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Патент USA US3042592

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3,5i42,584
ire States
Patented July 3, 1962
1
2
converted liquors, these consist essentially of reversion
3,ti42,5?4
TREATMENT AND USE 9F ENZYMES FOR THE
HYDRULYSIS 0F STARCH
Earl R. Kooi, La Grange, Clarence F. Harjes, Hinsdale,
and John S. Gilkison, Chicago, Ill., assignors to Corn
Products Company, New York, N.Y., a corporation of
Deiaware
No Drawing. Filed Dec. 22, 1960, Ser. No. 77,531
2 Claims. (Cl. 195—-31)
products and ash.
In liquors converted by means of
amylase preparations, the impurities include the synthetic
dextrose polymers resulting from the action of trans
glucosidase' and the incompletely hydrolzed starchy ma
terials.
.
An object of this invention is to remove trans~
glucosidase activity from amylase preparations in order
to increase the ef?ciency and usefulness of such prepara
10 tions. A further object is to provide an improved proc
ess for the hydrolysis of starch or conversion products
This invention relates to a method for treating starch
thereof to dextrose-containing liquors having an excep
hydrolyzing enzyme preparations and to an improved
tionally high concentration of dextrose by means of such
process for the production of hydrolyzates of starch con
improved amylase preparations. Other objects will ap
taining an exceptionally high concentration of dextrose
by means of such enzyme preparations.
15 pear hereinafter.
Fungal amylase preparations, particularly those derived
All of the fungal amylase preparations examined by us
contained appreciable. amounts of transglucosidase activ
ity, as measured by the extent of synthesis of unferment
genus, are known to convert starchy materials to sub
able sugars from maltose. We have found further that
stantial amounts of dextrose. However, experience has
shown that except in very dilute solutions, the conversion 20 the ratio of transglucosidase:glucamylase in the enzyme
preparation, measured as described below, greatly a?ects
stops far short of complete conversion of the starch to
the amount of dextrose that can be obtained from
dextrose.
amylaceous materials upon hydrolysis with enzyme prepa
Amylase preparations of microbiological origin, par
ration.
ticularly those derived from the members of the Aspergil
Determination of glucamylase aczivity.—The substrate
lus genus and the Rhizopus genus, contain three major 25
is a l5~l8 D.E. spray-dried acid hydrolyzate of corn
types of enzyme activity concerned with the hydrolysis of
from members of the Aspergillus genus and the Rhizopus
alpha-1,4-linked glucose polymers. These three types of
activity may be classi?ed as alpha~amylase activity, glu
camylase activity, and transglucosidase activity.
Alphaaamylase action on starch pastes causes consider
able reduction in viscosity. In the absence of appreciable
amounts of glucamylase (or maltase) activity, consider
able amounts of maltose are produced by alpha-amylase
action.
Glucamylase activity on starch, partially hydrolyzed
starch, or on maltose results in the formation of dextrose.
This type of action has also been referred to as maltase
activity, amyloglucosidase activity, glucogenic activity, or
starch-glucogenase activity.
Transglucosidase activity results in the formation, par
ticularly from maltose, of unfermentable dextrose poly
starch. This material is dissolved in water and diluted
to 4.0 grams of dry substance per 100 ml. of solution.
Exactly 50 ml. of the solution is pipetted into a 100-ml.
volumetric ?ask. To the ?ask is added 5.0 ml. of pH
‘4.3, 1.0 molar sodium acetate-acetic acid buffer. The
?ask is placed in a Water bath .at 60° C., and after 10
minutes, the proper amount of enzyme preparation is
added. At exactly 120 minutes after addition of the
enzyme preparation, the solution is adjusted to a phenol
phthalein end point with one normal sodium hydroxide.
The solution is then cooled to room temperature, and
diluted to volume. A reducing sugar value, calculated as
dextrose, is determined on the diluted sample and on a
control with no enzyme preparation added. Glucamylase
activity is calculated as follows:
mers containing alpha-1,6-glucosidic linkages. Pan et al.
(Arch. Biochem. Biophys, 42, 421-434 (1953)) tested
2H
the transglucosidase activity of various laboratory and
industrial enzyme preparations and found that fungal 45
» where:
amylase preparations exhibited considerable transglucosi
dase action. Pazur and French (J. Biol. Chem, 196,
A=glucamylase activity, units per ml. or per gram of
265-272 (1952)) showed that the most probable action
enzyme preparation.
of the transglucosidase was to transfer a glucosyl radical
from maltose to the 6-position of a glucose molecule, or 50 S=reducing sugars in enzyme converted sample, grams
per 100 ml.
to the 6-position of the non-reducing end of a maltose
B=reducing
sugars in control, grams per 100 ml.
molecule, resulting in the formation of isomaltose, (6—(a
E: amount of enzyme preparation used, ml. or grams.
D-glucopyranosyl)-D-glucose) and panose, (4~(6-(a-D
glucopyranosyl) - (alpha-D-glucopyranosyl)-D~glucose),
respectively.
We have now discovered that the transglucosidase con
tent of fungal enzyme preparations is a considerable fac
tor in limiting the extent to which amylaceous materials
can be converted to dextrose by the enzyme preparation.
When amylaceous materials in a reasonably concentrated
solution are hydrolyzed by fungal enzyme preparations,
the enzymatic resynthesis of carbohydrates which are not
hydrolyzed to dextrose at an appreciable rate by the
enzymes present in the enzyme preparation reduces the
extent to which the amylaceous material can be con
verted to dextrose.
The reducing sugar concentration in the enzyme-con
verted sample should be not more than 1.0 gram per
100 ml.
Determination of Transglucosidase Activity-A \solu- '
tion of maltose is prepared by dissolving 200 grams of
Pfanstiehl C.P. maltose in water and diluting to 500 rnl.
Exactly 50 ml. is pipetted into a 100-ml. volumetric ?ask.
To the ?ask is added 5.0 ml. of pH 4.3, 1.0‘ molar sodium
acetate-acetic acid buffer. After mixing, an amount of
enzyme preparation containing 2.8 units of glucamylase
activity is added. The ?ask is placed in a 60° C. water
65 bath. After 72 hours, the ?ask is placed in a boiling water
bath for 15 minutes, then cooled, and the contents are
transferred quantitatively to a ISO-ml. beaker. The solu
tion is adjusted to pH 4.8 with two normal sodium hy
obtainable by successive evaporations and crystallizations
of dextrose-containing liquors, resulting from. either the 70 droxide solution, transferred to a 500-ml. Erlenmeyer
?ask, and diluted to about 200 ml. Ten grams of Fleisch
enzyme or acid hydrolysis of starch is limited by the
mann’s active dry yeast is added, and the ?ask is shaken
buildup of impurities in the mother liquors. In acid
In prior art processes of obtaining crystalline dextrose
from amylaccous materials, the yield of pure dextrose
3,042,584
3
4
continuously for 5 hours at 30° C. The contents are
then transferred to a 250-ml. volumetric ?ask and diluted
to volume. Two-hundred ml. are then centrifuged at
2000 r.p.m. for 15 minutes, and the supernatant liquor is
decanted into a dry ?ask. Fifty ml. of this liquor is pipet
ted into a 70-ml. test tube, 5 .ml. of 5.0 normal hydro
crystallization from hydrolyzates of various dextrose con
tents has been found to be as‘follows:
Dextrose content of hydrolyzate, per
cent dry basis _____________________ ._
Recoverable dextrose, anhydrous basis,
percent of hydrolyzate dry substance
86
88
90
92
94
72
76
80
84
88
chloric acid is added, the test tube is stoppered loosely, and
is heated in a boiling water bath for 3 hours, then cooled
For each percent increase in the dextrose content of the
in an ice bath. The contents of the tube are transferred
hydrolyzate, a 2 percent increase in dextrose yield is ‘ob
to a 100-mi. volumetric ?ask, and adjusted with two nor 10 tained. The signi?cance of the increased dextrose con
mal sodium hydroxide to a phenolphthalein end point.
tent of the enzyme hydrolyzate is apparent.
A reducing sugar value, calculated as dextrose, is deter
The practice of treating enzyme preparations or at
mined on an aliquot of the ?nal solution. To obtain a
tempting to separate enzymes by the use of clay minerals
correction for reducing sugar contributed 'by the yeast, a
is not new. However, none of the prior art practices has
control sample is included in which 20 grams of pure 15 been directed toward solving our problem nor to our
dextrose are used in the place of maltose, no enzyme
method of solving it. The prior art practice relating to
being added. The reducing sugar value of the enzyme
the use of clay minerals in the treatment of fungal enzyme
converted sample, corrected for reducing sugars contrib
preparations has been directed toward the removal of ex
uted by the yeast, represents the unfermentable material
traneous protein, toward the separation of protease from
synthesized by the enzyme preparation. Results are calcu
alpha-amylase, or consists of strictly experimental iat
lated as grams of .unfermentable sugars synthesized per
tempts to isolate pure, single enzymes. None of the prior
100 grams of maltose hydrate added.
art has been directed toward the removal of transgluco
Since the two enzymes, glucamylase and transglucosi
dase, compete for the same substrate (maltose), the above
results may be expressed as the ratio of transglucosidase
activity to glucamylase activity. This ratio is obtained by
dividing the grams of unfermentable sugars synthesized
per 100 grams of maltose hydrate by 100 minus this
value.
'
‘We have found that in the enzymatic conversion of
amylaceous materials under practical conditions, the
'sidase from amylase preparations by the use of clay min
erals, nor does the prior art contain evidence that trans
glucosidase can be removed from amylase preparations by.
treatment with a clay mineral. None of the prior art has
been directed toward removal of transglucosidase from
enzyme preparations as a means of increasing the yield
of dextrose obtained upon use of the enzyme preparation
in the hydrolysis of amylaceous materials. In any event,
we are aware of no prior instance where transglucosidase
amount of dextrose ?nally formed is governed by this
activity has ‘been removed substantially quantitatively from
ratio of transglucosidase activity to glucamylase activity.
enzyme preparations without substantial loss of the de
We have found further that all fungal enzyme prepara
sirable enzyme activity, such as glucamylase and alpha
tions examined by us exhibit transglucosidase: glucamyl 35 amylase activity.
ase ratios in the range of approximately 0.2-0.1. These
In carrying out the invention, the enzyme preparation
fungal enzyme preparations, used under practical condi
is ?rst treated by adding a clay mineral to a solution or
tions of substrate concentration, amount of enzyme, and
time of conversion, convert partial hydrolyzates of starch
to hydrolyzates containing in the range of 84 to 90 per
cent dextrose, based on the dry substance content of the
hydrolyzate.
The disadvantages of processes proposed in ‘the prior
art for the production of dextrose involving enzymatic
suspension of the enzyme preparation. ‘The enzyme prep
aration may consist of the whole culture liquor obtained
from submerged growth of an amylase-producing micro
organism; the clari?ed liquor obtained from the sub
merged culture, a suspension of a dried or partially dried
preparation which may contain bran, starch, or various
other adulterants used in standardizing the amylase prep
hydrolysis of starchy materials are many fold. In order 45 aration, or a solution of a completely soluble enzyme
to obtain a high extent of conversion of the starchy mate—
preparation. Following addition of the clay mineral
rials to dextrose, such dilute concentrations of the starchy
to the solution or suspension of the enzyme preparation,
materials were used that the cost of evaporation ren
the mixture is agitated and then the solid and liquid phases
dered such processes uneconomicals. Other processes
are separated. The transglucosidase activity is retained
made use of highly puri?ed enzyme preparations, still 50 in the solid phase while the desirable carbohydrases, in
without attaining the improvements described herein.
We have made the startling discovery that we can re
move transglucosidase activity substantially completely
from fungal amylase preparations, thus remarkably lower
ing the transglucosidase:glucamylase ratio, by treating
cluding, for example glucamylase and alpha-amylase, re
main in the liquid phase.
Other methods may be usedfor bringing about contact
of the clay mineral with the liquid enzyme preparation,
provided that intimate contact between the clay mineral
and. liquid enzyme preparation occurs, and that the clay
the amylase preparation with a clay mineral. We have
also discovered that amylase preparations treated in this
mineral is then separated from the liquid phase containing
manner produce unexpectedly high yields of dextrose
the desirable enzymes.
when used to hydrolyze starchy materials under practical
The resulting liquid enzyme preparation in which the
conditions. Enzyme preparations which will convert 60 activity of the desired glucamylase and alpha'amylase is
partial hydrolyzates of starch to hydrolyzates containing
retained may then be used directly for the hydrolysis of
starchy materials, or may be processed in known manner
only 85 to 86 percent dextrose, dry basis, will after treat
to :obtain a solid enzyme preparation.
ment with a clay mineral, thereupon convert partial hy
In ‘general, any clay mineral may be used for the re
drolyzates of starch to hydrolyzates containing as much
65 moval of transglucosidase activity. The clay minerals,
as 93 to 94 percent dextrose, dry basis.
as a group, are described as “silicates of alumina.” The
The signi?cance of this improvement is apparent when
principal clay minerals are classi?ed as montmorillonite,
it is recognized that the yield of pure crystalline dextrose
attapulgite, kaolinite, and illite. The clay minerals are
obtainable from hydrolyzates of starchy materials is, in
“essentially hydrous aluminum silicates, with magnesium
general, governed by the amount of non-dextrose mate
70 or iron proxying Wholly or in part for the aluminum in
rial present in the hydrolyzates. Each part of non-dextrose
some minerals, with alkailies or alkaline earths present as
impurity in the hydrolyzate will prevent the recovery or"
essential ‘constituents in some of them.” .Some are fur
approximately an equal weight of dextrose. For example,
ther described as fuller’s earth, ?oridin, sub-bentonite,
the yield of pure crystalline dextrose, expressed on an
china clay, and ball clay. They may range in composi
anhydrous basis, which can be economically obtained by 75 tion from nearly pure magnesium silicate to aluminum
3,042,584
5
amples which follow. They are for illustrative purposes
silicate, and may also be synthesized from the component
oxides or salts, or by reaction of crystalline minerals
with chemical reagents. For details see data sheet No.
only and are not to be construed as limiting our invention.
204, American Colloid Company (1945); Industrial Min
erals and Rocks, Amer. Inst‘. Mining and Met. ‘Eng. (1949
ed.), and Clay Minerolog (R. E. Grim, McGraw Hill
A series of enzyme preparations containing various
ratios of transglucosidase: glucamylase activity was pre
EXAMPLE I
pared by ammonium sulfate fractionation, by solvent
(1953) pages 18, 19).
fractionation, and by clay mineral treatment.
The bene?ts of transglucosidase removal are obtained
when the clay-treated enzyme preparation is used to con
These
zyme preparation will convert the amylaceous materials
enzyme preparations, derived from cultures of the Aspar
gillus niger group, were used to convert a partial hydrol
yzate of starch as follows: A 35 percent by weight sus
to a higher dextrose yield than will the untreated enzyme
pension of corn starch was acid hydrolyzed to a dex
vert amylaceous materials to dextrose.
The treated en
preparation. The amylaceous material may range from
trose equivalent (D.E. value) of 16 (dextrose equivalent
gelatinized starch, partial acid hydrolyzates of starch,
refers to the reducing sugar content of the hydrolyzate,
CI: calculated as dextrose, and expressed as percent by weight
of the dry substance present), The thinned starch was
adjusted to pH 4.5, brought to 60° C., and an amount
partial enzyme hydrolyzates of starch, to pure maltose.
While the ?nal dextrose content attainable with a given
enzyme preparation will depend somewhat on the speci?c
substrate material, the ‘bene?ts of transglucosidase re—
moval will be apparent on all of the above mentioned
of enzyme preparation was added calculated to contain
14 units of glucarnylase activity per 100 grams of thinned
starch dry substance. After 72 hours of incubation at
60° C., the liquors were analyzed for DE. and dex‘
trose content, with the results shown in Table I. The rela
tion between the transglucosidase:iglucamylase ratio
and the extent of conversion is well established.
substrate materials. _
In the case of fungal amylase preparations, conversion
of the amylaceous material should be carried out under
conditions of pH, temperature, enzyme dosage, and time
of conversion conducive to the formation of the max
imum amount of dextrose by the enzyme preparation
used. ‘In the pH range of 3.5 to 5.5, at temperatures of
50°-70° C., it will be found that maximal dextrose yields
will be obtained in about 72 hours if the glucamylase
dosage is about 14 units per 100 grams of substrate dry
substance. It will be apparent tothose skilled in the art
that lesser enzyme dosages may be ‘used for longer periods
Table I
Composition of Enzyme
Converted Liquor
Ratio, Transglucosidase: Glucamylase
'
Dextrose,
D .E .
of time, and greater dosages for shorter periods of time
to obtain essentially equivalent results.
The bene?ts of transglucosidase removal will be most
readily apparent if the substrate concentration is ‘greater
percent
,
90. 1
91. 1
91. 2
90. 7
90. 9
than about 5 grams per 100 ml. The more concentrated
the substrate, the greater the bene?cial e?ect of transglu
91. 6
‘ccsidase removal.
The amount of clay mineral used in the treatment of
the enzyme preparation will depend to a certain extent
upon the species of clay mineral used, the amount of en
zyme activity present, the amount of extraneous material
present, and the pH value during treatment. In general,
a pH value of about 4 will be most suitable for treatment.
The time of treatment is not critical, as long as the clay
is well dispersed in the enzyme preparation.
D.
83.
85.
85.
85.
85.
5
6
6
6
8
86. 6
92. 1 ,
88.1
92. 2
92. 2
92. '7
92. 6
92. 8
93. 6
94. l
93. 7
95. l.
94. 7
95. 0
95. 4
87. 7
87. (i
S8. 7
S8. 5
88. S
89. 8
90. 9
90. 3
92. 8
Q2. 5
92. 8
93. 2
The elTects of the clay mineral treatment are the result
of the greater adsorptive capacity for transglucosidase
EXAMPLE II
activity than for glucamylase activity. While it may be
Clay minerals as a group are effective in the selective
desirable to use somewhat more than the minimum
removal of transglucosidase activity Without removal
of appreciable quantities of glucamylase activity. A
culture of Aspergillus niger Corn Products Company
amount necessary to effect substantially complete removal
of transglucosidase, in order to insure maximum conver
sion of the substrate to dextrose with the treated enzyme
preparation; in no case has it been found newssary or de
sirable to use more than the amount which will adsorb
more than 20 percent of the glucamylase activity.
Culture No. M-370, isolated from a soil sample from
Louisiana, was grown in a fermentor under submerged,
aerobic conditions on a medium composed of 14 per
cent ground corn and 1.0 percent corn steep liquor
’
The adsorptive preference of clay minerals is sur'li
ciently selective for transglucosidase so that transgluccsi
dase removal will be essentially complete at a ‘clay con’
centration which will adsorb less than 20 percent of the
glucamylase activity. In most cases, transglucosidase ac
tivity can be completely adsorbed with a loss of glucamyl
ase activity not greater than 10 to 15 percent, even a loss
of 20 percent of the glucamylase activity is not a substan
tial loss in view of the considerable improvement obtained,
particularly since it is true that eleven units of a clay
treated preparation per 100 grams of starch will give
hydrolyzates of higher dextrose contents than will four~
teen units of the untreated preparation.
It will be noted from the foregoing description and the
examples which follow that we have made a decided im~
dry substance.
After completion of the fermentation,
the liquor was ?ltered to remove the myceliurn and other
suspended matter. The ?ltrate was divided into several
I
parts.
To 100-ml. portions of the culture ?ltrate at pH 4.0
was added, with agitation, 5 grams of ?nely divided clay
mineral. After 30 minutes agitation, the clay mineral
was separated by ?ltration. The resulting ?ltrates were
examined for glucamylase activity, for alpha-amylase
activity, and for transglucosidase activity. There was
no appreciable decrease in alpha-amylase activity. The
treated ?ltrates, along with the untreated control, were
used to convert a 16 DE. hydrolyzate of corn starch
as described in Example I. In this example, the amount
of clay used was 2.4 grams per 100 units or‘ glucamylase
provement in the art by -a simple, inexpensive, yet highly
activity. The results shown in Table II demonstate the
effective expedient.
applicability of clay minerals as a group to the removal
The invention may be further illustrated by the ex
of transglucosidase, and the effectiveness of transgluco
she 2,58/l
8
sidase removal by clay minerals in increasing the dextrose
yield attainable by the enzyme preparation.
the ?ltrates from clay mineral treatment as described in
Example I. The amount of clay required for eifective
removal of transglucosidase, as shown in Table ill, will
EXAMPLE III
depend to a certain extent upon the species of clay min
While it may be desirable to ?rst remove any in— 5 61.211 Used‘
soluble material present in the enzyme preparation prior
Table. H]
to treatment with a clay mineral, it is not essential to
do so. A culture of Aspergillus niger M~370 was grown
in a fermentor as described in Example II, After completion of the fermentation, the whole culture liquor 10
was removed from the fermentor and or nded into .our
Amount 0; Clay Mmmn
Clay Mineral Trademark
Used
G
v 11
Ht 1 F1
P m
rams
0.0 ay
1
3.1)“ OI‘0X
8.21 101‘
Grams pm. 100
Damon Bentomte gas may XXF
Creek
100-ml. portions. The ?rst part was not treated in any
'
;
way. The second part was agitated at pH 4.0 with 2.0
ml. ofenzyme
re 3.1'3. 1011
P p
-
‘
"‘ '
‘I
41
glul
units
CBJHY 215G
Demos? ContentofEmymeaqnvcrtgd
grams of clay mineral, sold under the trademark ‘Volclay Bentonite.” The mixture containing the mold my-
Liquor, Percent Dry Basis
'
celium and clay mineral was ?ltered and the ?lter cake
was discarded. The third part was ?ltered without clay
treatment.
0 18
01;;
The fourth part was ?ltered; the ?ltrate
8‘
was agitated with 2.0 grams of “Volclay Bentonite” added.
2%
Table II .
Dextrose
Major
Clay Mineral Trademark
Glucatn-
Transglu-
Content of
Content of
G-lucam—
Converted
Units per
ml.
Ratio
ylase
Mineral
Type
Component
Treatment
of Clay
Filtrate,
Untreated Control __________________________________________________ ,_
Volclay Bentonite ________ __ MontmorilSwelling
None _____ __
lonite.
Bentonite.
'
2. 08
2.04
Panther Creek _________________ “do ____ ..
None _____ __
Calcined____
Impreg-
Nonswelllng
cosidase
ylase
Enzyme
Hydroly
zato,
percent
dry basis
0. 16
0.05
86. 4
93.0
2.01
0.05
93.3
2. 04
1.89
0.05
0.03
93.0
94.2
92.1
Bontonite.
Adsorbol A~565 ________________ __do __________________ __
AdsorbolA-46 _________________ __do __________________ __
hated
2. 04
0. 00
Pikes Peak 9877 _______________ __do_ _ -_..
Filtrol 105 _____________________ __do ____ __
None _____ __
Acid Acti-
1. 91
0. 03
93.1
Pikes Peak 9T77 _______________ __d0_ _ ___.
Galetnod__._
1. 93
0.07
91. 8
Florex XXF ______________ __ Floridin. _.
Caleined Florex XXIL __. _____do ____ __
None_______
Caleined____
1.96
1.80
0.05
0.05
93.0
93.3
_
vated
Attapulgus Clay __________ __ Attspulgitm
F1§lert’1.s__.-. None _____ __
Bandy Tan _______________ __
Ball Clay
1.91
0.05
93.8
1. 97
0.05
93. 1
1. 98
0.13
88. 7
1. 92
0.07
92.0
ar 1.
Illinois Fire Clay
_
Grundlte. _-_
i
_ _ _ . __ .
_ _ _ _ _ _ __
The suspension was then ?ltered. Conversion of :1
16 DB. thinned starch was, as described under Example
I, carried out with the four preparations. This example
shows that whole culture liquor may be e?ectively
treated with a clay mineral.
Dextrose
Clay Mineral Glueamylase
Filtration of Culture Liquor
Treatment
Activity,
Content of
Enzyme
Converted
Units per ml. Liquor, Per
cent dry basis
EXAMPLE V
While some transglucosidase can be removed over a
considerable range of pH values, it will be found that
the treatment is most effective in the pH range of 3 to 5,
and a pH value of about 4.0 is recommended. With
some enzyme preparations, it may be necessary to make
periodic adjustments of the pH value during the treat
ment period, since the addition of the clay may result in
a change of the pH value. At pH values above 6, either
transglucosidase removal will be incomplete, or such an
55 excessive amount of clay will be required to obtain sub
Un?ltered ________________ __
None
2%
2. 24
92. 3
Filtered __________________ __
None
2. 30
2. 3O
86. 1
84. 7
2%
2. 33
92. 8
EXAMPLE IV
stantially complete removal of transglucosidase that a
considerable loss of glucamylase and alpha-amylase ac
tivity will occur. At pH values below 3, substantial in
activation or adsorption of glucamylase may occur.
Separate portions of a culture ?ltrate from a sub
merged culture of Aspergillus niger were adjusted with
While clay minerals as a group are effective in the
hydrochloric acid or with sodium hydroxide to the pH
removal of transglucosidase from fungal enzyme prepara
values shown below. To each 100 ml. portion was added
tions, they are not all equally effective at a given amount.
2.0 grams of Volclay Bentonite. The mixtures were
In an Aspergz‘llus niger culture liquor ?ltrate containing 65 agitated for 30‘ minutes and ?ltered. Results of conver
about 2 units of glucamylase activity per mL, for ex
sions conducted with the treated enzyme preparations,
ample, it will be found that where 0.5 gram of Volclay
carried out as described in Example I, are shown below.
Bentonite will accomplish effective removal of trans
glucosidase, as much as 5 grams or more of other clay
pH value prior to addition of
________________________ __
minerals will be required to accomplish the same extent 70 pHclay
value of ?ltrate after treat
of removal.
ment _______________________ _.
Glueamylase Activity of ?l
Portions of a culture ?ltrate of Aspergillus niger M—370
trate, percent of original ____ _.
were treated at pH 4.0 with various amounts of several
Dextrose content of Enzyme
Converted Liquor, percent
clay minerals as described in Example II. Conversion
D.B ________________________ ._
of a 16 DE. hydrolyzate of starch was carried out with 75
2.0
2.4
3.0
4.0
4.3
2.2
2.5
3.1
4.0
4.6
5.0
5.2
26.6
42.8
75.5
100
96.0
93.0
91.9
92.7
93.2
93.0
92.1
87.9
3,042,584
The initial glucamylase content of the culture ?ltrate
was 2.33 units per ml. The dextrose content of hydroly
zate obtained with the untreated preparation was 85.9%,
eral dried preparations derived from Aspergillus ?avus
dry basis.
?ltered. The ?ltrates, as well as the untreated prepara
tions, were used to convert a 16 DE. hydrolyzate of
oryzae group were dissolved or suspended in water, treat
ed with Volclay Bentonite at pH 4.0 for 30 minutes, and
EXAMPLE VI
starch as described in Example I, except that the con
versions were carried out at pH 4.6-4.8, 50° C., for 72
hours, using an amount of enzyme preparation equivalent
Was vtreated essentially as described in Example V, but
to 14 glucamylase units per 100 grams of starch dry sub
with various amounts of Volclay Bentonite.
10 stance. Activity determinations were also carried out
Another sample of Aspergillus niger M-—370 culture
?ltrate, with a glucamylase activity of 2.20 units per ml.,
at pH 4.6-4.8, and 50° C.
Amount of Clay Mineral
Used:
g./100 ml ______________ __
0
0.5
1
2
5
g./100 units ____________ _.
0
0.23
0. 45
0.91
2. 3
Rhozymes
Enzyme Preparation
Mylase
Dextri
112158
GLUCAMYLASE ACTIVITY OF
FILTRATE, PERCENT OF
ORIGINAL
15 Glucamylase Activity of solution,
Units/ml:
PP max
Before Clay Treatment _____ __
After Clay Treatment ______ __
pH value during treatment:
3 ______________________ _.
100
98. 2
98. 2
98. 2
97. 9
100
100
100
100
100
98. 2
100
100
97. 8
100
100
98. 2
100
100
99. 0
Amount of Clay used:
g 100 ml. . _
g/100 units
cC0am1
10.
____________ ..
20 Dextrose Content of enzyme
converted liquor:
86. 6
92. l
Untreated preparation ______ __
DEXTROSE CONTENT OF EN
Clay-treated preparation _ _ . . _
ZYME-CONVERTED LIQUOR,
PERCENT D.B.
EXAh/IPLE IX
A dried enzyme preparation derived from a culture
of Rhizopus delemar was, dissolved in water. One por
tion of the solution was not treated, the other was treated
at pH 4.0 with 2 grams of Volclay Bentonite per 100 ml.
EXAMPLE VII
of solution. Conversions of 16 DE. thinned starch were
The cultures described as Aspergillus niger NRRL 330 30 conducted as described in Example VII. Dextrose con
and NRRL 337 have been studied very exhaustively for
‘tents of hydrolyzates resulting from conversion with the
production of amylases (Tsuchiya et al., Cereal Chem.
untreated and clay treated preparations were 85.1 and
27, 322 (1950)). These and other members of the Asper
93.3, respectively.
gillus niger group (see Thom and Raper, “A Manual of
EXAMPLE X
the Aspergilli,” Williams and Wilkins Co. (1945)) Were 35
In
the
case
of
substantially
puri?ed enzyme prepara
grown on a corn and corn steep liquor medium as de
tions, the pH range over which transglucosidase can be
scribed in Example II. The culture ?ltrates therefrom
e?ectively removed is extended up to at least pH 6.
were divided into two portions. One portion was un
However, it Will also be found that at a given glucarnylase
treated, the other portion was treated with Volclay
concentration, less clay will be required to e?ect sub
Bentonite at pH 4.0 for 30 minutes, then ?ltered. The
85.0
85.0
85. 0
85. 0
85. 8
88. 7
85. 5
84. 8
91. 3
91. 8
88. 2
84. 6
92. 5
92. 5
90.8
85. 5
92. 5
92. 5
92. 1
88. 7
25
stantially complete removal of transglucosidase.
untreated culture ?ltrates, and the‘ clay-treated ?ltrates
Also,
clay concentrations at which substantially complete re
moval of transglucosidase from an unpuri?ed prepara
drolyzate of starch, adjusted to pH 4.3, and held at 60°
tion is attained without substantial loss of glucamylase
C. for 48 hours. The enzyme dosage used was in each
case 28 glucamylase units per 100 grams of starch dry 45 activity may cause inactivation or adsorption of glucamyl
ase in the puri?ed preparation.
substance. The enzyme-converted hydrolyzates were an
An enzyme preparation derived from Aspcrgillus niger
alyzed for dextrose content with the results shown below:
M—370 was puri?ed by solvent precipitation. The puri
In all cases, the hydrolyzate from the clay ‘treated enzyme
?ed preparation contained essentially all of the glucarnyl
preparation had a substantially higher dextrose content
than that obtained with the untreated preparation, show» 50 arse, alpha-amylase, and transglucosidase activity of the
original preparation, ‘out only 5 percent of the total solids.
ing the general applicability of transglucosidase removal
The puri?ed preparation was dissolved in water to give
through clay-mineral treatment to members of the As
were then added to a 20% solution of a 16 DE. hy
a glucarnylase concentration of 1.76 units per ml. Por
tions of the solution were treated at pH 3, 4, 5, or 6 for
30 minutes with various amounts of Calcined'Florex
pergillus niger group.
Dextrose Content of
Enzyme-Converted
Culture
XXF, with the results shown below.
Hydrolyzate, Percent D13.
Untreated
Preparation
Amount of Clay
Clay treated
Mineral used;
Preparation
60
Aspergillus awamm'i NRRL 2042 ________ __
Aspcrgillus fonsecaerts NRRL 67
_
85.0
90. 4
Aspergillus
Aspergillus
Aspergillus
Aspergz'llus
_
_
r
__
S6. 0
86. 0
86. 7
87.0
92. 8
92. 8
92.1
92. 5
Aspergillus niger N REL 363 _____________ __
89. 7
95. 1
86.0
90. 8
84. 3
90. 4
88. 8
91.5
niger
niger
niger
nz'ger
GLPC M—370-_
NRRL 330..
NRRL 3.-.NRRL 337
>
89. 7
92. 8
merit:
____________________________________ __
Aspergillus phoem'cis CPC M-381.
__
84. 0
93.1
Aspergillus p/Loenicis ATCC 13156 _______ __
86.0
90. 4
0. 10
0. 057
0. 5
0.28
l. 0
0. 57
2. O
l. 14
5.0
2. 8
GLUCAMYLASE ACTIVITY OF FIL
TRATE, PERCENT OF ORIGINAL
100
98. 3
95. 5
89. 3
____ ._
76. 5
100
100
100
100
99. 0
100
93. 2
100
92. 5
96. 0
.... _ _
____ _ _
78. 3
84. 5
100
97. l
____ _.
94. 4
85. 4
100
Aspergz’llus niger mut Schismanii NRRL
Aspergz'llus phoenicis NRRL 1956.
0 0. O5
0 0. 028
ph value during treat~
Aspergz'llus m‘ger mut cinnamomeus NRRL
34 ____________________________________ ..
g/l00 11:11: _________ .t
g/100 uruts _______ __
____ _ _
____ __
DEX’I‘ROSE CONTENT OF ENZYME
70
CONVERTED
Lroguon
PERCENT D
EXAMPLE VIII
Enzyme preparations derived from the Aspergillus
?avus-oryzae group are also known to convert amyl
acc‘ous materials to substantial amounts of dextrose. Sev
75
88.0
92.3
____ _.
93. 3
86. 3
85. 5
91.8
88.0
93.0
90. 6
____ __
____ _-
.... __
93. 3
93. l’
85. 5
85. 5
____ _ _
89. 9
92. (l
3,042,584:
'11
ing from gelatinized starch to starch which has previously
EXAMPLE X
A culture ?ltrate of Aspergillus niger M~370, and a
puri?ed preparation like the one described in Example
been subjected to appreciable acid or enzyme conversion.‘
An 18% d.s. suspension of corn starch was ?rst gelati
nized and then cooked at 121° C. for one hour. One
1.66 units per ml. with, at pH 4.0, various amounts of UK portion of the cooked starch was converted for 90 hours
at pH 4.5 with an Aspergillus niger M-37O enzyme prep
Volclay Bentonite.
aration which had not been treated with a clay mineral.
X were each treated at a glucarnylase concentration of
Amount of Volclay
Bentonite Used
Results with Unpuri?ed Enzyme
I gap/100
g./l00 ml.
units
Loss of
Dextrose
Content
of
Loss of
Dextrose
Content
of
Glu-
Enzyme-
Glu-
Enzyme
camylase
Activity,
Percent
Ooncamylase
verted Activity,
Liquor, Percent
Con
verted
Liquor,
Percent
Percent
.B.
O
0
85.0
0
and 98.0% dextrose, respectively.
15
86. 0
__________________ __
0
0. 03
__________________ _ -
- l. 8
88. 4
4. 2
G. 0
10.8
27. 7
93.0
93. 3
93. 3
93. 3
0
0
0
O
0
1
2
hydrolyzates in conversions with the untreated and treat
ed enzyme preparations contained, on a dry basis, 93.3
.B
0. 015
0. G6
0. 15
0. 3
0. 6
1.2
2. 4
4. 8
Another portion was converted in the same manner with
a preparation which had been treated with Volclay Ben
tonite. In each case, the enzyme ‘dosage was 20 glu
camylase units per 100 grams of starch. The ?ltered
Results with
Puri?ed Enzyme
86. G
87.0
88. 7
92.1
92.
92.
92.
87. 0
EXAMPLE XII
In the case of dilute enzyme preparations, it may be
25
This application is a continuation-in-part of applica
tion Serial No. 666,469, ?led June 18, 1957, and now
abandoned.
We claim:
1. A process for selective removal of transglucosidase
activity from a fungal amylase preparation obtained from
an organism selected from the group consisting of mem
bers of the Aspergillus genus and members of the Rhizo
pus genus, said preparation containing transglucosidase
and glucamylase activities, which comprises treating said
amylase preparation in aqueous medium with a clay min
eral in an amount up to 50 grams of clay mineral per 100
units of glucamylase activity and not greater than that
a necessary to use a lesser amount of clay to remove trans
glucosidase selectively. For example, Aspergillus niger
which will remove 46 percent of the glucamylase ac
M-370 culture ?ltrate was diluted to glucamylase con
30 tivity to adsorb the transglucosidase on the clay mineral,
centrations of 1.7, and 0.1 units of glucamylase per ml.
separating the medium from the clay mineral, and re
covering said medium substantially free from transglu
cosidase activity, said recovered medium containing at
least 54 percent of the glucamylase activity of the original
Each portion was then treated with various amounts of
Volclay Bentonite at pH 4.0. For simplicity of expres
sion, the following values were read from plots of the
data. Values in parenthesis were obtained by extrapola 35
tion because of the di?iculty of dispersing these amounts
of Volclay Bentonite in the volume shown.
preparation.
’
2. A process for hydrolyzing starch to dextrose which
comprises partially hydrolyzing the starch to reduce the
The amount of alpha-amylase adsorption was nearly
viscosity thereof and thereafter subjecting it to the action
the same value as shown for glucamylase adsorption.
Similar results were obtained with the clay minerals 40 of a fungal amylase preparation treated by the process
of claim 1.
Florex XXF and Adsorbol A-420.
Initial Gluoamylase Concentration, Units/ml.
Amount of
0.1
1.7
‘
Volclay Used,
gJlOO units
glucamylase
Transgln-
Glue
Transglu-
Gluc
centration,
Clay con-
cosidase
amylase
centration,
cosidase
amylase
g./100 ml.
removed,
loss,
. g./10O ml.
removed,
1
2
3
0.17
0.42
0. 85
11
39
100
5
percent
Clay 0on4
percent
percent
loss,
percent
0
0
O
1. 7
__________ __
9
4. 2
_
l
13
8.5
0
19
31
46
67
(17)
(42)
(85)
(170)
_
_
_
.
(4)
(10)
(19)
(37)
2
EXAMPLE XIII
The bene?ts of transglucosidase removal from fungal
amylase preparations may be attained on substrates rang
References Cited in the ?le of this patent
60
Cereal Chemistry, vol. 26, No. 2, March 1949, pp. 98-;
1‘09.
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