CRUDE PAPAIN Preparation and Properties A. K. BALLS
R. R. THOMPSON
Bureau of Agricultural Chemistry and Engineering, U. S. Department of Agriculture, Washington, D. C.
Bureau of Agricultural Chemistry and Engineering, U. S. Department of Agriculture, Honolulu, T. H.
W. W. JONES Hawaiian Agricultural Experiment Station, Honolulu, T. H. The increase over the zero time titration represents the proteolytic activity of the enzyme used. In order to make the results easily comparable, the titration difference (in cc. of 0.1 N alkali) has been divided by the weight in milligrams of the enzyme sample that produced it. A rough correspondence may be assumed between these numerical values and the amount of proteolytic enzyme present. Since it is probable that two or more enzymes are present in crude papain and that they do not clot milk equally well in proportion to digesting casein @), there is no need for both methods to give the same result on the preparations here described. When an enzyme preparation was assayed after solution in water but without the addition of any reducing substance, the result is expressed as not activated. Activated preparations were made by adding neutralized cysteine-hydrochloric acid t o the solution of the enzyme in an amount equal t o twice the solid matter present in the enzyme preparation used. The mixture was then allowed t o stand 30 minutes a t room temperature before testing.
T
HE proteolytic enzyme papain is a constituent of the latex from the green fruit of Carica papaya. The dried powdered whole latex is commonly referred to as papain. Pure papain is apparently as strong a proteinase as any of the well-known enzymes. The fresh latex is extremely powerful proteolytically, and perhaps more than half of the total protein contained in i t is a t first active, though easily inactivated by oxidation. Air, probably catalyzed by other ingredients of the crude enzyme, causes rapid deterioration. Inactivation may at first be reversed by addition of reducing agents; hydrogen sulfide, hydrogen cyanide, sulfides, and sulfites are commonly used for the purpose. After continued exposure t o oxidizing influences, however, the protein is no longer capable of reactivation. Therefore, commercial preparations of papaya latex do not keep indefinitely and often lose their natural activity within a few months. Severtheless, papain is perhaps the cheapest form of proteolytic activity on the market today, and its use has increased greatly in recent years. The following observations were made during an investigation undertaken a t the suggestion of 0. C. lIagistad, then Director of the Hawaiian Agricultural Experiment Station, in order to learn more about the processes of obtaining papain from the fruit and to see if mechanical methods could be employed with profit. The results show that i t is not feasible to pick the fruit and obtain the papain by a process of maceration and juice extraction. On the other hand, it was found that the leaves and stems-in fact, the entire plant aside from the roots-contain considerable enzyme which may be removed as a press juice and subsequently purified. The yields of the product do not prove that this would necessarily be profitable, and there is still room for improving the product so obtained. Furthermore, the world demand for papain, although increasing, could be easily oversupplied. The results do show, however, that methods of extracting the enzyme from the whole plant are feasible. The product obtained from the stems and leaves of the papaya plant in these tests was not superior t o the average run of commercial papain samples nor was i t markedly inferior. However, there is reason t o suppose that the procedure in our brief experiments could be considerably improved should this ever become a matter of interest.
Coagulation of Papaya Latex Latex is obtained by making three or four longitudinal scratches in the skin of the green fruit while it still hangs on the tree. The latex is a t first liquid but rapidly forms a gel which, on standing in the cold, shrinks arid liberates a small amount of "serum". Coagulation is hastened by contact with water and greatly delayed by the addition of various substances t o the liquid. These substances are, in general, those which prevent the coagulation of blood-namely, fluoride, citrate, and oxalate. This behavior is shown for fluoride and citrate in Table I. Variations in the clotting time are also apparent between different-sized fruits, possibly because of differences in the water content. FLUORIDE AND CITRATE ON TABLEI. EFFECTOF SODIUM THE CLOTTIXG OF PAPAYA LATEX^ Time, minutes Na fluoride 0.002 i M 0.02 M 0.05 M
NaHa citrate 0.002 M 0.02
15
0 0 0
0
0 0 0
0
20 0
0 0
0
40 0 0
0
0
a 0
80 0
..
0 0
0
120 0
210
0
1200
0
~
0
~
..
..
..
0
5 0
..
0
0
0
~5
..
0.05 M 0 0 0 0 0 Formation of a clot. Latex clotted in 10 minutes when 0 2 24 NaCl or 0 . 0 4 M cysteine was added, or when no substance was added. a b
Methods of Assay Two rapid methods-namely, the milk clotting test (1) and the rate of digestion of casein a t pH 5 (8)-have been used for the
M
lob
assay of papain preparations in the work reported here. The results of the milk test are expressed as milk clotting units. The unit was taken as the amount of enzyme required t o clot 10 cc. of a standard milk preparation from dried milk in 1 minute a t 40" C. Units per milligram of a dry preparation are therefore equal to 1/(E X t ) , where E represents the weight of the enzyme in milligrams, and 1 the clotting time in minutes. The digestion of casein a t pH 5 was determined by titration in alcohol. The tests were incubated for 20 minutes at 40" C.
The coagulated latex imbibes water freely and retains its jelly-like consistency at a considerable dilution (3 volumes). The addition of sodium chloride (10 per cent of the tot,al volume) precipitates the solid in a compact form that still contains an appreciable amount of the enzymic activity. In an experiment in which 10 grams of wet latex were mixed with 20 cc. of water and 3 grams of sodium chloride, 26 cc. of solution containing about half the total enzyme were oh-
1144
AUGUST, 1940
INDUSTRIAL AND ENGINEERING CHEMISTRY
tained by centrifuging. After thorough washing, the insoluble portion of the latex contained 10.96 per cent nitrogen and was therefore apparently about two thirds protein.
Yield and Quality of Latex from Green Fruit Experiments on the yield of latex obtained a t any one time showed (Table 11) that the weight of dried latex obtained is a fairly constant proportion (about 0.10 per cent) of the weight of the fruit, irrespective of the quantity of wet latex secured. The latter varies widely, probably due to dilution. TABLE 11. LATEXOBTAIKED A T O N E BLEEDING F R O M TENTO TWELVE SCRATCHES IN A SIKGLE FRUIT Weight of. Fruit, Grams 1150 1000 1565 733 1470 1225
Weight of Fresh Latex, Grams 7.5 10.0 5.2 5.6 8.6 5.1
Fresh Latex, % of Fresh Fruit 0.65 1.0 0.3 0.77 0.58 0.42
Weight of Dried Latex, Grams 1.35 1.00
Dried Latex, % of Fresh Fruit 0.12 0.10
0:+3 1.6 0.92
0.11 0.07
o:io
The latex from different sized fruits and from two types of trees was measured and then dried in an ordinary vacuum oven a t 50" C. Assays were made on the dried material (Table 111). The differences are due more to the degree of activation than to the total enzyme (active plus activatable) present. Some latices appear to be inactivated more rapidly than others on drying and standing. It was noted also that a considerable part of the original activity was always irretrievably lost by drying, even under the mildest conditions. The vacuum-dried product is, however, superior to that dried in the air, from the standpoint of both appearance and activity. There seemed to be no apparent correlation between the type of tree and the activity of the enzyme, however. FRUITS TABLE 111. LATEXFROM DIFF~ERENT-SIZED Weight of Fruit Fruit, Tree Size Grams Female Large (old) 2250 Female Medium 500 Female Small (young) 50 Slightly male Large (old) 2000 Slightly male Medium 700 Slightly male Small (young) 250 Mixed latex from above 6 varieties (stood for 3 days before drying) Commercial papain for oomparison a Not activated. b Activated.
7p
...
So!ids in Latex 15.3 14.9 15.9 17.5 18.0 15.6 18.4
.. .
,..
MilkCasein Clotting Digestion, Units Cc. 0.1 per Mg.5 K O H 0.52 0.55 0.43 0.68 0.56 0.12 0.34 0.60 0.56 0.36 0.68 0.15 0.60 0.06 1.07b 1.2b 0.08 0.44b
0.16 0.32b
Effect of Press Juice on the Enzyme We next attempted to obtain the residual enzyme from the press juice of fruit already bled. The first experiment gave nothing. This was found to be due to the fact that the press juice is destructive toward the enzyme in the latex. When active latex was added to the press juice of the fruit, the additional enzyme was also destroyed (Table IV). Boiled juice was found to be far less destructive. Additional activator behaved in some degree as a protective agent. I n view of this behavior, i t seemed probable that the enzyme could be extracted from the fruit pulp after the addition of hydrogen sulfide, and this was found to be the case. The experiments, however, were not satisfactory. One of the best follows: One hundred grams of previously bled green papaya tissue was grated (not ground) and mixed with 100 cc. of 0.01 N
TABLEIv.
1145
IXHIBITION O F PAPAIN BY PAP.4YA PRESS
JUICE
Casein Digestion0 0 2.0 min. min.
Treatment 2.5 mg. latex Alone 0.3 0.3 f 2 cc. press juice 0.6 0 i 5 . 0 mg. cysteine (1 hr.) 1.3 1.3 + L O mg. cysteine & 2 cc. press juice 1.3 0 1 +5.0 mg. cysteine & 2 cc. boiled (3 hr.) juice ... 0.9 3.5mg. latex f3.5 mg. cysteine (72 hr.) 3.35 f3.5 mg. cysteine & 1 cc. juice 1.2 +3.5 mg. cysteine & 1 cc. satd. HIS water ... 1.6 +3.5 mg. cysteine, 1 cc. juice, & 1 cc. H2S water after . 1.4 20 hr. 0 I n cc. of 0.1 N KOH, per mg., measured a t p H 5 after the mixture had stood a t room temperature for the time indicated.
... ... ..
citrate (pH 5.0) to delay the coagulation of the latex as much as possible. The citrate solution had been previously saturated with hydrogen sulfide. The tissue was then strained and pressed in cloth, yielding 130 cc. of extract. Two cubic centimeters of this extract did not clot milk in 10 minutes or show any digestion with casein. The extracted pulp was thoroughly mixed with 15 cc. of 20 per cent sodium chloride solution and pressed dry. The press juice was a t once diluted with an equal volume of hydrogen sulfide water and thereafter measured 66 cc. Two cubic centimeters of this extract did not clot milk in 10 minutes, but it was active on casein. One cubic centimeter a t pH 5 gave 1.9 cc. of 0.10 A-potassium hydroxide in 20 minutes (and a t p H 6 gave 0.7 cc.). This is equivalent, as nearly as can be estimated, to 1.6 mg. of a good dried latex (when activated) or a total yield of 105 mg. from 100 grams of fruit (equal to 0.10 per cent). The yield of dried latex from this papaya, which was bled before being used, was also 0.10 per cent. The amount of enzyme recoverable from the fruit by grinding and extraction cannot, therefore, be much more than double the amount to be obtained in a single bleeding. Further efforts to increase the yield by extraction were not successful, Microscopic examination of the latex system of the fruit confirmed the opinion that the amount of latex remaining in the fruit after bleeding is small. It seems probable, therefore, that the enzyme exists only in the latex, and since no great quantity of latex remains in the fruit, there is also very little enzyme to be obtained. The amount of enzyme to be taken from the fruit after picking is therefore little or no greater than would be obtained by waiting a few days and bleeding a second time. It is clearly of no advantage to sacrifice the fruit and thereby the opportunity to bleed i t several times.
Extraction of Papain from Leaves Leaves, stalks, flower stems, and bark all yield enzymecontaining press juices. The roots yielded none (milk clotting data). Latices from young plants and from mature male trees were also rich in papain. Furthermore, the press juice of leaves and stalks appeared to contain less of the inhibitory or destructive substance than did the latex of the fruit. A possibility therefore exists of recovering the enzyme from the leaves and stalks, even from the otherwise useless male trees. There was little difference in the quality of the juice from leaves of different ages on a mature tree, but a great difference existed in the quantity of juice obtained. I n Table V the juices were examined as soon as possible after pressing, but nevertheless the influence of inactivating substances must be allowed for. The decrease of activity in the press juice from stalks and leaves, and particularly from leaves, is evidently due to inactivation of the enzyme and not to its destruction.
INDUSTRIAL AND ENGINEERING CHEMISTRY
1146
was 0.32 per cent of the juice, which is equivalent to 2.0 per cent protein. The leaf juice contained some plant pigments and also traces of the alkaloid carpaine, which gave it a bitter taste. Once acidified and centrifuged, the juice lost its milk-clotting power very slowly, as Table VI1 shows. The loss in activity was completely restored by the addition of cysteine in the case of the juice acidified with sulfuric acid. The juice could be filtered through paper, charcoal, or kieselguhr without material loss of enzymic activity.
TABLE Y. PRESSJUICE FROM LEAVES OF MATURE TREESAND FROM SEEDLIKG Milk-Clotting Unitsa Male tree Female tree
Mature trees Young leaves Medium leaves Old leaves
1.25 0.89 1.18
1.09 0.92 1.15
7 -
a
15-inch seedling Roots Stalks and leaves Not activated, per cc. of unfiltered press juice.
VOL. 32, NO. 8
0 0.70
Preparation of Enzyme from Press Juice
It was found that the enzyme could be removed from the acidified and filtered juice by precipitation. Two common Age of protein precipitants, alcohol and ammonium sulfate, gave Juice, Min. good results. 0.25 Alcohol precipitation was done a t room temperature by the 5.0 ... 0.40 0.80 addition of 5 volumes of 92 per cent alcohol. The precipitate 20.0 0:80 4.6 0.33 0.86 was found to deteriorate on standing in the alcoholic solution, Of a male tree. so it was removed as quickly as possible, pressed between folds of filter paper, and dried in a vacuum a t 48" C. The enzyme OF ACIDIFIED,FILTERED JUICE TABLE VII. KEEPINGQUALITY was completely precipitated by this concentration of alcohol. FROM LEAVES AKD STALKS Precipitation with ammonium sulfate was accomplished by of Juice, Acidified with: Milk-Clotting Units/Cc. Days of Storage the addition of 2 volumes of saturated ammonium sulfate a t 5' C., p H 4.0 HzSOi HC1 HAC solution to the juice. The precipitate was allowed to stand 3.5 3.0 3.0 3.0 3.0 ... overnight in the cold and then filtered, After pressing and 2.6 ... 2.4 1.4 ... ... drying in vacuum, the material contained about 50 per cent 1.4 ... ... of ammonium sulfate. The precipitation of enzyme was not 1.7 ... ... 3.50 ... ... quite complete a t the salt concentration used. a Activated with cysteine. A summary of the data obtained from a number of runs is shown in Table VIII. The ammonium sulfate precipitates show, on the whole, higher activity with a smaller yield. The addition of cysteine brought the preparation back to its Small additions of sodium sulfite were made to the juice original activity (Table VI). before precipitation in several cases, in an attempt to inWhen papaya leaves were ground in a Sepro-sieve apparacrease the yield of active enzyme in the precipitate. tus, the juice obtained was a thick green liquid containing Further purification of the juice before attempting to resolid particles of tissue. Seventy grams of average leaf tissue move the enzyme from it seems desirable but was not worked gave 25 cc. of this juice. On centrifugation 21.5 cc. of clear out in detail. Dialysis against water did not impair the enpress juice were later obtained. Clarification of the juice and zyme strength, but subsequent evaporation of the dialyzed inhibition of the oxidizing processes by which the enzyme is material did. About half the total solids were removed by inactivated were both aided by increasing the acidity to pH this means. Fermentation of the sugars by added yeast 4.0 with sulfuric, hydrochloric, or acetic acid. The acidified produced little or no change in the enzyme content and might filtered juice contained about 6 per cent of solid matter, offer a method of purification. Some method of avoiding including 2.6 per cent of substances that reduced Fehling the presence of the bitter alkaloid carpaine would be necessolution, when calculated as dextrose. The total nitrogen sary before the preparations could be used for food purposes, TABLE VI. DECREASE Ih' ACTIVITYO F PRESS JUICE WITH TIME -Juice from Leavesa- ,---Juice from Stalks=Not Not activated Activated activated Activated --Milk-clottinQ units/cc.-5.0 4.0 0.57 0.86
0
TABLEVIII.
yo!. Expt. A
of juice, cc. 100 60
D E
Acid HC1 HAC HAC HAC HAC
a
HC1
b
Hd30.1 HCI HCl
B C
Precipitant, 5 vol.
Dry ppt.. grams
92% alcohol
350 100 100
2 vol. (NHI)zSOI (satd. soln.)
1.40 1.14 1.73 18.35 7.95
0.28 2.29 0.76 1.97
e
HC1
100
0.84
f
HCl
100
0.90
g
HBc
1000
8.6
h a
... Per mg.
.. b
........
Cc. of 0.1 N KOH per mg.
...
JUICEO F LEAVESAND STbLKS, MALETREES Qualitv Assay Not Activated -ActivatedCasein Casein ReMilk digesMilk diges- Activity, covery, units" tionb unit" tionb per cent per cent 100 0.10 0.08 0.17 0.25 61
3
c
Yield
100 1000 600
25
PREPARATIONS FROM PRESS
7
Remarks
............
............
4 mg./cc. sulfite 2 mg./cc. sulfite
Juice was half-washines from pulp. 4 mg./&. sulfite
............ ............
Previous charcoal filter Previous H2S satn. Sulfite 2 mg./cc., ppt. filtered a t once Sulfite 4 mg./cc., ppt. filtered after 18 hrs. Sulfite 2 mg./cc., ppt. filtered after 12 hr. washed with sulfate: sulfite soln.
...........
Remarks
.............. ..............
0.06 0.20 0.06 0.06
0 04 0.20 0 08
0.20 0.24 0.13
0.60 0.80 0.32
32 85 50
110 100 80
Good product, not bitter Low grade, not bitter
0.13
0.10
0.46
0.44
35
120
Not bitter
0:30
25 36 33
74 56
0.15 0.14 0.05
..
..
..
0.49
0:08
..
0.22 0.22
..
..
...
147
0.24
..
..
..
66
...
0.22
0.32
0.35
1.1
66
105
0.15
0.28
0.17
0.32
43
47
0.08
0.16
0.40
0.32
..
..............
Bitter
..............
Probably an error in total activity
..............
Good product Good product but bitter, low yield Commercial comparison sample for
AUGUST, 1940
INDUSTRIAL A N D l3NGlKl
