CA1081633A - Heat and acid-stable alpha-amylase enzymes and processes for producing the same - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
Heat and acid-stable alpha-amylase enzymes having the following characteristics: (1) capable of retaining at least about 70% of their initial activity when held at 90°C
and at a pH of 6.0 for 10 minutes in the absence of calcium ion; (2) capable of retaining at least about 50% of their initial activity when held at 90°C at a pH of 6.0 for 60 minutes in the absence of added calcium ion; (3) capable of retaining at least about 50% of their initial activity at a temperature of 80°C and at a pH of 4.55 in the presence of 5mM calcium ion for 10 minutes; and/or (4) capable of retaining at least about 95% of their initial activity at a temperature of 80°C at a pH ranging from about 4 to about 6.5. The preferred alpha-amylases are prepared by culturing a strain of a Bacillus stearothermophilus microorganism in a suitable culture medium. The novel alpha-amylases are useful in hydrolyzing and/or liquefying starch and due to their stability at low pH values they can be used in con-junction with other acid stable amylases such as gluco-amylase in either a soluble or an immobilized form.

Description

16;~3 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.

:

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.

~ 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.

.

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%

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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.

. .

~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 ; - 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) (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) 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 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~.

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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.

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.

~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.

1~81633 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.

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~ ~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.

/:

~, ;

'' ~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 :

.

~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. ~
: ~:

' '~, , ' ''' ~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 , ~ .

, ~.~

,~ ~
,`' .
:

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).

~: .

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 , .
(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 ..
~' ' '` .

: 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.
~''-~::

~. : . . -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 -.,~, .

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'~

:

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 ~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.

-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.

Claims (15)

HEAT AND ACID-STABLE
ALPHA-AMYLASE ENZYMES AND
THE SAME

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:-

1. A heat and acid-stable alpha-amylase enzyme characterized as (1) capable of retaining at least about 70%
of its initial alpha-amylase activity when held at 90°C and at a pH of 6.0 for 10 minutes in the absence of added cal-cium ion; (2) capable of retaining at least about 50% of its alpha-amylase initial activity when held at 90°C 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 its initial alpha-amylase activity at a temperature of 80°C and at a pH
of 4.55 in the presence of 5mM of calcium ion for 10 minutes.

2. The alpha-amylase enzyme of Claim 1, wherein said enzyme has a molecular weight of at least about 90,000 as determined by SDS disc electrophoresis.

3. The alpha-amylase enzyme of Claim 1, wherein said enzyme is derived from a Bacillus microorganism.

4. The alpha-amylase enzyme of Claim 1, wherein said enzyme is derived from a Bacillus stearothermophilus microorganism.

5. The alpha-amylase enzyme of Claim 1, wherein said enzyme is capable of retaining at least about 50% of its initial activity at a temperature of 85°C and at a pH of 4.55 in the presence of 5mM of calcium ion and 22.5%, by weight, starch, d.s. for 30 minutes.

6. A heat- and acid-stable alpha-amylase enzyme characterized as being derived from a strain of Bacillus stearothermophilus which is a member selected from the group consisting of ATCC Nos. 31,195, 31,196, 31,197, 31,198, and 31,199, variants and mutants thereof and sub-mutants of said mutants.

7. The heat- and acid-stable alpha-amylase enzyme of Claim 6, wherein the Bacillus stearpthermophilus strain is ATCC No. 31,199 and mutants thereof.

8. A process for the preparation of a heat and acid stable alpha-amylase enzyme, comprising culturing a micro-organism derived from a strain selected from the group consist-ing of Bacillus stearothermophilus ATCC Nos. 31,195, 31,196, 31,197, 31,198, 31,199, variants and mutants thereof and sub-mutants of said mutants in a culture medium and recovering the enzyme produced.

9. The process of Claim 8, wherein the culture medium contains an assimilable carbon and nitrogen source.

10. The process of Claim 8, wherein the cultivation is conducted at a pH in the range from 5 to 9 at a temperature of 50 to about 70°C for 1 to 5 days.

11. A process for converting starch to a starch hydrolysate comprising:

(a) treating an aqueous slurry of starch with the alpha-amylase enzyme of Claim 1 at a pH of 3.5 to 6.5 to liquefy and convert the starch; and (b) obtaining a starch hydrolysate from the conversion of step (a).

12. The process of Claim 11, wherein the treatment in step (a) is conducted at a temperature in the range from about 50°C to about 100°C at a pH of 4 to about 5.

13. The process of Claim 11, wherein the starch hydrolysate is treated with a glucoamylase enzyme.

14. The process of Claim 11, wherein the starch hydrolysate is heated to a temperature above 100°C and thereafter cooled and treated with additional alpha-amylase.

15. The process of Claim 11, wherein the starch hydrolysate is treated with a glucose isomerase enzyme.