Note: Descriptions are shown in the official language in which they were submitted.
16;~3
D3147
HEAT AND ACID-STABLE
ALPHA-AMYLASE ENZYMES AND
PROCESSES FOR PRODUCING
THE SAME
BACKGROUND OF THE INVENTION
a) Field of the Invention
This invention relates to novel heat and acid-stable
alpha-amylase enzymes and processes for producing the same.
This invention is also concerned with processes ~or using
these novel alpha-amylase enzymes for hydrolizing, liquefy-
ing and/or converting starch containing materials into
starch hydrolysates.
b) Description of the Prior Art
Alpha-amylase enzyme preparations have been used to
hydrolyze, liquefy and/or convert starch containing ma-
terials into starch hydrolysates as well as being used in
detergent formulations. When alpha-amylase enzymes are used
; to treat starch containing materials, they are used as the
initial step in the production of a number of synthetic
starch hydrolysate materials, such as malto-dextrins, corn
syrups, dextrose, levulose, maltose and others. The alpha-
amylase enzyme hydrolyzes starch molecules to break them
down into a variety of intermediate molecular weight frag-
ments known as malto-dextrins. The malto-dextrins are
subsequently treated with one or more additional enzyme
preparations including glucoamylase, beta-amylase and
glucose lsomerase in order to produce the desired final
- 2 -
~.
~ 81tj;~3
product. Alternatively, a plurality of these enzyme prep-
arations may be introduced into a slurry of the starch
material simultaneously to directly produce the desired
starch hydrolysate.
Alpha-amylase enzymes are available from a wid'e variety
of sources. Most alpha-amylase enzymes are produced from
bacterial sources such as Bacillus subtilis, Bacillus
licheniformis, Bacillus stearothermophilus and others which
are cultivated in an appropriate culture medium, the cells
produced therefrom are then destroyed and the enzyme prep-
aration is thereafter separated from the broth and purified.
Many of the commercially available alpha-amylase
enzymes produced today are derived from Bacillus subtilis
microorganisms. When these enzymes are us'ed to convert
~15 starch to starch hydrolysates, they will generally have an
optimal temperature ranging from about 80 to about 85C,
and an optimal pH of about 6Ø The conditions of temper-
ature and pH necessary for efficient use of the enzyme have
two disadvantages. Firstly, if starch is converted with the
enzyme at a pH of about 6 and at a temperature of about 80
to about 85C, a part of the reducing end-groups of the
starch is isomerized, and in the subsequent conversion
process, maltulose is produced which reduces the degree of
recovery of the desired product, e.g., dextrose, levulose,
or maltose. Secondly, the optimum pH of glucoamylase used
in the conversion and saccharification process is generally
about 4.5 in the case of Aspergillus niger-type enzymes and
a pH of about 5.0 in the case of Rhizopus-type enzymes.
Therefore, upon completion of the liquefaction step using
~(~8~ 3
the alpha-amylase enzyme, it has been necessary to adjust
the pH from about 6 to 4.5 or 5Ø This pH adjustment
increases the lon concentration and as a result, increases
the load and consequent refining expense using the ion
exchange resins used in the purification of the final
product.
In recent years, various heat-stable alpha-amylase
enzymes have been developed. Examples of such heat-stable
alpha-amylase enzymes include those produced from micro-
organisms derived from Bacillus stearothermophilis as
described by Ogasawara et. al., J. Biol. Chem., 67, 65, 77,
and 83 (1970); G. B. Manning and L. L. Campbell, J. Biol.
Chem., 236, 2952, 2958 and 2962 (1961); S. L. Pfueller and
W. H. Elliot, J. Biol. Chem., 244, 48 (1969)~ More recent-
;15 ly, alpha-amylase enzymes having good heat-stability in
neutral or weakly alkaline solutions have been made avail-
able. These heat and alkaline stable alpha-amylase enzymes
have been market~d under the brand name "Thermamyl". They
are produced by c:ultivating microorganisms of the species
Bacillus licheniformis as described in British patent
specification No. 1,296,839, published November 22, 1972,
Madsen et. al., Die Starke, 25, 304, 305 and 308 (1973) and
Narimasa Saito, ABB, 155, 290 (1973). While the alpha-
amylase enzymes produced from Bacillus licheniformis have
relatively good heat-stability in neutral and weakly alka-
line solutions, they do not have suitable stability under
acidic conditions to make their use economical from a
commercial standpoint.
-- 4 --
:
3L~816;33
Accordingly, it is a principal object of the present
invention to produce alpha-amylase enzymes which have good
heat-stability as ~lell as good stability under acidic con-
ditions, particularly at pH values to render their use under
conditions compatible with other amylases such as glucoamylase.
SUMMARY OF THE INVENTION
The present invention is directed to novel heat and
acid-stable alpha-amylase enzymes which are characterized as
(1) capable of retaining at least about 70% of their initial
alpha-amylase activity when held at 90C and at a pH of 6.0
for 10 minutes in the absence of added calcium ion; (2)
capable of retaining at least about 50% of their initial
alPha-amylase activity when held at 90C, at a pH of 6.0 for
60 minutes in the absence of added calcium ion and (3)
capable of retaining at least about 50~ of their initial
alpha-amylase activity at a temperature of 80C and at a pH
of 4.55 in the presence of 5mM of calcium ion for 10 minutes.
The preferred enzymes of the invention are further characterized
as being capable of retaining at least about 50% of their initial
activity at a temperature of 85~C and at a pH of 4.55 in the pres-
ence of 5mM of calcium ion and 22.5%, by weight starch, d.s. Still
further the preferred enzymes of this inverltion are characterized
as being capable of retaining at least about 95% of their initial
alpha-amylase activity at a temperature of 80C and at a pH in the
range from about 4 to about 6,0. The enzymes of the invention are
preferably derived from a Bacillus microorganism and more prefer-
ably a Bacillus stearothermophilus microorganism.
The alPha-amylase enzymes of the present invention generally
have a molecular weight of at least about 90,000 as determined by
SDS disc electrophoresis and they are generally characterized as
being substantially free of protease activity, e.g., -they gener-
ally have a protease/alpha-amylase ratio of less than 3, and
preferably less than 1.
The novel alpha-amylase enzymes of the present invention
are produced by cultivating in a suitable medil~ a Bacillus
stearothermophilus microorganism, preferably culturing a strain
selected from the group consisting of sacillus stearothermophilus
ATCC Nos. 31,195, 31,196, 31,197, 31,198, 31,199, variants,
mutants and sub-mutants thereof and -thereafter recovering the
alpha-amylase enzyme produced.
According to another aspect of the invention there is provid-
ed a process for converting starch to a starch hydrolysate com-
prising treating an aqueous slurry of starch with an alpha-amylase
enzyme of the invention at a pH of 3.5 to 6.5 to liquefy and
convert the starch; and obtaining a starch hydrolyzate from the
conversion step.
BRIEF DESCRIPTION OF TME DRAWINGS
Fig. 1 illust:rates the ~elationship of the optimal pM of
two of the enzymes of the invention with Thermamyl alpha-amylase.
Fig. 2 illustrates the relationship of the optimal
temperature of two of the enzymes of the invention with
Thermamyl alpha-amylase.
Fig. 3 illustrates the relationship of the thermo-
stability of two of the enzymes of the invention with
Thermamyl alpha-amylase at 80C, pH 4.55 with 5mM of Ca
Fig. 4 illustrates the relationship of the thermo-
stability of two of the enzymes of the present invention
with Thermamyl al ~ -amylase at 90C and at a pH of 6 . O .
81~33
Fig. 5 compares the thermostability of two of the
enzymes of the invention with Thermamyl _lpha-amylase at
90C, a pH of 6.0 and in the presence of lmM Ca
Fig. 6 illustrates the relationship of the thermo-
stability of two of the enzymes of the present invention
with Thermamyl at 85C and at a pH of 4.55 in the presence
of 22.5% starch, d.s.
Fig. 7 illustrates the relationship of the thermo-
stability of two of the enzymes of the present invention
with Thermamyl alpha-amylase at 85C, a pH of 4.55 in the
presence of 22.5% starch and SmM Ca
'i
Fig. 8 illustrates graphically the determination of the
molecular weight of two enzymes of the invention by SDS disc
electrophoresis.
Fig. 9 illusl:rates the relationship between pH and
enzyme activity as to one of the enzymes of the invention
with prior art alpha-amylases derived from Bacillus stearo-
thermophilus microorganisms.
Fig. 10 illustrates the relationship between tempera-
ture of an enzyme of the present invention with a prior art
alpha-amylase derived from acillus stearothermophilus.
Fig. 11 illustrates the deactivation curves of an
enzyme of the present invention with prior art alpha-
amylases derived from Bacillus subtilis and Bacillus
licheniformis when treated at 80C and pH 4.5 in the pres-
ence of 5mM calcium ion.
-- 7 --
~ 8 ~
Fig. 12 illustrates the deactivation curves of an
,enzyme of the present invention with prior art alpha-
amylases derived from Bacillus licheniformis and Bacillus
stearothermophilus when treated at 90C and at a pH of 6.0
without calcium ion.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
' The production and thermal stability of alpha-amylase
from various kinds of microorganisms were screened under
various conditions such as growth temperature (37C, 45C,
55C, and 70C), pH (5.0 and 7.0) and media. It was found
that thermophilic ray fungi isolated at 55C and thermo-
philic fung'i at 45C produce thermolabile alpha-amylase in
high yields, whereas thermophilic bacteria at 70C produce
thermostable alpha-amylase in low yields. ,Based upon these
initial studies the screening of the microorganisms for '
acid- and thermostability of alpha-amylase was conducted by
culturing at 55C and 70C.
~ total of 550 samples of microbial sources including
soil, hot-spring water, compost, etc. were collected from
various locations.
The acid- and thermostabilities were determined by
assaying alpha-amylase activity before and after heat
treatment of culture filtrates at ~0C and pH 5.0 for 10
minutes in the presence of 10mM calcium ion.
~81~3
The _~e~-amylase activity was determined as follows:
0.2% soluble starch solution was prepared weekly as
follows: 200mg soluble starch in approximately 50ml of 0.2M
acetate buffer (pH 4.5) containing 0.013M CaCl2.2H2O was
heated to 10QC in boiling water, and the resultant solution
was made to 100ml with the same buffer. The tube containing
0.lml of enzyme solution and 0.3ml of 0.2% soluble starch
solution was incubated for 5 minutes at 60C. The reaction
was stopped by adding l.Oml of 0.SN acetic acid. After
3.Oml of 0.015% iodine solution was added and stirred, the
optical density was read at 700mu against H20. The tube
without enzyme served as a blank and its optical density at
700mu was designated as ODb. One unit of enzyme was that
amount which catalyzes a 10% decrease in blue value per
lS minute under the conditions described above.
NML units = (ODb ~ OD) x 100
ODb x 5 x 10
Except where indicated to the contrary, the alpha-
amylase activity reported was determined by the above
procedure (NML units). Where the alpha-amylase activity is
designated as CPC alpha-amylase units, the CPC units are
approximately 1/140 of the NML units.
The CPC alpha-amylase units of activity are determined
by the following procedure:
One milliliter of a properly diluted enzyme solution is
added to 10 milliliters of a 1% soluble starch - 0.0~M
_ 9 _
1~816;~3
acetic acid buffer solution (pH 6.0) and the reaction is
carried out for 10 minutes at 60C. One milliliter o~ the
reaction solutlon is put in a 100ml graduated flask con-
taining 50ml of 0.02N hydrochloric acid, and after adding 3
milliliters of 0.05% iodine solution thereto, the total
volume is made up to 100ml by the addition of water. The
blue color which develops is measured for absorbance a~
620mu. The amount of the enzyme required to decompos~ 10mg
of starch in one minute is defined as 1 CPC unit.
,.
1 CPC unit = D - D
o s x _ 50 x (dilution factor)
Do 10 x 10
where,
Do = absorbance of control solution (water is
added instead of the enzyme solution) -
Ds = absorbance of the reaction solution
The culturing conditions and media used in the screen-
ing study are summarized in Table 1.
The results of the first and second screening studies
are reported in Table 2.
.
-- 10 --
3~3
Table 1
Culturlng Conditions and ~edia
Conditions
Plate Culture( ) Slant Culture(2) Flask Culture(3)
; a) 55C pH 5 55C, pE~ 5 50C, pH 6
b). 55C, pH 7 55C, pH 7 50C, pH 7
c) 70C, pH 5 70C, pH 5 60C, pH 6
d) 70C, pH 7 70C, pH 7 60C, pH 7
(l) Medium for (2) Medium for ~3) Medium for
Plate Culture Slant Culture Flask Culture
(B-B) (B-D) (B-M)
Amylopectin 0.02% Soluble Starch 1.0%. 3.0%
(NH4)2HP4 0.025% Bacto-tryptone 0.5% 0.5%
(Difco)
i Yeast ext. 0.025%
Yeast ext. 0.5% 1.0%
Na-Citrate 0.01%
CaC12 2H2 O . 0596 , O . 05%
MgS047H20 0.O5%
MnC12 4H20 0.05%
KCl 0.15%
. MgSO 7H O - 0.05%
CaCl2 2H2 0.05% 4 2 : .
KH2PO4 0.1% 0.1%
Agar 2.0%
Agar 2.0%
~081~ 3
o _~_~ o o oU~ o o o o o o o
~:, o C
. U~
~ o E~ o o oI~ o o o o ~ o o r ~ ~
~,
~o .
~ o E ~o o ~ ~ ~r o ~ o o ~ o o , o
~ _1~ ~
;. c ~ ~
:1 E ~ o ~ ,1 o o ~ ~ ,~ _I o ~ ~ 3
.DI ~ 00 . ~C ' ~ .
E~ ~n .,~ ~ ~ E
., ~ c ~ o ~~D o ~ o v:, o~ ~r o r o u~ ~c u
~ ~ ~ i~ aJ
~a a ~
C ~I +I + I +I + I + I + .~ .
V~
U~ 01 U~U~ Ul .C
U~ ~ .,~ .C.~ C C .C
0
C ~ C ~ .,~
_~ ~ ~ h ~ ~ ~~ ~ ~ C :~
~ O ~ u~ o 0 ~ u~ o u~ o ~ C C-~
U~ H >~ U t~l ~alU a~ U _I U ~1 ,~
~11 o a ~ ~ D (a
~ m ~ ~ m ~ m ~ ~ ~
., ~ O O
.1 ~ O o O O
CJ C~ U~ I~ ~ I~ ~,
u 3 3 ~ ~ ~ iG
U~ ~ ~ ~ '
~ O ~ o~ C~ oC~ ~
U~ U~ O U~ In O O
_~ ~ tJ ~ U~ ~ ~
n o In o
~ 3
About 25% of thermophilic ray fungi isolated at 55C
showed 100-2,500 units of alpha-amylase production per
milliliter of culture broth, but no activity remained after
heat treatment (80C, lO minutes, pH 5.0).
A total of 35 strains from l,061 thermophilic bacterial
strains isolated at 55C showed lO0-1,000 units of alpha-
amylase production, and 10 of those strains produced a
thermostable alpha-amylase. Almost all of the alPha-
amylases from thermophilic bacteria isolated at 70C showed
acid- and thermostability; and the highest producers were
obtained from pH 5.0 isolates.
The relationship between activity and clear zone size
in the last 219 strains isolated at 70C and pH 5.0 was
examined. The results of this study are shown in Table 3
; 15 where it can be seen that 90~ of these strains gave clear
zone of less than 3cm in diameter and produced 0-200 units
of _~e~_-amylase. The highest production was obtained from
strains which gave a clear zone of more than 3cm in diameter,
but these represented only 10% of the tested strains.
. .
- 13 -
~081~;~3
Table 3
; Relationshin betwcen Activity and
Clear Zone Size of Isolated Strain
' '~
~., ' ' .
ActivityClear Zone SizP on
in Isolation Plate
Flask(cm in diameter)
(U/ml) ~ 2 - 3 -4 4 < Total
0 - 100 153 40 15 0 208
; - 200 3 2 1 0 ' 6
- soo 0 0 2 0 2
- 1, 000 0 0 1 0
- 1,500 0 0 1 1 2
.
Total 156 42 20 1 219
,,
Based on the above tests, it has been found that the
isolation of bacterial colonies having a clear zone of more
than 3cm in diameter on the plate medium used in the tests
at 70C and at a pH of 5.0 is an excellent method for screen-
ing for microorganisms capable of producing high yields of
acid- and thermostable alpha-amylase enzymes.
~(~81~33
As a result of this screening, five strains were
selected as the highest producers of acid- and thermostable
alpha-amyLase enzyme producers.
Each of the five strains in purified form as described
below have been deposited in the permanent collection of the
American Type Culture Collection (ATCC), 12301 Parklawn
Drive, Rockville, Maryland 20852. ATCC is maintaining
these strains pursuant to a contract between ATCC and CPC
International Inc., the assignee of this patent application.
.
The contract between ATCC and CPC International Inc.
provides for permanent availability of the cultures or sub-
cultures to the public, without restriction upon (1) issu-
ance of a United States patent describing and identifying
the subject deposits and disclosing the ATCC numbers as-
signed thereto; or (2) publication or laying open to public
inspection of any United States or foreign patent applica-
tion describing and identifying the subject deposits and
disclosing the ATCC numbers assigned thereto. CPC Inter-
national Inc. has agreed that, if any of these cultures on
deposit should die, or is destroyed, during the effective
life of the patent, it will be replaced with a living
culture of the same organism. In addition, CPC International
Inc. has authorized ATCC to grant the United States Patent
and Trademark Office and the West German Patent Office free
access to the cultures or sub-cultures at any time upon
request by an authorized official of such offices.
~81~33
Organisms Deposited
NML Strain No. ATCC No.
.
Bacillus stearothermophilus B-501 31,195
Bacillus stearothermophilus B-634 31,196
Bacillus stearothermophilus B-781 31,197
Bacillus stearothermophilus B-905 31,198
Bacillus stearothermophilus B-968 31,199
In addition, NML strains B-501 and B-781 have been
depcsited with the Fermentation Research Institute, Indus-
trial Technology Agency, MITI, as FRI Nos. 3389 and 3390,
respectively.
.
The bacteriological characteristics of the five (5)
selected strains deposited at ATCC are as follows:
(1) Morphologica:L Characteristics:
~15 A. Shape and size of cells: 0.6 x 2 - 3u; individual
rods seldom in chains
(all strains)
B. Pleomorphicity: Negative (all strains)
C. Motility: Motile and have flagella (all strains)
D. Spore: 0.6 x 1.0 - 1.5u, ovular shaped; racket-
shaped spore case (all strains)
E. Gram stain: Positive (all strains)
F. Acid fast: Negative (all strains)
- 16 -
1081633
(2) Growth on Various Media_
A. Nutrient agar plate: Active spreading colonie~
with coarse surface and
rough edge (all strains)
B. Nutrient agar slant: Good growth, white opaque,
active spreading, comb-like
outgrowths (all strains)
C. Nutrient broth liquid culture: Transparent brown,
white surface mat
(all strains)
D. Nutrient gelatin stab culture: Liquefaction
(all strains)
E. Nutrient gelatin agar plate: Wide clear zone
~ (all strains)
.L5 F. Salt-nutrient liquid culture: Growth inhibition
in 2% salt (all
strains)
G. Milk agar plate: Celar zone formation by hydrolysis
of casein (all strains)
' H. Glucose agar slant: Good growth, similar colonies
to those on nutrient agar
(all strains)
I. Proteose peptone agar slant: No growth (all
strains)
(3) Physiological Characteristics:
A. Nitrate reduction: Positive (all strains)
B. Catalase test: Positive (all strains)
C. Vogues-Proskauer reaction: Positive (all strains)
D. Utilization of citric acid: Positive (all strains)
E. Formation of hydrogen sulfide: Positive (all
strains)
- 17 -
1~)81633
; F. Formation of hydrogen sulfide: Positive (all
- strains)
G. Hydrolysis of starch: Strong hydrolysis (all
f strains)
- 5 H. Formation of acid and gas: Positive for acid but
no gas formation from
glucose xylose, arabinose,
mannitol (all strains)
~ I. Temperature and pH for growth:
.~, . . .
,. . .
ATCC No. 31,195 ATCC No. 31,197
: .
(B-501) (B-781)
37C no growth slight growth
~ - 42Cslight growth moderate growth
;~ 50 - 70Cgood growth good growth
pH range
f for growth 5 - 8 5 - 8
ir Optimum pH 6 - 7 6 - 7
,:, '
; The above t:ests were done in accordance with "Labora-
~! tory Methods in Microbiology" by W. F. Harrigan et. al., and
s 20 the "Manual of Microbiological Methods" published by the
American Bacteriological Association.
i,, :
, From the foregoing characteristics, the five (5) selected
strains were identified as Bacillus stearothermophilus in
accordance with Bergey's Manual of Determinative Bacteriology,
5 25 the 8th Edition.
These five strains were further purified by the plate-
streaking method. The results of the isolation, culturing
- 18 -
J ~81ti33
.,
and purification conditions with respect to the five selected
strains are summarized in Table 4. As it can be seen from
Table 4, the purified strain of ATCC No. 31,199 (B-968) was
the best of the five selected strains, producing 2,111 NML
units of alpha-amylase units of activity per milliliter of
cu ture broth (ab~ut 15 CPC unies~.
-- 19 --
. 1~81633
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, 1081633
,~
In the production of the ~ amylase enzymes of the
present invention a strain of a microorganism capable of
producing an acid- and heat-stable alpha-amylase enzyme such
as one which meets the tests in Table 3 (e.g., Bacillus
~i 5 stearothermophilus strains ATCC Nos. 31,195, 31,196, 31,197,
21,198 or 31,199) is cultivated in a nutrient medium known
for cultivating thermophilic bacteria. Such culture mediums
' should contain an assimilable carbon and nitrogen source
together with other essential nutrients.
Suitable assimilable carbon sources include carbohy-
drates such as starches, hydrolyzed starches, corn meal,
wheat flour, etc. The carbohydrate concentration to be used
in the medium may vary widely, e.g., it may range from about
1% w/v to about 25~ w/v, and preferable ranging from about
10% w/v to about 20% w/v, the percentages being calculated
as dextrose. The preferred assimilable carbohydrate is
starch or partially hydrolyzed starch (and when used on a
weight basis they are present in an amount ranging from 1 to
5%, by weight).
The nitrogen source ln the nutrient medium may be of
inorganic and/or organic nature. Suitable inorganic nitrogen
sources include ammonium salts, and inorganic nitrates, etc.
Suitable organic nitrogen sources include peptone, meat
extract, enzyme extract, casein, corn steep liquor, malt
extract, soybean flour, skim milk, etc.
'.
In addition, the nutrient medium should contain the
usual trace substances such as ~he inorganic salts which
include calcium chloride, magnesium sulfate, phosphates,
sodium chloride, potassium chloride, etc.
- 21 -
3~
.
These carbon sources, nitrogen sources and inorganic
salts can be used singly or in appropriate combinations. In
addition, a small quantity of metallic salts, vitamins,
amino acids, etc. can be used to promote the growth and
productivity of the bacteria.
The culturing conditions used to produce the alpha-
; amylase enzymes of the present invention are the same as
normally used in the cultivation of thermophilic bacteria.
Preferably, the strain is cultivated in a deep liquid
culture medium under agitation and aeration for 2 to 5 days
at 50C to 70C at a pH of 5 to 9. The enzyme accumulates
in the cultured medium.
Following the production of the alpha-amylase enzyme of
the present invention, the microbial cells are then removed
by conventional means such as centrifugation. The filtrate
is then preferably subjected to salting out by the addition
of organic salts such as ammonium sulfate, sodium sulfate or
magnesium sulfate, and/or by use of water miscible organic
solvents such as acetone, ethanol, 2-propanol, etc. to
precipitate out the enzyme so that it can be concentrated.
It is also possible to recover the alpha-amylase by adding
starch to the filtrate so that the alpha-amylase will sorb
to the starch.
A preferred means for purifying the enzyme from the
filtrate containing the enzyme includes the steps of treat-
ing the filtrate with cold acetone in twice the volume of
" the filtrate to precipitate the enzyme. The precipitated
enzyme is then dissolved in a 0.05M tris-hydrochloric acid
buffer solution (pH 8.5) and it is then passed through a
1(~81~;~3
DEAE-cellulose column which is equilibrated with the same
- buffer solution. At a pH of 8.5 the non-alpha-amylase
proteins, pigments, etc. are adsorbed to the DEAE-cellulose
whereas most of the alpha-amylase remains in solution. The
filtrate containing the alpha-amylase enzyme is referred to
~; herein as the "partially refined enzyme". The partially
refined enzyme can be further refined by dialysis against a
0.01 tris-hydrochloric acid buffer solution (pH 7.0) and
then passed through a hyrdoxylapatite column which is
equilibrated with the same buffer solution. The enzyme
' sorbs to the column in this step. The sorbed enzyme is
eluted out by linearly increasing the ammonium sulfate
concentration from 0 to 0.5 molar.
The partially refined enzyme thus obtained is, after
being concentrated, passed through a Sephadex G-150 column.
The enzyme is weakly adsorbed to the Sephadex and it is
eluted out in the fraction of below 10,000 molecular weight.
The activity of the refined enzyme obtained in this way is
increased by 100 times over the original filtrate, but in
disc electrophoresis, bands of a few other proteins are also
observed besides that of the alpha-amylase enzyme - and
crystallization of the enzyme has not yet been accomplished.
The following examples serve to more fully describe the
manner of making and using the above-described invention, as
well as to set forth the best modes contemplated for carrying
out various aspects of the invention. It is to be under-
stood that these examples in no way serve to limit the true
scope of this invention, but rather, are presented for
illustrative purposes only. It will be understood that all
proportions are in parts by weight, unless otherwise indicated.
- 23 -
~081~;~3
.
EXAMPLE I
A culture medium containing 3.0% (by weight) soluble
starch, 0.5% bactotryptone, 1.0% enzyme extract, 0.05%
calcium chloride, 0.05% magnesium sulfate, and 0.1% potas-
sium dinydrogen phosphate was adjusted to pH 7Ø A 50ml
aliquot of this medium was poured into a 500ml conical flask
and sterilized for 15 minutes at 121C. The sterilized
medium was inoculated with ATCC No. 31,195 (B-501) strain of
Bacillus stearothermoPhilus and cultured under agitation for
1~ 4 days at 60C. After the cultivation, the microbial cells
were removed by centrifugation. The enzymatic activity of
the filtrate per one milliliter was 10 CPC alpha-amylase
units (determined by CPC method described above). To this
filtrate, two volumes of acetone were added to precipitate
~5 the enzyme which was subsequently dissolved in a 0.05 molar
Tris-HCl buffer (pH 8.5). Then the solution was passed
through a DEAE-cellulose column which had been equilibrated
with the same buffer solution. The alpha-amylase enzyme was
not sorbed to the DEAE-cellulose while most other proteins,
pigments, etc., were sorbed to the DEAE-cellulose were thus
removed. The partially refined enzyme thus obtained was
first concentrated and then passed through a Sephadex G-150
column. The enzyme was weakly sorbed to the Sephadex and it
. was eluted out in the fraction of below 10,000 molecular
weight. The refined enzyme thus obtained was made into a
powdered, refined enzyme by freeze-drying and its relative
activity was about 200 CPC units/mg of protein.
- 24 -
1~81633
EXAMPLE 2
A culture medium which contained 3% corn starch, 0.5%
peptone, 1% corn steep liquor, 0.05% calcium chloride, 0.05%
.~
manganese chloride, 0.05% magnesium chloride, and 0.05%
potassium chloride was adjusted to pH 7.5. A 50ml aliquot
of this culture medium was placed in a 500ml conical flask
and sterilized for 15 minutes at 121C. The sterilized
medium was inoculated with ATCC No. 31,196 (B-781) strain of
' Bacillus stearothermophilus and agitated for 4 days at 55C.
~; 10 After the cultivation, the microbial cells were removed by
centrifugation. The alpha-amylase activity in the filtrate
was 14 CPC units/ml. The alpha-amylase was precipitated by
the addition of twice the volume of filtrate of 2-propanol.
The precipitate was formed into a dry powder by freeze-
;~ 15 drying. The activity of the crude enzyme powder was 3 CPC
,. .
units/mg. A refined, powdered enzyme having an activity of
230 units/mg was obtained from this crude enzyme powder by
~ refining by the procedure of Example 1.
j The five isolated and selected strains (i.e., ATCC Nos.
31,195, 31,196, 31,197, 31,198 and 31,199) were tested to
determine the effect of temperature and pH on alpha-amylase
production. The alpha-amylase activity in the experiments
f was expressed as the mean value of the peak activity of
j:
triplicate flasks. The medium composition used for both the
pre-culture and main-culture is the same shown in Table 1 (a
B-M medium). The results of these experiments are summarized
in Table 5.
i-
' '
I
- 25 -
,'. ~0~
:~
o o o o ~ ~ ~
,, O ~ cn ~ t:n
~;
$
~ ~ ~o ~ ~ ~ o ~
~ ~ ~l
~--~ --------
~ ~ ~ o o ~ ~
:~: In ~ ~ ~ ~ ~ ~ ~
`'~,, ' I` _ _ _ _ _ _ _
~ ~ co ~ Ln -I O ~ ~D
:~ ~ ~ n O ~ ~ d'
;; N r l O ~1 ~D
~, _ ~ _ _ _ ~ _ _ ~ _ _ _ _ _
O O O O O~ D O O ~ d' ~` t`
~ O ~ QO d~ D ~ N d~ 1
.~, . I I~ _ _, _, _ _ _ ~_ _ _ _ _ _ _ _
.. ~ ~ ~ o o
~ ~ O ~ t~
0 In ~ ~ ~1 ~ ~D O O ~D
' .
~ ~ ~ ----
aJ u~ ~
E ~o _ ~, _
~o ,~ ,, ,,
o ~ . ,i,i
~' ~
~_ _ _ _ ,~ _ _ ~ ~ _ _ _ _ ~ _
o o ~o o o~ U~ ,1 0
' E~ ~ ~1 O ~ ~ ~ ~ ~ D
. ~ ~ . _ _ _ _ _ _ _ _ _ _ _ _ _ _
. ~ ~D
.~ ~ ~ ~ LO ~ O . ~D 1` oD ~ ~ ~ ,1 1~ u~ t`
. :~ ~ ~ In U~ ~ ~I ~` O ~ O ~ In n CD 2D
` ~ I I o. l 1~ r 1`0~0~l
O In . .
.-. U
,,~, :,.
o In o u~ In O U~ O U~ O O U~ O U~
~ ~ ` u~
~. .
.' ~Z; u~ ~ 1~
~ !~ ,, ~1 ~1
26-
: ~ .
~L081~i33
,j~
OD~ U~
O ~ ~
:r: ~ o
,~
~,
,~, _~ _~
.'. ~ ~ ~ D
.~ ~ ~ O ~ ~0 ~ I` O
~ Lr) o~ O ~ ~D ~ ~r ~
. ~ 0~ ~D ~ ~ ~1
, . ~1 ~1
~t _ _ ~ ,_ CS~ n
~; rl O ~D d' d' d' ~ D
,, . O ~ O ~ <~ ~ U~ ~7 Ln ~ O
~ O~ ~ l Ln
~ ~ d' ~D 1` 1~ 'I d' O ~1 '
:~: h . ~1 ~1 . .
_ ~ _ _ ~ ~ _
., ~ ~ OD 0~ ~ ~ ~ a~ ~ ~ In
j; Q ~ E ~ ~ ~ ~ ~ ~ ~ ~ ~ ~)
E~ I ~ ~ t`~ In ~ ~ 0 1 O
~ Y ~ ~ r~
H ~ ~ I` ~ r` ~1
~" o~ ~ :l ,1 ~1
7 . U') 'a -- -- -- ~ ~ ~ ~ _ _ _
3 a) ~ o D ~ ~ ~ ~ D
¢ 7_~ ~ d' ~D ~1 o ~ 0 1` 0
E~ ~ ~ Ql ~D ~D ~ d' ~ ~ .
~ ~ ~ ~ " ,:;
" E ~ _ _ ~ _
~, ~o Ln ~
~ ~i . '
', .
E u o ~ o In u~ o 7n o ~ ~ ~
o u~ ~
' .
,. ~s
,. u ~ ~ ; ' '~ ~
6(a)- ~ :
~ ~08~6~3
. It can be seen from Table 5 that the optimum conditions
.~ for ATCC Nos. 31,195 and 31,196 were 60C, pH 7.0 and 60C,
pH 7.5, respectively. ATCC Nos. 31,197 and 31,198 produced
considerable amounts of alpha-amylase when cultured at 65C,
but this high temperature cultivation was not suitable for
producing good alpha-amylase activity for the other isolated
,.f strains. At 55C to 60C, the optimum medium pH was found
. to be 8.0 for alpha-amylase production for strain ATCC No.
31,198, and the optimum medium pH for strain ATCC No. 31,199
was 5.5.
~'
Tables 6 and 7, respectively, (1) summarize the alpha-
~ amylase production of the five isolated strains using
j repeatedly transferred slants (each strain was repeatedly
T transferred 9-11 times on two kinds of slant media, and then
~,15 liquid-cultured to find a suitable slant medium capable of
maintaining stable production of alpha-amylase) and (2)
~ determine the effect of medium volume on alpha-amylase
7; production (the medium volume in 500ml flasks was changed to
examine the aeration effect on the production of alpha-
amylase by each strain.
/:
~,
;
- 27 -
''
~08i~3
.
. Table 6
., _
~-Amylase P_oduction Using Re~eatedly Transferr
BD, 10 856 (90) 60 7.0
NA , 10 140 (72)
31,196 BD, 2 976 (26) 60 7
BD, 10 1,503 (26)
NA, 10 1,550 (26)
31,197 BD, 1 1,330 (27) 55 7
BD, 10 1,127 (26) " "
NA, 10 1,420 (26)
31,198 BD, 4 736 (44) 60 8.0
BD, 11 712 (44) " "
NA, 111,117 (44)
31,139 BD, 2
BD, 9 1,013 (68) " ';
! NA, 9 775 (68)
.
!
Soluble starch *2 Nutrient Agar Slant
Bacto tryptone (Difco) 0.5% Bacto-beef e co) 0.5%
CaC12 2H2 0.1% Agar 2.0%
Agar 2.0%
pH
5.0 :
- 28 -
.
~8~33
;
Table 7
-
Effect of Medium Volume on Amylase Productlon
I _ Flask Cul ture
Number of C ~ lons ~ed. volume ¦ Activity, U/ml
ATCC slant- I Temp. I ml/SOO ml
No. transfers ¦ (C) pH flask ¦ (culture time, hr)
._ , ~
150 (65)
127 (65)
31,195 BD-ll 60 7.0 50 1,192 (41)
! 60 790 (41)
146 (65)
954 ~26)
-, 40 966 (26)
31,196 BD-2 60 7.5 50 976 (26)
1,236 (26)
651 (43)
. _ _ _ _
1,435 (41)
l,S90 (41)
. . .
31,197 BD-3 55 7.5 50 1,510 (41)
1,380 (41)
, ::
840 (41)
. _
689 (44)
- 40 651 (44)
31,198 BD-4 60 8.0 50 736 (44)
939 (44)
777 (44)
1,131 (44)
1,240 (44)
31,199 BD-2 55 5.5 50 1,163 (44)
1,054 (68)
1,224 (68)
_ _ ~
s 1~81633
,j
Two strains of the present invention (ATCC Nos. 31,195
and 31,199) were purified and the alpha-amylase properties
,
produced therefrom were compared with purified Thermamyl
Liquid 60, Batch AN 1005 by the procedure described below.
Alpha-amylase enzyme was precipitated from the culture
; filtrates of ATCC Nos~ 31,195 and 31,199 or from Thermamyl
alpha-amylase (twice diluted) by adding two volumes of cold
acetone. The precipitate was collected by centrifugation
and dissolved in 0.025M calcium acetate solution. Soluble
starch was added to the solution so as to make a 20% starch
suspension. This suspension was heated at 85C for 30
; minutes and the precipitate was removed by centrifugation.
' Then, the solution was dialyzed against 0.05M Tris-HCl
buffer (pH 8.5) containing lOmM Ca++, replacing the buffer
, 15 twice. The dialyzate was applied to a DEAE-cellulose column
which had been equilibrated with the dialysis buffer. The
alpha-amylase was not adsorbed by the column and eluted with
the same bufPer. The fractions having alpha-amylase activity
were collected and the alpha-amylase enzyme was concentrated
by acetone precipitation. The alpha-amylase activity was
determined by the CPC method described above. The purifi-
cation process is summarized in Table 8. ~
: ~:
'
'~, ,
' '''
- 30 -
~l~381633
.,j. .
Table 8
Purification of U-Amylase
.~ .
;~ Purification Culture Broth Acetone
~: Process or Crude EnzymePrecipi- Heat- DEAE-
-Amylase Preparation tationTreatmentCellulose
~,' ' .
ATCC No. 31,195
~- Total Activity
(CPC Units) 9,870 8,6408,420 2,400
Total Protein
: (mg) 7,990 2,0161,4S9 15.7
Specific Activity
(Units/mg) 1.2 4.3 5.8 152.9
Yield (%) 100 87.5 85.3 24.3
A~ r~. 31,199
Total Activity
(CPC units) .9,910 8,2107,214 2,310
Total Protein (mg) 4914 1969 1206 33
Specific Activity
(Uni~cs/mg) 2.0 4.2 6.0 70
Yield ( % ) 100 82.8 72. 8 2 3.3
ThermamyI
Total Activity
(CPC units) 12,100 9,4009,540 2,410
Total Protein
(mg) 684 286 183 24
. Specific Activity
- ~units/mg) 17.7 32.9 52.1 100.4
,: .
Yield (%) 100 77.7 78.8 19.9
: .
The acid- and thermostability of partially purified
alpha-amylase preparations from ATCC Nos. 31,195 and 31,199
and Thermamyl alpha-amylase (having 7 units/ml) were deter-
mined by incubation for 60 minutes under the following
conditions:
(a) 90C, pH 6.0, Ca 0 or lmM;
(b) 80C, p~ 4.55, Ca++ 5mM; and
(c) 85C, pH 4.55, Ca lmM or 5mM, 22.5% soluble
starch.
;
5 ml o~ tne aLDha-amylase preparation was dialyzea in a
Visking 8/32 cellulose tube against O.OS ~ calcium acetate
buffer (pH 4.5-6.0) containing 0-5 ~M o' calcium acetate for
three hours at 4C, changing the buffer twice. Then 4 ml of
the dialyzates were put to small test tubes and incubated at
the designated temperature in a water bath. The tubes were
rapidly cooled in an ice water bath and the residual al?ha-
; amylase activity was determined.
**0.3 ml of a dialyzed enzyme solution of alpha-amylase,
0.5 ml of lM calcium acetate buffer, 0.2 ml of 0.; ~ CaC12,
'0 and 3.0 ml o.f 30% soluble starch slurry were pipeted into a
test tube. The mixture was then incubated 85C for 30 minutes
with continuous stirring. Then test tubes were rapidly cooled
in an ice water bath and the residual alpha-amylase activity
was determined.
,'.' .
.
~ ~ -32-
8 1 ~ ~ 3
' The residual alpha-amylase activities were determined
i after S, 10, 20, 30 and 60 minutes of incubation.
:
~ The optimum pH and temperature of the ATCC No. 31,195
f and ATCC No. 31,199 ~ -amylases and Thermamyl alpha-
~, 5 amylase were determined by the CPC assay conditions des-
cribed above except that the reaction pH or temperature was
changed. The results of the tests are shown in Figs. l and
2. The optimum pH of both ATCC No. 31,195 and ATCC No.
31,199 alpha-amylases was 4.0 - 5.2 and that of Thermamyl
alpha-amylase was 4.5. The Thermamyl alpha-amylase was
found to retain a high enzymatic activity over a neutral and
, alkaline pH range.
, .
~; The optimum temperatures were 75C for ATCC No. 31,199
alpha-amylase, 80C for ATCC No. 31,195 alpha-amylase and
~ .~
, 15 85C for Thermamyl alpha-amylase. The relationship between
enzymatic activity and reaction temperature was very similar
for the ATCC No. 31,195 and ATCC No. 31,199 alpha-amylases,
but that for Thermamyl alpha-amylase was very different. It
,
~ .
,
~.~
,~ ~
,`'
.
:
- 32A -
i33
was found that the alPha-amylase activity of Thermamyl
alpha-amylase was increased by 20-30% when it was incubated
at 85C and pH 6.0 in the presence of starch and Ca
This fact may explain the difference in the enzymatic
activity-reaction temperature relationship between the ATCC
No. 31,195 and ATCC No. 31,199 alpha-amylases and Thermamyl
alPha-amylase.
Fig. 3 illustrates the inactivation curves of the ATCC
No. 31,195 and ATCC No. 31,199 alpha-amylases and Thermamyl
0 alpha-amylases when they were incubated at 80C and pH 4.55
in the pxesence of SmM CaCl2. ATCC No. 31,199 alpha-
, amylase showed the highest thermostability followed by ATCC
No. 31,195 alpha-amylase. Thermamyl alpha-amylase was
j:
considerably inferior to the ATCC No. 31,195 and ATCC No.
,;5 31,199 alpha-amylases in thermostabiliity under these
conditions.
Fig. 4 illustrates the inactivation curves of the ATCC
, No. 31,195 and ATCC No. 31,199 alpha-amylases, Thermamyl
alpha-amylase and Bacillus stearothermophilus alpha-amylase
0 described by Ogasawara et. al. (J. Biochem., 67, 65, 77,
83 (1970)) when they were incubated at 90C, and pH 6.0 in
the absence of Ca (the incubation conditions described by
Ogasawara et. al. were the same as used in this test).
',.' '
i ~ As it can be seen from Fig. 4, the alpha-amylases of
~5 the present invention showed much higher thermostability
, than either Thermamyl alpha-amylase or the Ogasawara et. al.
f alpha-amylase (even though the alpha-amylases of thç present
invention tested also belong to Bacillus stearothermophilus).
- 33 -
~: .
1(1181~
Fig. 5 illustrates the inactivation curves of the ATCC
No. 31,195 and ATCC No. 31,199 alpha-amylases and Thermamyl
alpha-amylase under the same ~onditions as described above
, .,
for Fig. 4 (e.g., 90C and pH 6.0) except that the medium
contained lmM Ca . The alpha-amylases of the present
invention still showed higher thermostability than Thermamyl
alpha-amylase, but the difference was not as great as in the
A7 case of no added Ca~+. These facts tend to indicate that
the ATCC No. 31,195 and ATCC No. 31,199 alpha-amylase bind
Ca+ more firmly than the Thermamyl alpha-amylase, therefore
their requirement for Ca to stabilize the protein molecule
is less than that of Thermamyl ~e~_-amylase.
Figs. 6 and 7 illustrate the inactivation curves of the
ATCC No. 31,195 and ATCC No. 31,199 alpha-amylases and the
Thermamyl alpha-amylase when they were incubated at 85C and
at a pH of 4.55 in the presence of soluble starch (22.5%,
d.s.) and Ca (lmM Ca + as illustrated in Fig. 6 and 5mM
Ca as illustrated in Fig. 7). The ATCC No. 31,195 and
ATCC No. 31,199 alpha-amylases showed higher thermostability
'7 20 than Thermamyl alpha-amylase under the conditions of both
Figs. 6 and 7. Since the conditions of the above tests
illustrated in Figs. 6 and 7 are similar to many industrial
liquefaction conditionsj it is to be expected that the
~ alpha-amylases of the present invention would demonstrate
.~. .
higher thermostability at acidic pH values in the industrial
7~'. liquefaction and conversion of starch.
t~ The molecular weights of both ATCC No. 31,195 and ATCC
No. 31,199 alpha-amylases were determined to be 96,000 as
measured by the method of Weber and Osborn, J. Biol. Chem.,
244, 1406 (1969) by the use of SDS disc electrophoresis.
The marker proteins were albumin (M. W. 67,000), ovalbumin
- 34 -
1633
, .
(M. W. 45,000), chymotrypsin (M. W. 25,000) and cytochrome C
(M. W. 12,500). The position of the alpha-amylases of the
`~ present invention on the polyacrylamide gel was determined
,
by putting the gel on an Amylose-Azure agar plate and
incubating it at 37C. The results of this test are illus-
, trated in Fig. 8.
:. .
The value of 96,000 for the molecular weight of the
alpha-amylases of the present invention is much larger than
those for the Bacillus stearothermophilus alpha-amylases
reported by Ogasawara et. al., J. Biochem., 67, 65, 77, 83
, (1970) (M. W. reported as 48,000) and by Manning et. al. J.
s Biol. Chem., 236, 2952, 2958, 2962 (1961) (M. W. reported as
~ ~ 15,600).
.''' ~ ,:
The five isolated strains were tested for protease
s 15 activity since the presence of protease or proteolytic
enzyme in alpha-amylase enzymes tends to react with various
proteinaceous materials present in many starchy materials to
produce water soluble protein hydrolysates such as amino
acids. For this reason, the presence of the protease enzyme
contaminant in alpha-amylase enzymes is detrimental to the
efficient hydrolysis of starchy materials. The protease
activities in the alpha-amylase enzymes produced from the
:~
five isolated strains were determined from acetone pre-
cipitates of culture filtrates at the stage of maximum
alpha-amylase production using a modified Anson-Hagiwara
method.
., .
The results obtained were compared with enzyme prep-
arations of Thermamyl alpha-amylase and a CPC bacterial
alpha-amylase. As shown below, Thermamyl and the culture
s
- 35 -
..
~' ' '` .
: L~81~33
, filtrates of isolated thermophilic ray fungi contained large
quantities of protease. On the other hand, the thermostable
alpha-amylase-producing strains of the present invention
produced no significant amount of protease.
PROTEASE ACTIVITY
OF
i,: _
ALPHA-AMYLASE
Protease/Alpha-Amylase
Enzyme Ratio
ATCC No. 31,195 0.06
ATCC No. 31,196 0.17
ATCC No. 31,197 2.35
ATCC No. 31,198 0.08
ATCC No. 31,199 0.06
CPC-BLA 6.2
Thermamyl 60 36.5
B-1721 109.0
Thermophilic ray fungi isolated at 55C at a pH of 7Ø
:~ '
As it can be seen from the above results the alpha-
amylases of the present invention do not contain any signif-
icant amounts of protease activity, i.e., a protease~alpha-
5' amylase ratio of less than 3 and generally less than 1.
, This is a significant advantage when using the enzyme to
7,` hydrolyze starchy materials.
~''-
~::
- 36 -
~. : . . -
lV81~i33
; EXAMPLE 3 ~
:,
.,.
The following example illustrates the use of the alpha-
amylase enzymes of the present invention in liquefying
~ starch in the manufacture of high D. E. starch hydrolysates.
.', .
Thirty grams of potato starch were suspended in 70ml of
water, 75mg of calcium chloride dihydrate was added and the
pH was adjusted to 4.5. To this slurry there was added 50
CPC units of the refined, powdered enzyme obtained in Example
~ ~ 2 to liquefy the starch at 85C for 30 minutes. The D. E.
;~; 10 of the liquefied solution was about 21 and the pH was 4.3.
When the temperature had decreased to 60C, a glucoamylase
3 enzyme derived from Aspergillus ni~ was added and the
solution was saccharified and converted at 60C for 48
hours. The D. E. of the saccharified and converted solution
was 97.5 and its dextrose content was 96.0%.
,~:
As shown in the~preceding example (Example 3), the
alpha-amylase enzyme of the present invention hydrolyze and
liquefy starch. They can be used to convert soluble starch,
amylase, amylopectin, glycogen, etc., to abruptly reduce the
!0 viscosity of these substrates. When the enzymes of the
present invention react with soluble starch at pH 4.5 and
. ~ :
60C, the starch-iodide reaction disappears at a hydrolysis
o rate (D. E.) of about 15 and the ultimate rate is a D. E. of
,i~
. 3~ to 36. The sugars of the hydrolyzed product were analyzed
as maltose, maltotriose, maltotetrose and other malto-
~ oligosaccharides together with a small quantity of glucose.
;; The mutarotation of the reducing sugar product has been
found to be negative. Accordingly, the enzymes of this
invention are alpha-amylase enzymes of the liquefying type
) and freely hydrolyze the alpha- 1, 4 bonds of starch.
~- - 37 -
.,~, .
33
The relationship between liquefaction using the enzyme
, of the present invention (relative value) and the operational
, pH is shown in Fig. 9 and it is contrasted with the known
alpha-amylase enzymes derived from Bacillus stearothermophilus.
(Ogasawara et al J. Biochem. 67 (1970) and Campbell et al
J. Biol. Chem. 236, 2952 (1961)). As shown in Fig. 9, the
, optimal pH for the enzymes of the present invention is pH 4.2
'; to pH 5.2. The preferred enzymes of the present invention do
'~ not lose their activity even if they are left for 24 hours at
, 10 room temperature at pH's in the range from 3 to 11.
'~ The relationship between liquefaction by the enzymes of
i the present invention (relative value) and the operational
temperature is shown in Fig. 10 and contrasted with that of
the alpha-amylase derived from Bacillus stearothermophilus
~, 15 described by Ogasawara et. al. (J. Biochem. 67, 65 (1970)).
As shown in Fig. 10, the preferred operational temperature
,' for the enzymes of the present invention is about 80C.
As apparent from the foregoing, the alpha-amylase
enzymes of the present invention can be used in the lique-
faction and conversion of starch in the starch saccharification
industry, desizing process in the textile industry and as
~; additives in detergent formulations similarly to the conven-
tional uses for bacterial alpha-amylase enzymes.
'~
The alpha-amylase enzymes of the present invention are
particularly suited in the liquefaction and conversion of
starch in the production of maIto-dextrins, subsequent
production of dextrose using glucoamylase since isomeriza-
tion of the end group of the molecules can be avoided
, because the enzyme can be efficiently used at an acidic pH
! 30 (i.e. a pH of 4.5 - 5.0), thereby increasing the dextrose
s'~
-38 -
:
1~8~633
yield. The use of these novel enzymes also reduces the ion
exchange load in the refining process because no pH adjust-
:; .
ment is required prior to saccharification and conversion
with the glucoamylase enzyme.
:,,
In one preferred manner of using the alpha-amylase
i enzymes of the present invention, the enzyme is used to
s convert starch to a starch hydrolysate wherein the residual
unconverted starch remains in its granular form. These
processes are described and claimed in U. S. Patent Nos.
, 10 3,922,196; 3,922,197; 3,922,198; 3,922,199; 3,922,200 and
, 3,922,201, all issued November 23, 1975. In
these granular starch procedures, at least the initial
, solubilization of the starchy material is carried out at
relatively low temperatures, i.e., below the initial gela-
tinization temperature of the starch up to the actual
initial gelatinization temperature of the starch. In a
preferred manner of carrying out these processes, the
glucoamylase enzyme is used concurrently with the alpha-
s amylase enzyme in the initial solubilization stage. The
; 20 enzymes of the present invention are particularly suited for
this process because their optimal pH range is compatible
with glucoamylase.
~'
In another preferred manner of using the alpha-amylase
enzymes of the present invention one can use the processes
described in U. S. Patent No. 3,853,706, issued December
~; 10, 1974 and U. S. Patent No. 3,849,194 issued November 19,
1974.
;
~. .
- 39 _
1~81~3
In still another preferred manner of using the alpha-
amylase enzymes of the present invention one can use the
process described in U. S. Patent No. 3,91~,590, issued
October 14, 1975 to Slott et. al. and assigned to Movo
Industri A/S. By use of the alpha-arnylase enzymes of the
present invention with the Slott et. al. process, a slurry
of starch, such as corn starch having at least 25% by weight
starch material is treated with the alpha-amylase enzyme at
a temperature in the range from about 100C to about 115C,
preferably from 105C to about 110C for 1 to 60 minutes,
' and preferably 5-10 minutes to liquefy the starch and
thereafter reduce the temperature to 80-100C and preferably
90C to 100C when the viscosity of the thinned solution is
less than 300 c.p.s. measured at 95C. The unique advantage
of applying this temperature,profile procedure with the
alpha-amylase enzymes of the present invention is that a
lower pH can be used so that little or no pH adjustment is
needed in a subsequent saccharification procedure with
glucoamylase.
The most preferred use of the alpha-amylase enzymes of
the present invention for converting starch include subject-
ing a starch slurry to the action of the enzyme at a p~ in
the range from about 3.5 to about 6.5, preferably from about
4 to about 5, at a temperature ranging from about 50C to
about 100C, and preferably 60,C to about 95C to liquefy
the starch. In the case of the granular starch hydrolysis
processes described above the temperature will range from
the normal initial gelatinization temperature to the
actual initial gelatinization temperature of the starch,
i.e. about 60C for corn starch. In the case of a direct
liquefaction procedure, the starch slurry containing the
- 40 -
~81~
enzyme is preferably heated ~e.g. by a jet heater) to a
temperature ranging from about 85C to about 95C and more
preferably from about 90C to about 92C to liquefy the
starch. Following the initial solubilization (as in the
granular starch hydrolysis) or liquefaction, the slurry is
preferably subjected to a "heat-shock" treatment at a
temperature above 100C and perferably ranging from about
110C to about 150C to liquefy any residual starch gran-
ules. Thereafter, the liquefied starch slurry is cooled and
preferably treated with additional al;eha-amylase, alone or in
combination with other enzymes such as glucoamylase, beta-
amylase, pulluanase, glucose isomerase, sequentially or in
combination. If alpha-amylase is used alone in the second
enzyme stage the temperature will preferably range from
about 80C to about 90C and most preferably about 85C, the
optimum temperature for the enzymes of the present invention.
If other enzymes are present such as glucoamylase and/or
glucose isomerase, the temperature will be somewhat lower,
i.e., 55-75C and preferably about 60C.
CONCLUSION
The alJeha-amylase enzymes of the present invention can
be clearly differentiated from the prior art alpha-amylases
obtained from animals, plants, yeasts, imperfect fungi, and
molds inasmuch as these prior art enzymes have such a low
heat-stability that they completely lose their activity upon
5 minutes treatment at 70C and pH 6Ø By comparing the
heat-stability of the _lph_-amylases of the present inven-
tion as shown in Fig. 12 (the data for preparing Fig. 12 is
substantially the same as that used for Fig. 6) it is clear
that the alpha-amylase enzymes of the present invention are
far superior as to acid- and heat-stability compared to the
~ 41 -
:~38~6~33
prior art alpha-amylase enzymes compared. It is also
apparent from Fig. 12 that the alpha-amylase enzymes of the
present invention are characterized as capable of retaining
at least about 70% and preferably at least about 90% of
their initial activity when held at 90,C and at a pH of 6.0
for 10 minutes in the absence of added calcium ion and
capable of retaining at least about 50% of their initial
alpha-amylase activity when held at 90C at a pH of 6.0 for
60 minutes. As seen from Fig. 11, it is apparent that the
alpha-amylase enzymes of the present invention is further
characterized as capable of retaining at least about 50% of
their initial al~ha-amylase activity at a temperature of
80C and at a pH of 4.55 in the presence of 5mM of calcium
ion for 10 minutes.
Table 9 shows the relationship between the alpha-
amylase enzymes of the present invention and the alpha-
amylase enzymes Bacillus subtilis and Ba_ llus li_hen rmi~
which are employed industrially, and those of Bacillus
stearothermophilus described in the literature. They are
compared with respect to optimal operational pH, proper
operational temperature, and molecular weight. Figs. 11 and
12 contrast their properties of heat- and acid-stabilities.
~ 3
Table 9
Proper
- Optimal Operational Molecular
Enzymes TestedOperational pH Temperature Weiqht
Alpha-amylase
enzymes of this
invention 4.0 - 5.2 80C 96,000
Alpha-amylase
of B. subtilisl4.5 - 6.5 45 - 60C 49,0005
Alpha-amylase of
~2 5.0 _ 9 0 76 - 78CC 22,soo5
Alpha-amylase of
B. stearothermo~hilus
a) Ogasawara et. al.l 5.0 - 6.0 65 - 70C 48,000
b) Campbell et. al. 3 4.8 55 - 70C 15,6004
Ogasawara et. al., J. Biochem., 67, 65 (1970).
Shigemasa Saito, ABB, 155, 290 (1973).
3Campbell et. al, J. Biol. Chem., 236, 2958 (1961).
4Campbell et. al, J. Biol. Chem., 236, 2958 (1961).
5British Patent Specification reports the molecular
weight of alpha-amylase from B. licheniformis to be
18,000-20,000 and B. subtilis 96,000.
When the alpha-amylase enzymes o~ the present invention
are compared with the alpha-amylase enzymes from Bacillus
subtilis, the two are seen to be remarXably different, as is
clear from Figs. 9 and 11, in their optimal operational pH,
proper operational temperature, molecular weight and heat-
and acid-stabïlity. This indi$ates that this enzyme is
quite different from the alpha-amylase of Bacillus subtilis.
0 When the alpha-amylase enzymes of the present invention
and the alpha-amylase enzymes from Bacillus licheniformis
are compared, they are remarkably different in their optimal
operational pH, molecular weight and heat- and acid-stability
as is shown in Table 9, and Figs. 11 and 12.
-
- 43 -
As it will be apparent to those skilled in the art, the
strains used to produce the novel alpha-amylase enzymes of
the present invention may be subjected to mutagenic agents
known using known techniques, such as ultra-violet light,
S chemical treatment and the like. Accordingly, the present
invention contemplates alpha-amylase enzymes produced from
the strains of ATCC Nos. 31,195, 31,196, 31,197, 31,198,
31,199, variants and mutants of these strains, and sub-
mutants of said variants and mutants.
Like some of the other known al~ -amylase enzymes, the
enzymes of the present invention are inhibited by mercury
and EDTA, but are stabilized by calcium.
:
It will be understood by those skilled in the art that
various modifications of the present invention as described
in the foregoing examples may be employed without departing
from the scope of the invention. Many variations and
modifications thereof will be apparent to those skilled in
the art and can be made without departing from the spirit
and scope of the invention herein described.
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