Dec., 1921
T H E JOI1RiV.4 L OF INDUSTRIAL A N D ENGINEERING CHEMISTRY
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The Properties and Action of Enzymes in Relation to Leather Manufacture' By Joseph Turney Wood TVENBV BP-OIBBBS, LTD.,N O ~ ~ C N O ENDLAND HAM,
TNESUAKING PROCESS Enzymes occur in and influence almost all the operations of ta.nning from the moment that the skin is flayed until i t is dried out in the form of leather. The fresh skin contains a number of enzymes about which very little is known and other enzymes are soon formed by bacterin. We shnll uot consider these in any detail, as we may assume that the raw skin has hecn properly preserved after flaying and before coining into the hands of the tanner, hut in the snaking process we meet with enzymes secret,ed by a variety of species of bacteria. Most of these bacteria liquefy gelatin by meanz of the proteolyt.ic enzymes u.hich they secrete, and consequently they attack the skin. So far as the miter knows, the bacteria of the soaks have not been specially studied except by Andreascl1,2 who isolated a nuFber of specics of bacteria from the soaks. He identified the followina:
THELIMELianoas
In a lime liquor t.hrougli which skins have passed, there are enzymes present. The writer is inclined to believe that sonic of these are from the skin it.self, at least when fresh market skins are worked, as, for example, the tissue enzymes, but nothing definite is known of these. Bacteria, however, develop in tho limes. B. prodiyioaus and ~2licrococcusj'lauus liquefaciens, both of mhich are known to produce prot,colytic enzymes, have been identified. These enzymes decompose the dissolved skin substance int.o gelatones (gelatin-peptones) and eventually into amino acids, caproic acid, and ammonia. I t is very probable that the cnzymes in the limes act in the same ivay a3 the enzymes in the sweat.ingprocess of unbairing, but in t,he limes the process st,ops short of putrefaction, whereas in t.he sweating stove it may go on until the skin is run on the grain. TIE I~ATING I'riocms White bacillus (Mschek) B. fluorerrens liovefaricnr (F1"g~eJ The investigation of the action of the bates or pners has r r o t i u r zu1goris a. mcgaurium (de Beryl B~ sublilis Prnirur mirobilis Tiroved of great, interest and has resnlted in the use of scien8.mrrenlcriGus *.Ulgo1us B bu*).rirus (Nueppel tificmethodsdepending White slreptorurcirr (Maschrk) R. mcrcnlrrirur f u c u r on the use of commerXVorm shaped strentococcus B. myioidrr (FlQpe) cia1 enzymes in lentlier a.lipuidur (Frankland) (iMarchek) Gray CDCCUQ (Marhek) a.garoformans (Einenbcrg) manufacture. Theobject. of hating or puering is to render the skins, and the rcsulting leather, soft and supple. Skins which have undergone the liming ptocess must be thoroughly freed from linre hefore going into ihr tan liquors, and for light and soft leather they must he reduced or "brought do-iMI" so that, the eiast,icity or resilience of the s k i n fibers is got. rid of, and the skin when tanned can be stret,clied without F ~ C . I-TYPICAL PLATS C U L ~ R I : O N GBL- application of formalin springing back. In the case of the old manure hates Eor light A T C X os S o ~ a USBD90% SOPT&NING DRY vapor before many of leathers, this is done by pa?sing t,he skin t,Iirougli a bat! or SXeeP S K I N S t'le species to puer composed of an infusion of dogs' dung in uvitcr a t a develop; othernisc the whole plate w-odd have heen liquefied. t,cmpcrnture of 3 5 to ~ 4 n ~ t,hc desired result is obtaiued, I n view of the fact that the enzymes in the soaks are nearly This condition is knolvn to n.orkman by the feel f, the all derived from ba.ct.eria, it, is evident that the sonking skin, ~ 1 process , ~ lias heen in use from the uery shonld he carricd out under antiseptic conditions as far as times, but until comparatively recently no explanation E, t ~ l e possible. act,ion of the dung bates was satisfactory. Rideal and Orchard3 examined the action of B. $?~~wescins T,lirty years ago, u,hen tho author the in. k&ciens on fielatin f.0 ~ h i c l iwas added 10 per cent of T,cstigat.ionof the action of the dung bate in manufRcturo Pasteur's solution to serve as nutrient medium. The gelat,in of light, leatllers, to the eonc,usion tliat a peat part was completely liquefied in 3.5 days. It WRS shown that t.he of tire bating effpct was due to action ,E proteolyt,ic liquefaction of gdatin was doe to an enzyme, or enzvmes, 'se- enzymes on the skin in conjunction with ammonium salts creted by the bacteria. The liquefied gclntin was alkaline and in the hat,c. ~h~ lather not only Tvith the liadaslight put,rcfact,iveodor,but eontninedno hydrogen sul- lime remaining in ttre s,lrin, hnt as a,ct.ivators for fide. Snotahlc featurewas the small amount of ammoilia and the enzymes, N~~~~~~~~~ were made on skins volatile bases produced; only 0.2 g. of ammonia w 100 (:e. botl, in the presence of growiilg ilacteria anri of thr products were produced even after 16 days' incubstion. The l i w f a c - of the bacteria, precipit.atcd alcotlol, a.nt~it ,vas found that tion of gelatin by pancrcatic trypsin takes place ~ i t , fmidi l suit.able ammolljllm sal+,~ \,.ere present the bating action greater rapidity; 0.2 mg.of the enzyme preparation will liq- in both cases was nefy 5 cc. of 4 per ccnt Relatin in 30 min. a t SO". There is little doubt that evcry variety of enzyme, hydrolyeing, oxidizing, ammoniacal, etc., is produecd by bacteria. The 1 Presented before the Division of h a t h e r Chemistry at the 62nd Meeting of the American Chemical Society, New Yo& N Y.,September 6 produrtion of enzymes by bacteria was ohseryed by Wortto I O , 1921. mnnii in 1882, and Ferini and ofhers have investigated the * Cerbcr, 1896-6. enzymes of the bacteria which liqnefy 4elat.in. Since the I Analyrl, October 1891.
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bacteria in the bate produced these enzymes, the first idea was to use them in practice in a scientific way. In conjunction with Prof. H. Becker, we manufactured a bacterial bate “Erodin,” which-the writer believes was the first attempt to apply the use of pure cultures of bacteria in the leather industry. The bacterium used was a variety of B. coli isolated from dog dung. Harden’ has shown that this bacterium produces a t least three enzymes. An investigation of the enzymes contained in dog dung showed that five different enzymes were present :2 1-A 2-4 3-A 4-An 5-A
peptic enzyme resembling stomach pepsin. tryptic enzyme, or enzymes, resembling pancreatic trypsin. rennin (coagulating enzyme) amylolytic enzyme. . lipase.
I n view of the fact that the bate solution is alkaline, it seemed pretty certain that trypsin must be the principal enzyme acting, and it is this enzyme which has been applied practically in the manufacture of artificial bates. It must be clearly understood that, although the enzymes and ammonium compounds are the chief bodies acting on the skin in the dung bate, they are not the only bodies taking part in the process. As the writer has shown, there are many other bodies in the dung which act in a way a t present not understood. Among these the fats, soaps, and phosphates are, in his opinion, important, Among other enzymes present in the dung, the lipases have a considerable effect in emulsifying and hydrolyzing the fats. Skins which have been overlimed are bated more effectively in a dung bate than in an artificial, enzyme bate, and this must be due to some constituents in the dung bate which are absent from the enzyme bate. It has further been shown that excessive liming so alters the chemical constitution of the hyaline membrane as to render it capable of being attacked by trypsin. Rosenthals states that elastin is digested in the bating process; and more recently Seymour-Jones has been able to show that the elastin is contained principally in the grain layer. Elastin is the characteristic constituent of the elastin fibers which remain after treating the skin with boiling water, caustic alkali, dilute hydrochloric acid, alcohol, and ether. It is of pale yellow color and is insoluble in any menstruum which does not act on it chemically. When boiled with strong hydrochloric acid and stannous chloride, leucine (30 to 40 per cent) and a small quantity of tyrosin (0.25 per cent) are found among the products of its decomposition, together with ammonia! glycine, and aminovalerianic acid, but not aspartic or glutamlc acid. This behavior distinguishes elastin from both proteids and gelatin, since the former yield aspartic and glutamic acids but no glycine, while gelatin never yields a trace of tyrosin. Elastin is digested by the body enzymes. It is allied to the fibroin of silk and spider web. Quite independently J. A. Wilson‘ has shown definitely that the elastin of the skin is removed by the tryptic enzymes of the bate. In the unhated skin the elastin fibers are fairly dense in the grain layer. As the bating proceeds, they gradually disappear, and a t the end of 24 hrs. the grain is quite free from elastin. In skin delimed with ammonium chloride only, the elastin was left apparently unaltered. Wilson concludes that the mechanism of bating consists of two distinct parts: (1) reducing the limed skin to a condition of minimum swelling, and (2) digesting the elastin fibers present in the outer layers of the skin. It may be added that Mr. SeymourJones considers the so-called hyaline layer to be really the Chcm. World, 1912,403. J . SOC.Chem. Ind , 81 (1912), 1105. 8 “Biochemical Studies of Skin,” J . A m . Leather Chem. Assoc., 11 (1%l6), 463. 4 THISJOURNAL, la (19201, 1087. 1
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deepest layer of the epithelium, and proposes to call it the grain membrane, as being more in harmony with the nomenclature of the leather industry. I n light leather manufacture it is what is called a “flywing” and yields practically no gelatin on boiling, whereas the split flesh is wholly transformed into gelatin. Although the elastin is completely removed from the skins bated by trypsin for a sufficiently long time, about 24 hrs., in practice the bating is not continued to this point, but is carried on for only about 2 to 6 hrs. The elastin is only partly removed in this time, but the skins make good leather. The writer believes that it is not necessary, or even desirable, for the whole of the elastin to be removed or dissolved in order to let the skin down, but that it is sufficient for the elastic fibers to be broken up or weakened, in order that the desired suppleness may be obtained. There still remain many unsolved problems in the bating process; these are being pursued with vigor, and the writer looks forward confidently to the time when we shall be able to bate any kind of skin by means of artificial bates suitable to the particular leather required.
DRENCHING In the manufacture of many kinds of light leathers, skins are drenched after bating. This process usually consists in placing the skins in a mixture of bran and water (5 to 10 g. per liter) a t a temperature of 30”to 35“. This ferments vigorously for 18 to 24 hrs., with evolution of a considerable quantity of gas and the formation of weak organic acids. In a drench taken in actual work, the gases had the following composition: Carbon dioxide Hydrogen sulfide Oxygen Hydrogen Nitrogen
Per cent 26.2 Trace 2.5 46.7 26.0
The acids produced per liter were: Formic Acetic Butyric Lactic
Gram 0.0306 0.2042 0.0134 0.7907
Of other bodies formed during drenching, the quantity is insignificant, trimethylamine being the chief. It has been shown that the starch of the bran is converted into glucoses and dextrin by the action of an amylolytic enzyme, cerealin, allied to diastase. This enzyme was discovered by Mege Mouries.1 It resembles the diastase of translocation described by Brown and Morris2 in their work on the germination of grass seeds. It transforms starch into dextrin and glucose, whereas ordinary malt diastase transforms starch into dextrin and maltose. The action of cerealin is much slower than that of diastase. Dr. W. H. Wilcox and the author found that in 12 hrs. at 40” about one-half the starch is transformed. With malt extract, the whole of the starch disappears in about 2 hrs. The glucoses are then fermented by bacteria (Bacillus furfuris) with the formation of the organic acids above mentioned. The principal acid produced is lactic; the acetic acid is produced directly from the glucoses without any preliminary alcoholic fermentation by yeasts. The mode of action of the drench on the skins is as follows: 1-Solution of the last traces of lime not removed by the bate and the subsequent swelling action of the organic acids on the skin fibers. The acids also dissolve a small amount of skin substance. 2-Distension and floating of the skins by the gases produced by the fermentation. 3-Mechanical absorption of dirt by the particles of bran or flour in the drench. 1 Comfit. r e n d , 87 (1853),351;88 (1S54), 505; 43 (1855), 1122; 48 (1859). 431; 60 (1860),467. J 1 J . Chem. SOC.,67 (1890). 458.
Dee., 1921
THE JOURNAL OF INDUSTRIAL A N D ENGINEERING CHEMISTRY
It mil1 thus be seen that the action of enzymes in the drench is an indirect one.
TANLIQUUR~ In the tan liquors changes go on which azo brought about by enzymes; some of these are secreted by molds and yeasts and others are present in the plants from which the tanning materials are extracted. Unfortunately very little is kuowu about these enzymes, but attention should be called to the work of Fernhach’iu France, who prepared an enzyme which he called tannase from the mold AspergiZZza niger, obtained from gall nu&. He cultivated the mold in Raulin’s liquid, using tannin in place of the sugar. The product vas maccrated in water, tbe maceration concentrated a t a low temperature in vacuo, the liquid precipitated by alcohol, and the precipitate treated exactly as in Lintner’s method for the preparation of amylase. The F a y powder thus obtained, when dissolved in water, acted rapidly on tannin a.t 50”,
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converting it into gallic acid. The solution of tannase fittered through a Chamberland fitter into a sterilized solution of tannin acted just as effectively, proving that the action was due to an enzyme and not to a fermentation through the agency of organized cells. H. Polt,evin’ prepared a solution of tannase by a process similar to that of Fernbach, but independently of him. Along with the gallic acid formed there is also a variable amount of glucose in the case of commercial tannins, but he considers these commercial tannins as mixtures. From a purified tannin he obtained gallic acid corresponding to 9S.5 per cent of t,he tannin. The fact that tannase also hydrolyzes phenyl and methyl salicylates lends support to Schiff’s formula for In nature tantannin, CsH2(OH)3.CO0.CsHz(0€~)~.~O~H. nin is often accompanied by gallic aeid. No douht the latter is formed by the action of tannase, which Polteviu found bo exist in sumac leaves, and which no doubt occurs in other substances dontaining tannin.
A Critical Study of Bating’,‘ By John Arthur Wilson and Guido Daub L ~ n o a ~ r o a r nOF s A F. GALLON& Soms Co.. MILWAURBB, Wiscolisr~i
In an earlier paper‘ it was shown that the mechanism of bating limed skins in the tannery by means of pancreatin and ammonium chloride consists of two distinct parts: reducing the skins to a condition of minimum swelling, and digesting the elastin fibers which are present only in the outer layers of the skin. The progress of this digestion was shown by means of photomicrographs of cross sections of the skin taken a t intervals during bating. The examination of more than 200 sections under the microscope failed to reveal any other function of the bating process, even when a great variety of stains was employed on the sections. It therefore would appear t,hat the primary function of bating is the removal of elastin, since the skins may bc brought to a condition of minimum swelling simply by regulating the hydrogen-ion concentration of the solution with which they are in contact and, furthermore, this condition is only a temporary one, while the removal of elastin is permanent. The work described in this paper was confined to a study of the removal of elastin from calfskin and its effects. PREPARATION OF SKINFUR EXAMINATION The most reliable method we know for studying the progress of bating is by means of the microscope. The per cent of elastin’removed by any treat,ment is estimated by ohserving under the microscope cross sections of the samples taken before and after the treatment. For this purpose the sections must be suitably prepared, and the following procedure has been found quite satisfactory. Strips of the sample, about 2 in. X 0.5 in., are dehydrated with alcohol and soaked successively in (1) a mixture of equal volumes of alcohol and xylene, (2) a mixture of 1 volume of melted phenol to 3 of xylene, (3) pure xylene, and (4) melted paraffin, after which they are imbedded in paraffin and sectioned at 4 0 ~ . The sections are then stained either hy means of Weigert’s stain as described by Gages or by the following method which the writers have found convenient: The section is washed on a slide with xylene, then with the alcohol-xylene mixture, and finally with pure alcohol. The whole slide is covered with alcohol saturated with Bismarck brown and rend., 56 (1848).1214: J . Soi. Chrm. I n d . , 80 (1901), 137. Presented belore the Section of Leat er Chemistry at the BZnd Meeting of the American Chemical Smiety, New ark, N. Y.,September 6 to 10, 1921. I Received A u ~ u s t3, 1921. 6 ~ a . 3 JO~=W., ia (1s2o). 1087 6 ‘“The Xicroscope:’ 1911, 355. I Compf. 9
a,
kept over night in an airtight jar containing alcohol. Next day the section is rinsed with alcohol, alcoholxylene, phenol-xylene, and finally with pure xylene. A drop of Canada balsam and a cover g h s are then put aver the section, which is now ready for examination and may be kept ingefinitely for record.
W c . 1-Cnoss SSCTZOXOB Ssrlr
OP Y O V N HEIPE. D CALB. M ~ C N ~ I C I T I26 ON DIAMBTERS ,
Fig. 1 is a photomicrograph of a cross section of the skin of a healthy young heifer calf. A strip from the butt was taken an hour after the death of the animal and fixed in Erlicki’s fluid. A section was stained first with Weigert’s stain and then with an alcoholic solution of picrc-red. It was photc1
Compt. rend.. 26 (1848), 1215; J . Soc Chrm I d . . 80 (1901). 137.
