2 :2:i2:33;; =2;

 

“we .
a...»
h. .
w .
.U.
i. o
m .
A w
2.1.

..n

o (\3

5.:‘J‘Tf: ‘1‘ .1 '\.-' If; 2 ’78
. i I
K“ o o

c. 1m

.9": ’x
c {‘50 5|

a i
36“,.
a ,0
~ .
r. u
o.:'
bk‘
.1.”

a. e?
a. c.

'\ II.

 

 

vferv»

 

 

 

 

      

 

I .--‘-- " .4! " ."- . . 1 .~, 4‘» t"  .~.
,.» u. 4.. owg""-*;=*~tq a, ,- ,13' A; kg: cloth-v. x... Av 4:44”
‘- ‘ ' . ' «a ' '&K. I'. '.. .
, i, _
I “I

‘
4‘ .‘-
L. w.
qixi‘a’yr

LIK

    
 

THEIPHOTEDLYTIG AJTIVITX OF TAKA-DIASTASE
FROM ASPPBGILLUS ORYZAE ON CASEIN

 

By

Robert Clement Lieaer

A THESIS
Submitted to the school of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE

Department of Chemistry
1952

,4 "'"-—. ‘\
I
C“ 3-

\Nn"
‘0

AQKNOWLEDGMENT

The author wiehee to expreee hie gratitude to Dr.
H. A. Lillevik and other members of the faculty of the
Chemistry Department of Michigan State College, to when
he ie greatly indebted for the help and encouragement

' received during this work.

nr' 4" mi? s‘
:'.,, .?&3 I t;';

TABLE OF CONTENTS

Introduction . . . . . . . . . .
Hietoricel . . . . . . . . . . .
Experimental . . . . . . . . . .
A. “nipment 089d 0 e e e e
B. Materiele and Solutions
0. Experimental Methode . .
De Tablefl Of Resal‘. e e e
E. Figure. 0 e O o e o e e
Discu'alon O O 0 O O O O O O O 0
Summary . . . . . . . . . . . .

Bibliography 0 e e e e e e e e e

Page

I. INTRODUCTION

The enzyme catalyzed hydrolysis of proteins has been
studied since the early 1900's. The objectives of such work
have included those of seeking information on the structure
of proteins and the nature of the proteolytic reaction.

The following observation was made in this laboratory.
Tana-Diastase when introduced into a clear, neutral solution
of casein. produced after several hours at room temperature‘
a white, turbid, cloudiness. It had the appearance of milk!
This transformation could be construed as the result or the
influence of proteolytic enzymes in Take-Diastase.

It is not known that any systematic studies have been
reported on the factors influencing the activity of this
particular combination of substrate and enzyme. Hence, the
following investigation was undertaken to contribute infor-
mation concerning such factors, as well as, the physical

and chemical nature of the transformation.

II. HISTORICAL

A. The Source 23 Proteolztic Enzym .
TakapDiaetase is prepared from a culture of the mold

Aspergillus orzzae according to the process patented by
Takamine (1923). The sold is grown on wheat bran, the
moldy bran is extracted with water, and alcohol is then
added to a final concentration of 70 S by volume. The pre-
cipitate thus obtained is dried and marketed under the name
Take-Diastase. This product has been found to consist of a
number of enzymes which can be demonstrated by their cats-
lytic action on various substrates. fauber (1949) lists at
least twenty-three enzyme systems as being present.
Recently, attempts have been made to isolate a crys-
talline proteolytic enzyme from TakapDiastase. Crevther and
Lennox (1950) using a combined alcohol and salt precipita-
tion method, obtained such a crystalline fraction. Its
proteolytic activity on gelatin was comparable to that of
crystalline trypsin. The preparation, though crystalline.
was not pure. It could be demonstrated that solutions of
the crystals contained at least 2 proteolytic enzymes; one
reduced the viscosity of gelatin, and the other acted on the
lower molecular weight components of gelatin. Esterase
activity was also associated with the crystals. Whether this
indicated the presence of a true esterase or the unspecific

action of a peptidases was not determined.

Gillespie. Jermym and woods (1952) employed paper
chromotgraphy and paper electrOphoresis to achieve a partial
separation of the proteolytic enzymes present in Aspergillus
oryzae cultures. These workers obtained four main fractions
and at least three subsidiary components. The four main
fractions exhibited a number of activities among which were
two proteinase activities on gelatin.

Astrup and Alkjaersig (1952) have attempted to classify
enzymes by the effect of cationic and anionic detergents and
natural inhibitors on the proteolytic activity of a number
of enzymes. They observed that all the enzymes studied were
inhibited by blood serum and mentioned that the proteolytic
activity from Aspergillus oryzag in fibrinolysis was inhi-
bited by laurylamine and activated by laurylsulfonate and
cetyl-pyridinium chloride.

B. The Substrate Casein.

The nature and properties of casein have been well
summarized by Sutermeister and Browne(1959) and since that
time by Momeekin and Polis (1950).

Casein. a phosphOprctein, was long considered to be a
I'pure" protein. However, it became apparent, particularly
from the studies of Linderster-Lang (1925), (1929) and
others that a modification of this view was necessary. The
electr0phoretic investigations of Hollander (1959) demon—
strated that casein is composed of, at least, three indepen~

dently migrating components, which he designated st 3?)“

and YC-casein in order of their decreasing mobilities.
Warner (1944) discussed previous methods for fractionating
casein and devised an improved chemical method for separa-
ting electrophoretioally homogenous 0(- and fi-casein.
Gordon, Bemmet, Cable and horris (1949) published on a
thorough analysis of the amino acid composition of 4- and .
(5- casein, prepared according to Warner. The s(- fraction
was found to contain a greater number of amino acids with
polar side groups whereas the 6- component possessed the
more non-polar type. In addition,the ratio of phosphorus to
nitrogen in the aL-component was appreciably greater than
that of E's-casein.
Warner and Polls (1945) made a viscosimetric study on

a proteolytic enzyme contained within casein which previously
had been called 'Galactase.‘ They stated that the viscosity
change occurring at an optimum pH of about 8.5 could be
attributed to an enzyme catalyzing the slow hydrolysis of
casein in solution. It was also noted that the enzyme
within casein could be inactivated by heating a solution to

80° C. for ten minutes.

0. Proteolztic Activity 25 fake-Diastase from Aspergillus

0r: 280s
Since the terminology used by enzyme chemists has

 

varied, the term proteolytic activity as used herein means:
any catalytic action of an enzyme that causes a detectable

physical or chemical change which leads to or results in

hydrolysis of the intact protein.

Vines (1910) found that Takapniastase had a marked
proteolytic effect upon fibrin and Witte peptone at 67° 0.
Upon fractionating the crude enzyme material with 50‘s ethyl
alcohol, there resulted an extract that catalyzed the
hydrolysis of fibrin, which was termed as having ”ereptase“
activity. The alcohol insoluble residue, when extracted
with water, showed activity upon peptone only and was
characterized as having “peptase' activity. The results
were arrived at by use of a tryptophane color test on
filtrates of the digests.

Wohlgemuth (1912) carried out similar studies and
included skim milk as substrate for investigating rennin
activity contained in TakapDiastase. In this connection
it was observed that at 38° 0. milk clotted. Using the
Gross-Fuld method of analyzing (described by Tauber (1949))
other protein digests at 58° 0., he concluded that 1 gram of
Take-Diastase was equivalent to 100 c.c. of human or canine
pancreatic Juice. In general proteolytic action upon 5-6 %
protein substrates was. like that of trypsin. strongly
inhibited by blood serum of dog and horse. In contrast, an
activation resulted upon digestion of peptone substrates and
was greatest in neutral or slightly alkaline media.

Szanto (1912) made a comparative study of the effect
of weak and strong acids, bases. and neutral salts on
proteolytic activity, in Which TakasDiastase was used as one

source of enzyme. It was concluded that compared to trypsin

and pepsin, Taha-Diastase activity was: 1- not materially
influenced by neutral salts, 2- more sensitive to mineral
acids (e.g. in their presence it was irreversibly inacti-
vated), 5- less sensitive to organic acids, 4- not destroyed
by alkaline treatment but diminished, and 5- generally not
affected by carbohydrates, except slightly by fructose.
These conclusions were based upon digesting l S casein at
38-90 C. for 1 hour and measuring the activity by the afore-
mentioned Gross-Enid method.

Okada (1916) incubated 4 % Witte-peptone and 10 %
Take—Diastase solutions at 87-39° O. and found an optimum
pH 5.6. The result was based upon Sorensen (1908) formol
titration analysis of the digest.

Cabins and Church (1923) reported that the optimum pH
for Take-Diastase digestion of 0.5 % casein.depended on the
method of following activity. If casein disappearance was
measured by the Gross-Fuld method, the optimum was pH 8.0.
On the other hand, liberation of amino nitrogen as determined
by Van Slyke analysis exhibited an optimum at pH 6.2.

Digesting 5 % gelatin or fibrin or skimmed milk with
1 % Take-Diastase at 30° 0., Nishikawa (1927) confirmed the
observations of wohlgemuth that trypsin and rennin type
enzymes were present. He stated that the optimum pH for
rennin activity was 5.2-6.7 and that metal ions such as Zn”;
Cu“, and Hg}¥inhibited the tryptic-like activity of Tatar
Diastase on gelatin. Activity was measured by the alcohol
titration method of Willstatter and Persiel (1925).

Kawakami (1929) stated that at digestion temperature
of 65° 0., Take-Diastase was inactivated as shown by no
increase in alkali or formol numbers. It was noted that
during the first 1 to 2 hours, the digestion was most rapid
at 55° 6., and after 6 hours had nearly ceased. At 45° C.
the change was very slow with reaction still in progress
after 80-90 hours. No inhibitory effect could be detected
by ultra-violet light irradiation of the enzyme in solution
if kept cooled during treatment.

Berger, Johnson and Peterson (1937) tested the protec-
1ytic activity of a number of molds among which was included
_§spergillus oryzae. Digesting 4 % gelatin at 40° C. and
measuring activity by the Linderstrsm—Lang (1927) acetone
titration method, they concluded the enzyme had an Optimum
pH of approximately 7. Similarly, from synthetic polypep-
tides there was found amino-peptidase, carboxypeptidase and
dipeptidase activity.

By extracting powdered mycelia from aspergillus organs
with water for 4 hours at 40° 0., Otani (1940) found in the
extract the presence of pepsin-type, rennin-type, and pepti-
dase enzymes.

Lichtenstein (1947) demonstrated that dialyzed solutions
of TakerDiastase were capable of catalytically hydrolyzing
casein, gelatin, and a number of synthetic polypeptides.
These results confirmed the findings of Berger, Johnson and

Peterson (1937).

III. EXPERIMENTAL

A. Eguipment Used:

 

Thermostat .-The constant temperature bath was equipped
with a reservoir bottle to automatically maintain a constant
level of water. The thermoregulator (8.3 Instrument 00.,
Inc.) controlled the temperature at 29.93t-0.l°0.

Glassware.-All pipettes and volumetric glassware were
the Kimble Glass brand. A one milliliter burette, used for
alcoholic titrations, made by Kimble Glass 00., was graduated
to 0.01 ml. and could be read to i 0.002 ml,

Timers.-Ths reaction periods were timed with either a
heylan st0pwatch or a Precision Scientific Co. timer, cali-
brated to tenths of a second,

Viscometer.-Its flow time for 10 ml. of water at 29.9°0.
was found to be 75 seconds.

Polarimeter.-A Precision Polarimeter, model 169, manu-
factured by 0. C. Rudolph and Sons, provided with a sodium
vapor lamp as light source, was employed,

Refractometer.~Tbe Abbe’ refractometer used in these
measurements was manufactured by Carl Zeiss, Germany.

Turbidimeter.-A Model 400, Hellige-Diller Photo-electric
Nephelometer was used. The instrument was calibrated for
percent transmittance or optical density units.

pH Meter.-A Beckman model G, glass electrode, pH meter

was used in making pH measurements.

Dialyzer.-All dialysis were carried out with a rotating
external liquid dialyzer constructed in this laboratory.

Semi-micro IJeldahl Apparatus.-The 100 ml. digestion
flasks and distillation apparatus as modified in this
laboratory was used in the determination of total protein

and non-protein nitrogen.

B. Materials‘ggg Solutions!

.zhg‘gggzgg Sourcs.-Taka-Diastass (marketed by Parke,
Davis d 00.) a yellow, amorphous, non-hygroscopic powder
analyzed for 1.51 x N. It was readily soluble in water and
produced a clear yellowbbrown solution.

333;; Casein Preparatign.~ Casein was prepared by the
directions of Cohn and Hendry (1943). The white product
contained 15.6 i N and 7.52 1 moisture (determined by drying
overnight in an oven at 106° 0.). Electrophoretic analysis
revealed that the material was comparable to that of Warner
(1944) both in number of components and their mobility. No
calcium ion could be detected in the outside liquid of a 6 %
casein solution which was dialyzed against redistilled
water.

‘Egggh Casein gtggg Solution.-Six grams of air dry casein
was weighed into a 100 ml. volumetric flask. Beventy~fivs
_ml. of glass redistilled water was introduced in small
portions until a smooth paste formed. To this, 20 ml. of
0.2 N NaOH was added gradually, with shaking, until a clear
solution of pH 7 was obtained. The liquid was made to

volume, filtered and a crystal of thymol added as preserva-
tive. The stock solution prepared in this manner was stored
in the refrigerator for immediate use.

The immediate use of the stock solution is imperative.
It was observed by electrophoretic analysis that the protein
in solution underwent a change if allowed to stand for a
period of a week or more. This was particularly true if the
solution was allowed to come to a temperature other than that
of cold storage (5° 0.).

Taka—Diastase §§225 Solutione.-The pro-determined amount
of dry powder was weighed on an analytical balance, then it
was dissolved in redistilled water and filtered clear. The
final concentration was eXpressed in terms of milligrams
per milliliter. The solutions were stored in a refrigerator
when not in use. Fresh solutions were prepared each day
prior to use.

0.05 N Alcoholic was.” grams (Baker) potassium
hydroxide (EOE) was dissolved in 62.5 ml. of redistilled
water, and diluted to 1 liter with 95 % ethanol. After
‘ separating from precipitated carbonates the reagent was
standardized against 0.1052 N 301 with methyl red indicator.

Thymolphthalein Indicator Bolution.-The indicator
solution for the Willstatter and Waldschmidt-Leitz (1921)
titration was prepared by diluting 6 ml. of 0.5 % thymol-
phthalein in 95 % ethanol to 100 ml. with absolute ethanol.

‘Q;;_§ Standard fig; §£g_§gg§.-These solutions were

prepared according to accepted standard procedures in

-10-

quantitative analytical chemistry.

Kaeldahl ReagentsIggd Solutions.-These were prepared,
with minor modifications, as described by Clark (1943).

Buffers.- Buffer solutions were prepared according to
the tables in Gortner (1949).

Activator §gg_lnhibitor Reagentg.-All chemicals used to
prepare these solutions were from EKCo., white label, a.0.8.
specifications or C.P. grade reagents. Any not so labelled

are indicated.

0. ggperimenta; Methodg:
General Digestion Procedure.-An appropriate volume

(usually 3 ml.) of 6 1 casein was pipetted into one are

(10 ml. capacity) of a bifurcated test tube, and into the
other arm was placed an appropriate amount of enzyme solution.
Usually the volumes of substrate and enzyme solution were
chosen so as to give upon mixing a resulting digest concen-
tration of enzyme in terms of mg./ml. digest. The reaction
vessel containing the unmixed solutions was placed in the
thermostat for 20 minutes prior to mixing. Digestion was
commenced by tilting the two-branched tube back and forth
10 times. The time of initial contact was taken as zero
digestion time and noted by starting the stop watch. Suit-
able aliquots of digestion mixture (usually 1 ml.) were
removed and quenched at specified intervals for the types
of analysis to be described.

Controls using enzyme solution previously heated in

-11-

a sealed tube to 1000 C. for 1 hour were subjected to diges-
tion in the same manner.

1. The Influence 2; Enzyme Congentration.-A series of
digestions were carried out where the initial concentration
of casein was always 3 X. Take-Diastase concentrations were
taken at 0.5, 1.0, 1.5, 2.0, 5.0, and 4.0 mg./ml. of digest
for each run, respectively. Several types of analysis were
periodically performed upon digest aliquots and are described
as follows:

‘ghg Alcoholic~KOH Titratign'ggg‘thgl Acidity
Change.~0ne ml. digest aliquots were removed at intervals
and total acidity was immediately titrated in the manner
described by Willstatter and Valdschmidt-Leitz (1921). This
method is a modification of Foreman's (1920) original alco-
. holic sodium hydroxide titration. To arrest the digestion,
the aliquots were pipetted directly into 2.5 m1. of an abso~
lute alcoholeindicator mixture already contained in a 25 x
100 mm. test tube. This sample was then titrated to a
distinct blue color; 7.5 ml. of absolute ethanol was added
and the sample again was titrated to the appearance of a
blue color in the solution. During titration the solution
was kept well mixed with the aid of a motor driven glass
rod stirrer. The titer obtained from the aliquot taken
immediately after mixing was first recorded. This initial
titer was subtracted from subsequent titers and gave the
increment in mi. (£>m1.) of standard alcoholic KOH titrated

per ml. of digest. These results representing increase in

-12-

titratable acidity or liberation of acid groups are the
values reported in the subsequent tables as indicated.

It should be emphasized that the digest aliquots were
titrated immediately. It was observed that the alcohol—
indicator mixture did not completely quench the reaction
if it stood around for several hours. When a number of
simultaneous digestions were performed, they were started
at 15 minute intervals, allowing ample time for immediate
titration. The results are reported in Table I and repre-
sented in Figure 1.

Change 331 pg 9}; Hydrogen $93; Activitz.~The
prescribed digestion mixtures were started in an open glass
cup adequate to receive the electrodes of a pH meter with
its temperature adjustment set for 50° 0. Measurements
were made directly on the digestion mixture clamped in the
thermostat. The resulting changes in hydrogen ion activity
are reported in Table II and shown in Figure 2.

Non-Protein Nitrogen LN.P.NL) Formed.-After starting
digestion, aliquots were periodically pipetted into an
equal volume of 20 5 (w/v) Trichloroacetic Acid (T.C.A.).
These samples were shaken occasionally during a period of
1 hour and were then filtered through Whatman #40 filter
paper. Total nitrogen was determined on the filtrates as
described by Clark (1943). Blanks were run on reagents with
the substrate alone. The rate of the increase in non-protein
nitrogen with digestion can be seen in Table III and Figure
3.

-13-

Optical Densitz for Change in Turbiditz.-Five ml.
digests were prepared by the previously described procedure

and placed into 6-inch test tubes and kept in the thermostat
between turbidity readings. At the specified time intervals
the tubes were momentarily placed in the turbidmeter for
Optical density readings. The readings obtained during
digestion are shown in Table IV and plotted in Figure 4.

2. Influence of Substrate Concentration.-A series of
digestions were conducted where substrate concentrations
of 1.0, 2.0, 3.0, 4.0 and 5.0 percent were prepared by prOper
dilution of the 6 f stock casein solution. IJeldahl total
nitrogen analysis were made upon the digests to more exactly
define the substrate concentration. The digest concentra-
tion of Taka-Diastase was always 2.0 mg./ml. One ml. ali-
qucts were titrated with alcoholic KOH according to the
preceding directions. Activity measured by increase in
titratable acidity is given in Table V and illustrated in
Figure 5.

3. Influence ggwpfl‘gg Activitz.~The substrate solutions
in one branch of the digestion tube were adjusted to the
desired pH, using 0.1 N 801 or Neon. The volume required
was predetermined from a titration vs pH curve obtained
from a 3 ml. sample of 6 % casein. One ml. of enzyme
solution containing 12 mg. of TakapDiastase was added to
the other arm of the bifurcated tube and diluted with redis-
tilled water to a volume, which when mixed with the substrate,

would give a casein concentration of 3 S and enzyme

-14-

concentration of 2 mg./ml. with respect to the digest. The
initial pH of the digest was determined with the Beckman

pH meter and 1 ml. aliquots removed for titration. At
subsequent digestion times pH readings on the digest were
taken 30 seconds before the aliquot was removed and titrated.
The results are reported in Table VI and demonstrated in
Figure 6.

4. Influence‘gg Temperature.-To observe the effect of
temperature upon activity, the following procedure was
adOpted. Thermostatically controlled digestion was carried
out at approximately 10° temperature intervals up to 600 0.
One ml. samples were removed and titrated with alcoholic
KOH. The digest concentration in all cases was 3 f with
respect to casein and 2 mg. per ml. with respect to Take-
Diastase. The increase in titratable acidity was recorded
at specified digestion times and data from these experiments
are recorded in Table VII and represented in Figure 7.

5. Influence‘gghégggg’Electrolyte.-The previously
described digestion procedure was employed, with the following
modification. From a l R solution of sodium chloride
(Merck), appropriate aliquots were added to the substrate
before mixing. When enzyme and substrate were mixed. the
resulting digest contained 3 % casein. 2 mg./ml. of Taker
Diastase, and according to the salt added was 0.05, 0.10,
0.15. and 0.20 molar respectively. After equilibrating to
temperature and mixing, 1 ml. digest alquots were titrated
with alcoholic KOH. The results demonstrating the effect

of added ionic strength to the medium are given in Table
VIII and Figure 8.

6. Aotivators and Inhibitors 2£_thg’Enzzme.-Stock
solutions (0.1 H) of the compounds listed in Table IX were
prepared. The solutions were apprOpriately diluted to
10"3 M and allowed to react with 8 mg./m1. of TakapDiastase
for exactly 1 hour in the thermostat. Twenty minutes before
the hour was up an equal volume (2 ml.) of 6 S casein was
introduced into the other side arm.of the bifurcated vessel
used.a At the end of the hour, the contents were mixed for
digestion and aliquots of digest were removed at the begin-
ning and after 1 hour of digestion. A similar digest
without enzyme treatment was used as control. The acti~
vation or inhibition result was arbitrarily taken as the
increase or decrease in titratable acidity over the control.
and is expressed as percent in Table IX.

Hydrogen 133 Treatment 2!. L133 Enzzm .-To study the
effect of added hydrogen ion upon the enzyme, 5 ml. of
solution containing 40 mg. of TakapDiastase. pH 6.85 was
adjusted to pH 3 with 0.6 ml. of 0.1 N H01. The solution
was placed in a constant temperature bath at 30° C. for
exactly 1 hour, after which 0.6 ml. of 0.1 N NaOH was
added to restore the original pH. 1.24 ml. of this solution
was taken and diluted to 2 ml. and mixed with an equal volume
of 6 % casein. Thus the digest was 3 S with respect to
casein and contained 2 mg./ml. of enzyme material. Aliquots

for alcoholic titration were removed at the beginning and

after 1 hour of digestion. A.oontrol was run on an untreated
sample. The results are listed in Table IX.

gxdrole igg'Treatment‘gf Enzzm .-The effect of
hydroxyl ion upon the enzyme was determined in a similar
manner. Five ml. of enzyme solution containing 40 mg. Taka~
Diastase was adjusted to pH 11 with 0.6 ml. of 0.1 N haOH.
At the end of the hour the pH was restored to 6.85 with
0.8 m1. of 0.1 N H01 and 1.32 ml. of this solution was
diluted to 2 ml. The sample was assayed for activity as
previously described. The results are shown in Table IX.

Ultraeviolet Irradiation agiEnzzme Bolution.-Three
10 m1. volumes of Tats-Diastase solution (4 mg./m1.), each
contained on watch glass covers. were placed one at a time
beneath a Kupper-Hewitt ultraeviolet lamp. The samples
were exposed at 40. 50, 60 cm. distant from the light source.
A 2 ml. aliquot of each irradiated sample was digested with
2 m1. of 6 % casein according to the prescribed procedure.
The increase in titratable acidity after 1 hour of digestion
was measured and compared to a control. See Table IX.

Dialysis 25.322 Enzyme Solution.-Fifteen ml. of
a TakaPDiastase solution (8 mg./ml.) was dialyzed against
four 50 ml. changes of redistilled water. Total dialysis
time was 14 hours. Two ml. of 3 % casein was digested with
2 ml. of the dialyzed enzyme solution. The increase in
titratable acidity after 1 hour of digestion was compared

to a control. See Table IX.

-17-

7. PhysicooChemical Changes During Digestion.

Viscosity Change .-The samples were prepared by
the general digestion procedure. The digest concentration
was 3 x with respect to casein and 2 mg./m1. with respect to
enzyme. Immediately upon mixing, 10 ml. of the 'digest was
pipetted into the 0stwald viscometer. Flow times were
determined as rapidly as possible during the early stages
of digestion. For results see Table X and Figure 9.

Polarimeteric Change .-A digest was made 3 5 with
respect to casein and 2 mg./ml. with respect to enzyme. It
was immediately transferred into a 100 mm., Jacketed, polari-
meter tube. The Jacket was maintained at 30° C. by circu»
lating water through from a water bath. Readings were
taken as rapidly as possible until turbidity obscured the
readings. The instrument may be read to 320.002 angular
degrees. For results see Table XI.

Refractometeric Change .-A 3 % casein digest
containing 2 mg./ml. of enzyme was prepared. Immediately
after mixing and at subsequent intervals samples were taken
and read on the refractometer at room temperature. For
results see Table XII.

8. Electrophoretic Analysis.~Two m1. samples of 3 %
casein were pipetted into 4 ml. of phosphate buffer pH 7.0
and ionic strength 0.1 with respect to phosphate plus 0.05
M NaCl. The sample was dialyzed against 100 m1. of buffer
for 1 hour, against 100 ml. of fresh buffer for 2 hours, and
finally for 14 hours against 300 m1. of new buffer. The

sample was then subjected to electrOphoretIic analysis with

a.Perkin~Elmer, Model 38, Tissilus Electrophoresis apparatus.

-19-

TABLE I.

The Effect of ENZYME CONCENTRATION Upon Activity.
TakaeDiastase and 3 1 Casein. 1. Increase in H1. 0.0483 N

Alcoholic X03 Titrated per Ml. of Digest.

 

Diges- TakaeDiastase Concentration, mg./ml. digest
tion' 0.5 1.0 1.5 . a. 4.0
Time, gave gave gave gave gave gave
15 0.001 0.008 0.011 0.015 0.025 0.055
50 0.007 0.017 0.026 0.059 0.050 0.062
45 0.012 0.029 0.059 0.055 0.071 0.085
60 0.024 0.058 0.055 0.075 0.086 0.105
75 0.054 0.052 0.066 0.082 0.098 0.111
90 0.041 0.058 0.072 0.086 0.104 0.146
105 0.045 0.065 0.076 0.091 0.115 0.175
120 0.050 0.068 0.081 0.098 0.159 0.200
155 0.055 0.075 0.092 0.112 0.165 0.225
150 0.054 0.085 0.109 0.155 0.181 0.244
165 0.056 0.094 0.125 0.157 0.205 0.252
180 0.062 0.112 0.150 0.185 0.224 0.265
195 0.070 0.129 0.165 0.210 0.255 0.271
210 0.082 0.145 0.182 0.214 0.246 0.277
225 0.098 0.161 0.195 0.227 0.254 0.282
240 0.116 . 0.167 0.200 0.254 0.260 0.285

 

lInitial pH 7.0.

2All values in this table, except in column 6, represent
the average of at least 2 runs.

-20~

TABLE II.

The Effect of TakapDiastase Concentration Upon Activity
With 3 5 Casein. 2. Net Change in Free Hydrogen Ion
Activity.

-___4.__._

 

1

1Beckman Mbdel G pH meter set at 30° C.

Diges- TakapDiastase Concentration, mg./ml. Digest
tion 0.5 1.0 1.5 . . 4.0
Time, gave gave gave gave gave gave
.._...._.....m1n- .231: .222. .222. .26.. .96. .26.
0 7.10 7.10 7.10 7.05 7.02 7.00
15 7.05 7.05 7.05 6.98 6.94 6.90
50 .7.00 6.99 6.96 6.92 6.88 6.85
45 6.99 6.94 6.92 6.89 6.83 . 6.78
60 6.98 6.91 6.90 6.88 6.80 6.75
75 6.98 6.90 6.88 6.86 6.78 6.70
90 6.96 6.90 6.86 6.85 6.75 6.69
105 6.94 6.87 6.82 6.80 6.72 6.66
120 6.91 6.85 6.79 6.76 6.70 6.64
155 6.90 6.85 6.77 6.74 6.68 6.64
150 6.89 6.81 6.75 6.72 6.68 6.60
165 6.89 6.81 6.75 6.70 6.68 6.60
180 6.89 6.80 6.72 6.70 6.68 6.60
195‘ 6.89 6.80 6.72 6.70 6.65 6.59
210 6.89 6.80 6.70 6.69 6.62 6.57
225 6.89 6.79 6.69 6.67 6.60 6.55
240 6.89 6.77 6.68 6.67 6.59 6.55

TABLE III.

The Effect of Taka-Diastase Concentration Upon Activity
Fith)3 % Casein. 3. The Formation of Non~Protein Nitrogen
NPN .

 

 

 

Diges- Digest T.C.A. 0.0212 NPN/ml. Increase
tion' Ali- Filtr. N n01 of di- - hPfl/sl.
Time, cuot, Aliquot Titer gest, digest,
min. ml. ml. ml. mg.2. amp
Take-Diastase Concentration of 1.0 ma./ml. digest
0 10 14 3.32 0.137 -
30 10 14 5.56 0.244 0.087
60 5 6 5.20 0.509 0.172
90 5 6 5.81 0.575 0.258
120 5 5 2.18 0.416 0.279
150 5 5 2.64 0.507 0.570
180 2 l 1.04 0.570 0.435
210 2 l 1.15 0.655 0.498
240 2 1 1.19 0.659 .52
Concentration 2.0 mg/ml. digest
0 10 12 5.22 0.149 -
30 10 14 7.50 0.313 0.164
60 5 5 4.08 0.470 0.521
90 5 5 5.12 0.594 0.445
120 5 5 5.77 0.722 0.575
150 5 5 4.40 0.847 0.698
180 2 2 5.48 0.997 0.848
210 2 2 5.78 1.090 0.941
240 2 2 4.06 1.170 1.021
Concentration 4.0 mg./m1. digest
0 10 14 4.09 0.170 -
50 10 14 11.14 0.469 0.299
60 5 6 7.45 0.726 0.556
90 5 6 9.91 0.971 0.880
120 5 5 6.14 1.21 1.040
150 5 5 7.05 1.58 1.210
180 2 1 2.86 1.67 1.500
210 2 l 5.07 1.78 1.610
240 2 1 5.29 1.84 1.670

 

lInitial pH 7.0.

2Milliequivalent of H01 x 14 . mg of N.P.N /m1

Til t rat e aliquo t
2"x digest aliquot x digest aliquot of digest

 

TABLE IV.

The Effect of Taka-Diaetase Concentration Upon

Activity With 5 X Casein. 4.

The Change in Optical

 

 

Deneity.‘
Diges- TakavDiastase Concentration, mg./m1. Digest
tion 1 0.5 1.0 1.5 2.0 3.0 4.0
Time, gave gave gave gave gave gave
min. 6.11. 3 (1.11. 6.1:. £1.11. d.u. d.u.
0 0.0 0.0 0.0 0.0 0.0 0.0
15 0.7 0.9 1.0 1.1 1.2 1.5
50 1.4 1.6 1.9 2.0 2.1 2.5
45 1.5 2.0 2.5 2.8 2.9 5.5
60 2.0 2.2 2.6 5.2 5.7 4.5
75 2.1 2.5 2.8 5.6 4.4 6.0
90 2.4 2.6 5.0 5.9 4.6 7.9
105 2.8 2.9 5.2 4.5 5.2 11.2
120 2.8 5.0 5.6 4.7 5.9 16.9
155 2.9 5.2 5.9 5.5 7.1 25.8
150 2.9 3.4 402 509 900 -
166 300 3.7 406 607 12.5 "
180 3.0 4.0 5.0 7.9 18.0 "
196 8.2 4.2 5.6 90', 26.0 "’
210 594' 405 6.5 1208 "’ -
225 5.5 5.0 7.9 16.5 - -
240 3.5 5.5 908 2504 ‘ "
256 5.6 6.1 15.0 - ~ -
270 5.8 7.0 17.2 - - -
285 4.0 9.0 24.7 - - -
300 496 1200 "’ "' "' "'
315 500 1605 " " "' "’
550 6.8 25.8 - - - -
345 8'5 - " - - -
560 11.2 - - ~ - -
375 1408 " " "" - -
690 21.7 " " - "’ -

 

 

1Measured by Hellige—Diller, Model 400,

Colorimeter.

21n1t1a1 pH 7.0.

3Increase in density unite after zero setting.

Photoelectric

TABLE V.

The Effect of SUBSTRATE (Casein) CONCENTRATIOH Upon
Activity Using 2.0 mg. Takaeviastase per £1. of Digest.
The Increase in Ml. of 0.0485 N Alcoholic KOH Titrated per
Ml. of Digest.

“W

 

Diges- Concentration of Casein, % ae gr./ 100 m1.
tioni 1.1 2.2 . . 5.4
Time, gave gave gave gave gave
._._.._..m1n- A921... .4111... .421. 91-11.. 9.2.1...
15 - 0.010 0.015 0.025 0.056
50 0.015 0.025 0.059 0.055 0.062
45 - 0.056 0.055 0.070 0.094
60 0.027 0.048 0.075 0.087 0.109
90 - 0.056 0.087 0.107 0.140
120 0.059 0.065 0.098 0.124 0.155
150 - 0.078 0.155 0.155 0.195
180 0.046 0.099 0.185 0.190 0.240
240 0.065 0.126 0.254 0.254 0.516

 

llnitial pH 7.0..

.. 24..

- TABLE VI.

The EFFECT OF pH on the Activity of 2 mg. of Take-
Diastase per Ml. of Digest on 5 i Casein.

 

W

Increase in m1. 0.0485 N 110. XOR/m1. digest

 

 

 

Initial

pH of At 60 min., At 120 min., At 240 min.,
Digest pH éifil; EH '11211 pH :}§mli
6.51 6.28 0.046 6.26 0.082 6.22 0.150
6.50 6.44 0.086 6.58 0.124 6.50 0.200
6.70 6.64 0.109 . 6.60 0.140 6.62 0,242
6.66 6.89 0.080 6.79 0.114 6.70 0.250
7.05 6.66 0.073 6.76 0.105 6.67 0.225
7.50 7.44 0.055 7.57 0.062 ' 7.26 0.126
6.10 8.07 0.015 6.01 0.058 7.98 0.062

TABLE‘VII.

The Effect of Digestion TEMPERATURE'Upon the Activity
of 2.0 mg. of Take-Diastase per M1. of Digest on 5 % Casein.

W

 

 

 

 

 

Diges- Increase in mi. 0.0485 N alcoholic KOH/ml.
tion Tem- digest after digesting:
perature'. 50 min., 60 min., 120 min., 240 min.,
00 Am. Amle Amle Amle___
1.2 0.0 0.005 0.005 0.008
5.8 0.0 0.005 0.005 0.008
10.2 0.0 0.008 0.014 0.025
20.0 0.018 0.040 0.072 0.100
29.9 0.059 0.075 0.098 0.252
40.2 0.070 0.100 0.150 0.260
50.1 0.110 0.118 0.122 0.122
60.5 0.060 0.070 0.070 0.072

 

lInitial pH 6.8 to 7.0.

TABLE‘VIII.

The Effect Of Added ELECTROLYTE Upon Activity of 2
mg. of TakaeDiastase per Ml. of Digestl with 5 1 Casein.

 

 

WW

 

 

Conc. Change in mi. 0.0485 R alcoholic XOR/m1 digest,
NaCl. at 50 min., at 60 min.. at 120 min.. at 240 min.
moles Aml. Ami, Ami, A611,
0.00 0.040 0.072 0.098 0.255
0.05 0.019 0.051 0.072 0.150
0.10 ~0.050 0.050 0.055 0.108
0016 “00080 “00002 0.024 00062
0.20 ~0.122 -0.085 -0.067 -0.009

 

1Initial pH varied from 6.8 to 7.0.

-27-

TABLE IX.

3 The Effect of ACTIVATORS AND INHIBITORS Reacting in
10' H Concentration on 4.0 mg. Take-Diastase per Ml. for
1 Hour and Then M1xed With an Rani-volume of 6 S Casein.

 

 

Cations Source and Brand Result2
ff“ 3 4. 9 erck) None
Zn*"* Zn804.7Hé0 (85A) None
Ag+ AgNOs (Schaar) None
Cu+~+ Cu804 (Baker) _ Inhibits 84 %
Mn4‘* Hn804.H20 (Baker) Activates 17 5
Co:**' Co(N05) .6H60 (Baa) Activates 29 %
064”? CaCl .6 20 (Fischer) Activates 54 %
Nif-f Ni(0 CCHB) .4H20 (Baker) None
HgI—f- HgCl (x baum) Inhibits 98 5‘
8+3 0.1 E 601 (Ben) Inhibits 98 %
Anions
CN- NaCN (Merck) None
2'“ Na? (Baker) Inhibits 42 %
06-4“ 0.1 N NaOH (6&1) Inhibits 50 5
Carbon 1 Bee ents
fisfi§53 Bodium bisulrite (Merck None
116208.801 Hydroxylamine.HCl (EKCo None
Oxidizin agent
H202 Superoxol (Merck) None
Suifh dr 1 Rea ent
10320503 Iodoacetic acid (EKCo) None
Amino and Carbofi
Ninhydrin r ketohydrindene
hydrate (E500) None
Deter ent
5531mm IEuryl Bulfonate (EKCo) None
thsical égents Ex 6 re Distance
tra-v o e 560 cm.; I5 mIn. Inhibits 50 i
irradiation 50 cm., 15 min. Inhibits 40 1
(40 cm., 15 min. Inhibits 70 S
Dialysis For 14 hrs. Inhibits 50 %
Heat At 1060 0., 1 hr. Inhibits 100 %

1A11 digests had initial pH 6.8—7.0.

Esased upon increase in m1. 0.0505 s 810. xon titrated/m1.
digest after 1 hr.. compared with non-treated control.
3Brought to pH 5 for 1 hr. and neutralized before digesting.
4Brought to pH 5 for 1 hr. and neutralized before digesting.

TABLE X.

The Change in VISCOSITY When 2.0 mg. TakasDiastase per
in. of Digest is Acting Upon 5 Z Casein.

 

 

 

 

Diges- Flow Reins Diges- Flow Rela-
tion Timel, tive tion Time, tive
Time, sec. Vis- Time, sec. Vis-
min. cositx2 min. cositz
2.5 147.1 0.96 1.65 149.6 1.09
7.1 144.0 0.92 4.65 145.4 0.94
10.0 157.9 0.84 7.51 140.4 0.87
15.1 154.8 0.80 10.5 156.8 0.82
16.1 151.2 0.75 15.1 154.6 0.79
18.9 129.0 0.72 16.1 151.0 0.75
21.7 127.0 0.69 18.7 129.2 0.72
24.5 125.0 0.67 22.5 127. 0.70
26.8 124.0 0.65 25.9 125.4 0.67
29.5 122.0 0.65 26.5 124.6 0.66
52.2 121.0 0.61 29.0 125.2 0.64
54.7 119.6 0.60 51.5 121.2 0.62
57.2 119.2 0.59 54.8 120.2 0.60
59.8 117.4 0.57 56.5 119.0 0.59
42.8 116.6 0.56 41.5 117.4 0.57
4408 11506 0054 4601 11600 ’055
49.0 114.2 0.52 50.7 114.4 0.55
117.0 105.8. 0.58 55.4 115.8 0.52
120.0 105.2 0.58 59.8 112.4 0.50
164.0 100.4 0.54 79.0 110.5 0.47
9002 107.5 -343
110.0 105.2 0.40
155.0 105.0 0.57
180.0 100.2 -.54
198.0 99.2 0.52
225 98.2 0.51
240 97.4 0.50

 

laeaeured in 75 880.. 0stwa1d Viscometer at 29.60 0; initial
pH 700s

2Values from: (Flow time of digest/Flow time water) -1)

-29-

TABLE XI.

The Change in OPTICAL ROTATION When 2.0 mg. Take-
Diastase per Ml. Digest is Reacting with 5 fl Casein.

m

 

 

 

 

IInitial pH 7.0.

Diges- Reading, Diges- Reading,
tion| Time, angular tion Time, angular
min. degrees min. degrees
5.5 -2.962 17.9 -5.268
4.5 ~2.920 18.5 ~2.982
5.0 -2.970 19.4 -5.012
5.4 -2.950 20.0 -2.982
6.0 ~2.910 25.0 -2.992
6.5 -2.952 50.7 -2.964
7.0 —2.958 54.7 -5.000
7.4 -2.940 40.0 -2.994
7.8 ~2.846 45.0 ~2.956
8.5 -5.016 50.0 -2.972
9.0 -2.992 55.4 -5.022
9.4 -5.000 60.0 «2.954
10.0 -2.992 70.7 -2.954
10.9 -2.992 79.2 -2.998
11.5 ~2.998 89.4 ~2.980
12.0 -2.998 99.4 ~2.971
15.0 ~2.990 109.4 ~2.958
15.2 -5.296 117.4 ~2.981
15.8 ~5.194 151.0 -2.955
16.5 -5.218 149.4 -2.970
17.2 -5.222

TABLE XII

The Change in INDEX OF REFBAUTION when 2.0 mg. Tata-
Diastase per M1. Digest is Reacting on 5 S of Casein.

“:1:
w.

Diges-
tion
min.
1.0
5.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
15.0
14.0

15.0

Time,

Refractive

Index
25

.__JE.___._

1.5584
1.5590
1.5582
1.5590
1.5590
1.5590
1.5390
1.5588
1.5580
1.8390
1.5590
1.5590
1.5592
1.5592

Diges-
tion Time,
1111“ e

 

17.0
19.0
21.0
26.0
51.0
41.0
60.0
90.0
120.0
154.0
213.0
280.0
520.0
1.440

Index
n25#

Refractive

 

1.5588
1.5588
1.5590
1.5388
1.5590
1.5590
1.5580
1.5380
1.5575
1.3380
1.5580
1.5579
1.5579
1.5585

 

1Initiai pH 7.0.

-31-

w—Vr

mEHH onemmoHQ mo mmhszfla

 

 

 

ow...“ . fifl . Lama 8 a
fi 1 s w m o
o 0 .N
. . Q 5
..io._,.flpongnn_ fawn \\ \ ‘\ Wm
.So .3 5,254 n t . 0
13.25-1250 O 1 G o 0 .. +03
0.0 3,. \J 0 ‘0 \§ .
n .- .. c ‘ w ‘\
60 A: 0 0 v 0 ~ s
.cé A3 1 D 0 n s I .2.»
a..- . 1 - \ r. (
m. C :v \\ \ C \
“amino \ 0 1 0
\
\ x Q I
2.0
\s
. Q
. \
0 .
0 t ‘\ v C . .4 0H o
.888 52 0
e a 0
$3 285 .8 a
O a \s c 1 00.0
onadmmqu mug -
Q. m
0 o s
0 Q
a o 1 3.5
fl 0.
a a
0 v o
.H :mzuHm \
. . J 3-- .1
o a. (

 

 

 

”IN?

010

a

;-.\G(\
1' .3

Ti

~58-

8

N?

r” 31V

HO)!

II’Z-

 

 

 

 

 

l {.4 .-;k 5
F4 :5 iit (J 'f‘“ .‘ .
H :L“ L1G C1 C3 +1
(_ {—4 Ir a o- a- 0 F4 (_'.
[I] C:) 2 .. L.\ f". .z F, P m (a! {.4 C,
a CD .1; m (f) A o 1 “) 0 1) i O H-
(U 2?. (3 Q Li: ("2 r1 r-1 “1" m <9- 4: 3)
<2: >1 2 7" ix”. 2*:
[z] 7:". fr: C) mAAA/xz—\A¢£H ,‘_.‘
(I: U 0 ° .2 ("1 {\J ”3'? ‘1\\O {‘4 E
:3 [3'] Z UVVVquv \ a
U m [13 z O O (14. +)
H :1: LL 0 H :00) 01
[1. E“. [3" H E“ E5 <1 m
"* r‘J m0 1n \0
0
'fi 0
N
O
"'" a)
H
d
_ O
N
H
C}
d \o
O
H 1“
0 o
L\ \0

pH

~55-

MINUTES OF DIGESTION TIME

OF D IGEST

N ~/ML.«

N ..P.,

A MG

1.6

 

 

 

 

— 15
3 ,
_ C)
'0
_ {I
40
CD
1 l 1 l L l l l
O 60 120 180 240

MINUTES OF DIGESTION TIMES
FIGURE 3. THE FORMATION OF NON=PROTEIN NITROGEN. CURVES:

(l) 1.0 , (2) 2,0and (3) 400 mg. TAKAaDIASTASE/mlo of digest,
refractivnly; '

-34-

 

.»H1>fiuoaw an mumcrflc we .HE ucu mm¢9w¢wow¢m<a .mE 0.5 on ngm

 

 

.o.m Amy .o.m oqv .m.H Amv .o.H Amv .m.o AHV "wm>m:o .on922mom wHHonmze .0 mmpon
mzHa onemmuHa no mmeasz
00m 00m oma oma oo o
A 1‘ _ _ d H7 0‘ _ «a a _ Mfiw
mwuu www mmwm\\\
o .lolb Iolmilhmum“w.m®\\\@w\ Nm\
nY\\ \\.O\\.O .O\\LU\\. .\\0..\\.O..\..G\\O
nu\\v \\Au \\Au\\. nwxxnv n%\\ .L
QW\\_ Au\\Au \\\nv .ux\. nfl\\ .
H \m 0 m 0 0 \ m 0 0
Au \\\ Q\\\ n0 \\\
\ o 1
\\\ \\\. \\\\ nv
nu
Au n0 n0
\ \ L
O O O O
1 my mu 1
m .10

 

 

OH

ma

om

mm

SiINfl KLISNEG TVOIldO

.35-

KOH

OF 0.0483 N ALC.

ML.

A

0.32

0.28

0.24

0.12

0.08

 

 

 

 

FIGURE 5
(1)0(2)’(3)3

cent (w/v) casein.

 

34)

5
C)
_ C3 4
3
% c) C)
C)
L / /
////,»43//////C3
O///’0 2 C)
//0 o/
//’//'Cj/////’
,..———o /19
/o
1:O I 150 l 2:0

MINUTES OF DIGESTION TIME

EFFECT OF SUBSTRATE (CASEIN) CONCENTRATION. CURVES:
and (5) represent 1.1, 2.3, 3.1, 4.3 and 5.3 per:

(\

.mao>uuuwgmmu .coflumamfln mo 00:02 0 Gem V .H pm mmfiufipwuom

:\

.0. Amv on. Amv .AHV .mm>m:o .q¢sz 02¢ a<HBHzH .ma zpsHemo .8 mmaon

ma
o.w m.u 0.5 ..m m.m o.u «.0 0.0 0.8 m.o

 

 

 

— a A _ a u q _ .

 

 

O O ,x/ \
I / \\
/ \
// O O

x, g. o
I/ l

/ 5

/ \

,./ \

/ \

II \
/
/
I ll
mm ,mm C O
/ \
/ \\
x 0 \
/ \
0 , x
O O

 

 

wo.c

Nfi.o

caoo

VNOO

HO)! °O"IV N {BVC°O JO ""va

. 87.

43 ML. OF 3.3483 N ALC. KOR

0.32

0.08

.01

C)

 

 

 

 

 

.:

‘.
.gggeagfl 1 1 1 1 1
O 10 2O 30 40 SO 60

TEMPERATURE, DEGREES Co
FIGURE 7. OPTIMUM TEMPERATURES. CURVES: (l), (2),
(3) and (4) are activities at 0.5, 1, 2 and 4 hours of

digestion, respectively.

~58-

0.1-

3.04

-0004

-0.08

~3.12

 

 

 

 

 

 

_ i.
i.
5
L
1.
l l 1 l 1 l #1 l
O 60 I20 180 240
MINUTES OF DIGESTION TIME
FIGURE 8. EFFECT OF ADDED ELECTROLYTE. CURVES: (I), (d), (3),

(4)9 and (5) represent 0, 0.05, 0.10, 0.15 and 0.20 M N301 added.

-39-

RELAT IVE V ISCOS ITY

 

9.4

0.3

9.2

 

 

 

 

composite of 2 determinations.

-40.

K
b 1
k“
.2
~ \.
— 3
f)
._ \
1 l l I 1 l g l
0 60 120 180 240
MINUTES OF DIGESTION TIME
FIGURE 9. THE CHANGE IN VISCOSITY. The values are from a

IV. DISCUSSION

This discussion will be an attempt to evaluate the data
and results by considering the scape, significance and.
limitations of the experimental methods employed. In
such manner it is preposed to present information about
the following factors that influence the proteolytic acti-
vity of Take-Diaetase on casein: l- enzyme concentration,

2- substrate concentration, 5- optimum pH, 4- optimum
temperature, 5- ionic strength. and 6- activators and inhi-
bitors. Additional physio-chemical information about the
over all reaction and remarks concerning the nature of the

reaction will be presented.

A. ‘ghg Influence 2f Varying Enzyme Concentration.

Change £2.2222l Aciditz.-The increment obtained by
alcoholic KOH titration may be considered a measure of the
increase in total acidity, due to formation of hydrogen ion
donating groups. The theoretical considerations involved in
this analytical method have been discussed by Richardson
(1954) and others. Such acidic groups are presumed to arise
from protein hydrolysis of predominantly peptide bonds. But
this may not be the sole source of acid groups liberated from
native proteins. There may be an additional contribution
of phosphoric acid residues formed as a result of phos-
phoamide and/or phosphoeeter hydrolysis. Proteolytic enzymes
are known to catalyze the hydrolysis of such types of bonds,
as evidenced by the work of Holter and Li (1950) and Smoke,

Schwert and Neurath (1948) on synthetic peptides. However,
one would expect that the data of this measurement is
principally the result of peptide bond cleavage, particularly
in the more advanced stages of the reaction.

Referring to the results in Table I and Figure l, the
following observations are apparent. The total acid groups
liberated depends upon the enzyme concentration. All curves
seem to support the hypothesis of a two stage reaction. But.
as enzyme concentration is increased.the transition from the
first to the second is less apparent and approaches a condi—
tion where it would become indistinguishable. 0n the basis
of these experiments digestions run at constant enzyme
concentration were chosen as 2 mg. of Takapbiastase per ml.
of digest. This was the enzyme concentration best repre—
senting the two stage nature of the reaction.

Change‘ig Active Aciditz‘gg Measured Potentiometricallz.~
The net change in hydrogen ion activity as brought about by
protein hydrolysis would be due to the type of acid groups
liberated. Carboxylic acid groups from peptide bond cleavage
being of the weak type, would not contribute as many free
hydrogen ions as the stronger phosphoric acid groups.
Further, proton accepting groups present could associate
with the liberated free hydrogen ions to maintain their
respective equilibria. Thus a partial buffering effect
could be eXpected to be involved in the net change. This
may explain the buffering effect apparent in Figure 2.

-42-

All digestions were unbuffered, in spite of the usual
practice to do so. Sumner and hyrbfick (1950) point out that
buffers may exert specific as well as a general ionic
strength effect on enzymatically catalyzed reactions. There
are cases known where common buffer substances acted as
substrates, especially when crude enzyme preparations were
used. Hence it could be difficult to distinguish between
the influence of free hydrogen ions formed from buffer and
that of the substrate. Buffer components may also interfere
with analytical methods employed for following enzymatic
activity. Consequently. the initial pH was always checked
either colorimetrically or potentiometrically at the begin-
ning of all digestions. All results are recorded with the
initial pH defined. The data of Table II and Figure 2
demonstrates an increase in active acidity during the
reaction and dependency on the enzyme concentration.

‘223 Liberation 23.Egg-Protein Nitrogen.-Non-protein
.nitrogen is that nitrogen of hydrolysis products not precipi-
tated by 10 f trichloroacetic acid. The upper limits of
molecular weight of such products is not well defined. In
general, it has been considered to consist of mainly low»
molecular weight polypeptides and free amino acids. Such
measurements would be indicative of the extent to which these
types of products are liberated. It can serve to demonstrate
the fraction of the total protein nitrogen that has undergone
extensive hydrolysis. The results in Table III and Figure 5

show linear dependency between such activity and enzyme

-45-

concentration during the early stages of the reaction. At
the highest enzyme concentration under the defined condi-
tions, 6.1 % of the total nitrogen was thus liberated.

Turbidity.-One cf the preliminary observations leading
to this study was that Take-Diastase produced a cloudy solu-
tion when digested with casein. It was desired to evaluate
this observation on a quantitative basis. Therefore, a
method of analysis that would register any change in the
transmissibility of light through the solution was devised.
Before digestion the solution was a clear, slightly amber
colored liquid.l This would indicate that the substrate was
well dispersed and stabilized by hydration and charge.

whereas when proteolytic action set in and had pro-
ceeded for a period of time, a turbidity deve10ped. This
might be interpreted as being the result of altered charge
and hydration capacity of the disperse phase due to proteo-
lytic activity. The data of Table IV and Figure 4 show a
direct relationship between enzyme concentration and the
period of time for maximum change in Optical density. It
seems that there is an induction period proportional to the
enzyme concentration. This would suggest that a preliminary
or a series of reactions must have taken place before the
cloudiness set in.

Comparison and Information from Results.-In enzymati-

 

cally catalyzed reactions the usual behavior anticipated
has been sumnarized by Somers (1950). He states that in the

presence of a sufficiently large amount of substrate, such

that the rate of reaction is independent of the substrate
concentration, the rate of reaction may be changed by
changing the concentration of enzyme present.

In all these studies, it may be concluded that the
data discussed in this section fulfills the above condition.
The relationship, at least in the early stages of the reaction.
holds true. Table I and Figure I show that the relationship
does exist. In addition the rate curves in this figure show
that the digestive process is not a single stage reaction,
but is indicative of at least two stages. It seems that each
stage initially is independent of the substrate concentration.
The slow transition from one stage to the other at the lowest
enzyme concentration indicates the possibility that there
is not sufficient new substrate to completely saturate the
enzyme of the second reaction. This transition effect varies
from 1 to 2 hours of digestion in the enzyme concentration
ranges studied.

If the non-protein nitrogen curves are compared with
those of titration, there does not seem to be any distinct
correlation between the activity plots of Figures 1 and 3.

In Figure 5 no transition is apparent between 1 and 2 hours
of digestion, but dependency upon enzyme concentration is
exhibited. The liberation of non~protein nitrogen may not
necessarily follow that of total acidity, inasmuch as
measurements of two different entities are involved.

The net change in hydrogen ion activity curves of

Figure 2 exhibit no intercorrelation with those of

-45-

non-protein nitrogen or total acidity. Dependency upon
enzyme concentration is evident.

The optical density curves of Figure 3 demonstrate
dependency upon enzyme concentration. If one inspects the
data, the digestion time at which the turbidity approaches
maximum rate of change corresponds favorably with the time
at which transition step is noted in the titration curves.
Such a relationship is not apparent with the non-protein

nitrogen or hydrogen ion liberation.

B. 233.1nfluence 2; Variable Substrate Concentration.

This factor was studied by measuring the increase in
alcoholic KOH titrated or total acidity. The significance
of the titration has been discussed under the previous
section on the influence of enzyme concentration. It is
usually found that the reaction rate is a linear function of
the initial substrate concentration. At relatively higher
substrate concentrations less or no influence is exerted on
the rate, since the enzyme becomes sufficiently saturated
with substrate. In addition a substrate concentration may
be reached where the rate may decrease.

Figure 6 demonstrates the dependency of acid group
liberation rate upon substrate concentration. It is apparent
that the substrate concentration at which the rate would
become uninfluenced was not attained. A correlation between
Figure l where enzyme concentration was varied and Figure 5

where the substrate concentration was varied is apparent.

-45-

The break in the curve of the latter figure, which appears
after 2 hours of digestion. is quite pronounced since the
points that would more clearly define the transition were
not measured. However, a correlation in favor of a 2-stage

reaction is observed between the two graphs.

0. Egg Optimum Llnitiall 2.3-

HOst all enzymes exhibit a maximum activity at some pH
value, known as the pH Optimum. In some cases this may be at
a rather sharp maximum and means that the enzyme shows its
greatest activity over a restricted pH range. In other cases
a rather broad miximum zone is exhibited indicating less
sensitivity to hydrogen ions. But pH optima cited for an
enzyme may depend on other factors. The optimum may be
influenced by the concentration and nature of the substrate
temperature and concentration of enzyme. Oshima and Church
(1925) pointed out that the method of measurement as well.
exerts an influence on the result.

From Figure 6, the reaction appears to take place most
favorably in slightly acidic media, with an optimal (initial)
pH range 6.6.6.8. This agrees closely with the pH value of
6.7, reported by Nishikawa (1927). He stated that this
value was also the upper limit of the optimum pH observed for
rennin activity. The value pH 7.0. reported by Berger,
Johnson and Peterson (1957). for TakaPDiastase activity on
gelatin also agrees. As mentioned the substrate and method
of assay may influence this factor. By the conditions of

these eXperiments, the optimum value appears to be between
6.6-6.8. The increase in titer at l, 2 and 4 hours of
digestion is plotted against initial pH on curve (a) of
Figure 6 and similarly final pH’s at the time of measurement
is plotted on curve (b) of Figure 6.

The optimum pH range broadens as measurements on longer
digestion periods are made. The direction seems to be towards
a less acid solution for the longer times of digestion. A
similar effect was reported about urease by Van Slyke and
Zacharias (1914). They concluded that the optimum pH value
was a result of two reactions: one which could be attri-
buted to the addition of neutral salts and the other by the
concentration of substrate. and that Optimum pH was a compro-

mise between their respective optima.

D. ‘2h3_02timum Temperature.

Optimum temperature, like Optimum pH, is influenced
by concentration and nature of substrate, the method employed,
and the time of digestion. It also varies widely with the
enzyme being studied and source of the enzyme.

Below certain characteristic values, temperature
influences the rate of enzyme-catalyzed reactions in much
the same way it does comparable non—catalyzed reactions.
That is, an increase in temperature increases the effect of
catalysis. It is generally known that most enzymes being
thermolabile are inactivated at elevated temperatures. As

a result, the point of balance between the accelerating

~48-

influence of temperature and the inactivating influence of
heat is designated as the optimum temperature.

Figure 8 indicates a shift from an optimum of 50° C. in
the initial stages of digestion toward a lower optimum
around 40° C. in the later stages.

Since the substrate used by Kawakami (1929) is not
mentioned in the literature available, no significant

comparison of results can be made with this work.

E. The EffeotIgg Added Ionic Strength by Neutral 5 t.

w

 

Ionic strength influences charge. hydration and solu-
bility of protein dispersions. This could show alteration
of the rate of reaction either as a direct result or as an
influence upon the mode of activity measurement. The
influence of such an effect was tested by increasing the
ionic strength of the digestion medium. The results are
tabulated in Table IX and shown in Figure 9.

The effect of added salt (Neel) appreciably decreases
the activity at all salt concentrations as compared to the
control. azanto (1912) reported that the presence of 0.1 M
neutral salts did not exert appreciable influence on the
activity of Take-Diastaee in a 1 % casein solution. The
results were based upon an isolectric precipitation of
undigested casein. The apparent contradiction may be due
to the methods of assay and thus a strict comparison of
results may not be valid.

From Figure 9. the NaCl concentrations of 0.1 H and

above show greatest activity change over all stages of the

-49-

digestion. The titers for digests with 0.2 M haul never
reached the initial value showing the anomaly of a negative
increment. Among various considerations, the salt effect

on the indicator might account for this, but other obser-
vations led to a more tenable eXplanation. At all salt
concentrations, the digest solubility was altered as evidenced
by a rapid turbidity formation. No precipitation occurred
during the digestion period, but a distinct difference was
noted when the samples were titrated. This was most evident
at the higher salt concentrations. On adding digest aliquots
to the alcohol-indicator mixture, a heavy white flcculation
appeared. This was not completely redissolved when the

final end point was reached. Such behavior was not evident
at 0.5 M salt concentration or in the control which contained
no added salt. Hence the anomalous character of the initial
part of the curves. 1. 2 and 3 of Figure 9, may be due to
such circumstances. In the later stages of digestion when

titrating aliquots this flcculation behavior disappeared.

F. Activatorslggg Inhibitors.

Massart (1950) points out that activator and inhibitor
reagents when allowed to react with the enzyme and then
testing its catalytic power may give information about the
‘active centers" which were associated with the normal
enzymatic activity. Experiments were designed to provide,
if possible, some information concerning the type of groups
in the enzyme that are associated with its activity. The

results are in Table IX.

~50-

Cations.-The strong inhibition exerted by reacting
Cu”:- and Hg#vith the enzyme compares with work of Nishikawa
(1927), who reported that tryptic action upon gelatin was
inhibited by on”: Hgfi‘and 2n”? Perhaps the 2 ions form
weak dissociating complexes with substrate binding groups
of the enzyme. Table IX shows no inhibition by 2n”f‘ The
activating effect of Mn”: Co”? and Ca may be due to
supplementing a ccfactcr requirement or to an unspecific
catidn effect. Enzymes which are metallo-proteins respond
similarly.

The strong irreversible inactivation caused by hydrogen
ion and the partial inactivation with hydroxyl ion confirm
the report of Seantc (1912) about the behavicr'cf strong acids
and bases.

Anions.- The inhibition by flouride ion may possibly
be due to its high complexing ability with cations. Since 0a .
Mn and Co exert an activating effect, the flouride ion may
complex these metallic ions of a metallo-protein enzyme.

Qarbonyl Feagents.~$ince the carbonyl reagents
tested do not influence the activity, it would seem that
carbonyl centers of the enzyme sensitive to these are not
essential for activity.

Oxidising égents 223 Sulfhzdryl Reagent .-1f the
enzyme depended on sulfhydryl groups for activity, inactiva-
tion should be expected by oxidizing agents or sulfhydryl
reagents. From Table IX it would appear that sulfhydryl
groups may not be necessary for activity, since both hydrogen

peroxide and iodoacetic acid exerted no noticeable influence
on activity.

M mailinhydrin can react with free «-
amine, cx—carboxyl groups. Any effect of ninhydrin upon
such was not detected in these experiments.

Petergents.-Denaturation or other chemical effects
of sodium lauryl sulfonate on Take-Diastase was not evident.
This is contrary to that reported by Astrup and AlkJaersig
(1952), but their observations were applied to its fibrino-
lytic activity.

Physical Agents.-Inhibition by ultrapviolet
irradiation night b3 expected to be due to a photochemical
reaction on the enzyme. But Kawakami (1929) stated that it
might be due to a heat effect unless the solutions being
irradiated were cooled. The results reported in Table Ix
were obtained upon solutions that had not been cooled during
irradiation, but indicate that inactivation decreased as
the samples were further removed from the light source.
Under the conditions of the experiments it seemed logical -
that the results were as representative of irradiation effects
as that of heating. Nevertheless a more careful examination
should be made.

Dialysis.-Since an estimation of the dilution
resulting upon dialysis of the enzyme solution was not made,
it is not possible to state with certainty that the loss
in activity was due entirely to a removal of a co-factor or

essential prosthetic group. However the cation activating

~62-

3

.-=‘
i {'3‘

iti’ef?‘ ,. VP‘VIJT .

III! ..

effect lends substantiation to such a conclusion.
Eggwlieating Taka-Diastaee solutions at 100° 0.

for 1 hour was found to completely inactivate the proteolytic

enzyme. This preperty of thermo lability was employed in

preparing inactive enzyme solutions for control eXperiments.

G. Physico-Chemical Studies on the Nature 9;. £13 Reaction.

Viscosity Change.-The colloidal nature of a protein
solution lends itself to the possibility of studying visco-
sity changes due to reactions leading to or resulting in
hydrolysis. This change should be quite sensitive to
physical and chemical effects upon the state of aggregation,
nature of associated complexes or any physical alteration
of the initial condition of the substrate. Figure X
demonstrates a rather striking initial decrease in viscosity.
and subsequent independency of further reaction. This
supplements information on nature of the initial stages of
the digestion previously discussed from titration data. The
drop in viscosity almost parallels the first stage of the
reaction.

Optical Rotation Chang .oIt might be anticipated that
a change in protein structure which would lead to or
result in hydrolysis could be directly followed by change
in optical rotation.

The results in Table x did not meet this expectation,
at least in the manner the method was employed. The specific

rotation for casein as calculated from the initial rotation

observed is [“1303 -98.6 and agrees favorably with [4330:
~95.2 reported by Lone (1905). Among others,Winnick and
Greenberg (1941) applied optical rotation change to follow
protein hydrolysis. But in all cases, the rotation was
determined upon filtrates of trichloroacetic acid after
precipitation of the unreacted protein. The experiments
should be repeated along these lines. It is possible that t
the rotations reported in this work were a result of compen-
sating effects induced by the products formed. But the fact
remains that there was a pclarimetric change in the presence
of the enzyme; however, it did not yield a significant trend.
Perhaps higher substrate concentration would have improved
the results.

Refractive $2225 Change.~As light passes from one media
to another it is refracted. If any change takes place in the
composition of media, it is conceivable that this would
manifest a change in refractive index. The results in Table
XII do not demonstrate a significant change. Again, this
may be due to an inadequate substrate concentration or other
conditions requiring further investigation.

Effect gg‘égg gg.£h3 Substrate.-When a casein stock
solution (up to 5 weeks old) was not carefully refrigerated
when not in use, the following observations were made.
Initial titer of alcoholic KOH on digest aliquots varied
from one day to another. The electrOphoretic patterns were
altered. Duplication of Proteolytic activity measurement

was not possible from one period to the next. The acid or

-54-

a

,‘

‘glrieb . exitrwflt .. 5;... .. . «sm,,l.an|...w
a

n

 

baee binding capacity was shifting. The pH of the stock
solution itself was shifting towards s more acidic condition.
To test the stability of a stock casein solution

carefully maintained under refrigerator conditions, elec-
trophoretic analyses were run weekly from date of prepara-
tion through 6 weeks. The patterns showed no alteration.

It is quite likely the proteolytic enzyme within casein as
mentioned by Warner and Falls (1945) brought about the above

noted gradual change.

H. Remarks 9}; £133 14933 23‘. Reaction:

The increase in free hydrogen ions, viscosity decrease,
liberation of acidic groups, formation of non-protein nitrogen,
increased optical density, change in Optical rotation. change
in refractive index. all indicate that during digestion the
substrate was undergoing physical and chemical alteration
that resulted in hydrolysis.

The liberation of acidic groups is representative of
principally peptide bond cleavage, but in addition may
include phosphoamide or phosphoester linkages. The curves
of Figure 1 suggest two stages for this type of change.
Perhaps stage 1 reaction may be associated with an initial
attack upon one of the fractions of casein, followed by a
reaction of stage 2 upon the remaining components or pro-
ducts of the initial reaction. ‘The change in Viscosity
during the first hour of reaction shown by Figure 9 corres~

ponds favorably with stage 1 of curve 3 in Figure 1. It

-55-

suggests that the initial stage is the most significant
hydrolytic step affecting viscosity.

The rapid increase in turbidity corresponds with the
digestion time of stage two. Figure 3 shows that turbidity
becomes most pronounced after 5 units of Optical density
have been attained. This occurs between 1 to 2 hours of
digestion, depending upon enzyme concentration or appears
at the end of stage 1 and the beginning of stage 2. Tur-
bidity suggests change in solubility cf'an altered component
of casein or a remaining unattached fraction with more hydro-
phobic preperties. The fact that only up to 1 % nonpprotein
nitrogen was formed during stage 1 lends support to this
contention. At present it is not possible to ascertain
whether the products or the insolubility of one of the
components of casein was contributing to the turbidity.

Eon-protein nitrogen may be the result of over-all
hydrolytic changes in the media. since its formation rate
showed no deviation at the times of stage 1 and stage 2 of
reaction.

Direct correlation could not be made between the
change in hydrogen ion activity and the preceding obser-
vations. This may be the result of buffering in the medium
which arises from the products or remaining substrate. It
would seem that the more strongly acidic phosphoric acid
groups were being liberated during the initial stage, since
the hydrogen ion activity increased most rapidly at this
time.

-55-

The shifting of the pH Optimum, to a less acidic OOH!
dition, and the shift in the Optimum temperature require-
ments during the second stage of reaction may indicate the
presence of more than one proteolytic enzyme. That is,
the initial reaction may involve an enzyme system different
from that in Operation later'on.

The foregoing remarks point out that further investi-
gations on casein fractions as substrates, using the crys-
talline proteolytic enzymes described by Crewther and Lennox
(1960), would be highly desirable. However, the data pre-
sented here gives some indication of the circumstances lending

to turbidity formation.

l. The liberation of acidic groups, turbidity formae
tion, hydrogen ions and non-protein nitrogen are dependent
on the concentration of TaksvDiastase during casein digestion.

2. Acidic groups are liberated in s two stage reaction.
This is most pronounced with 5 % casein digesc:containing
2 mg./ml. of Take-Disstase. Transition from one step to the
other takes place between 1 to 2 hours of digestion.

3. The onset of rapid turbidity formation corresponds
with stage 2 of acidic group liberation.

4. The maximum drop in viscosity during the first hour
Of digestion parallels the first stage Of acid group libero-
tion in the reaction.

5. The formation of non-protein nitrogen shows a
dependency upon enzyme concentration, but gives no parallel
correlation with a 2 stage liberation of acidic groups.

6. At all substrate concentrations up to 5 S the rate
of reaction was dependent on the concentration of substrate.

7. An Optimum initial pH 6.6-6.8 was found for l to 2
hours of digestion. A broader Optimum range extending to
pH 7.0 was observed with 2 to 4 hours of digestion.

B. The Optimum temperature was found to be 50° C. for
l to 2 hours of digestion. A shift down towards 40° 0. was
observed for 4 hours of digestion.

9. Sodium chloride added to the digest decreased

-53-

activity at all concentrations. Titration anomalies were
observed at added salt concentration of 1.0, 1.5, 2.0 molar.
lo. Digests exhibited no significant change in optical
rotation or refractive index during the first 4 hours.
11. All chemical reagents tested as activators or
inhibitors were added in l x 10""3 M concentration for 1
hour of reaction with the enzyme. The effect upon digestion
with casein was tested by measuring the increase in titra-
table acidity after 1 hour and showed these results:

a. 00,;1— Mn” Gaff accelerated the rate of reaction,

29,17, and“ M} respectively.

b. cw" gflgfl, r , inhibited the reaction rate, 94, as.
firespectively.

0. Reaction with HCl at pH 5 for 1 hour and restoring
to the original pH. gave irreversible (98 %)
inactivation. Similar treatment with NaOH to pH
11.0 resulted in 30 % inactivation.

d. All other chemicals tried had no noticeable effect
on the enzyme activity.

e. Depending upon distance, ultrasviolet irradiation
for 15 minutes resulted in up to 70 % inhibition.

f. Dialysis for 14 hours against 20 volumes redistilled
water with 4 changes resulted in 50 % inactivation.

g. Heating the enzyme solution at 100° c. in a sealed
tube for 1 hour produced irreversible inactivation.

12. Some effects of age on casein stock solutions are
presented and warn of the necessity for fresh solutions and

low temperature storage.

VI. BIBLIOGRAPHY

Astrup, T. and N. Alkjaersig
1952 Classification of Proteolytic Enzymes by Means
of Their Inhibitors.
Nature, 169: 314-616.

Barman, E. and K. Hyrback
1345 Die Methoden der Fermentforschung, Vol. I-IV.
Academic Press Inc., Publishers, New York.

Berger, J., M. J. Johnson, and w. H. Peterson
193'? The Proteolytic Enzymes of Some Cerzamon Holds.
J. 8101. Chemo. 117: 429-4360

Clark, E. P.
1945 Semiuhicro Quantitative Organic Analysis.
Chapter III
Academic Press Inc., Publishers, New York.

Cohn, E. J. and J. L. Hendry
1943 Organic Syntheses, 0011. Vol. II; pp. 120-122.
Edited by Blatt, H. A.,
John Wiley e Sons, Inc., New York

Crewther, w. G. and F. G. Lennox

1950 Preparation of Crystals Containing Protease
from As er illus orzza .
Nature, 6 3 e

Foreman, F. W.
1920 Rapid Volumetric Method for the Estimation
of Amino-acids, Organic Acids and Organic Bases.
BiOChem. J0. 14: 451‘473s

Gillespie, J. H., H. A. Jermyn and E. F. Woods
1952 The Multiple Nature of Enzymes of Aspergillus
orvzae and of Horse-Radish.
Reture, 169: 487-489.

 

Gordon, W. 6., V. F. Semmet, R. B. Cable, and M. Morris
1949 Amino Acid Composition of of. Casein and <3 Casein.
Jo Ame Chem. 300.. 71: 3298‘32g7s

Gortner, R. A.
1949 Outlines of Biochemistry.
3rd Edition, Chapter IV
John wiley s Sons, New York.

~60-

Holter, H. and Bi-OH Li
1950 Phosphoamidase Activity of Some Proteolytic
Enzymes and Rennin.
Acta. Chem. Scand., 4: 1321—1322.

Kawakami, J.
1929 Chemical Change of Taka-Diastaee Solutions on
Heating.
J. Pharm. Soc. Japan, 49: 346-55,
cited from C.A., 26: e238 (1929).

Lichtenstein, N.
1947 - The Proteolytic Enzymes of Taka-Diastase.
Empt. Med. and Surg., 5: 182-190.

Linderstrdm-Lang, K.
1925 Studies on Casein 11. Is Casein a Homogenous
Substance?
Compt. rend. trav. lab. Carlsberg, Ser. Chim.,

Linderstrdm-Lang, K.

1927 Titrimetrish Bestemmelse Af Aminokvaelstoff.
heddelelser fra Carlsberg Laboratoriet 17x
4 1-16s

Linderstrpm-Lang, K.
1929 Studies on Casein III. On the Fractionation
of Casein.
Compt. rend. trav. lab. Carlsberg, Ber. Chim.,

Long, J. H.
1905 On the Specific Rotation of Salts of Casein.
J. Am. Chem Soc., 27: 563-566.

mas sart , L.
1950 Enzyme Inhibition.
The Enzymes, Vol. 1, Pt. 1, pp. 307-342.
Edited by Sumner, J. B. and Myrback, K.,
Academic Press Inc., Publishers, New York.

ficheeklin, T. L. and B. D. Polis
1950 Milk Proteins.
Advances in Protein Chem., Vol. V, pp. 202-225,
Academic Press, Inc., Publishers, New York.

1939 ElektrOphoretische Untersuchung von Casein.
Biochem. 2., 300: 240-245.

-61-

Eishikawa, K.
1927 Zur Kenntnis der Takadiastase.
Biochen. 2., 188: 586-404.

Chadd, S. '
1916 On the Optimal Conditions for the Proteoclastic
Action of Takadiastase.
BiOChBMa Jo. 10: 130.136.

Oshima, K. and M. B. Church
1923 Industrial Mold Enzymes
Ind. mg. Chemo, 15: 67-700

(Wmu,Y. .
1940 Enzymes in Young myselia of Aspergillus oryzae.

Bull. Agr. Soc. Japan, 15: 59-64,

cited from C. A. 54: 132 (1940).

Richardson, G. .
I934 The Principle of Formaldehyde, Alcohol and
Acetone Titration.
Proc. Roy. Soc., 115 B: 121-199.

Snoke, J. E., B. W. Schwert and H. Neurath, H.
1948 The Specific Esterase Activity of Carboxypep-
tidase.
J. Biol. Chem. 175: 7-13.

Somers, G. F.
1950 Agricultural Chemistry a Reference Text, Vol. I.
pp. 135-171, Edited by Freer, D. E. H.,
D. Van Nostrand Co., Inc., New York.

sprensen, 3. P. L.
1908 Etudes Enzymatiques.
Compt. rend. trav. lab. Carlsberg 7: 58-136.

Sumner, J. B. and E. Hyrback
1950 The Enzymes, Vol. I, Part I, pp. 1-27,
Academic Press Inc., Publishers, New York.

Sutsrmeister, E. and F. L. Browne
1939 Casein and Its Industrial Applications.
A.C.S.Nonograph, No. 30
Reinhold Publishing Corp., New York.

Szanto, 0. -
1912 Zur Kenntnis der Proteolytischen Wirkung
der Takadiastase.
Biochem. 2., 43: 51-43.

-52-

Takamine, J.
1925 Enzymic Composition.
U. 8. Patent No. 1,460,756, March 5.
Cited from C. A., 17: 2894 (1925).

Tauber, H.
1949 The Chemistry and Technology of Enzymes.
John Kiley & Sons, Inc., New York

Van Slyke, D. D. and G. Zacharias
1914 The Effect of Hydrogen Ion Concentration
and of Inhibiting Substances on Crease.
J. Biol. Chem., 19: 191-185.

Vines, S. H.
1910 Protesses of Plants.

Warner, R. C.
1944 Separation of 0(- and 6'- Casein.
J. Am. Chem. Soc., 66: 1725-1751.

Warner, Rs Ca and E. P011.
1945 On the Presence of a Proteolytic Enzyme in
Casein.
J; Am. Chem. 800s. 67: 529*5320

willstatter, R. and H. Persiel
1925 Trypsinbestimmung.
Hoppe-Seylers Z. physiol, Chemie., 142: 245-256.

Willstatter, R. and E. Waldschmidt-Leitz
1921 Alkalimeterische Bestimmung von Aminosauer
und Peptiden.
Ber, 54 B: 2988-2995.

Winnick, T. and D. M. Greenberg
1941 The Use of Optical Rotation in the Study of
Protein Hydrolysis.
J. Biol. Chem., 157: 429-442.

Wohlgemuth, J.
1912 Zur Kenntnis der Takadiastase.
Biochem. 2., 59: 524-558.

THE PROTEDLYTIO ACTIVITY OF TAKA-DIASTASE
FROM ASPEHGILLUS ORYZAE ON CASHN

By
Robert Clement Lieeer

AN ABSTRACT

Submitted to the School of Graduate studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Chemistry
1952

 

 

ABSTRACT

It was observed that a clear, neutral solution of
casein became turbid, within several hours in the presence
of TakarDiastase.

This study was undertaken to determine the Optimal
conditions and the nature of the reaction involved in the
formation of these turbid solutions.

The rate of liberation of acidic groups,ae determined
by alcoholic potassium hydroxide titration, was the principle
method of measuring activity. The influence of enzyme
concentration upon the liberation of acidic groups, non-
protein-nitrogen, free hydrogen ions, as well as the rate
of turbidity formation, was measured by titration, micro-
Kjeldahl analysis, pH measurements and change in optical
rotation.

The rate at which acidic groups were liberated at
different substrate concentrations was determined by titration.

The influence of initial pH, temperature and added
neutral salt upon the rats of acidic group liberation was
studied. The digestion mixtures used for these measurements
were 5 S with respect to casein and contained 2 mg. of Taka’
Diastase per ml. of digest.

A number of possible activators and inhibitors of the
enzyme were tested. Their effect was compared with the rate
of acid group liberation of untreated enzyme. Viscosity,

Optical rotation and index of refraction measurements were

made on digests containing 5 % casein, and 2 mg./m1. of

Take-Diastase. Only viscosity measurements gave significant
results.

The results of these eXperiments are as follows:
The liberation of acidic groups, turbidity formation, free
hydrogen ions, and non-protein-nitrogen during casein
digestion are dependent upon the concentration of Taka~
Diastase. Acidic groups are liberated in a two-stage reac-
tion. The time at which the most rapid change in turbidity
is observed corresponds with the second stage of acid group
liberation.

The rapid drop in viscosity during the first hour of
digestion parallels the first stage of acidic group liberation.

Liberation of non-proteincnitrogen and changes in hydro-
gen ion activity exhibit a dependency upon enzyme concentra-
tion. But, no direct correlation can be drawn between their
rate of change and that of acidic groups and turbidity.

For digestion mixtures containing 5 % casein and 2 mg./ml.
of TakapDiastase, the Optimum pH appears to be 6.6-6.8 for l
to 2 hours of digestion.

Under the same digest conditions,initia1 pH 7.0,the
Optimum temperature for 1 to 2 hours of digestion appears
to be 50° C.

At all salt concentrations tested, the rate of libera-
tions of acidic groups was decreased.
In concentrations of l x 10—5 Helar, 06*, Mn“, Ca”

fl' e-
accelerate the rate of casein digestion. Cu“, Hg , F ,

 

dialysis and ultraviolet irradiation inhibit the liberation
of acidic groups. If the enzyme solution is kept at

pH 3.0 for for 1 hour the enzyme solution is irreversibly
inactivated. Similar treatment at pH 11 results in 30 %
inactivation. If the enzyme solution is heated at 100° C

for one hour, total inactivation occurs.