INDUSTRIAL A N D ENGINEERING __ CHEMISTRY
1424
Vol. 23, No. 12
Preparation of Aspergillus oryzae Enzymes' Taichi Harada DEPARTMENT OF BIOCHEMISTRY, NEWYORKPOST-GRADUATE MEDICAL SCHOOL, COLUMBIA UNIVERSITY. NEWYORE,N.
Y.
Important points for cultivation of Aspergillus HE a p p l i c a t i o n s of his commercial m e t h o d f o r oryzae fungus on cooked wheat bran for the preparaAspergillus oy z a e enthe preparation of the ention of diastatic enzyme and the use of sodium chloride zymes in the t e x t i l e z y m e s of Aspergillus oryzae, as antiseptic for the water extract of the diastatic world and in the food indusand for purification by means enzyme in connection with food industries are briefly tries, especially the latter, are of alcoholic p r e c i p i t a t i o n . described. Sodium chloride at the saturation point v e r y important in the F a r Hence, further description is gives the most satisfactory results as an antiseptic. East, as in the manufacture of not n e c e s s a r y here. HowCertain properties of the diastatic enzyme of the soy (Shoyu) sauce, miso, and ever, essential factors in the Aspergillus oryzae were studied. Diastatic activity sak6, since the preparation of production of the enzymes undergoes little change when heated at low temperathe enzymes contains two imby Aspergillus oryrae fungus tures, such as 30" and 37' C., for 5 and 2.5 hours, rep o r t a n t enzymes-namely, growth may be specified as spectively, whereas at relatively high temperatures diastase and protease. The follows: (1) quality of bran; (above 45" C.) it was greatly affected even when heated present paper will d e s c r i b e (2) water c o n t e n t of bran; for only 30 minutes. certain properties of Aspergil(3) hydrogen-ion concentraThe relation of temperature to optimum pH does not lus oryzae e n z y m e s which tion; (4) time of incubation; follow Olsen and Fine's linear equation. It appears were obtained by Takamine's ( 5 ) t e m p e r a t u r e ; (6) huthat the optimum pH does not vary with temperature method (27). midity of chambcr; and (7) below 5 0 " C. The optimum temperature at pH 5.4 An important part of the sterilization. was found to be a narrow range between 55" and 60" C. industry is the culture and Antiseptic Used under the conditions stated. growth of the fungus on which Hydrolysis of starch by the enzyme appears to be a the production of e n z y m e It is extremely difficult to bimolecular reaction, and the value of K decreases as quality depends. k e e p t h e e n z y m e in liquid the time of incubation proceeds. It is probably due The enzyme action depends form (water extract) because to a partially reversible reaction. of heat, b a c t e r i a , etc. It upon the following important The presence of certain enzymes associated with the factors: temperature, time, is, therefore, n e c e s s a r y to diastatic enzyme preparationlis demonstrated. employ s o m e s u b s t a n c e s hydrogen-ion concentration, which will Drevent bacterial buffers. and concentration of the substrate. The enzyme is sensitive to heat, acid, and growth, as an antiseptic. A great numger of substances alkali; for example, when heated to approximately 100"C. in have been suggested (3, 11, 1.4, 24)) including chloroform, toluene, phenol, tricresol, and various salts. the moist state, the activity is completely destroyed. Precipitation of the diastatic enzyme from a colloidal Power of Sodium Chloride for Water Extract solution of water extract of Aspergillus oryiae tissue by the Table I-Preservative of Diastase a t Room Temperature addition of alcohol to the extent of 70 per cent does not yield a NaCl LINTNER'S VALUE IN: Odays 2days 26 days 48 days pure product, but a mixture of many enzymes of which the % following will be mentioned: maltase ( 2 , 12, IS, 18, 29); No strength No strength No strength 6 123 N o strength No strength h'o strength 8 123 dextrinase ( 8 ) ; invertase (2, 6, 12, 18); lipase (18, 19, 29); 10 123 121 119 81 peptase ($9); ereptase (29) ; rennet (5, 18, 29) ; trypsin (69); 20 123 121 119 115 24 123 117 111 111 amylase (29); catalase (18); lactase (18); inulase (18, 26); 27 123 113 106 103 36 123 111 100 07 sulfatase (16, 18); amidase (17, 18); glycerophosphatase ( 1 ) ; emulsin ( I S ) ; and esterase ($2). I n addition to the above, As shown in Table I, that concentration of sodium chloride the author found evidence of the presence of small amounts most nearly approximating the saturation point (20 per cent) of alcoholoxidase and phenolase in the preparation. Certain properties of the amylase (diastase) were studied gives most satisfactory results for the preservation of diastatic by Sherman and his co-workers (23, 2 4 , and others (9, 10, activity in such a dilution of the enzyme. It is interesting to note that the diastatic enzyme passes 21). through a parchment paper. When water and the enzyme The amount of ash in the preparation of enzymes was found to be about 15 per cent, sometimes over 28 per cent, solution are separated by a membrane, passage of the enzyme and consisted chiefly of magnesium, phosphorus, and small through the membrane is accelerated by the presencc of an electrolyte, such as sodium chloride. This will be discussed in amounts of calcium. a subsequent paper.
T
Culture and Preparation
The fungus of Aspergillus oryzae was cultured on steamcooked wheat bran containing about 50 per cent of water a t 32" C. It took about two days to get the maximum growth of the fungus. Then the cake obtained from the bran was crushed and extracted with water, yielding 100 grams of extract from 100 grams of the cake. Such an extract gives in the neighborhood of 200 Lintner's value (L. V.) of diastatic strength by the author's scale (4),depending upon the growth conditions of the fungus. Takamine (27) described in detail 1
Received August 19, 1931.
Heat Resistance of Diastatic Activity
A 0.06 per cent solution of the purified enzyme obtained by alcoholic precipitation, whose strength is 4500 L. V. a t 50' C., was heated a t varying temperatures and each strength determined a t definite time intervals a t a temperature of 35' C.by the method which is fully described by the author (0,using 10 cc. of 0.2 N sodium hydroxide as the inhibiting agent for the enzyme activity, During the period of incubation a t 30", 37", and 40" C., the strength of the enzyme was slowly diminished. However, when incubating a t the temperatures
INDUSTRIAL A N D EIVGINEERING CHEMISTRY
December, 1931
1425
of 45", 50", and 55" C., the enzymatic strength was rapidly destroyed as time progressed, as shown by Table 11. Table 11-Effect TIME Hours 3OOC. 0 n.5
1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
of Heat on Diastatic Enzyme Solution LINTNER'SVALUESAT: 37'C. 40'C. 45OC. 5 0 ° C . 55OC. 4000 3033 2383 1900 1500 1283
and therefore (28).
n=l+
... ... ... ... ...
2383 2383
... ...
.
I
.
...
... ...
...
...
Relation between Temperature and Optimum pH
The optimum temperature of the enzyme under certain conditions-namely, pH 5.4 and a digestion time of one-half hour-was found to be between 55" and 60" C., as shown in Table IV.
If a
=
=
K(o
- x)
(b
- x ) (c - x )
(1)
b = c . . . . . . . . ., the equation becomes
To find the value of la, let C1 and C, represent the concentration of the reacting substance in the solution a t the end of certain time tl and tt. Equation 2 becomes, if C = CIwhen t = tr, etc., a - x = C; d C = - d x ;
-dC = KCn at
2
- x)Z
(5) of Hydrolysis of Soluble Starch by Aspergillus oryzae Diastase a t 33O C. STARCE TITRATION x
n
cc.
15 20 30 45 60 90 Av.
2.41 2.24 2.39 2.46 2.22 2.34
19.25 15.25 10.75 8.00 6.50 5.05
a
-x
2.0000 0.1508 1.8492 0.1847 1.8153 0.2560 1.7440 0.3440 1.6560 0.4190 1.5810 0.5346 1.4060
K
GLUCOSE Gram
0.00272 0.00254 0.00245 0.00231 0.00221 0.00211
0.1677 0.2052 0.2814 0.3822 0.4656 0.6940
It may be seen that the constant K decreases as the time of incubation is prolonged. According to Hill (7), the reaction of the hydrolysis is always partially reversible. Therefore, the actual velocity of the hydrolysis by the enzyme is equal to the difference between the velocity of the hydrolytic and of the synthetic process a t any given time. The relation between the amount of hydrolyzed starch by the enzyme and the corresponding time necessary for its hydrolysis is represented by Figure 2. of Ratio of Diastase to Starch on Rate of Hydrol yair RIIDUCINQ R A ~OQO RATIO OF S T A R C E SUGARS AS HYDROLYZED STARCH TO ENZYME GLUCOSE TO ENZYME Gram 2000 0.150 137.1 1000 0.245 110.3 500 0.404 96.2 70 4 333 0.469
Table VI-Influence
where n is the number of molecules taking part in the action. From Equation 3,
= K(a
where K is the velocity constant of the reaction, or on integration we have
therefore,
(3)
l-m=s%l-lJR€ and Optimum pH Curve
any given time by 2 in grams, the velocity of hydrolysis is If this quantity is proportional to the quantity of starch remaining, which may be represented by a; - 2,where a is the original quantity of starch, then the velocity of hydrolysis will be given by the equation,
0
According to the law of mass action, the velocity of a reaction may be expressed by the following general equation:
(4)
dx/dt,
TIME
Velocity of Hydrolysis of Starch
$
Figure 1-Temperature
Table V-Rates
Table IV-Optimum Temperature a t pH 5.4 55 60 70 Temp., a C. 25 40 50 1360 2816 3566 4000 4000 900 1. v.
- log h
- log c1
Substituting the data in Table V for Equation 4, "n" is found to be about 2.34. Therefore, the reaction appears to be bimolecular. Now, assuming the hydrolysis of starch by the enzyme to be a bimolecular reaction, if the time is expressed by t in minutes, and the quantity of starch hydrolyzed after
Table I11 shows that there is a definite relationship between the temperature and the optimum pH of the reacting substances for a given enzymatic activity. At 30" and 37" C., the values for the optimum pH were found to be pH 5.2; at 50" C., pH 5.4; a t 60" C., pH 6.2; and a t 65" C,, pH 6.6. The elevation of temperature raises the optimum pH markedly. This holds true for temperatures above 50" C. However, a t temperature8 below 50' C. (for example, 37" and 30" C.) the optimum pH is practically unchanged, provided there is half an hour of digestion under the given conditions. The relations are shown in Figure 1. Thus, the result does not appear to be a linear function of the temperature as found by Olsen and Fine (20) with the mixture of wheat and malt flour. Table 111-Relation between Temperature and pH AT 50' C. AT 30' C. AT 37' C. AT 60' C. AT 65' C. pH L . V . pH L.V. pH L . V . pH L.V. pH L.V. 5 . 4 2850 5 . 8 2033 6 . 4 1466 4 . 3 3033 6 . 8 3291 5 . 4 2066 6 . 2 2160 4 . 8 3200 6 . 2 3566 6 . 2 3062 5 . 2 2108 5 . 6 2216 5 . 0 3383 6 . 1 3493 6 . 4 3275 4 . 8 2060 5 . 2 2300 5 . 2 3475 5 . 6 3383 6 . 6 3325 4 . 2 2033 4 . 8 2216 5 . 4 3566 5 . 2 3200 0 . 8 2250 3 . 9 1900 3 . 9 2183 5 . 8 3383 6 . 0 2000 7 . 0 1476 . . . . . . . . . . . . 6 . 2 3200 . . . . . . . . . . . . . . . . . . . . . . . . 6 . 4 3033 . . . . . . . . . . . . . . . . . . . . . . . . 6 . 6 2583 . . . . . . . . . . . . . . . . . . . . . . . . 7 . 0 2300 . . . . . . . . . . .
log t l log cz
I N 10 CC. ENZYME OF SOLN.
Gram
0.001 0.002 0.004 0.006
INDUSTRIAL A N D ENGINEERING CHEMISTRY
1426
The ratios of the amount of substrate starch (2 per cent solution) and of starch hydrolyzed by diastase to reducing sugars to the amount of enzyme after incubation for 30 minutes a t 50" C., a t a p H of 5.4, is given by Table VI. Protease
For the formol titration (the demonstration of the existence of proteolytic enzymes), three different solutions were used. I n the k s t experiment, 100 cc. of a 4 per cent solution of casein in 0.5 per cent sodium carbonate were digested with
Vol. 23, No. 12
incubated a t a temperature of 37" C. for 30 minutes. This enzymatic activity was then stopped with 10 cc. of 0.4 N sodium hydroxide (this amount of alkali was found by experiment to be sufficient to stop the activity for this buffered solution). The inversion capacity of the enzyme preparation is shown in the last column of Table IX, indicating that the preparation possesses only 2.27 to 3.14 times its own weight of sucrosehydrolyzing activity a t given conditions. of Concentration of Invertase upon 2 Per Cent Sucrose Solution a t 3 7 O C. TREATED SUGAR RATIO TOTAL INVERT SOLN. REQUIRED OP SUSUGAR PRORATIOOF HYENZYME IN 10 FOR 5 c c . FEECROSE TO DUCED AS DROLYZED Succ. OF SOLN. LING'S SOLN. ENZYME GLUCOSE CROSE TO ENZYME Gram CC. Cram 0.2 8.2 10.00 2.27 0.15 7.95 13.33 2.44 0.1 11.5 20.00 2.54 0.075 15. 28.08 2.04 19. 0.08 33.33 2.89 22.75 0.05 40.00 2.81 42.5 0.03 2.82 86.00 0.025 50. 80.00 2.94 0.02 58. 100.00 3.14
Table IX-Influence
I
The kinetics of .sucrose hydrolysis by invertase from different sources has been studied extensively (7, 16, 25), and the reaction was clearly shown to be monomolecular. Catalase TIP€ IN MlMlTES Figure 2-Reaction
Velocity Curve
10 cc. of 2 per cent enzyme solution a t 40" C. A portion of 25 cc. of the mixture together with 10 cc. of 40 per cent neutralized formaldehyde was titrated a t the end of each time interval with 0.1 N sodium hydroxide, using phenolphthalein as indicator. This test was repeated using solutions of 1 per cent fibrin in sodium carbonate and 2 per cent peptone in sodium carbonate, both these solutions being adjusted to a pH of 8.3. A few drops of toluene were added to each solution before incubation in order to inhibit bacterial growth. The results shown in Table VI1 represent the corrected figures after the subtraction of the blanks. TIME
Hours 0 1 3 22
Fifty cubic centimeters of approximately 2 per cent hydrogen peroxide solution were treated with 5 cc. of a 1 per cent enzyme preparation of Aspergillus oryzae a t 24' C. The solution had previously been buffered with dihydrogen potas-
Table VII-Formol Titrations 0.1 N SODIUM H Y ~ R O X ISDOE L N . TO 25 cc. OF: Casein mixture Fibrin mixture Peptone mixture CC. CC. CC. 0 0 0 4.1 0.0 1.8 7.9 1.9 2.9 18.8 12.4 5.9
The titration curves are shown in Figure 3. Rennet
Milk-coagulating power by the enzyme preparation was determined a t 37" C. The results are tabulated in Table
VIII.
T I E Figure 3-Form01
IN MXRS Titration Curves
Table VIII-Coagulation of Milk 1% enzyme (4500L. V. at 50' C.),cc. 0.5 0.5 0.5 0.5 0.5 5 8 10 12 14 Milk (sp. $r. 1.03). cc. 30 40 45 Time required to coagulate at 37' C.,minutes 15 25
sium phosphate and sodium hydroxide to a pH of 6.9. The actual results after subtraction of the blank are given in Table X. (The blank, which was run for 60 minutes, amounted to 0.2 cc.)
However, a t 57" C. the enzymatic activity almost disappeared.
Table X-Oxygen Evolved from Hydrogen Peroxide by Catalase Time, minutes 0 5 10 15 20 25 30 35 40 45 50 55 00 2.0 3.8 5.0 8.0 8.0 7.3 7.8 8.0 8.9 9.2 9.5 Evo1vedoxygen.c~.0
Invertase
The results show that the diastatic enzyme preparation contains a considerable amount of catalase.
The presence of invertase in 4500 L. V. diastatic enzyme preparation a t 50" C. was determined by the same method as the estimation of diastase (4, but with the following conditions: 100 cc. of 2 per cent cane sugar solution buffered by means of a mixture of 25 cc. of 0.2 M acid potassium phthalate and 10 cc. of 0.2 M sodium hydroxide to p H 4.9, which was found to be optimum for this enzyme, were
..
Phenolase
Fifty cubic centimeters of 0.1 per cent hydroquinone were treated with 5 cc. of 2 per cent enzyme solution a t 37" C., the pH being 6.4 by electrometric determination. After three hours it was found that the solution with the enzyme
INDUSTRIAL A N D ENGINEERING CHEMISTRY
December, 1931
produced 0.0054 gram of quinhydrone as compared with the blank. This reaction is evidently due to the oxidation of the hydroquinone in the presence of phenolase. Alcoholoxidase
To 100 cc. of a 4 per cent ethyl alcohol solution neutralized with calcium carbonate to pH 7.2, 10 cc. of 0.5 per cent enzyme solution which had been boiled were added and incubated for 20 hours a t 35’ C. This experiment was also run using an active enzyme solution of the same strength, and the results were as follows: 1 (Boiled enzyme s o h ) 2 (Unboiled enzyme soln.)
pH 6.9 6.9
pH after 20 hrs.’ treatment 6.9 6.4
The change of the acidity is apparently due to the formation of acetic acid by the action of alcoholoxidase on the alcohol. Literature Cited (1) Akamatsu, S.,Biockem. Z., 142, 184 (1923). (2) Atkinson, R. W., Proc. R o y . S O L .(London), 32,311 (1881). (3) Chittenden, R. H . , and Painter, H. M., “Studies from the Laboratory of Physiological Chemistry,” p. 52, Sheffield Scientific School, Yale, 1885.
(4) Harada.
1427
T.,IND.ENG.CHEH.,Anal. Ed., 3,1 (1931).
;1: ::::try; ~:,~~~12$.;::q’l”g~:f.’”).
(7) Hil!, C. A., J . Ckcm. Sor.. 83, 578 (1903). (8) Hudson, C. S., J . A m . Chcm. Soc., SO, 1160 (1908). (9) Kawakam, J., J . Pharm. S O C J. a p a n , 49, 346 (1929). (IO) Kobayashi, Y . , Biockem. Z . , 203, 334 (1928). (11) Kopaczewski, W., Ibid., 44, 349 (1912). (12) Leibowitz, J., and Mechlinski, P., Z . pkysiol. Chem., 154, 64 (1926). (13) Matsumoto, A., Acto Sckol. M e d . Unio. I m p . Kiofo, 2S6, 10 (1928). (14) Myers, R. C., and Scott, L. C., J . Am. Chem. Soc.. 40, 1713 (1918). (15) Nelson, J. M., and Vosburgh, W. C., Ibid., 39, 790 (1917). (16) Neuberg, C., and Kurano, K., Biochem. Z.,140, 295 (1923). (17) Neuberg, C., and Linhardt, K., Ibid., 142, 191 (1923). (18) Nishimura, S.,Chem. Zellc Gcwebe, 12, 202 (1925). (19) Ogawa, I., Biochem. Z.,149, 216 (1924). and Fine, M. S., Cereal Chem., 1, 215 (1924). (20) Olsen, A. G., (21) Oya, T., Biockem. Z., 207, 410 (1929). I b i d . . 217, 42 (1930). (22) Rona, P., Ammon, R . , and Werner, M., (23) Sherman, H. C., and Tanberg, A P., J . A m . Chem. Soc., 38, 1638 (1916). (24) Sherman, H. C., and Wayman, M., Ibid., 41, 231 (1919); 43, 2454 (1921). (25) Suzuki, B.,J . Chem. SOL.J a p a n , 44,231-96 (1923). (26) Takahashi, Y . , Biochem. Z., 144, 199 (1924). (27) Takamine, J.,J. IND.ENG.CIIEM.,6, 824 (1914). (28) Van’t Hoff, J. H., “Studies in Chemical Dynamics,” p. 103,Chemical Publishing Co.,Easton, Pa., 1896. (29) Wohlgenmuth, J., Biochem. Z., 39, 324 (1912).
Effect of Fine Inerts on Agglutinating Power of Pittsburgh Coal’” Joseph D. Davis and W. D. Pohle PITTSBURGH EXPERIMENT STATION, U. S. BUREAUOF MINES,PITTSBURGH, Pa.
A study of the effect of addition of fine inerts on the HE term inert, asused The purpose of the work agglutinating power of Pittsburgh coal has been made h e r e , will be underwas to apply a l a b o r a t o r y in which the test as modified by Marshall and Bird was stood to refer to subtest to various s y n t h e t i c used. A further modification of the method used by stances s e p a r a b l e from the mixtures of Pittsburgh coal these writers in which electrode carbon was substituted coal by m e c h a n i c a l means with inerts occurring natufor sand was also tried. w h i c h d o n o t form a coke rally in the coal and to deterIt is concluded that by the agglutinating-power test, on d e s t r u c t i v e distillation. mine, if possible, the applias used by Marshall and Bird, the strength of the coked Thus a l l m i n e r a l m a t t e r cability of the test for estibuttons obtained is increased by substitution of fine occurring in the coal is inert mating their effect in coking inert material up to 25 per cent of the coal under test. in t h i s s e n s e , as is a l s o power. The Marshall-Bird The effect varies somewhat with the character of inert fusain, or mineral charcoal. test (3) was selected for the used, but in every case an increase was found. It is possible that there are work because it has b e e n With the substitution of electrode carbon for the sand o r g a n i c constituents, other shown to give an approximate prescribed in the Marshall-Bird test, mixture of fine than fusain, making up the measure of the comparative mineral inert with the coal used produced a weakening coal s u b s t a n c e , which are coking power of coals. If this of the coked buttons obtained in the test, which, in the inert, but so far this has not is true, then the test should case of fusain, however, was slight for percentages under been proved. It is not necesalso show the effect on coking 20 per cent. This behavior of fusain was checked by sarily true that such materials power of adding different inactual coking tests on SO-pound charges. are inert in thoir effect on the erts in varying amounts to strength of coke o b t a i n e d one and the same coal. from coal containing them; in fact, they may even improve the Coal and Inerts Used strength of the coke when admixed with the coal and coked The coal used in this work, as well as the samples of hard under favoring conditions. Unquestionably it is of practical importance to know the effect of inerts on the coking property, and soft fusains, was obtained from the Banning No. 1 mine because it is possible to regulate their amount in coal sup- of the Pittsburgh Coal Company, located near Van Meter, plied for coke-making. The desirability of finding a simple Fayette County, Pa. The proximate and ultimate analyses laboratory test capable of measuring the effect of inerts on of the coal and the proximate analysis of the fusians are coke strength is thus evident. It has furnished the incentive given in Table I, for the work now to be described. Table I-Analysis of Banning (Pittsburgh Seam) Coal a n d Fusains
T
Occurring Therein
August 6, 1931. Presented before the Division of Gas and Fuel Chemistry a t the 80th Meeting of the American Chemical Society, Cincinnati, Ohio, September 8 to 12, 1930. 2 Published by permission of the Director, U. S. Bureau of Mines, the Carnegie Institute of Technology, and the Mining Advisory Board. (Not subject to copyright.)
VOLA-
1 Received
FIXED MOIS MAT- CAR-
HY-
TILE
SAMPLE Coal Softfusain Hardfusain
TURE
%
0.9 2.1 0.4
TER
%
33.7 11.0 17.3
BON
%
ASH
%
NI-
DRO-
CAR-
TRO-
GEN
BON
DEN
OEN
%
% 7.1
%
59.0 6.4 5.5 78.9 8.0 54.8 27.5
%
78.4
1.5
O X Y - SULFUR
%
1.1
