Note: Descriptions are shown in the official language in which they were submitted.
~1311'.~Z
BACKGROUND OF THE INVENTION
At present, when producing dextrose indus-trially
from starch, the principal glucoamylases employed for the
saccharification process are those produced by micro-
organism belonging to the genera Rhizopus and Aspergillus.
The conditions under which these glucoamylases are employed
are pH 5.0 and 55C for the enzyme of the Rhizopus micro-
organism, and pH 4.5 and 60C for the Aspergillus micro-
organism's enzyme. In addition, maximum dextrose content
of the hydrolyzate is about 96% (dry solids basis) when
these glucoamylases react with enzyme liquefied starch at
a 30% concentration. One reason that the dextrose yield
does not reach 100% is that isomaltose accumulates due to
a reverse reaction by these glucoamylases. However, there
was recently published a report (U.S. Patent 3,897,305)
that the reverse reaction of glucoamylses is extremely
small in the vicinity of neutrality and that the dextrose
yield can thus be elevated to about 98% by carrying out
the reaction at about a neutral pH with the joint use of
pullalanase. The pullalanase acts to debranch the starch
and increases the rate of glucoamylase action under these
nearly neutral conditions. As far as neutral glucoamylases
are concerned, only one has ~een reported to date, that
being the glucoamylase produced by the rice blast-causing
fungus (Piricularia oryzae; Kazuo Matsuda, et al:
Amylase Symposium, Vol. 9, 1974), but t~ls glucoamylase
possesses low thermostability and so cannot be employed
under industrial conditions.
SUMMARY OF THE INVENTION
It is therefore a primary object of the present
invention to provide a glucoamYlase that is active at
nearly neutral pH.
It is another object of the invention to provide
a glucoamylase that possesses enough thermostability so
that it can be employed under industrial reaction
conditions.
It is yet another object of the invention to
provide a glucoamylase that reacts with a starch hydrolyzate
to give high yields of dextrose.
A microbial strain has been discovered which
produces a new glucoamylase having optimum activity at
a pH of 6.o to 6.5 and good thermostability. The new
glucoamylase is capable of converting a 30% by weight
solution of a 10 D.E. (dextrose equivalent) liquefied
starch to a product containing at least about 96% dextrose
when reacted with the starch hydrolyzate at pH 6.0 to 6.5
at 55C. This invention includes the method for the
production o~ this glucoamylase wherein the microorganism
11;31142
of the genus Stach,ybotrys, which produces the glucoamy,ase,
is cultured in a medium and the enzyme is recovered from
' the culture broth.
.
BRIEF DESCRIPTION OF THE DRA~INGS
Figure 1 shows the relationship between the pH
and the enzyme activity in the cases of the enzyme of the
present invention and the conventional glucoamylases
produced by _. niveus and A. niger microorganisms.
Figure 2 shows the relationship between the
temperature and the enzyme activity in the cases of the
present enzyme and the glucoamylase from P. ~
Figure 3 presents the inactivation curves for
the enzyme of this invention when it is treated at various
'pH levels.
Figure 4 provides a comparison of the present
enzyme and the conventional glucoamylases produced by the
R. ni'veus, A. niger and P. oryzae microorganisms in terms
of their relative thermostabilities.
DETAILED DESCRIPTION~ OF THE INVENTION
The properties of the novel neutral gluco-
amylase of the present invention are presented in detail~
and their properties are contrasted with those of the
previously-known glucoamylases.
The term "D.E." is an abbreviation for "dextrose
equivalent", and these terms are used interchangeably to
refer to the reducing sugar content of a material calculated
as dextrose and expressed as percent of total solids.
The term "starch hydrolyzate" is used in a
general way to refer to a syrup or dry product that is
made by the partial hydrolysis of starch. Such a product
may be made by acid or enzymic hydrolysis.
The term "liquefied starch" is used to refer
to a low D.E. (D.E. from about 2 to about 20) starch
hydrolyzate.
1. Activity and Substrate Specificity
The present enzyme is able to hydrolyze such
carbohydrate compounds as starch, soluble starch, amylose,
amylopectin and glycogen, and to produce dextrose from
them. The yield of dextrose from each of these substrates
is 100% when the substrate concentration is 1%. The muta-
rotation of the produced dextrose is positive. This enzyme
is thus a glucoamylase. The reaction velocity of this
enzyme was compared to the rates shown by the glucoamylases
produced by microorganisms belonging to Rhizopus and
Aspergillus in relation to various substrates. The results
.
are presented in Table I. As can be seen from this table,
the activity of the present enzyme is notably higher than
the activities of the other two glucoamylases especially
in relation to the hydrolysis of pullulan.
Z
TABLE I
SUBSTRATE SPECIFICITY
; Reaction Ratea)
AspergillusRhizopus
niger niveus
Present Gluco- Gluco-
SubstrateEnzymeamylaseb)amylaseC)
Dextrin (D.E. 10) 100 100 100 .
Amylpectin 104 113 91
Soluble Starch 122 95 112
Pullulan 9 2 2
Glycogen 102 100 91
Maltotriose 6 12 8
'! Maltohexaose~1 100 146
Panose 44 47 48
Maltose 14 26 19
' . , ,
a) The enzymatic activitie$ of each glucoamylase were
determined with the sub.strates present at a 1% con-
centration; each enzyme t S acti.vity in relation to
dextrin was assigned the value 100, and the activities
on the other substrates are presented as relative
- values.
b) Available from Enzyme Development Corporation,
- 2 Penn Plaza, New York, N.Y.
c) 5umyzyme available from Sumitomo Shoji Kaisha, Ltd.,
l, Kanda Mitoshiro-Cho, Chiyoda-ku, Tokyo, Japan.
2. Optimum pH and Stable pH Range
The relationship between the enzymatic activity
(relative value) of the present enzyme and the reaction
pH were investigated and then compared with the corre-
sponding relationships for the conventionally-known
glucoamylases produced by the Rhizopus and Aspergillus
microorganisms. The results are presented in Figure 1.
114;~
As shown in the figure, the optlmum pH of this enz~me at
60C is 6.0 to 6.5, considerably higher than the pH optima
of the other enzymes. In addition, this enzyme shows its
best stability in the vicinity of pH 6.o, but no inacti-
vation of this glucoamylase is seen even when it is left
sitting for 24 hours at room temperature, over a pH range
of 4 to 11.
3. Determination_of Potency
A 0.5-ml aliquot of a suitably diluted enzyme
solution was added to 0.5 ml of a 2% solution of a spray-
dried maltodextrin (D.E. about 10) in 0.1 M acetate buffer
solution (pH 6.o) and this was incubat~ed at 60C for
precisely 10 minutes. The enzyme reaction was then stopped
by heating the mixture for 5 minutes in a boiling water
,bath. The amount of dextrose produced was determined by
the glucose oxidase method. The amount of enzyme producing
1 micromole of dextrose per minute was defined as 1 unit.
4. Optimum Reaction Temperature Range
The effect of temperature on the relative enzymatic
activity of the present enzyme at pH 6,o was compared with,
the relative activity for the knowr. glucoamylase from the
rice blast fungus, Piricularia oryzae. This comparison
is shown in Figure 2. It is evident that the optimum
temperature for the reaction of the present enzyme under
these conditions is ~5C, about 10C higher than that of
the enzyme from Piricularia oryzae.
ll~ Z
5. Inactivation Due to pH and Tem~erature Conditions
.
- Figure 3 presents inactivation curves of the
relative enzymatic activity of the present enzyme when it
was treated for 60 minutes at 60C over a pH range of 3 to
8. As is clear from the figure, this enzyme is most stable
at pH 6, and it is completely inactivated by this treatment
for 30 minutes at pH 3 and for 1 hour at pH 4. In addition,
Figure 4 shows a comp~rison of the thermostability of the
present enzyme and the glucoamylases from the Rhizopus,
Aspergillus and Plricularia microorganlsms. Namely,
Figure 4 presents the inactivation curves obtained for
these enzymes when they were treated at 60C while being
held at their respective optimum pH's for stability. It
can be seen that the thermostability of the present enzyme
is inferior to that of the glucoamylase of Aspergillus
- origin, but is superior to the thermostability shown by the
glucoamylases from the Rhizopus and the Piricularia micro-
organisms.
6. Inhibition, Activation and Stabilization
This enzyme does not require any special acti-
vating or stabilizing agents. However, the same as in the
case of most of the other glucoamylases, this enzyme is
inhibited by mercuric chloride, potassium manganate,
ferrous chloride, other metal salts and ~ris.
7. Purification Procedure
The present enzyme can be purified by means of
a combination of any of the ordïnary purification methods
~13,1~
such as ammonium sulfat~ fractianation, organic solvent
fractionation, starch adsorption, and various chromatog~
raphies. An lllustrative example ~f such a purification
procedure is presented next.
The cells and other insoluble material are
eliminated from the cultured material and then the
culture fluid is frozen overnight at -20C. This is
then melted at room temperature and the insoluble matter
is removed by centrifugation. Next, two volumes of cold
isopropanol is added to this and it is left standing for
one night at 4C. The enzyme precipltates and the
supernatant is removed by decantation, The precipitate
is then dissolved in a 0.05 M tris-HCl ~uffer solution
(pH 7.5) containing 1 mM EDTA, and the dissolved material
is next dialyzed for one night at 4C against the same
buffer. DEAE-cellulose which has been equilibrated with
the same buffer solution is next added to this dialyzed
enzyme solution so that the enzyme is adsorbed thereto.
After washing this DEAE-cellulose with the same buffer,
enzyme is eluted from the resin with a preparation of the
same buffer containing 0.3 M NaCl. The enzyme is then
precipitated by the addition of two volumes of cold iso-
propanol to the eluate, and this precipitated material
is recovered by centrifugation. The precipitate is
dissolved in the 0.05 M tris-HCl buffer solution (pH 7.5)
containing 1 mM EDTA, followed by overnight dialysis
Z
against the same buffer. The dialyzed enzyme solution is
; next applied to a DEAE-cellulose column which has been
equilibrated with the same 0.05 M tris-HCl buffer (pH
7.5) containing 1 mM EDTA. The enzyme is then eluted
from this column by passing through it a linear concen-
tration gradient of the same buffer containing MaCl up
to 0.5M. The eluted fractions which contain the enzyme
are pooled and the enzyme is concentrated by means of the
isopropanol precipitation technique. This concentrated
enzyme is then applied to a column of Sephadex* G-150
which has been equilibrated with a 0.05 M tris-HCl buffer
(pH 7.0) containing 1 mM EDTA, and elution is carried
out with the same buffer solution. When this procedure
was followed, the purified enzyme which was obtained
showed a single band in disc electrophoresis.
8. Molecular Weight
The molecular weight of the present enzyme was
investigated using a Sephadex G-150 column in accordance
with the procedure of Andrews, P., Biochem J. 96, 595
(1965). The results indicated that this enzyme's molecular
weight is about 50,000.
Next, the points of difference between the
present enzyme and the conventionally-known glucoamylases
will be presented, and an explanation will be made of the
reasons that this enzyme is to be considered a new enzyme
having its optimum pH in the vicinity of neutrality.
* trade mark
.
Regarding the optimum pH.of enzymes, it can be
seen from the data presented in Figure 1 and Table II that
the only glucoamylases which have their optimum pH's near
the neutral zone are the present enzyme and the glucoamylase
produced by the rice blast microorganism, Piricularia
oryzae. However, as is clear from Figure 2 and Table. II,
the present enzyme and the rice blast glucoamylase have
optimum reaction temperatures which are extremely different.
In addition, the curves presented in Figure 4 indicate that
the thermostability of the present enzyme is vastly superior
to that of the rice blast glucoamylase Moreover, Table II
shows that the molecular weight of the present enzyme is
much smaller than the molecular weight of the other kno~n
glucoamylases.
TABLE II
COMPARISON OF VARIOUS GLUCOAMYLASES IN TERMS
OF OPTIMUM pH, OPTIMUM TEMPERATURE
AND MOLECULAR WEIGHT
Optimum
Optimum Temp. Molecular
Glucoamylase ~Ha) oCa~ Weighta)
Present Enzyme 6.o-6.5 65 50,000
(Stachybotrys subsimplex)
Rhizopus sp. (Sumyzyme)5.0 60 70,000b)
Aspergillus niger 4.5* 70* 97,000C)
Endomyces sp.d) 5.0 - 64,ooo
Endomyces fibuligerae) 5.5 60
Trichoderma viride ) 5.0 60 75,000
Cephalosporium charticolag) 5.4 60 69,000
Piricular~a oryzaeh) 6.5 55 94,000
(rice blast org.
--10--
a) All values except those marked with an asterisk C~
were taken from the references.
b) Hiromi, et al: Biochem. Biophys. Acta 302, 362 (1973)
c) J. H. Pazur, et al: J. Biol. Chem. 237, 1002 (1962).
d) Hattori, et al: Agr. Biol. Chem. 25 ~ 95 ~1961).
e) Harada, et al: J. Ferment. Tech. 53, 559 (1975).
f) Okada; J. Jap. Soc. Starch Sci. 21, 283 (1974).
g) H. Urbanek, et al: Appl. Micro. 30, 163 (1975).
h) Matsuda, et al: Amylase Symposium 2, 105 (1974).
On the basis of the above facts, it can be con-
cluded that the glucoamylase produced by the method of
the present invention is a new neutral glucoamylase
which has been totally unknown to date.
An explanation will now be made of the method
for the production of the present enzyme.
As a desirahle example of the glucoamylase-
producing microorganism to be used in the present inven-
tion, there is strain G30-1140, which was isolated from
the soil by the present inventors. The identification
of this strain will be presented first.
T~emorphological properties of tne present
strain were determined in accordance with the methods
described by the researchers listed below:
Gilman, J. C. A MANUAL OF SOIL FUNGI. The Iowa
State University press, Ames. 1971.
Clements, F. E. and Shear, C. L. THE GENERA OF
FUNGI. Hafner, New York. 1964.
Barnett, H. L. ILLUSTRATED GENERA OF IMPERFECT
FUNGI. 2nd ed. Burgess, Minneapolis. 1968.
--11--
Z
.
3isby, G. R. Trans. Br l~lycol. Soc. 26,
133-43 (1943).
Ainsworth, G. C. DICTIONARY OF THE FUNGI.
6th ed. Commonwealth Mycological Institute,
Kew, Surrey. 1971.
9. Morphological Properties of Strain-G30-1140
The present strain was cultured on five kinds
of media in Petri dishes. The following sections present
the morphological characteristics which were observed
for isolated colonies.
a) Czapek Agar Medium
When incubated at 30C for 10 days, the
colonies are thin and round, with a diameter of 4 to
5 cm. The vegetative hyphal are hyalin and show poor
growth with hlack conidial clusters scattered like powder
over the surface of the colonies. T~e undersides of the
colonies are a brown color, and a tan pigment is secreted
into the medium.
The vegetative hyphal consist of branched
fibers which rarely possess any septa; the conidial
structure is uniformly supported by the fibers. The
conidiophores which have septa protrude from this at
right angles. The length of the conidiophores is usually
from 40 to 60 ~, but sometimes they attain more than 100 ~.
The diameter of these is about 3 to 6 ~, and although
there are cases when the basal area of these is smooth,
most of their surface is verrucose, being covered with
fine granular projections. These conidiophores are
hyalin, and most are not branched.
-12-
Z
On the apex of the cGnidiophores, hyalin
phialides form whorls of 3 to 8 units. The shape of the
ph~alides is ovoid or flask-like; they are 8 to 15 ~
in length and have a diameter of 2 to 6 ~; their surface
is smooth. The conidia are formed at the apex of the
phialides and are oval shapes of 3 to 5 ~ x 5 to 10 ~,
and have a smooth surface. These are hyalin at the time
of formation, but become blackish green as they mature.
The surface of the conidia is covered with a
large amount of viscous material. For this reason, the
conidia stick together and form large conidial clusters
at the apex of the conidiophores. The viscous material
is transparent at the time that it is formed, but then
gradually becomes black.
b) Modified Czapek Agar Medium
Percent
Soluble Starch 1.0
Corn Steep Liquor (dry solids basis) 0.1
NaN03 0.2
K2HP0 4 - 0 . 1
KCl O 05
MgS04 7H20 ~ 0.05
FeS04 7H O 0.001
Agar 2.0
pH to 7.0 with NaOH
The growth of colonies on this medium is a
bit slower than on the previously described Czapek
medium, reaching about 3 cm when incubated for 10 days
at 30C.
The colonies are circular and thin, and their
surfaces have radiating from their centers a black viscous
material which is in the form of oil-like drops having
diameters reaching 1 to 3 mm. These arise from the
gathering together of clusters of conidia which are
enclosed in the viscous material and then form oil-drop-
like bodies. The undersides of the colonies show a
darker brown color than is seen with the previous Czapek
medium, and a small amount of brown pigment is secreted
into the medium.
c) Potato-Dex rose Agar Medium
The growth of colonies on this medium is a bit
slower than on the previously-described Czapek medium,
reaching 3 to 4 cm when incubated for 10 days at 30C.
These colonies are also circular, but they have a
somewhat greater thickness than the colonies on the
Czapek medium. The growth of the vegetative cells is
good, developing in a radiating pattern. The surfaces
.
of the colonies are black with a slightly green luster,
and are rich in hyphae, conidial clusters and so on.
After 14 days of incubation, the surfaces of the old
colonies have radiating formations of synnemata standing
-14-
42
about l to 3 mm erect. The undersides of these colonies
show a blackish-brown color, and a large amount of brown
pigment is secreted into the medium.
d) Special Agar Medium
Percent
Soluble Starch 1.0
Corn Steep Liquor (dry solids basis) 0.2
Cottonseed Oil Dregs 0.1
Yeast Extract 0.1
K2HP04 0.1
MgS04 7H20 0.05
- Agar 2.0
- pH to 7.0 with NaOH
- The colonies on this medium after 10 days of
incubation ~t 30C have diameters of 5 to 6 cm, and are
round and thin. The vegetative hyphae show good growth
and have a black luster. The growth of Gonidia is worse
than on the above-mentioned media. The undersides of
the colonies are tan in color, and a tan pigment is
released into the medium.
e) Davis's Yeast Salt Agar Medium
The colonies on this medium after 10 days of
incubation at 30C have diameters of 2 to 3 cm znd are
more oval than round in shape. The hyphae are tan with
~13~Z
a touch of white and form somewhat thick colonles which
are velvety. The undersides of the colonies are tan
in color, but absolutely no pigment is secreted into
the medium.
10. Physiological Properties of Strain G3~114Q
a) Growth Temperature
This strain is capable of growth over a
temperature range of 10 to 37C, but its optimum growth
temperature is in the vicinity of 30C.
b) Growth pH
This strain is capable of growth over a pH
range o~ 3 to 10, but its optimum growth pH is in the
vicinity of pH 7.
c) Carbon Source
This strain i5 capable of using such carbon
sources as dextrose, fructose, galactose, mannose,
saccharose, maltose and starch in order to support its
growth.
On the basis of the above microbiological
findings, strain G30-1140 was identified as Gliobotrys
alboviridis after consulting the GENERA OF FUNGI and
A MANUAL OF SOIL FUNGI. However, according to the
DICTIONARY OF THE FUNGI and G. R. Bisby (Trans. Br.
-16-
~L~llL~Z
Mycol. Soc. 26, 133-43 (1943)), this organism is the
same as Stachybotrys subsimpleX, and for this reason
strain G30-1140 was identified as Stachybotrys subsimplex.
Strain G30-1140 has conidiophores which stand
erect from its vegetative hyphae, branching is almost
nonexistent, and they have septa. There are occasions
when the basal part is smooth, but the apex is covered
with projections. At the apex,a level of phialides form
a whorl of 3 to 8 units. Conidia having smooth oblong
surfaces divide from these phialides, and they are
enclosed in a richly viscous substance. These properties
agree well with those described for Stachybotrys
subsimplex by G. R. Bisby (Trans. Br. Mycol. Soc. 26,
133-43 (1943)).
This Stachybotrys subsimplex strain G30-1140
is being stored at the Fermentation Research Institute,
Agency of Industrial Science & Technology, Chiba City,
Japan, as Deposit No. 4377.
Regarding the cultivation of the microorganism
to be employed in the present invention, the general
knowledge and techniques used in the culture of molds
are appllcable.
Namely, as the nutritional source medium, it
is possible to employ the media which are used for the
culture of ordinary molds. For example, various starches,
starch hydrolyzates, corn meal, wheat flour, final
molasses, etc., can be employed as carbon sources, while
~17-
~ ~ 31~
the nitrogen requlrement can be supplied in the form of
peptone, cottonseed oil dregs, meat extract, yeast
extract, casein, corn steep liquor, malt extract,
soybean dregs, skimmed milk~ inorganic ammonium sa~ts,
inorganic nitrates, etc. As the inorganic salts, it is
possible to employ calcium chloride, magnesium sulfate,
phosphates~ sodium chloride, potassium chloride, and so
on. Furthermore, these carbon sources, nitrogen sources
and inorganic salts can be used either singly or in
appropriate combinations. In addition, when it is
desired to promote the growth of the microorganism and
bring about an increase in its enzyme production, it is
possible to employ trace amounts of metallic salts,
vitamins, amino acids, and so forth.
~ The culture conditions usually employed for
molds are also applicable to the cultivation of this
microorganism. Namely, in liquid culture, if this
mlcrobe is cultured for 7 to 14 days at pH 5 to 8 and
20C to 37C together with agitation to provide aeration,
the enzyme of the present invention is accumulated in
the culture fluid. In addition, if solid materials such
as bran are employed, it is possible to carry out solid
culture.
Next, an example will be presented of a method
whereby the new neutral glucoamylase which is the
objective of the present invention can be recovered from
the cultured material. In the case of liquid culture,
the mycelia are eliminated by any of the publicly-known
-18-
142
methods; then the filtrate can be concentrated under
reduced pressure, or the enzyme can be salted out with
the other proteins by adding inorganic salts such as
ammonium sulfate to the filtrate, or the enzyme can be
precipitated out and concentrated by the addition of an
organic solvent such as acetone or isopropanol.
.
In the case of solid culture~ the e.nzyme is
first extracted from the. cultured material by the use
of water or a buffer solution. Then, as in the case of
liquid culture, it is possible to obtain the enzyme in
a concentrated form.
The crude preparations of this new neutral
enzyme obtained in this way can then be purified by
carrying out the previously-mentioned purification
techni~ues.
It is possible to employ this new neutral
glucoamylase of the present invention for the saccharl-
fication of liquefied starch when producing dextrose
from starch. Especially, if the present enzyme is used
and the saccharification is carried out at pH 6.o to
6.5, ~here is, as was mentioned earlier, little reverse
reaction occurrence, and this results in an increased
yield of dextrose being obtainable in comparison with
the cases of employing the conventional glucoamylases
and carrying out the saccharification under acidic
conditions.
--19--
Z
The invention is further illustr~ted by reference
to the following examples in which all parts and percentages
are by weight unless otherwise noted.
EXAMPLE 1
A liquid culture medium containing 5% soluble
starch, 2% corn steep liquor, 0.5% cottonseed oil dregs,
0.5% yeast extract~ 0.1% dipotassium phosphate, 0.05%
magnesium sulfate and 0.01% calcium chloride was adjusted
to pH 7.0 and 100 ml of this was placed in a 500-ml
Erlenmeyer flask. This medium was sterilized at 121C
for 10 minutes, inoculated with Stachybotrys subsimplex
strain G30-1140, and incubated at 30C for 7 days on a
shaker. After the culture was completed, the mycelia were
eliminated from the culture fluid by filtration. The
filtrate was found to contain 70 units of glucoamylase
activity per milliliter.
This filtrate was next frozen for one night at
-20C and then thawed at room temperature. The insoluble
matter was removed by centrifugation. Two volumes of
cold isopropanol was then added to this solution and it
was left standing at 4C for one night so that the enzyme
would be precipitated out. The supernatant was removed
by decantation and the precipitate was dlssolved in a
0.05 M tris-HCl buffer solution containing 1 mM EDTA
and having a pH of 7.5. This enzyme-containing solution
-20-
142
was then dialyzed against the same buf~er at 4~C for one
night. DEAE-cellulose which had been equilibrated with
the same buffer solution was then added to the dialyzed
enzyme solution and the enzyme was adsorbed to this
carrier. After washing this DEAE-dellulose with the
same buffer, the enzyme was eluted from it using a
solution of the same buffer containing NaCl at a con~
centration of 0.3 M. Next, two volumes of cold
isopropanol was added to the eluate to cause the enzyme
to precipitate, and the precipitate was collected by
centrifugation. This precipitate was then dissolved in
the 0.05 M tris-HCl buffer (pH 7.5j containing 1 mM
EDTA, followed by overnight dialysis against the same
buffer solution. The dialyzed enzyme solution was next
applied to a column of DEAE-cellulose which had been
equilibrated with the same 0.05 M tris-HCl buffer (pH 7.5)
containing 1 mM EDTA. Elution of the enzyme from this
column was carried out by linearly increasing the con-
centration of NaCl in the same buffer solution up to
0.5 M. The fractions of the eluate which contained the
enzyme were then pooled and two volumes of cold iso-
propanol was added in order to precipitate the enzyme
out of this solution and concentrate it. The concentrated
enzyme was next applied to a column of Sephadex G-150
which had been equilibrated with a 0.05 M tris-HCl buffer
solution (pH 7.0) containing 1 mM EDTA, and elution was
carried out using the same buffer. The eluted fractions
1~311~2
which showed enzyme ac~ ity were then pooled, and two
~olumes of cold isopropanol was added to this to preciPi-
tate out the enzyme. This resulted in the recovery of
the enzyme in a purified and concentrated form. The
specific activity of this purified enzyme was found to
be 127 units per milligram of protein.
EXAMPLE 2
To a 30% solution of a spray-dried maltodextrin
(D.E. about 10) in 0.05 M acetate buffer at pH 6.5 was
added the purified glucoamylase of Example 1. The enzyme
was added at a dosage of 0.20 units of enzy~e per gram
of substrate on a dry solids basis. After the solution
had been incubated at 55C for 72 hours, the dextrose
content of the filtered hydrolyzate,as determined by high
performance liquid chromatography, was 96.5% of the total
carbohydrate.
.
EXAMPLE 3
Starch was converted to a 10.2 D.E. starch
hydrolyzate using bacterial alpha-amylase from B.
licheniformis according to the general procedure given
in U.S. Patent 3,912,590. The solution was boiled for
5 minutes after adjusting the pH to 2.0 with 2 N HCl to
inactivate the residual alpha-amylase. The starch
hydrolyzate solution was then adjusted to pH 6.2 and
diluted to the desired concentration before treatment
with 0.20 units of the purified glucoamylase of Exam?le 1
per gram of substrate (dry solids basis). The solution
-22-
~3114Z
was incubated at 55C in a stoppered tube. The pH was
ad~usted to 6.2 a~ter 5 hours and 48 hours. After the
solution had been incubated for 72 hours, the dextrose
content of the filtered hydrolyzate,as determined by
high performance liquid chromatography, was 97.6p of the
total carbohydrate. The final concentration of the
solution was 31.2% on a dry solids basis.
When saccharification tests at the same substrate
concentration were carried out with commercial glucoamylase
from A. niger under its optimum conditions (pH 4.3 at 60C)7
- the corresponding dextrose yield was 96.5%. Similarly,
the glucoamylase from R. niveus at pH 5.0 and 55C gave a
dextrose yield of 97%. Dextrose yields were about 1~ lower
when the saccharification tests were carried out with the
commercial glucoamylases under the conditions used for the
new enzyme. These results show that the new glucoamylase
of this invention gives higher yields of dextrose than do
the commercial glucoamylases even when each enzyme is
utilized under its optimum reaction conditions.
