THE PROPERTIES 0%‘ POLYZIME.
1261
[CONTRIBUTION PROM TAKAMIKE LABORATORY. ]
THE F ~ O F ~ OF ~ ~A ~SPECIALLY E § P EPARED ENZYMIC NG ITS STARCH LIQUELT DIASTASE. KOKICHIO S H l M h . Receive3 March 24, 1920.
The aim of this study and article is to compare certain properties of ?olyzime, an enzymic extract of Aspergillus Oryxae prepared by special proccsses, with those of standard mait extract.
A. Literature. “Polyzime,” recently invented by Dr. J. Takamine, is an aqueous extract of diastatic enzymes, containing many other enzymes, made by a specially prepared culture of the fungus Aspergillus Oryxae on media consisting mainly of wheat bran. As many reports have been published ab out the enzymes produced by this fungxs (generally using Taka-Diastase as sample) a briei: abstract will therefore first be given of the important literature directly pertaining to this subject. J. Wohlgemuthl found in a sample of Taka-Diastase amylase, maltase, trypsin, lab. erepsin, lipase and haemolysin. This amylase can resist ger acid than can pancreatin diastase. C. Sherman and A. P. Tanberg2 showed that the amylase of Aspergillus Oryzae exerts its maximum activity9 both amyloclastic arid saccharogenic, in a very slightly acid medium; and as prepared by them i t has higher amyloclastic, but lower saccharogenic power than the most active malt amylase preparations yet recorded. He C. Sherman, A. W. Thomas and MI. E. Baldwin3 reported that the amylase of AspergiUus Oryzae showed activity from PH 2 .6 With optimum at PH 4.8. Pancreatic amylase was active between the limits of P s 4 aiid io with greatest activity at about 7, the solutions commonly considered neutral showing under similar conditions a PHvalue of 5.8. Malt amylase was active between PH 2 . j and g with optimum activity a t 4.4 to 4 .5. The influence of the concentration of electrolytes, as distinguished from concentration oi hydrogen alone, appeared greatest in the case of amylase of pancrealin, and least in the case of the amylase of Aspergillus Qryzae.
Experimental. The rcsults of our numerous experiments on Polyzime preparations and malt extract are as follows. “Zur Kenntnis der Taka-Diastase,” Biochem. Z., 324 (1912). “experiments upon the Amylase of Aspergillus Oryzae,” THISJOURNAL, 38, 1638 (19aG). 3 Influence of hydrogen concentration upon enzymic activity of 3 typical amylases: THISJOURNAL, 61, 231 (191.9). 2
JOKICNI TAILAMINS, JR., ANI) KOKICHI OSEIIMA.
2262
B. General Properties of Polyzime. a. Sp. gr., 1.03-1.06.
b. Amyloclastic (starch liquefying) power by Wohlgemuth's method: D 3,000;
40' 30 min. =
40
s 24 hours = II5iOOO.
Saccharogenic (starch saccharifying) power by Lintner's method : Lintner's Vdue (21') = 43. 1,intner's Value ( 5 0 " ) = 150. d , Chemical Composition: Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87.5% Solid Matter.. ......... ............ 12.5 Mineral Matter.. .......................................... I .5 Acidity with rosolic acid as indicator: I O cc. Polyzime required 5 cc. 0.1N acid to neutralize. . . . . . Total nitrogen.. , . .......... 0.5 Protein precipitated with Stutzer's reagent. . . . . . . . . . . . . . . . . . . . 0 . Reducing sugar as &glucose.. .. 2 .oo/o G.
.
1.5
Dextrin .......... I .3 e. Destructive influence of heat. By placing Polyzime on a water bath the following decreased percentages of diastatic power after 3 hours were obtained: Temperature of Water Bath. "C.
Decreased Diastatic Power.
%. 0
0
55.0
95.0 98.0
Prom the above experiments and many others, i t is evidently necessary to preserve Polyzime a t a temperature lower than 40" (104"F.) ; if the temperature is higher, the diastatic power is decreased proportionately. It is also shown that the optimum ternperature for diastase conversion has a destructive influence upon Polyzime itself during time period of 3 hours or over. f. Preservation of strength: If Polyeime is kept in a closed barrel a t ordinary temperature it can be preserved for half a year with practically no change of the diastatic power.
C. Amyloclastic Power of Polyzime Compared with Malt Diastase. X. Method of Experimentation.-There are quite a number of methods for this purpose, such as, for example, Dr. J. Takamine's simple quantitative method of diastatic power determination ;l William A. S. A. 'IVaksman's.4 Among these, the Johnson's;2 J. Wohlgem~th's;~ most accurate appears to be that of Wohlgemuth, and to make subseuent data clear, we give here a condensed description of it, as follows. ' J . Soc. Chem. Id.,17,437 (1898). "A Proposed Method for Routine Valuation of Diastase Preparation," TRIS 30, 801 (1908). 8 Biochem. Z., 9, I (1908).
2
IJOURNAI;, 4
THISJOURNAL, qa,
293 (1920).
THE PROPERTIES
OF POLYZIME.
1263
Wohlgemuth's Method and Scale.-Place in a series of test-tubes diminishing amounts of the enzyme solution t o be tested. Introduce into each tube 5 cc. of a I % solution of soluble starch and place each tube immediately in a bath of ice-water. Transfer the tubes one a t n time to a water bath a t 40' for 30 minutes (or 24 hours with toluol). At the end of this time transfer again immediately t o the ice-water bath to stop the action. Dilute the contents with 20 cc. of water, add one ce. of 0.01N solution of iodine and shake well. One usually obtains colors from blue through violet, red and yellow t o colorless, indicative of starch, erythrodextrin, achrodextrin, ete. Take the tube in which the blue or violet has entirely disappeared, giving place t o either red or orange-red color as judged by Mulliken's color standard sheet C;note the amount oE enzyme solution in this tube and calculate the power of the enzyme as the number of cubic centimeters of one per cent. starch solution which is digested t o this stage in a given time by one cc. of enzyme solution. Thus if 0.2 cc. (or gram) of the substance completely digests 5 cc. of starch solution in 30 minutes a t 40'. then one cc. of that solution will be able t o digest 2 5 0 cc. of one per cent. starch solution; or its power is then stated: 40° D 30 d n . = 250 (Wohlgemuth Scale) Here the time of reaction is brought into the final expression of diastatic powers that the method may be applied to substances whose power is very slight by allowing them t o act a long time.
11. Optimum Reaction of Starch Solution.--We tried to find the best degree of acidity or alkalinity of starch solution for liquefaction with Polyzime. For this purpose we always used Wohlgemuth's method with m e per cent. souble starch solution, but with different concentrations of acid or alkali and a t different temperatures. The soluble starch used was a standard preparation and the acidity or alkalinity of the solution was regulated by adding hydrochloric acid or sodium hydroxide, with rosolic acid as indicator. The malt diasiase used was one of the a.
20'
200 and 2 hours D2h. -
Malt Extract.
Reaction of starch soln.
0.0004 M HC1
430 660 930
0.0003 0.0002 0.0001
0.nom 0.001
(neutral) MNaOH
0.0002
0.0003
b. 50' and Reaction of starch soh. 0.0004 0.0003 0.0002 0.0001
M HCl
2
hours.
Pnlyzime.
5600 6000
6000 6240
930 930 730
4200
660 530
2500
5000
3600
DiEY
Malt Extract.
Polyzime.
960
3320
I700
I 6660
2800
0.0002
1870
16660 16660 r 6660 14000 I 2400
0.0003
1670
IT200
o.oooo (neutral) a. 0001 M NaOH
2800 2 800 2 800
best malt extracts oii the market with a, Lintner's value (saccharifying power) of 380 (50'). The Polyzime used was a standard strength factory product, neutralized with acid. Lintner's value = 150 (50'). These results indicate that Polyzime and malt diastase show a parallel behavior with changing acidity of a starch solution. Their maximum activities are shown in neutral or very slightly acid solutions. Rowever, Polyzime exhibits a 3 to 5 times greater acidity. 11%.Optimum Temperature of ~ ~ ~ ~ ~ e ~ ~ temperature c ~ ~ ~ nac-, , - ~ ~ g celerates diastatic action, a t the same time destroying the diastase itself, Therefore, to determine the optimuni teinperature, it is necessary to con: sides the duration 0.1 digestion. Wohlgemuth's method was used with one per cent. soluble starch solution, the reaction of which wa,s that of o.ooox M hydrochloric acid, with rosolic acid as indicator. The malt extiact and Polyzime were the same as be€ose. a.
2
Hours and Different Tempera.ture.
Tcmperature. 0
c:.
Malt IZxtract.
20
40 50
60
Polyzirne.
4540
660 27 0 0
15000
2 yo0
I
70
1870 833
80
200
1000
357 100
DlOh. = Temperature. C. Malt Extract. I'olyzime.
Duration.
Somin.. .....................
40
700
55 40 hours.. . . . . . . . . . . . . . . . . . . . 58 55
1070 2 700 2 800
24 hours..
5000
6250
b. Different Times and Temperatures.
2
IO" D2h.
..................
3000 4820 16000 I 6660 I 6000
50
2770 1870
40
6600
jj
3700
1 I5000 3 I 600
6250
'The conclusion to be drawn from the above is that the optimum temperature is about 55' for 30 minutes to 2 h.ours digestion and 40' for 2 4 hours. Here also the same relation exists between malt extract and Polyzime diastase..
IV. ~
~ ~ ~ 04 ~ther Arny1oclastic ~ s Q nPowers of Pslyzime and
Diastase Samples. The following results were obtained wi.th Polyzime (dry Taka-Koji and Taka-Koji extract) and malt diastases (the best malt flours and extracts) with the Wohlgemuth method and for saccharogenic power tested by Lintner's method:' I
J. prukt. Chem., 34, 386 (1886).
1265
TI38 ESTERIFICATION OP ALPHS AMINO ACIDS.
The samples tested (all lately manufactured). a. Polyzime “D” (dried Taka-Koj: flour, Takamine Laboratory manufactured).
b. Polyzime (Taka-Koji extract, Takamine Laboratory manufactured), c. Malt flour I. d. Malt flour 11. e. Malt flour 111. f. Malt extract I. 4. Malt extract 11.
D
a . . b..... C.....
40‘ 30 inin.
40’
D 2 h. 2 4000
. . . 4700 ..... 3000 . . . . . 1053
d . . . . . . .... I O 0 0 e . ......... 850
j.... . . . . . . 700
-
D
I
I
-
Zintner’s vaiue. 200.
1,intner’s value. 500.
1~0000
96
I 6000
I I 5000
43
4000
IO000
156
550
..
I 4000
I39
‘.
15000 6600 8632
128
4‘0 400
2 yao
g . . . . . . . . . . 400
400
24 h.
IOOC
100
98
250 150
380 340
Summary. The diastatic power of Polyzirne does not decrease a t temperatures lower than 40’. Below that temperature it preserves its enzymic activities for more than half a year with practically no change. 2. The optimum reaction of starch solution for liquefaction by Polyzime Es neutral or very faintly acid. 3 . Polyzime is 3 to 5 times stronger than ordinary malt extract in its amyloclastic power as indicated by testirig according to Wohlgemuth’s method. 4. The optimum temperature of starch liquefaction by Polyzime is 50’ for a 30 minute to 2 hours digestion and 40’ for 24 hours digestion, althoiigh it shows weaker saccharifying power than malt extract tested by Wntner’s method. 1.
CLIFTON, N. J.
[~ONTRIBUTZON FROM THE
DEPARTMENT OF AXIMAL HUSBANDRY,
UNIVERSITY OP
1 THE ESTERIFICATION OF ALPHA AMINO ACIDS. ILLINOIS.
BY I$. A.
SHONLE AND
H. H. MITCRELI,.
Received March 25, 1920.
In the preparation of amino acids, or in the analysis of proteins by Fischer’s ester method, some means of measuring the extent and rate of esterification would be of value. The rate of hydrolysis of a protein can he accurately follcwed by the Van Slyke amino nitrogen determiiiation (as well as by other excellent methods), but in so far as we are aware, no accurate method has been devised for following quantitatively the subsequent esterification. Such a method would permit a direct determination of the relative merits of the different methods of esterification and
