Fischer when he began his classical studies of the enzymic hydrolysis of glucosides reported in 1894. The stage was also set by another enzyme known as emulsin, which Fischer purchased from E. Merck, Darmstadt, and which was known to hydrolyze several natural aromatic glucosides such as salicin, coniferin, arbutin, and the synthetic phenyl glucoside. These aryl glucosides were already known to not be cleaved by invertin. 

At this point Fischer concluded that the enzymes, in terms of the configurations of the substrates, are as fastidious as yeast and other organisms. He then returned to the above-mentioned hypothesis that he and Thierfelder had proposed (30) and concluded (32) that the protein substances known as invertin and emulsin, like the substrates whose hydrolyses they effected, were asymmetrically formed molecules. On the basis of this consideration, he came to the momentous lock and key concept for enzyme activity and commented as follows ... 

The controversy between the views of Liebig, Schwann and Pasteur over the nature of ferments was reviewed in Chapter 2. In part, this arose from confusion between organized ferments, the living cells themselves, as exemplified by yeast, and unorganized ferments such as emulsin or pepsin which could work independently. The term, enzyme, was coined by Kuhne in 1878 to describe the agent... which occurs [in yeast] to exert this or that activity which is considered to be... fermentation. The study of enzymes has formed an important bridgehead for physicochemical exploration of biological systems from the time of Berzelius. 

From about 1894 onwards Emil Fischer investigated in a series of experiments the action of different enzymes using several glycosides and ohgosaccharides the results revealed specificity as one of the key characteristics of ertzymes. In 1894 he compared Invertin and Emulsin. He extracted Invertin from yeast, a usual procedure, and showed... 

In 1896, Fischer developed the phenylhydrazine test" for the detection of hydrolytic scission of disaccharides, especially by enzymes this depends on the fact that the phenylosazones of disaccharides are soluble in hot water, whereas those of the monosaccharides are not. Lactose is hydrolyzed by emulsin (1894) and by lactase it is not fermentable by yeast, and is unaffected by invertase (1894). An extract of the small intestine of horses and cattle, especially from young animals, hydrolyzes lactose (1896). The action of enzymes on lactose allowed it to be classified, along with cellobiose and maltose, with the normal (and not the y-type of) methyl glucoside (1914). In the discussion of maltose, the relationship of lactose to the /9-series will be mentioned later. 

Sucrose is fermented by nearly all yeasts (1894,1898). Inversion precedes fermentation (1895). Of various animal secretions tried, the only one effective in cleaving sucrose was that from the mucous membrane of the small intestines of several animals (1896). Sucrose was found to be unaffected by emulsin (in 1894). An approximately correct formula for sucrose had been published in 1883 its shortcomings were the incorrect ring size for the glucose residue and the uncertainty regarding configurations at the interlinked carbonyl groups. 

Cellobiose and its /3-glycosides were hydrolyzed to glucose by emulsin. Cellobiosone and hydrocellobial were also cleaved by this enzyme. Extracts of AspergiUus niger and Kefir (a Caucasian micro-organism that ferments milk) hydrolyzed cellobiose yeast extract was inactive. 

The use of ester-cleaving enzymes is probably going to be one of the most useful biological-chemical methods in the synthetic laboratory. No example of this type of reaction has hitherto been published in the Organic Syntheses series of procedures. So far, the only biological-chemical Organic Synfheses-procedures are two yeast reductions,4 5 one oxidation with horse-liver-alcohol-dehydrogenase,6 and a disaccharide synthesis catalyzed by emulsin.7 The procedure described here is... 

In further work, Strepkov obtained99 labiose in crystalline form, of m.p. 126-8°, [c ]d + 136.7° (in water, air-dry basis). Labiose is completely hydrolyzed by invertase at 47-8° in 20 hours, and by 1% hydrochloric acid at 68-70° in 10 to 12 minutes, to give one molecular proportion of D-galactose and two molecular proportions of D-fructose. Labiose is not hydrolyzed by almond emulsin, and it is fermented but little by pressed yeast. Strepkov concluded that labiose is related to the alpha series ( ) and that, probably, the D-galactose unit is in the center of the molecule. 

The hydrolysis of yeast nucleic acid by sweet almond emulsin has been found26 to give high yields of guanosine and adenosine and forms a practical basis for the preparation of D-ribose.26... 

A solution of 1.65 kg. of crystallized glucose or 1.5 kg. of anhydrous glucose (8.3 moles) is prepared by heating the solid on a steam bath with 1.35 1. of distilled water. The solution is cooled and placed in a 2.5-1. glass-stoppered bottle. Fifteen grams of emulsin (Note 1) is added, then 20 cc. of toluene, and the flask is closed and allowed to stand at room temperature, with occasional shaking, for five weeks. The solution is then boiled, diluted with 8.5 1. of water, and filtered. To the filtrate is added 56 g. of baker s yeast in 650 cc. of water, and the temperature of the mixture is maintained at 28-32° for twelve to fourteen days... 

Pigman has prepared crystalline u-altrose from neolactose by cleavage of the disaccharide with sweet almond emulsin, followed by removal of the D-galactose by fermentation with yeast. This procedure avoids the transformation of any n-altrose to u-altrosan, the non-reducing anhydride which is formed to the extent of 57% in the hydrolysis of neolactose by acids. 

In these various ways, the anomeric forms of methyl, ethyl and benzyl thioglucosides and n-propyl -thioglucoside were all prepared in pure crystalline form. They are typical glucosides, non-reducing, stable toward alkalies, hydrolyzed by acid, but not by emulsin, myrosin or yeast maltase. 

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