SEPTEMBER, 1940
INDUSTRIAL AND ENGINEERIKG CHEMISTRY
1277
Literature Cited Budenholeer, Sage, and Lacey, IND. EFG. CHEM.,32,384 (1940). Deming and Shupe, Phys. Rev., 37, 638 (1931). Kay, IND. EKQ.CHEM.,30,459 (1938). Keyes and Burks, J . Am. Chem. Soc., 49, 1403 (1927). Kvalnes and Gaddy, Ibid., 53,394 (1931). Lewis, J. Am. Chem. SOC., 30, 668 (1908). Lewis, PTOC.Am. Acad. Arts Sci., 43, 273 (1907). Lewis and Randall, “Thermodynamics and Free Energy of Chemical Substances”, p. 205, New York, McGraw-Hill Book Co., 1923. Noyes and Bray, J. Am. Chem. SOC.,33,1693 (1911). Roozeboom, “Die heterogenen Gleichgewichte”, Vol. 11, part 1, p. 288 (1904). Sage. Hicks, and Lacey, IND. ENG.CHEM., 32, 1085 (1940). Sage and Lacey, Ibid., 31, 1497 (1939). Sage, Webster, and Lacey, Ibid., 28, 1046 (1936). Ibid.. 29, 658 (1937). Ibid., 29, 1188 (1937). Ibid., 29, 1309 (1937). Vold, J. Am. C h a . SOC.,57, 1192 (1935). WEIGHT
FIGURE 15. EFFECT OF
FRACTION METHANE
COMPOSITION UPON THE FUG.4CITY O F ?%-BUTANE I N
THE
GAS PHASEAT 160’ F.
Drying of Papaya Latex STABILITY OF PAPAIN Reasons for the relative inactivity and poor keeping quality of commercial papain have been sought. It has been found that the enzyme loses much of its activity on drying and shortly thereafter, and still more of i t on being diluted just prior to use. Methods for minimizing these losses are suggested. Papaya latex is shown to contain a thermostable factor that destroys enzyme activity, apparently through an oxidative process.
R
E C E N T experiments with purified (6) and with crystalline ( 2 ) papain have shown that the proteolytic activity of the enzyme itself, measured under optimum conditions, is of the same order of magnitude as the activity of the pancreas proteinases. Furthermore, there is reason to think that, of the total solids in fresh papaya latex, over half is enzyme protein so t h a t fresh latex is an extremely active proteolytic agent. On the other hand, the activity observed generally for commercial preparations of papain is relatively so low that a heavy loss of enzyme must have occurred in preparing the latex for commercial use. The methods employed by industry for this purpose consist of drying the wet latex (which contains 80-85 per cent of water) in the air or in a vacuum oven. The dried latex is subsequently pow-
A. K. BALLS, H. LINEWEAVER, 4ND S. SCHWIMMER Food Research Division, Bureau of Agricultural Chemistry and Engineering, U. S. Department of Agriculture, Washington, D. C.
dered and sieved in some cases, and in this form appears on the market as papain. It is well known t h a t oxidizing agents, including air, inactivate papain under suitable conditions. Some of the deterioration that occurs in commercial preparations is doubtless due t o oxidation, although Frankel, Maimin, and Shapiro (4) showed that part of the enzyme in fresh latex exists in a form more labile than the rest. It may be t h a t the decomposition of such a labile fraction during and directly after the drying process is responsible for large losses in activity. As evidenced by the following experiments, the deterioration of papain is rapid during and soon after drying. It is also faster in air than in vacuum, and particularly rapid in dilute solutions. Only part of the inactivated enzyme may be reactivated by cyanide.
Method of Assay The milk-clotting method of Balls and Hoover (1) was employed, except t h a t the temperature was 30” instead of 40” C., and 5 instead of 10 cc. of “Klim” were used. Experience has shown that some samples of dried milk behave ir-
1278
INDUSTRlAL AND ENGINEERING CHEMISTRY
regularly. To avoid this error, a sample of dried papain was kept in cold storage as a reference preparation. It was assigned an activity of 7.4 units per mg. of protein nitrogen. The results obtained with each new specimen of dried milk were corrected by direct proportion when the assay of the reference preparation differed slightly from 7.4, but "Klim" samples that differed greatly in clotting time from the general r u n were discarded. The reference preparation of papain is not permanent, but its rate of deterioration is slow in the cold. I n subsequent work a crystalline preparation suspended in sodium chloride and dilute cyanide will probably be used.
Loss of Activity Caused b y Drying Papaya Latex Experiments were made in which wet latex was dried in a vacuum oven a t 45" C. after various additions. Roughly one hour was required for complete drying of the 1.0-gram samples. The results are given in Table I. Dilutions for assay were made with boiled distilled water and were then assayed quickly so that conclusions drawn from Table I should be valid.
TABLE I. EFFECTOF DRYIXGIN VACUUMON PAPAYA LATEX
Loss of Activity from Dilution of Papaya Latex
7cof T o t a l Activity
Moist latex one month old was homogenized mechanically and kept cold a t all times until diluted. About half of the total enzyme in this latex was active a t the start. The emulsified latex mas then diluted 250 times. One cubic centimeter of the dilution was assayed a t once and a t short time intervals thereafter. The results are shown in Figure 1. It is apparent that dilution with distilled water (containing air) was very destructive t o the enzyme. The natural activity (originally half of the total) was completely lost in a little more than an hour, whereas the total activity was reduced proportionally-that is, to about half the original value. Dilution with 20 per cent sodium chloride solution was less destructive, dilution with recently boiled distilled water was still less so, and dilution with 0.02 M sodium cyanide prevented any serious loss in total activity, although in this case the natural activity still suffered somewhat. Other experiments showed that greater dilution does not necessarily lead to more rapid inactivation, nor do different latices all behave the same way. This is not surprising, for latices after a long shipping and storage time vary in the amount of inactive enzyme present. Part of this may be again reactivated. Furthermore, it is shown later that some destructive factor also exists in latex, and this mould also
75 90 63
100
63 GO
77
46 37
100 100
99
77
~
L a t e x B 1 gram" Dilutdd with 50 00. of water while wet Dried 1 hr. a t 40' C., diluted with 50 cc. of water
of 0.3 per cent gave similar results. The sulfide-treated latex has the further disadvantage of turning a rather unattractive green color on drying. Sodium chloride with subsequent complete drying yielded a product t h a t resembles the untreated latex in that it is very active a t first but deteriorates rapidly. The most stable preparations were made by the addition of sodium chloride, with partial removal of the water subsequently. The result is a gray paste of considerable stability. Material similar to No. 4, Table 11, has been exposed to the air a t room temperature (about 30" C.) for 14 days with no appreciable loss of enzymic activity. It has also been kept in sealed metal tubes and in lacquered tins for as long as 50 days a t room temperature without appreciable alteration, and less than 10 per cent in 190 days. It is believed that by means of this treatment the original high proteolytic activity of the latex can be retained for a long time in a form easily used by industry. Weight for weight, the papain pastes have about half the activity of the best dried latex, although they were made from lcas than one fourth the amount of wet latex. For practical purposes it appears that the productivity of a papaya plant in terms of proteolytic enzyme may be roughly doubled by this means.
ACTIVITYOF
THE
Remaininga As natural As total activity activity L a t e x A 1 gramb Dilutdd with 50 cc. of w a t e r while wet Diluted with 50 cc. of 0.02 M N a C N while wet Dried 1 hr. a t 40° C., diluted with 50 cc. of water Dried 1 h r . a t 40' C . . diluted with 50 cc. of 0.02 M NaCS Dried 1 hr. a t 52' C., diluted with 50 cc. of water
VOL. 32, NO. 9
G5
a T o t a l activity is t a k e n a s t h a t shown b y t h e latex diluted with 0.02 M N a C N , activated b y t h e addition of 5 drops of 2 M K a C X per cc. of enzyme solution, a n d assayed after standing f o r 3 minutes. Comparisons of different e n z y m e preparations were made on t h e bsms of t h e protein N. b L a t e x A was from a freshly opened enameled can of latex previously k e p t i n t h e ice box. Latex B was from a similar can t h a t had been opened a n d t h e contents exposed t o t h e air for a b o u t 2 m o n t h s in t h e ice box. Latex A contained 16.2 milk-clotting u n i p per mg: of protein nitrogen without activation a n d 21.6 units after,activation with cyanide. Latex B contained 7.0 units before a n d 15.2 units after activation.
hiany times it had been observed that on drying, papaya latex lost roughly half its initial activity, whereas the resulting dried product was then relatively stable. Both results were obtained before the detrimental effect of dissolving and diluting the enzyme was understood. It is evident that in this experiment the loss attending the dryinn Drocess was much less severe than that . . observed before, but the subsequent deterioration of the dried enzyme was very rapid so t h a t in about a month the samples again showed only approximately half the original strength. Whether the latex loses most enzyme during drying or only on subsequent standing is perhaps of no practical moment, but the latter case now appears the more probable. I n any event, much less enzyme is available after even careful drying. By the addition of sodium sulfide to the latex before drying, it was hoped to increase the amount of activator available and decrease the concentration of possibly harmful heavy metals. Although the results were better T I M E AFTER t h a n those obtained with untreated latex, they still show considerable loss. Sodium FIGURE 1. cyanide added to wet latex in the amount . a 1
DILUTION (MIN)
EFFECT O F
TIME AFTER DILUTION ( M I N ~
250-FOLD DILUTIONO
S PAPAYA
LATEX
SEPTEMBER, 1940
TABLE 11.
IXDUSTRIAL AKD EKGINEERIKG CHEMISTRY ACTIVITY OF ~ UXDRIED LATEXREMAISISG AFTER STORAGE O F VACUEM-DRIED DRIEDPREPARATIOX~ OF LATEXAT ROOMTEMPERATURE
PERCEKTAGE O F TOTAL
D a y s of storage: Dried 1. 2. 3.
0
latex Y o additions Contained 10% XalS Contained 10% NaCl
T o t a l Enzyme (.ictivated) 3:3-36 45-50 130-135 41
92 85 78
Partially dried latex 4. Papaya solids 35YC,NaCl 20, water 4 5 5,
1279
P a p a y a solids 3.5'3, KazS 10, N a C l 10, water 45
82 8% 100
49
50
..
80 89 58
SO
..
..
100
0
..
..
.. ..
91 97 76
79 74
75
73 73
..
30
..
..
..
ASD PARTI.4LLY
Xaturally Active Enzyme 33-36 45-50 130-1,'35
190
17
811
DJ
.. 74
74
.. ..
73 19
io
67
6.5
..
..
..
a T h e activity of t h e original latex was 1.1-1.3 milk-clotting units per nig. of dry weight without activation and 1.2-1.5 units a f t e r activation with cyanide. T h e per cent activity is calculated on t h e weight of papaya solids only, which excludes such additions as salt, sulfide, etc.
be diluted. The data merely represent the resultant of such changes. This is illustrated by the behavior of two latices when diluted 250 times with 0.004 M sodium cyanide (Figure 2). I n one case slow activation occurred, in the other, enzyme destruction. These observations also illustrate the difficulty of making a direct determination of the natural activity of crude papain. If additional activator is used, the result may be too high; if none is used, further inactivation will occur after dilution. Extrapolation of a curve similar to those in Figure 2 has given satisfactory values vihich should approximate the truth. Almost as good results can be obtained by diluting with airfree water and then testing as quickly as possible. The effect of dilution on the activity of papain appears to be due t o oxidation, for it may be greatly reduced by the removal of air. This was also shown by keeping an emulsified undiluted latex for a month in vacuum at 5-8" C. Under these conditions it lost 20 per cent of the original (unactivated) activity, whereas the same latex kept in air a t this temperature for 12 days lost 83 per cent. Xevertheless, the presence of dissolved oxygen cannot be held solely accountable for the effect, as the behavior of the crystalline enzyme showed.
observed that the press juice of the papaya fruit contains a substance that rapidly inactivates the latex enzyme (s). Hydrogen sulfide apparently inhibits this inactivation; a t least there is less loss of activity in its presence. The crystalline enzyme has now been found to lose activity n-hen it is mixed with latex. Several mixtures of latex and crystals were incubated a t 30" C. in the air for 20 hours, and the mixtures assayed a t intervals. The results are shown in Table 111. Khere the proportion of latex to crystalline enzyme n a s large (experiments 2 and 3 in Table 111),it is n o t possible t o say more than that considerable inactivation of the total enzyme occurred. Where the proportion wts ?mall (experiment 4), it is evident that some crystalline enzyme was inactivated. Inactivation also occurred when latex heated a t 90" C. for 30 minutes was used. This heated preparation vias itself inactive.
TABLE 111.
MIXTURES OF LATEX .4XD CRYSTALLIKE PAPAIN AFTER STASDISG IS AIR^
ACTIVITY OF
0 Hr. 1 Hr. 3 Hr. 13 IIr. 20 EIr,
Mixture 1. 2. 3. f. a.
6. 7.
Crystals alone Crystals and freshly opened latex Crystals and partly oxidir;ed latex Crystals and freshly opened latex Crystals and heated fresh latex (in same amount a s 2) Latex alone, same as 2 Heated latex alone
100 470 340 140
120 370 0
...
,
..
440 260 135 114
2iO
190 95 94
210 170 85 77
290
190
14.5
. . . . . . . . .
120 190
loo 85
50
115 0
1 cc. of a suspensiun of crystals, containing 1.09 mg. protein S , was used in each experiment. T h e final volume, after addition of latex, was 20 co. T h e activity of t h e crystals used is taken as 100, t h a t of t h e mixture (assayed wit,hout additional activator) is shown proportionately in t h e table.
Acknowledgment
TIME AFTER DILUTION (MIN)
FIGCRE 2. VARIATIONIN LATICESWHES DILUTED WITH 0.004
SODIUM CY.4NIDE
The behavior of crystalline papain on dilution is in marked contrast to that of the crude material. Crystals of papain in which only one third of the total enzyme was active showed no loss of activity on standing for 90 minutes after a dilution in ordinary distilled water (not air-free) corresponding to a 1300-fold dilution of latex. On the other hand, dilution with 0.02 3 ' sodium cyanide produced marked activation during the same period. The relative stability of the crystalline enzyme when compared with latex was noticed elseIThere ( 2 ) . It was also
Our thanks are due the Hawaiian Agricultural Experiment Station a t Honolulu for their support and cooperation in this investigation. The enzyme material used was entirely of Hawaiian origin. This work was done under Special Research Fund KO.SRF 2-9 provided by the BankheadJones Act.
Literature Cited (1) Balls, d. K., and Hoover, S.R., J . B i d . Chem , 121,737 (1937). (2) Balls, A. K., and Lineweaver, H., I b i d . , 130, 669 (1939). (3) Balls, A. K., Thompson, R. R., and Jones, IT. W., ISD. ENG. CHEhf., 32,1144 (1940). (4) Frankel, M., Maimin, R . , and Shapiro, B., S u t u r e , 139, 249 (1937). ( 5 ) Okumura, S., Bull. Chem. SOC.Japan, 13, 534 (1938); Maeda. S., I h i d . , 12,319 (1937). FOOD RESEARCH Division Contribution 474,
