February 1951
INDUSTRIAL AND ENGINEERING CHEMISTRY
The kind cooperation of Stanley Siege1 and Irene Corvin of the Armour Research Foundation in obtaining a portion of the x-ray data is acknowledged.
Literature Cited (1) Bum, C. W., Rubber Chem. and Technol., 15,704 (1942). (2) Hendricks, 8. B., Wildman, S. G., and Jone8, E. J., Arch. Biochum., 7 , 427 (1945); Rubber Chem. 4 Technol., 19, 501 (1946). (3) Lee, T. S., Kolthoff, I. M., and Mairs, M. A., Zbid., 21, 835 (1948).
403
(4) Mark, H., “Physical Chemistry of Hlgh Polymeria Substanoes,” p. 175, New York, Interscience Publishers, 1940. ( 5 ) Meyer, K. and High Polymers,,, p. 119,New York, Intersoienoe Publishers, 1942. (6) Prochazka, G.A., and Endemann, H., Pharm. J . (3rd series), 9, 1045, 1067 (1879). (7) Sands, G . D.,and Johnson, B. L.,Anal. Chern., 19, 261 (1947). ( 8 ) Saunders, R. A., and Smith, D. C., J . Applied Phys., 20, 953 (1949). (9) Schlesinger, W., and Leeper, H.M., Science, 112,51 (1960). (10) Wagner, R. H., IND.ENQ.CHEM.,ANAL.ED., 16,520 (1944). RECEIVED October 16, 1950.
Esterase in Latex of Hevea bradliQns8s I . N. S. De Haan-Homans IndonesZech InetZtuut Voor Rubberonderwek, Bogor, Indoneeia
L
b
*
which naturally contain good T h e investigations described here were for the greater ITERATURE does not emulsifiers, by vigorously part carried out during the Japanese occupation of Java. mention the presence shaking i t by hand for 3 Because of shortages of materials at that time, it was imof an esterase in the latex of minutes in a small glasspossible to perform the experiments as elaborately as was Hevea braeilim‘s, but the stoppered Erlenmeyer flask. desirablefor example, ethyl and methyl butyrate were presence of a lipase in the At first, 2 to 5 ml. of a buffer unobtainable. seeds of Hevea brasiliensis has solution were added (acetate No fat-splitting enzyme-that is, no l i p a s e w a s found been proved (1,8). Furtherbuffer, but generally phosin the latex of Hevea brasiliensis. I t was proved, howmore lipases have been found phate buffer), but later on ever, that this latex contains an esterase which decomposes in the latices of other plants this was discontinued as latex, low esters (acetates above all and ethyl acetate in particu-for e x a m p l e , E u p h o r yellow fraction, or serum lar) rapidly and to a high degree. biaceae, Ficus carica, Maclura buffer is satisfactory. One Experiments carried out at the Indonesian Rubber ReMoms, Carica papaya (7, 8). drop of toluene was added to search Institute also demonstrated that the pH of the Gerber (2) studied the acthe emulsion to prevent baclatex, about 7 on exudation from the tree, decreases raptivity of enzymes, including terial activity. The contents idly immediately after exudation, and within a short time a lipase, in the latex of Browof one Erlenmeyer h s k of reaches 6.2 to 6.5. The esterase may be involved in this sonetia papyrifera. in the each series were examined process. Further work will be necessary to find an answer various seasons. immediately after emulsificato this question. When investigating the tion; the remaining flasks spontaneous coagulation of were incubated a t 37” C. for latex, v a n Gils ( 8 ) and periods of 0.5 to 48 hours and then examined. These were rinsed Altman arrived at the supposition that a fat-splitting enzyme with 25 or 50 ml. of alcohol, if necessary, and titrated with 0.1 N might be involved in the process. van Gils (4) succeeded in sodium hydroxide. I n control experiments, in which Ricinus oil accelerating the spontaneous coagulation by adding a prepwas investigated with pancreas lipase, the oil was prepared acaration of pancreas lipase to the latex. This lipase preparacording to Willstlltter (9) and emulsified in a buffer solution by tion had only been very roughly purified, however, and it shaking it with albumin. Other emulsifiers, such as Exanol, probably contained a large portion of the other pancreas enzymes, lissolamine and triethanolamine oleate were found to inhibit the such as maltase, amylase, proteases (trypsin), and possibly leciactivity of the enzymes too much. thinase. The two last-mentioned enzymes in particular may also There are difficulties attached to the use of latex and yellow have influenced the stability of the latex. The lipase promoting fraction as the rubber coagulates during shaking or when alcohol the spontaneow coagulation might be present naturally in the is added. Before titration the coagulum was squeezed out and latex, or it might be formed by growth of bacteria. It was consesubsequently removed. quently highly desirable to determine first of all whether the Inasmuch as the serum obtained by freezing gave the best latex of Hevea brasiliensis naturally contained a fat-splitting results, this serum or the yellow fraction was generally used. enzyme. By way of control, a few mixtures to which no substrate had been Methods added were made with each experiment (to determine the selfacidification of the latex or the serum) as well as emulsions withInvestigations concerning the presence of the enzyme were out enzyme solution or with boiled serum (to trace the autolysis carried out with fresh latex and with the components obtained of the substrate). from it, such as serum obtained by coagulating latex with acid (coagulation serum) or by freezing the latex and thawing i t after Experiments with 6 c L ~ tFat,” e ~ Ricin- Oil, 5 to 7 days (serum o.b.f.), the yellow fraction, and coagulation Arachis Oil, and Monobntyrin serum and serum by freezing from the yellow fraction. The If the assumption of van Gils and Altman that a lipase is inp H was increased by the addition of a small quantity of sodium volved in the spontaneous coagulation of latex is correct, the subhydroxide solution or decreased by a little sulfuric mid. strate for the lipase must also be present in the latex. The accurately weighed quantity of substrate (0.26 to 1 gram) Owing to the lack of good agents for the extraction of fats dur-. was emulsihd in 2 to 5 ml. of the components mentioned above,
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
404
Vol. 43. No. 2
Investigation of Lower Esters Table I.
Decomposition of Esters by Esterase-Active Latex Components
(Unless stated otherwise the active latex components were derived from latices; a drop of toluene was added to each emulsion)
Remarlis Substrate, amyl acetate Serum 0.b.f.u Phosphate Serum 0.b.f. Phosphate Yellow fraction Phosphate Yellow fraction Phosphate Yellow fraction Acetate
6.6 6.6 6.6 6.6 5 7 5.7 5.7 5.7 5.7 5.8
3 2 3 2 3 2 3
6
Yellow fraction Phosphate
7.3 7.3
2 3
18.2 23.8
7
Serum 0.b.f. yellow fraction Boiled serum 0.b.f. yellow fraction Serum 0.b.f. Boiled serum 0.b.f.
7.6
2
23.0 33.8
7.6 8.5
3 2
13 3
8.5
3
1
2 3 4 5
8
9 10
11 12 13
14
Substrate, ethyl acetate Serum 0.b.f. 8.5 Boiled serum 0.b.f. 8.5 Serum o.b.f. yellow fraction 8.8
Serum 0.b.f. yellow fraction 15 Boiled serum 0.b.f. yellow fraction Substrate, ethyl butyrate 16 Serum 0.b.f. 17 Serum 0.b.f.
10.0
14 8
2
16.1
24 0
11.2
14.8
8.8
16.9
12.2
22.1
2
Yellow fraction used from same portion and experiments carried out simultaneously Yellow fraction froin same portion used in 14
20.4
31.4 44.5
3 5
49.8
5
55.1
67.8
5
56.0
3
24.7
69.3 28.3
7.3 7 3
2
8.0
10.4
7.6
2
15.d
21.8
7.6
3
6.5 8
5
B Y
9 2
5
3.8
7 . 1 Serum from same portion used in 13 and 19
Serum 0.b.f. yellow fraction 8.6 Serum 0.b.f. yellow fraction 7.2 Serum 0.b.f. 8.0 Substrate butyl butyrate Yellow fraction Phosphate
a
3
3
Serum from same portion used in 17 and 19
Yellow fraction froni same portion used in 6
19
Serum 0.b.f. Serum 0.b.f.
2
3
4
5
6
7
8
9
1011
PH
Figure 1
Substrate, methyl butyrate
18
The indication obtained with monobutyrin led to the investigation of lower esters. At first only amyl acetate and ethyl acetate were available but later butyl butyrate was made in the laboratory and finally also ethyl and methyl butyrates. The acetates have the disadvantage that in decomposition the liberated acetic acid causes the pH of the medium to decrease to a considerable degree. It was found that latex contains an esterase which more or less decomposes all the esters mentioned here but which has a special preference for acetates. The well known variability of latex was evident also with respect to esterase activity, but it was obvious that of the esters mentioned ethyl acetate was decomposed most rapidly and most completely. Table I gives some of the results; in calculating the decomposition percentages the quantities of acid decomposed in the mixtures, in which the latex substances had been boiled brfore emulsification, were taken into account.
6.5 8
5
13.5
18.1
5
12 8
1 7 . 1 Serum froni same portion used in 13 and 17
Obtained by freezing latex.
ing the Japanese occupation it was only possible to try to extract the substrate from the latex with alcohol. By this method it was possible to extract a substance resembling fat and feeling fatty. For the sake of convenience this substance was called "latex fat." Latex fat, Ricinus oil, arachis oil, and monobutyrin were investigated in emulsions in latex, yellow fraction, and various serums. All experiments gave negative results except those in which monobutyrin was used as a substrate and where a slight splitting of the ester could be demonstrated. Latex fat was not affected by a preparation of ox pancreas in ,which a very active lipase could be demonstrated with the other substrates.
The results of experiments 4 and 5 indicate that acetate8 exeiciar an activating influence on the esterase.
Determinationof Optimum pH of Latex Esterase The determination of the optimum p H of the latex esterase vas carried out provisionally with ethvl acetate as substrate as butyrates &-erenot available. As already stated, when acetates are used the liberated acetic acid causes a rapid decrease of the pH of the medium. On the other hand, ethyl acetate is decomposed so rapidly that, with an active serum, reliable titrations can be carried out after an incubation period of half an hour, when the effect of the initial pH is still very great. The experiment mas carried out as follows: Serum obtained by freezing the yellow fraction was divided into seven portions. The pH values were fixed a t 3.6,4.3,5.8, 7 . 2 8.6, 10.1, and 11.0. I n 5-ml. portions of this serum 0.27 gram of ethyl acetate was emulsified, and 1 drop of toluene was added to each portion. Titrations were made before and immediately after shaking and after incubation periods of 30 minutes, 1, 2, 3, 5, 8, 12, 24, and 48 hours a t 37" C. Corresponding portions, the serum of which had been boiled beforehand, were also put into the incuba-
February 1951
INDUSTRIAL AND ENG INEERING CHEMISTRY
tor. The resulting autolysis, which was considerable only at p H values higher than 8, was taken into account in the calculation of the decomposition by the enzyme of the active serum. The results are shown in Table 11. I n considering the figures it should be borne in mind that for pH values higher than 8 the decomposition as a result of the esterase was influenced to some extent by the autolysis. After the incubation period of half an hour, however, the autolysis was still very slight even a t pH values of 10 and 11. Table 11. Decomposition of Ethyl Acetate at Different pH Values
c
Incubation Period, Hours 3 min. 0.5 1 2 3 5 8 12 24 48
(Serum obtained by freezing yellow fraction) Hydrolysis, Initial p 2 5.8 7.2 8.6 3.6 4.3 0 0 1.2 0.5 0 11.2 12.6 5.3 0 0.5 15.0 18.1 0.2 4.8 8.6 19.3 24.1 10.9 1.1 6.4 23.9 27.7 10.2 15.2 1.2 2 9 . 6 31.8 1.6 13.4 22.3 37.1 38.3 3.3 19.4 28.9 44.7 42.3 4.7 24.9 36.0 54.9 55.1 7.0 34.2 47.8 6 8 . 2 6 7.8 4 4 . 8 6 2 . 8 12.3 ,-pH after 24-hour incubation 3.6 3.8 3.9 4.1 4.4 I
10.1 1.1 10.4 15.7 22.7 25.9 31.3 37.7 43.9 52.7 64.7
11.0 1.9 7.5 12.4 16.8 20.7 25.7 29.2 36.1 37.6 44.8
4.5
4.7
The results obtained after 0.5-hour incubation show that this esterase with ethyl acetate as substrate reaches its optimum pH a t about 8.6. Figure 1 shows the influence of the p H graphically.
405
6. Fraction A 6 wa,s precipitated between the proportions of serum to alcohol at 1:4 and 1:8. 7. A proportion, A 7, was made by directly adding 8 parts of alcohol to 1 part of serum. Each of the fractions, 1 to 6, was dissolved into 1/12 part of water, whereas 1 part of water was added t o preparation 7 .
The activities of the solutions were determined and the results reported in Table 111. Table 111 shows that all esterase may readily be separated from the serum by the addition of a double quantity of alcohol (96y0)to the serum. Fraction 4 is by far the most active as well as the most pure, since the volume of the precipitated proteins in this fraction is small-much smaller than that of fractions 1, 2, and 3. I n another experiment the proteins were salted out with nmmonium sulfate in a similar way: serum obtained by freezing yellow fraction was, first, one-third saturated; after centrifuging off the deposit, it was one-half saturated and ultimately saturated completely. I n this way fractions Z 1, Z 2, and Z 3 were obtained. A preparation, Z 4, was made by saturating the serum completely. To fractions Z 1 to Z 3 were added 2/1,) parts of water; to preparation Z 4, 2/5 parts of water (calculated on the original quantity of serum) were added. The results of the investigations are shown in Table IV. The accuracy of these figures is doubtful because the Z preparations buffered very strongly, and the titrations were less accurate. The color of the solutions, moreover, was very dark as a result of the action of oxidizing enzymes (6) which are also present in latex and which may be very active especially in thr yellow fraction and its serum obtained by freezing.
Purificationof Esterase
.
. *
Some experiments were carried oue in which the esterase wafi somewhat purified by precipitating the proteins of serum obtained by freezing latex or yellow fraction by adding alcohol (96y0)or ammonium sulfate. After precipitation with alcohol the proteins were separated by centrifuging, dried by evaporating the alcohol, and then water was added to the proteins. After standing for 1 night the deposit was centrifuged off and the clear solution examined, In these solutions the esterase was still active but to a lesser degree than in the original serum. I n another experiment alcohol in gradually increased quantities was added to the serum, in the following way: 1. One uarter part of alcohol was added to 1 part of serum, the depositqfraction A 1 ) was centrifuged off. 2. Another quarter part of alcohol was added to the serum, so that the ratio of serum to alcohol became 1 : 0 . 5 ; the precipitated proteins were again centrifuged off (A 2). 3. Fraction A 3 was the protein proci itated between the proportions of serum to alcohol at 1:0.5 a n 2 1 :1. 4. Fraction A 4 was precipitated between the proportions of serum to alcohol a t 1:1 and 1:2. 5. Fraction A 5 was precipitated between the proportions of serum to alcohol at 1:2 and 1:4.
Table 111. Decomposition of Ethyl Acetate by Preparations of Latex Esterase Partially Purified with Alcohol Emulsion composition: 0.27 gram ethyl acetate, 2 ml. preparation (or original serum), 3 ml. boiled serum obtained by freezing; pH = 8.0; 1 drop of toluene Hydrolysis at 37' C.. % Enzyme After After After Solution 20 min. 2 hours 24 hours Fraction A1 0 1.9 8.3 0 10.8 2.7 A2 1.7 A3 0 7.4 10.8 25.3 A4 2.8 0.9 A.. 5 0 0 . A6 0 0 0 Preparation 1.35 7.3 19.5 Original serum 10.1 19.9 54.2
k?
Table IV. Decomposition o€ Ethyl Acetate by Latex Esterase Partially Purified with Ammonium Sulfate (Emulsion composition: 0.262 grain of ethyl acetate, 2 ml. of preparation plus 3 ml. boiled serum, or 0.262 gram of ethyl acetate plus 5 ml. original serum obtained by freeaing yellow fraction) % Hydrolysis afterEnzyme Solution 3 hours 21 hoiir3 Fraction 21 10.8 22.9 z2 49.6 21 .s 23 14.4 23.9 14.1 Preoaration 24 29.8 Original serum obtained by freezing yellow fraction 18.3 40.3
Dialysis of the solutions of the Z-fractions did not make the color any lighter; buffering capacity decreased considerably but remained nevertheless fairly strong, whereas the esterase activity decreased strongly.
Conclusions No fat-splitting enzyme (no lipase) was found in the latrx of Hevea brasiliensis. However, this latex contains an estcrase which decomposes low esters-acetates above all and ethyl acetate in particular-rapidly and to a high degree. The optimum pH of this esterase is about 8.6. It is not known from the literature whether latex contains a lower ester which might serve as a substrate for this esterase. According to C. K. Sekar of the Rubber Research Institute of Malaya, the volatile acids which are formed during deterioration of latex consist for the greater part of acetic acid; this indicates the presence of acetic acid esters. The great activity of the latex esterase towards acetates is in agreement with this theory. Experiments carried out a t the Indonesian Rubber Research Institute further demonstrated that the pH of the latex, which is about 7 on exudation from the tree, decreases very rapidly immediately after exudation until within a short time, a t any rate before decomposition by bacteria can have taken place, i t reaches
406
INDUSTRIAL AND ENGINEERING CHEMISTRY
Vol. 43, No. 2
6.2 to 6.5. The esterase might be involved in this process, Further experiments will have to be carried out in order to elucidate this matter.
( 5 ) Haan-Romans, L. N. S.de, ibid., XXV, 346 (1950). (6) Iwamoto, J., J. sot. Chem. Ind. Japan, 33, mppl. 409-11 (1930). (7) Klein, G., “Handbuch der Pflanzenanalyze,” Vol. IV., Part 2,
Literature Cited
(8) Oppenheimer, “Die Fermente und ihre Wirkungen,” p. 510, Leipzig, Georg Thieme, 1924. (9) WillstBtter, R., Waldschmitt-Leita, R., and Memmen, F., 2.physiol. Chem., 125,93 (1923).
Dunstan, H. W., Proc. Chem. Soc., 23, 168 (1907). (2) Gerber, M., Compt. rend., 152, 1611 (1911). (3) Gils, G. E. van, Arch. Rubbercult. Nederland.-lndit?,25,383 (1941). (4) Gils, G. E. van, Trans. I n s t . Rubber Ind., XXIII, 74 (1947). (1)
Vienna, J. Springer, p. 942, 1933.
RECEIVED October 3, 1950.
Creaming Latex with Ammonium Alginate INFLUENCE OF PARTICLE SIZE lEErnst Schmidt and R. H. K e l s e y The Firestone Tire & Rmbber Co., Akron, Ohio
T
the various synthetic latices. IIE process of conoenI n view of the great variety of particle sizes exhibited by The first requirement in the modern synthetic latices it was desirable to obtain more trating natural rubber study of creaming as a funcextensive and systematic information regarding the inlatex with the aid of creamtion of particle sine wm a set fluence of particle size on aided creaming than is available ing agents is of considerable of latex sample8, which varied in the literature. technological importance on in particle size but in no other It is shown that the concentration of ammonium algithe rubber plantations in the respect. nate required forreversible clustering and creamingof GR-S Middle and Far East. This It has been shown (1, 8, 9, latex increases regularly with decreasing particle size of process has gained interest in f f ? ) that the particle size of the latex. Modifying the stability of the latex without recent years in this country GR-S latex can be increased changing the particle size is of little consequence in creamas a practical means for conafter polymerization by addiing. The observed relation between particle size and centrating synthetic latices, tion of e l e c t r o l y t e s . The creaming agent requirement holds for GR-S latices made many of these cannot be conelectrolyte concentrations rein different emulsifier systems, for ammonia-preserved centrated by centrifugation quired to produce the desired Hevea latex, and for silica hydrosol, over a wide range of on a practical scale, primarily particle sizes (200 to 9000 A. in diameter). It is shown changes in particle size of the because of their small particle G R S latex used in the greater that Hevea latex can be fractionated according to particle size. The manner in which part of this study, were desize by successive creaming operations. creaming of natural latex is termined in a preliminary exThe results of this study suggest the general principle influenced by various factors, periment. Theresultsshowed that the influence of particle size on the reversible agglomsuch as time, mechanical that the desired variation of eration and creaming of colloidal dispersions is predomiagitation, temperature, connant over that of stability and the chemical nature of the particle size could be acconicentration and type of creamplished by using a constant dispersion. ing agent, rubber content of quantity of salt and varying the latex, pH, and addition of only the contact time. various substances, has been A 60140 butadiene-styrene GR-S latex D-229 (Type X-435) the subject of a great number of investigations (10). However, emulsified with 6.6 parts (per 100 polymer) of sodium Dresinate less extensive information has been published regarding the in731 (the salt of a disproportionated wood roein) was used in the fluence of particle size on the creaming of latex, particularly the following experiments. This latex, produced on the Firestone creaming of synthetic latices. Defense Plants Corporation plant, was polymerized a t 5 ” C. in That the creaming of natural latex by means of creaming agents is influenced by particle size was recognized by McGavack (4), the presence of cumene hydroperoxide, iron pyrophosphate, and dextrose. Portions of the latex were mixed with increasing quanwho studied the particle size distribution of concentrates of pretities of sodium chloride and simultaneously diluted to a constant served Hevea latex prepared by creaming with different amounts polymer content of 10.4%. Samples were withdrawn after varyof creaming agent. ing time intervals and analyzed for particle size by means of the McGavack ( 4 ) found that incomplete creaming of normal electron microscope. It was found that particle growth had taken Hevea latex gives concentrates of larger average particle size than place in those samples which contained more than a certain critical that of the original latex and that the average particle size of the concentration of sodium chloride. The resulting particles were cream is increased as the amount of creaming agent is reduced. spherical in shape and showed no evidence of being simple cluster8 A number of recent patents ( 1 , d , 7, 9) reveal that increasing of the original small particles. the particle size of synthetic latices leads to creamed concentrates The rate of particle growth was slow a t about the critical salt of increased rubber content. concentration and increased as the salt concentration was inIt was believed that further systematic contributions to the creased. Below the critical salt concentration no particle growth knowledge concerning the influence of particle size on creaming of ww observed over a period of several weeks. These results are latex, should be of interest, particularly in view of the great illustrated in Figure 1. variety of particle sizes and particle size distributions exhibited by
