DE2717333A1 - HEAT- AND ACID-RESISTANT ALPHA AMYLASE ENZYME, METHOD FOR ITS MANUFACTURING AND USE - Google Patents

Description

MÜLLER-BORE DEUFEL SCHÖN UERTEL PATE NTAN WALTE

DR. WOLFGANG MÜLLER-BORE (PATENT ADVERTISER FOR IM7-I97S) DR. PAUL DEUFEL. DIPL.-CH EM. DR. ALFRED SCHÖN. DIPL.-CHEM. WERNER HERTEL. DIPL.-ΡΗΥβ.

C 2999

CPC INTERNATIONAL INC.

International Plaza, Englewood Cliffs, New Jersey O7632, USA

Heat and acid resistant Oi - amylase enzyme, Process for its manufacture and its use

7 09845/0838

β MÜirCHSir ββ · SIXBXRTSTH. 4 ΡΟβΤϊΑΟΒ ββΟΠΟ CABLE: MUXBOPAT TXL. (Μ ·) 47 «000 · TSLXX S-MSM

The invention relates to a heat and acid-resistant amylase enzyme, a process for its preparation and its use in processes for hydrolyzing, liquefying and / or Converting starch containing materials into starch hydrolysates.

0 (.- Amylase enzyme preparations have been used for some time to hydrolyze, liquefy and / or convert starchy materials into starch hydrolysates, as well as in detergent mixtures in the first stage of processes for the manufacture of a range of synthetic starch hydrolyzate materials, such as maltodextrins, Corn syrup, dextrose, levulose, maltose and the like are used. <X-amylase enzymes hydrolyze starch molecules that do so split into a variety of different medium molecular weight fragments known as maltodextrins. The maltodextrins are subsequently given a or several other enzyme preparations, including glucamylase, β-amylase and glucose isomerase are treated to produce the desired end products. Optionally, several of these enzyme preparations can be incorporated into a slurry of the starch material at the same time, in order in this way directly to produce the starch hydrolyzate desired as the end product.

Amylase enzymes can be obtained from a wide variety of different sources. Most <X amylase enzymes are produced from or from bacterial sources such as Bacillus subtili3, Bacillus lichenifortnis , Bacillus stearothermophilus and other strains of microorganisms that are cultivated in a suitable nutrient medium. whereupon the cells produced are destroyed and the enzyme preparation is then separated from the culture broth and purified.

Many of the <X amylase enzymes currently available on the market are made from Bacillus subtilis microorganisms. When using these enzymes to convert starch into

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As a rule, starch hydrolysates have an optimal temperature in the range from about 80 to 85 ° C. and an optimal pH of about 6.0. These temperature and pH conditions, which are necessary for efficient use of the enzyme, have two disadvantages. First, when the starch is converted with the enzyme at a pH of about 6 and a temperature of about 80 to 85 C, a portion of the reducing end groups of the starch is isomerized, and then in the course of the subsequent conversion process, maltolose is produced, thereby increasing the yield in the desired end product, e.g. dextrose, levulose or maltose. Second, the optimum pH of glucamylase used in the conversion and saccharification process is typically around k _t 5 for enzymes derived from Aspergillus niger and around 5.0 for enzymes derived from Rhizopus microorganisms. Therefore, after completion of the liquefaction step carried out using the o (—amylase “enzyme, the pH must be lowered from about 6 to about k, 5 or 5» 0. This pH adjustment increases the ion concentration, thereby reducing the burden on the The ion exchange resins used in the purification of the end product and, consequently, the refining costs increase.

Various heat-stable d-Amy-1 ase enzymes have been developed in recent years. Examples of such α-amylase enzymes include the enzyme preparations produced from microorganisms of the species Bacillus stearothermophilus (cf. Ogasawara and coworkers, J. Biol. Chem . 67, 65, 77 and 83 (1970); GB Manning and LL Campbell, J. Biol Chem. , 236 , 2952, 2958 and 2962 (1961); SL Pfueller and WH Elliot, J. Biol. Chem . 2kk , k8 (1969)). For even more recently, cX-amylase enzymes with good heat stability have been available in neutral or weakly alkaline solutions. These heat and alkali-resistant Ot-Ataylase enzymes are represented under the brand name "Thermamyl" . They are produced by cultivating microorganisms of the species Bacillus licheniformis (cf. GB-PS 1 296 839, Madaen and coworkers, Die Starke, 2 £, 304, 305 and 308 (1973) and Narimasa Saito, ABB, J_5J>, 290 ( 1973)) S From Bacillus licheni-

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Form! 3 generated Ot-amylase enzymes have good heat stability in neutral and weakly alkaline solutions, but are not stable enough under acidic conditions to be economical.

The invention was therefore based on the object ° (-Ainylaseenzyme to create or make available that both a have good heat resistance as well as good stability under acidic conditions, and especially at pH values, which make it possible to use them under conditions that are compatible with other amylases such as glucamylase.

According to the invention, this object is achieved by a heat-resistant and acid-resistant o (-Amylase enzyme dissolved, which is characterized by the fact that it is able to

1. to retain at least about 70 % of its initial Λ-amylase activity if it is kept in the absence of added calcium ions for 10 minutes at 90 C and a pH of 6.0,

2. to retain at least about 50 % of its initial & -amylase activity when held in the absence of added calcium ions for 60 minutes at 90 C and a pH of 6.0,

3. at least about 50 # to keep its initial Ot-amylase activity, if one in the presence of 5 mM calcium ion for 10 minutes at 80 ° C and a pH of k, 55 holds.

Preferred enzymes according to the invention are also capable of retaining at least about 50-6 of their initial activity at a temperature of 85 ° C. and a pH of 4.55 in the presence of 5 mmol calcium ions and 22.5 percent by weight starch, dry matter. Preferred enzymes according to the invention are further characterized in that they are able to retain at least about 95% of their initial α-amylase activity at a temperature of 80 ^° C. and a pH in the range from about k to 6.0. These enzymes are preferably derived from a microorganism of the genus Bacillus and in particular

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in particular from a microorganism of the species Bacillus stearothermophilus .

Preferred Ci-amylase enzymes according to the invention have a through SDS disk electrophoresis determined molecular weight of at least about 90,000 and are characterized by the fact that they are essentially free of protease activity. For example, they usually have a protease / ct-amylase ratio of less than 3 and in particular less than 1.

The new α-amylase enzymes of the invention are preferably prepared by treating a microorganism of the species Bacillus stearothermophilus and in particular a microorganism strain from the group Bacillus stearothermophilus ATCC No. 31 195, 31 196, 31 197, 31 198 and 31 199, including the variants, Mutants and submutants of the strains mentioned are cultivated and then the oC-amylase enzyme produced thereby wins.

In the drawing shows:

Figure 1 is a diagram showing the relationship between residual activity and the pH value for two enzymes according to the invention and, for comparison, for Thermamyl-öf-amylase explained;

Figure 2 is a graph showing the relationship between relative activity and temperature for two of the present invention. moderate enzymes and, for comparison, Thermamyl-Ot-amylase explained;

Figure 3 is a graph showing the thermal stability in terms of the relationship between the residual activity and the incubation time at 8O ⁰ C and a pH of 4.55 in the presence of 5 mM calcium ion for the two according to the invention enzymes, as well as, for comparison, Thermamyl- (A-Amylaee explained;

Figure 4 is a diagram showing the thermal stability at 90 ⁰ C and

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a pH of 6.0 based on the relationship between the Residual activity and the incubation time for two enzymes according to the invention and, for comparison, at Thermamy 1- <X-Amy read explained;

Figure 5, is shown in each case at 90 ⁰ C, pH 6.0 and in the presence of 1 mM calcium ion, a diagram in which the heat resistance of two inventive enzymes and were reading for comparison, of Termamyl-OC- Amy;

FIG. 6 is a diagram showing the heat resistance of two enzymes according to the invention and, for comparison, of Thermamyl, each at 85 ° C. and a pH of 1.55 in the presence of 1 mmol of calcium ions and 22.5 hours of starch, dry substance, explained;

FIG. 7 © in a diagram showing the heat resistance of two enzymes according to the invention and, for comparison, of Thermamyl-od-Amylase, each at 85 ° C., a pH of 4.55 as well as illustrated in the presence of 22.5 # starch and 5 mmol calcium ions;

FIG. 8 is a diagram in which the determination of the molecular weight of two enzymes according to the invention by SDS disk electrophoresis is graphically represented and explained;

FIG. 9 shows a diagram which explains the relationship between the pH value and the enzyme activity in an enzyme according to the invention and, for comparison, in known <X amylases derived from microorganisms of the species Bacillus stearothermophilus;

Figure 10 is a graph showing the relationship between temperature and activity for an enzyme of the invention

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and, for comparison, a known σί-Amy läse from Bacillus stearothermophilus explained}

FIG. 11 shows a diagram which shows the deactivation curves of an enzyme according to the invention and, for comparison, two known o (amylases derived from Bacillus subtilis or Bacillus licheniformis , each at 80 ° C. and pH 4.5 in the presence of 5 mmol of calcium ions;

FIG. 12 shows a diagram in which the deactivation curves of an enzyme according to the invention and, for comparison, two known cfcr amylases originating from Bacillus licheniformis or Bacillus stearothermophilus , each at 90 ° C. and a pH of 6.0 without calcium ions.

Different kinds of microorganisms were treated under different conditions such as growth and cultivation temperature (37 ° C, 45 ° C, 55 ° C and 70 ° C), pH (5.0 and 7.0) and culture media the generation of Λ-amylase and the thermal stability of the enzyme formed. It was found that thermophilic ray fungi (ray fungi) isolated at 55 ° C and thermophilic fungi at 45 ° C produce OC-Amy läse in high yields, which, however, is not heat-resistant, while thermophilic bacteria at 70 ° C heat-resistant oil-amyl & ae however, only produce _{f in low yields.} On the basis of these preliminary investigations, the further screening of microorganisms with regard to their ability to produce acid- and heat-resistant oC-amylase was carried out by cultivation at 55 and 70 ° C. A total of 550 samples of microorganism sources, including soil samples, water from hot springs, compost samples, etc., were collected from various locations

The acid and heat resistance was determined in each case by the OEG-amylase activity was determined before and after a 10-minute heat treatment of the KuIturfilträte at 8O ⁰ C and pH 5 »0 in the presence of 10 mM calcium ions, respectively.

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The oC-amylase activity was determined as follows:

A 0.2 # solution of soluble starch was prepared weekly as follows: 200 mg of soluble starch was dissolved in about 50 ml of 0.2 molar acetate buffer solution (pH 1.5) containing 0.013 mol of CaCl ₂ ^# 2H ₂ O in boiling water 100 ° C, whereupon the resulting solution was made up to 100 ml with the same buffer solution. 0.1 ml enzyme solution and 0.3 ml 0.2 # starch solution containing the sample tube was in each case 5 minutes. Incubated at 60 ° C. The reaction was then stopped by adding 1.0 ml of 0.5 normal acetic acid. 3 »0 ml of 0.015% iodine solution were then added and stirred in, whereupon the. optical density was measured at 700 mu against HO as a standard. The tube without enzyme served as a blank. Its optical density at 700 µm was called OD. One enzyme (activity) unit was defined as the amount of enzyme which, under the conditions described above, leads to a decrease in the blue value of 10 Ji per minute.

NML units = (0D, - OD) χ 100

0D _b χ 5 x 10

Unless expressly stated otherwise, the ct-amylase activities indicated below were each determined in the manner described above (NML units). Where the Ot-amylase activity is given in CPC-λ-amylase units, it should be noted that the CPC units are about i / i * K> the NML units.

The CPC-o (-amylase activity units were determined according to the following Method determined:

One ml of an appropriately diluted enzyme solution becomes 10 ml of a 0.3 molar acetic acid buffer containing 1.0% soluble starch He solution (pH 6.0) was added, whereupon the mixture was added Allowed to react for 10 minutes at 6o ° C. Then 1 ml of the reac-

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tion solution is placed in a 100 ml volumetric flask in which 50 ml of 0.02 normal hydrochloric acid are placed, whereupon 3 ml of 0.05% iodine solution is added and the contents of the flask are then made up to 100 ml with water. The blue color that develops is determined by measuring the absorbance at 620 μm. One CPC unit is defined as the amount of enzyme that is required to break down 10 mg of starch in one minute under the specified conditions.

1 CPC unit = D _q - D _g χ 50 χ (dilution factor);

D 10 χ 10 ο

D = absorption of the reference solution (instead of the enzyme solution water is added);

D = absorption of the reaction solution

The culture conditions and media used in the screening tests are summarized in Table X below .

The results of the first and second series of screening tests (1st and 2nd screening) are shown in Table II below .

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Culture conditions and media

conditions

Plate culture * ¹ 'Slant agar culture' ² 'Flask culture * ³ '

a) 55 ° C / pH 5 55 ° C / pH 5 50 ° C / pH 6

b) 55 ° C / pH 7 55 ° C / pH 7 50 ⁰ C / pH 7

c) 70 ⁰ C / pH 5 70 ° C / pH 5 60 ° C / pH 6

d) 70 ⁰ C / pH 7 70 ⁰ C / pH 7 60 ⁰ C / pH 7

(1) Medium for plate culture

Cb ^ bJ

Amylopectin 0.02 96 (NH ^) ₂ HPO 0.025

Yeast extract 0.025 Na Ci joined 0.01 96 MgSO. · 7HO 0.05 96

KCl 0.15 96

CaCl ₂ * 2H ₂ O 0.05 96 agar - 2.0 96

(2) medium for the (3) medium for the Agar slant culture (B-D) Flask culture (B-M)

Soluble starch 1.096 3.0 96

Bacto-trypton (Difco) 0.5 # ° »5 #

Yeast extract 0.5 # ¹ »° #

CaCl ₂ »2H ₂ 0 0.05 96 ° tO5%

• 4H 0 0.05

• 7H ₂ 0 - ° t ° 5 96

_k 0.1 96 0.1 #

Agar 2.0

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Table II

1 . Sighting

Isolation- Isolated conditions. Tribes

Screening result 2nd sighting

Distribution of OC amylase producers

100-200 ~ 500 ~ 1000 -2000 ~ 2500 U / ml U / ml U / ml U / ml U / ml

55 ° C / pH 5.0 Ray mushrooms
(Ray fungi)
503 tribes + 16
O O 46
O 34
O 1
O Bacteria
292 tribes + 9
O k
1 2
2 O
O O
O 55 ° C / pH 7.0 Ray mushrooms
(Ray fungi)
629 tribes + 16
O 50
O 34
O 27
O 5
O Bacteria
769 tribes + 5
k 3
3 2
O O
O O
O 70 ° C / pH 5.0 Bacteria
841 tribes + O
7th 1
1 O
1 O
3 O
O 70 ° C / pH 7.0 Bacteria
1326 tribes + O
15th O
2 O
O O
O O
O

RA: Remaining activity and residual activity after 10-minute heat treatment at 8O ⁰ C and pH 5.0. All RA positive strains had about 100 # residual activity

About 25 96 of the thermophilic ray fungi isolated at 55 C produced 100 to 2500 units of Λ-amylase per ml of culture broth, however, after the heat treatment (10 minutes at 80 ° C. and pH 5 »0) there was no longer any residual activity.

From a total of 1061 thermophilic bacterial amines isolated at 55 ° C generated a total of 35 strains of oi-amylase in one between 100 and 1000 units per ml of culture broth, with 10 of these strains being a heat-resistant oC-amylase generated. Almost all of the oc-amylases produced by thermophilic bacteria isolated at 70 ° C were acid and heat resistant. The largest amounts of (X-amylase were produced by bacterial stenunas that had been isolated at pH 5 * 0.

In the latter 219 strains were isolated at 70 ⁰ C and pH 5.0, the relationship between the activity and the clear zone size was investigated. The results of this study are shown in Table IXX, from which it can be seen that 90% of these strains gave clear zones less than 3 cm in diameter and produced 0 to 200 units of d- amyase. The highest Ot-amylase production was achieved in the strains that gave a clear zone with a diameter of more than 3 cm, but this was only 10 # of the strains tested.

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Table III

Relationship between activity and clear zone size of the isolated strain

Activity clear zone size on the insulation

ira plate

Piston (diameter in cm)

(U / ml) <2 -3 15th 0 total 0-100 153 ko 1 0 208 - 200 3 2 2 0 6th - 500 0 0 1 0 2 - 1000 0 0 1 1 1 - 1500 0 0 2

Total 156 k2 20 1 219

Based on the above experiments, it was found that the isolation of bacterial colonies with a clear zone with a diameter of more than 3 cm on the plate medium used in these experiments at 70 ° C and a pH of 5.0 a
excellent method of sighting . or the search for microorganisms which are capable of producing acid- and thermostable egg-amylase enzymes in high yields.

As a result of this screening, 5 strains were selected as the best or most efficient producer of acid- and heat-resistant Ct-Amylaae enzymes selected.

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-Vt-

Each of these five strains was described in as follows purified form in the permanent collection of the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, USA. The ATCC preserves and receives these logs on the basis of a contractual agreement of CPC International Inc ..

The contract between the ATCC and CPC International Inc. provides that the cultures or subcultures of the general public after

1) Grant of a US patent describing and identifying the deposits in question and those assigned to them ATCC no. disclosed

or

2) Notice or disclosure of a US or foreign Patent application describing and identifying the deposits in question and the ATCC no. disclosed, · «

are always available. CPC International Inc. has contacted any of these deposited cultures during Should the patent die or be destroyed during the term of the patent, this is bound by a living culture of the same organism to replace. In addition, CPC International Inc. has authorized the ATCC, the United States Patent and Trademark Office and the Patent Office of the Federal Republic of Germany by an authorized member of the named authorities To make cultures or subcultures accessible at any time upon request.

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Deposited organisms NML-Staroma No. ATCC No.

Bacillus stearothermophilus B-501 31 195

Bacillus stearothermophilus B-63k 31 196

Bacillus stearothermophilus B-781 31 197

Bacillus stearothermophilus B-905 31 198

Bacillus stearothermophilus B-968 31 199

In addition, the NML strains B-501 and B-781 have been deposited with the Fermentation Research Institute, Industrial Technology Agency, MITI, under FRI numbers 3389 and 3390.

The five (5) selected strains deposited with the ATCC exhibit the following bacteriological. Features on:

(1) morphological features;

A. Shape and size of cells: 0.6 χ 2 - 3 p »individual

Chopsticks, rarely in chains (all stems)

B. Pleomorphism: Negative (all tribes)

C. Agility: Agile and flagellated (all tribes)

D. Spores: 0.6 χ 1.0 - 1.5 ρ »ovoid; club-shaped

Spore casing (all trunks)

E. Gram stains: Positive (all strains)

F. Acid Fastness: Negative (all strains)

(2) growth on different media

A. Nutrient Agar Plate: Active Colonies Spreading

with a grainy surface and rough edges (all trunks)

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B. Nutrient slant agar plate: good growth, white-opaque »

active spread, honeycomb-like outgrowths (all Tribes)

C. Liquid broth culture: Transparent brown, whiter

Surface turf (all trunks)

D. Nutrient Gelatin Stitch Culture Liquefaction (all strains) (Nutrient gelatin stab culture)

E. Nutrient gelatin agar plate: wide clear zone (all

Tribes)

F. Salt Broth Liquid Culture: Growth Inhibition

in 2 % salt (all strains)

G. Milk agar plate: clear zone formation by hydrolysis

of casein (all strains)

H. Glucose Agar Slant Plate: Good growth, similar

Colonies as on nutrient agar (all strains)

I, proteose peptone agar slant plate: no growth

(all stamines)

(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. Hydrogen sulfide formation: Positive (all strains)

F. Hydrogen Sulphide Formation: Positive (all strains)

G. Starch hydrolysis: Strong hydrolysis (all strains)

H. Formation of acid and gas: Positive for acid, however

no gas formation from

709845/0838 ^glucose > xylose, arabinose

and mannitol (all strains)

* 0

I, temperature and pH conditions!

37 ° C

50-70 C.

pH range for growth

optimal pH

ATCC No. 31 195 (B-5 01)

ATCC No. 31 197 (B- 781)

no growth weak growth weak growth moderate growth good growth good growth

5-8
6-7

5-8 6-7

The above tests were carried out in accordance with "Laboratory Methods in Microbiology ¹¹ by WF Harrigan et al. And the" Manual of Microbiological Methods "published by the American Bacteriological Association.

On the basis of the above characteristics, the five selected strains were identified as Bacillus stearo-thermophilus in accordance with Bergey's Manual of Determinative Bacteriology, 8th edition.

These five strains were further processed by the plate-strip method cleaned. The results of the isolation, cultivation and purification conditions on the five selected strains are summarized in Table IV below. As As can be seen from Table IV, the purified strain was ATCC No. 31 199 (B-968), the Öf amylase in one activity of 2111 NML units / ml of culture broth corresponding amount (about 15 CPC units) produced the best of the five strains selected.

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Table IV

Overview of the selected strains

ATCC No. 195 1. Sighting 5.0 2. Sighting 1 152 89 Culture
condition
gene cleaning 72 Culture
condition
gene 196 Isolation
conditions
Temp. PH 5.0 Origin activity culture
or (U / ml) time
Source (h) 700 112 Activity cultural
(B / ml) time 65 O 31 197 ( ^c ) 5.0 591 concentration camp 26th CO 31 198 5.0 1 217 60 ° C / pH 6 65 60 ° C / pH 6 cn 31 199 70 5.0 Arable land 1 098 50 ° C / pH 6 1 372 50 ° C / pH 6 / 0831 31 55 Floor off
Starch deposition
pool 60 ° C / pH 6 687 60 ° C / pH 6 31 70 Floor off
hot spring 60 ° C / pH 6 1 002 60 ° C / pH 6 70 Fermented
Oats in silo 60 ° C / pH 6 859 55 ° C / pH 6 70 compost 2 111 IS5 fl7333
I.
I.
I.
I.
I.

In the production of the <X -amylase enzymes according to the invention, a microorganism strain capable of producing an acid- and heat-resistant d-amylase enzyme, e.g. a strain which meets the test requirements specified in Table III, for example Bacillus stearothermophilus strain ATCC No. 31 195t 31 196, 31 197, 31 198 or 31 199 cultivated in a nutrient medium known for cultivating thermophilic bacteria. These culture media should contain an assimilable source of carbon and nitrogen as well as other essential nutrients.

Suitable assimilable carbon sources include carbohydrates, such as starches, hydrolyzed starches, corn flour, wheat flour, etc. The carbohydrate concentration in the nutrient used medium can vary within wide limits and for example between about 1 and 25 and preferably in the range from about 10 to 20 pounds (weight / volume) calculated as dextrose, lie. Preferred assimilable carbohydrates are starch or partially hydrolyzed starch, which when put on weight- base, in amounts ranging from 1 to 5 Weight percent should be present.

The nitrogen source in the nutrient medium can be inorganic and / or organic. 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, soybeans flour, skimmed milk, etc.

In addition, the nutrient medium should contain the usual trace substances, e.g. inorganic salts, including calcium chloride, magnesium sulfate, Phosphates, sodium chloride, potassium chloride, etc. The above carbon sources, nitrogen sources and inorganic salts can be used singly or in appropriate combinations. You can also use small amounts of metal salts, vitamins, amino acids, etc. can be used to promote the growth and productivity of bacteria.

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In the preparation of the CV amylase enzymes according to the invention the same cultivation conditions are used as those normally used for cultivating thermophilic bacteria can be applied. Preferably, the strain is in a deep liquid culture medium with stirring and aeration two to cultured for five days at 50 to 70 ° C and a pH of 5 to 9. The enzyme accumulates in the cultured medium.

After the generation or formation of the O ^ -amylase enzyme according to the invention the microbial cells are separated in a conventional manner, e.g., by centrifugation. The filtrate is then preferably by adding inorganic salts such as ammonium sulfate, sodium sulfate or magnesium sulfate, salting out and / or by adding water-miscible organic solvents such as acetone, ethanol, propan-2-ol, etc., added to the enzyme precipitate so that it can be concentrated. The ft-amylase can also be obtained by adding to the filtrate Starch adds so that the OC-amylase adsorbs to the starch will.

A preferred method of purifying and separating the enzyme from the filtrate is to add twice the volume of cold acetone to the filtrate in order to precipitate the enzyme. The precipitated enzyme is then dissolved in 0.05 molar Tris-hydrochloric acid buffer solution (pH 8.5) and then fed through a DEAE cellulose column equilibrated with the same buffer solution. At a pH of 8.5, the non-ÖG amylase proteins, pigments, etc. are adsorbed on the DEAE cellulose, while most of the OC amylase remains in solution. The filtrate containing the it-Amy-lase enzyme is hereinafter referred to as "partially refined enzyme ^11. The partially refined enzyme can be further purified by dialyzing it against a 0.01 M Tris-hydrochloric acid buffer solution (pH 7» 0) and then through a Hydroxyapatite column, which is equilibrated with the same buffer solution, and the enzyme is adsorbed onto the column.

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The adsorbed enzyme is eluted by increasing the ammonium sulfate concentration steadily from 0 to 0.05 molax *.

The partially refined enzyme thus obtained is after one concentrated it, passed it through an ion exchange resin column (Sephadex G-150), whereby the enzyme weakly binds to the Ion exchange resin is adsorbed. The enzyme is eluted in the fraction with a molecular weight below 10,000. The activity of the refined enzyme obtained in this way is indeed 100 times higher than in the original filtrate, however, on disk electrophoresis, besides that of the QC amylase enzyme, there are also strips of a few other proteins and the enzyme has not yet crystallized. The following examples illustrate the invention and are in no way to be understood as limiting. Information on the proportions or proportions relate to Unless expressly stated otherwise, always based on weight.

example 1

A 3.0 wt. # Soluble starch, 0.5 wt. ~ # Bactotrypton, 1.0 wt. # 96 enzyme extract, 0.05 wt. # Calcium chloride, 0.05 wt Culture medium containing wt .- # potassium dihydrogen phosphate was adjusted to pH 7. 50 ml of this medium was poured into a 500 ml conical flask and sterilized at 121 ° C. for minutes. The sterilized medium was inoculated with Bacillus stearothermophilus strain ATCC No. 31 195 (B-501) and then incubated for hours at 60 ° C. with stirring. After culturing, the microbial cells were centrifuged off. The enzymatic activity of the filtrate was 10 CPC-o <-amylase units / ml (determined according to the CPC method described above). Two times the volume of acetone was added to this filtrate in order to precipitate the enzyme, which was then dissolved in a 0.05 molar Tris-HCl buffer solution (pH 8.5). This solution was then passed through a DEAE cellulose column containing

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the same buffer solution has been equilibrated was. The ji-amylase enzyme was not attached to the DEAE cellulose adsorbed, while most of the other proteins, pigments, etc. were adsorbed and thus separated. The partially refined enzyme thus obtained was then concentrated first and then sent through a Sephadex ~ G-150 column. Included the enzyme was weakly adsorbed on the ion exchange resin and in the fraction having a molecular weight of less eluted than 10,000. The purified enzyme thus obtained was then worked up by freeze-drying to give a refined enzyme powder preparation, the relative activity of which is about. Was 200 CPC units / mg protein.

Example 2

Containing a culture medium containing 3% corn starch, 0.5% peptone, 1 # corn steep liquor, 0.05 ° fa calcium chloride, 0.05% manganese chloride, magnesium chloride and 0.05 # 0.05 # of potassium chloride, was adjusted to pH 7 »5 set. 50 ml of this culture medium was placed in a 500 ml conical flask and sterilized at 121 ° C. for 15 minutes. The sterilized medium was inoculated with Bacillus stearothermophilus strain ATCC No. 31 196 (B-781) and stirred at 55 ° C. for k days. After the cultivation, the microbial cells were separated by centrifugation. The i-amylase activity in the filtrate was 1 ^ CPC units / ml. The oC-amylase was precipitated by adding propan-2-ol in an amount corresponding to twice the volume of the filtrate. The precipitate was worked up to a dry powder by freeze drying. The activity of the crude enzyme powder was 3 CPC units / mg. A refined enzyme powder with an activity of 230 units / mg was obtained from this raw enzyme powder by purification as in Example 1.

The five isolated and selected strains, i.e. ATCC No.

31 195, 31 196, 31 197, 31 198 and 31 199 have been modified with respect to the

Influence of temperature and pH on the formation of

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θί-amylase checked. The <X-amylase activity was found in these Tests expressed as the mean value of the respective maximum activity in or for three flasks or parallel tests. The composition of the culture medium for both the preculture and the main culture was as shown in Table I. specified composition (B-M medium). The results of these tests are summarized in Table V below .

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ATCC No. Influence of temperature and 055 pH Tabel pH 6 V 1 (Culture time) 7.0 1 PH 7.5 pH 8 , 0 IS? 31 195 355 on the 1 pH (90) 100 (90) 942 activity 514 (90) 120 (90) 50 pH 5.5 pH 6.0 0 334 (90) 1 1 040 (90) co
CO 55 1 418 (90) 490 (90) 1 30th (90) 31 196 60 1 477 (90) 100 (89) 1 O 65 608 (48) «X-amylase production 368 (41) co
co 45 932 (90) U / ml 681 (41) I _, η. 50 403 (89) , 5 916 (68) 168 (43) 1 051 (43) ! £ 55 24 (65) 975 (20) 262 (20) 191 (68) 60 751 (41) 1 356 (20) 155 (20) 59 (43) 31 197 65 057 (68) 720 1 224 (44) 001 (44) 70 085 (43) 1 155 679 (44) 330 (27) 50 687 (43) 1 166 012 (27) 139 (27) 55 (44) 615 084 (27) 848 (27) 60 1 (44) 815 65 1 (44) 1 (44) (44) (27) (27) (27)

Temp.

Continuation of Table V

Activity U / ml (culture time)

ATCC No. (C) pH 5.5 pH 6.0 pH 6.5 pH 7.0 pH 7.5 pH 8.0

31 198 50 184 (68) 272 (68) 480 (68) 352 (68) 152 (68)

55 768 (44) 752 (68) 832 (44) 880 (44) 955 (44)

60 621 (44) 675 (44) 739 (44) 1 104 (44) 1 123 (44)

65 1 340 (68) 782 (44) 776 (44) 698 (44) 661 (44)

Os 31 199 45 143 (139) 133 (139) 113 (139) 115 (139) 86 (139)

£ 50 686 (139) 663 (139) 543 (139) 493 (139) 491 (139)

Q 55 1 608 (43) 1 365 (43) 1 267 (43) 1 025 (43) 946

⁰⁰ 60 1 485 (43) 1 457 (43) 1 295 (43) 1 154 (43) 1 117

ο »65 0 (65) 0 (65) 0 (65) 0 (65) 0 (65)

From Table V it can be seen that the optimal conditions for the strains ATCC No. 31 195 and 31 196 60 ° C and pH 7.0 and 7.5. ATCC Nos. 31197 and 31198 strains produced significant amounts of .X-amylase when at 65 ° C were cultivated, but the cultivation at this high temperature was not suitable for the production of the other strains good Λ-amylase activity suitable. At a cultivation temperature from 55 to 60 ° C, the optimal pH of the culture medium for ot amylase production was 8.0 ATCC No. 31,198 strain and 5.5 to ATCC No. 31,199 strain.

The following Table VI summarizes the results with regard to the <X-amylase production of the five isolated strains Using repetitively transferred agar slants (each Strain was inoculated and inoculated several times (9-11 times) on two different types of agar slant culture media then cultivated in a liquid nutrient medium in order to find a suitable agar slant medium which contains a - stable OC amylase - production can be maintained) again, while the following Table VII shows the most important data of the experiments reproduces those used to determine the influence of the medium volume on Λ-amylase production (the medium volume in 500 ml flasks was varied in order to assess the influence of the aeration effect on Qi-amylase production by the particular strain examined determine) have been carried out.

709845/0838

lo

Cultures , 1 strength Flask culture conditions
Temp. PH
( ^c ) for screening , 0 70 0.5 ic 7.0 ATCC medium and 10 Activity, E / ral
(Culture time / ~ "h__7) 60 1, 0? Έ agar , 0 0.05 % η Table VI No. number of
Plate change
sel , 10 856 (90) Il Bacto-Tryptone (Difco) 0.5 # pH 0.05 # η 31 195 BD * ¹ 2 910 (43) Il Yeast extract 0.1 # 7.5 BD, 10 i4o (72) 60 CaCl ₂ '2H ₂ 9845/0838 η ίΧ, amylase production using repetitively vaccinated NA * ² , 10 976 (26) η MnCl ₂ '4H ₂ η Oblique agar - 31 196 BD, 1 1 503 (26) It KH ₂ PO ₄ 7.5 BD, 10 1 550 (26) '55 η N / A, 10 1 330 (27) Il η 31 197 BD, 4th 1 127 (26) Il 8.0 BD, 11 1 420 (26) 60 η N / A, 11 736 (44) η Il 31 198 BD, 2 712 (44) Il 5.5 BD, 9 1 117 (44) 55 M. N / A, 9. 1 163 (44) η η 31 199 BD, 1 agar slant medium 1 013 (68) η BD, Soluble 775 (68) 2.0 j, N / A, 5.0

1"

* 2 nutrient slant agar medium

Bacto-Pepton (Difco) 0.5 Bacto-Beef Extra.

(Difco) 0.3

Agar 2.0

PH 7.2

709845/0838

Table VII Influence of medxum volume on * amylase production

: Flask culture

number of

ATCC No. plate change

Conditions

Temp.

(° C) H medium volume activity E / ml ml / 500 ml culture time £ λ / flask

31 195

BD-11

60

7.0

50
60
70

150 127

1 192

790

(65) (65)

(41) (65)

31 196

BD-2

60

7.5 30
40
50
60
70

954 966, 976 1 236 651

(26) (26) (26) (26)

31 197

BD-3

55

7.5 30
40
50
60
70

1 435

1 590

1 510

1,380

840

(41) (41)

(41) (41)

31 198

BD-4

60

8.0 30
40
50
60
70

689

651 736 939 777

(44

(44)

(44)

(44)

(44)

31 199 BD-2 55 5.5

709845/0838 30th 4o 50 60 70

1 131

1 240

1 163

1 054

1 224

(44) (44) (44) (68) (68)

Two strains of the invention (ATCC No. 31 195 and 31 199) and the properties of the OC-amylase produced by them according to the procedure described below with those of a commercially available enzyme (Thermamyl Liquid 6O, Batch AN IOO5) compared.

OC-amylase enzyme was precipitated from the culture filtrates of the strains ATCC No. 31 195 and 31 199 or from the Thermamyl-λ-amylase preparation (diluted twice) by adding 2 volumes of cold acetone. The precipitate was collected by centrifugation and dissolved in 0.025 molar calcium acetate solution. Then enough soluble starch was added to the solution thus obtained that a 20% starch suspension was obtained. This suspension was heated to 85 ° C. for 30 minutes, after which the precipitate was centrifuged off. The solution was then dialyzed against 0.05 molar Tris-HCl buffer solution (pH 8.5) containing 10 mmol of calcium ions, the buffer solution being renewed twice. The dialysate was fed to a DEAE cellulose column which had been equilibrated with the dialysis buffer solution. The ok-amylase was not adsorbed in the column and eluted with the same buffer solution. The fractions exhibiting Ci-amylase activity were collected, whereupon the θί- amylaseenzym was concentrated by precipitation with acetone. The Λ-amylase activity was determined by the CPC method described above. The essential data of the cleaning process and the tests carried out afterwards are summarized in Table VIII below.

709845/0838

Table VIII * "

o < .-Amylase Emission Acetone-
precipitation Heat-
treat-
lung DEAE-
cellulose Cleaning
,. - Procedure
c amylase Culture broth
or raw
Enzyme preparation ATCC No. 31 195 8 640
2 016 8 420
1 450 2,400
15.7 Total activity
(CPC units)
Total protein
(mg) 9 870
7 990 ^, 3 5.8 152.9 Specific activi
ity (unit / mg) 1.2 87.5 85.3 24.3 Yield (#) 100 ATCC No. 31 199 8 210 Z 214 2,310 Total activity
(CPC units) 9 910 1 969 1 206 33 Total protein (mg) 4 914 * .2 6.0 70 Specific activi
strength (units / mg) 2.0 82.8 72.8 23.3 AiB booty (#) 100 Thermamyl 9 400 9 540 2 410 Total activity
(CPC units) 12 100 286 183 24 Total protein (mg) 684 32.9 52.1 100.4 Specific activi
strength (units / mg) 17.7 77.7 78.8 19.9 Yield ($) 100

709845/0838

The acid and heat resistance of the partially cleaned tf amylase preparations from ATCC No. 31 195 and 31 199t strains as well as Thermamyl-O-amylase with an activity of 7 units / ml was determined by incubating for 1 hour under the following conditions:

(a) 90 ° C, pH 6.0, Ca ⁺⁺ 0 or 1 mmol;

(b) 80 ° C, pH 4.55, Ca ⁺⁺ 5 mmol;

(c) 85 ° C, pH 4.55, Ca ⁺ * 1 mmol or 5 mmol, 22.5 # soluble starch.

5 ml of the rt-amylase preparation were dialyzed in a Visking 8/32 cellulose tube for 3 hours at 4 ° C. against 0.05 molar calcium acetate buffer solution (pH 4.5 to 6.0) which contained 0 to 5 mmol calcium acetate, the buffer solution was changed twice. Then h ml of the dialysate were placed in small test tubes and incubated at the specified temperature in a water bath. The test tubes were then rapidly cooled in an ice water bath, whereupon the remaining (X-amylase activity (residual activity) was determined.

** 0.3 ml of a dialyzed <X-amylase enzyme solution, 0.5 ml a 1 molar calcium acetate buffer solution, 0.2 ml of a 0.5 mo- lar calcium chloride solution and 3.0 ml of the above slurry soluble starch were pipetted into a sample tube. The mixture was then stirred for 30 minutes Incubated at 85 C. Then the sample tubes were placed in an ice water bath rapidly cooled and then the residual activity determined.

The residual amylase activities were determined after each incubation period of 5 »10, 20, 30 and 60 minutes.

The optimal pH value and the optimal temperature were determined for the oC-amylases from the strains ATCC No. 31 195 and 31 199, as well as the Thermamyl-OC-amylase according to those described above

709845/0838

CPC experimental conditions were determined, but of course either the pH or the temperature was varied. the Results of these tests are shown graphically in FIGS. The pH optimum was with the two (X-amylases from ATCC 31 195 and 31 199 strains at 4.0 to 5.2 and the commercially available ^ (- amylase (Thermamyl) at 4.5. It was found that the commercially available C (-Amy läse retains high enzymatic activity even in a neutral and alkaline pH range.

The optimum temperature was at the Ot-Amy were reading from the strain ATCC no. 31199 at 75 ° C, in which from the strain ATCC no. 31 at 80 ⁰ C and in the commercial öt-amylase (Termamyl) at 85 C. The relationship between the enzymatic activity and the reaction temperature was very similar for the o (-amylases from the strains ATCC 31 195 and ATCC 31 199, while the course for the commercially available rt-amylase (Thermamyl) differed considerably. It was found that the Ot-amylase activity in the commercial Oi-amylase (Thermamyl) were increased by 20 to 30 i "when the enzyme at 85 C and pH was incubated in the presence of starch and calcium ions 6.0. This fact can be the difference between the inventive Explain Λ-amylase enzymes from the strains ATCC 31 195 and ATCC 31 199 on the one hand and the commercially available Λ-amylase (Thermamyl) on the other hand with regard to the course of the enzymatic activity as a function of the reaction temperature.

FIG. 3 gives the inactivation curves for the Öt-amylases according to the invention from the ATCC strains 31 195 and 31 199 as well for the commercially available <X-amylase (Thermamyl) during incubation at 80 ° C and pH 4.55 in the presence of 5 mmol calcium chloride again. The Cf amylase from strain ATCC 31 199 had the highest thermal stability, followed by the tt amylase off the strain ATCC 31 195 · The commercially available Ä-amylase (Thermamyl) was the two A-amylase enzymes according to the invention from the Inferior to strains ATCC 31 195 and ATCC 31 199 in terms of heat resistance under the conditions mentioned.

709845/0838

Figure k git) * the inactivation curves for the cX-amylase enzymes according to the invention from the strains ATCC 31 195 and 31 199, as well as for a commercially available-amylase (Thermamyl) and another known cC-amylase, namely that of Ogasawara and coworkers in J_z. Biochem. . 67 , 65, 77, 83 (197O) described oil amylase from Bacillus stearothermophilus when incubated at 90 ° C. and pH 6.0 in the absence of calcium ions (the incubation conditions correspond to those of Ogasawara and colleagues, conditions given for this).

As can be seen from Figure h , the Ot-amylase enzymes of the invention had a significantly higher heat resistance than both the commercially available oil amylase (Thermamyl) and the well-known <? L-Amy lase described by Ogasawara and colleagues, which is to be regarded as particularly surprising because the OC-amylases according to the invention as well as the last-mentioned known enzyme originate from Bacillus stearothermophilus.

Figure 5 shows the inactivation of the (X-amylase enzymes of the invention from strains ATCC 31195 and ATCC 31199, as well as, for comparison, the commercial Λ-amylase (Termamyl) among the above for FIG k specified conditions (for example 90 ⁰ C and pH 6.0) again, although, unlike this, the medium contained 1 mmol calcium ions The facts suggest that the Ct-amylases according to the invention from the ATCC 31 195 and 31 199 strains bind calcium ions more tightly than the commercially available flt-Amy lase (Thermamyl), so that their calcium ion requirement to stabilize the protein molecule is lower than that of Thermarayl - OIL amylase.

Figures 6 and 7 give the inactivation curves of the invention CF amylase enzymes from the strains ATCC 31 195 and 31 199 »as well as, for comparison, the commercially available Oi-Amy läse

709845/0838

it

(Thermamyl) when incubated at 85 C and a pH of 4.55 in the presence of soluble starch (22.5 % TS) and calcium ions, namely FIG. 6 with a calcium ion concentration of 1 mmol and FIG. 7 with a calcium ion concentration of 5 mmol. The Ot-Amy according to the invention from the ATCC 31 195 and 31 199 strains had a higher heat resistance than the commercially available ΰί-amylase (Thermamyl) both under the conditions of FIG. 6 and under those of FIG. The conditions in the above experiments, the results of which are shown graphically in FIGS. 6 and 7, are similar to the working conditions used in many large-scale liquefaction processes, so that it is to be expected that the Ci-amylase enzymes according to the invention will be used in the industrial liquefaction and conversion of starch in acidic pH values will have a higher heat resistance than previously known enzymes.

The molecular weight of the df-amylase enzymes of the invention from strains ATCC 31195 and 31199 were each determined by SDS disc electrophoresis method described by Weber and Osborn (J ^ Biöl. Chem., 244, "14O6 (1969)) by the method described 96 000. The labeling proteins used were albumin (molecular weight 67,000), ovalbumin (molecular weight 45,000), chymotrypsin (molecular weight 25,000) and cytochrome C (molecular weight 12,500). The position of the Λ-amylases according to the invention on the polyacrylamide gel was determined by placing the gel on an amylose azure agar plate and incubating it at 37.degree. The results of this test are shown graphically in the figure.

The molecular weight of the cC amylases according to the invention determined in this way, at 96,000, is much higher than that of Ogasawara and colleagues (J. Biochem . 67 , 65, 77, 83 (197O)) and Manning and colleagues. (J. Biol. Chem. 236. 2952, 2958, 2962 (1961)) for C (-Amy reads given molecular weights of 48,000 and 15,600, respectively, derived from Bacillus stearothermophilus.

709845/0838

The five isolated strains or the Ä-Al sen were checked for the presence of protease activity, since the presence of protease or proteolytic enzyme in «χ-amylase enzymes causes the enzymes to do so tend to make water soluble with various protein materials that are present in many starch materials Protein hydrolysates »e.g. amino acids to react. Aue this Reason is the presence of protease enzyme as an impurity in «X-amylase enzymes in terms of economic and efficient hydrolysis of starch materials is disadvantageous. The protease activities of the invention, isolated from the five OC-amylase enzymes obtained from strains were after a modified Anson-Hagiwara method on preparations determined by precipitation with acetone from nitrates of cultures was obtained at the stage of maximum oC amylase production was.

The results obtained in these experiments were compared with those of enzyme preparations from commercially available # -amylase (Thermamyl) and a bacterial Ot-amylase from CPC. As shown below, Thermamyl and the culture filtrates of isolated thermophilic ray fungi contained large amounts of protease, whereas the heat-resistant 01-Amy reads producing strains of the invention do not show any appreciable amounts of protease generated.

709845/0838

Mon

Protease activity

by c (. -Amy läse

enzyme

Protease ^ C-Amylase- relationship

ATCC No. 31 195

ATCC No. 31 196

ATCC No. 31 197

ATCC No. 31 198

ATCC No. 31 199 CPC-BLA Thermamyl 60

0.06 0.17

2.35 0.08 0.06 6.2

36.5 109.0

1)

Thermophilic ray fungi, isolated at 55 ° C and pH 7.0.

As can be seen from the above test results, the tf-amylase enzymes of the invention do not contain any significant ones Protease activity, i.e. having a protease / it-Amylaee ratio of less than 3 and usually even less 1 have. This is a significant advantage when using the enzyme to hydrolyze starch materials.

Example 3

The following example explains the use of the flf-amylase enzymes according to the invention for liquefying starch in the production of starch hydrolyzates with high OE values.

of starch hydrol

709845/0838

«Μ

In 70 ml of water, 30 g of potato starch were suspended, was added followed by 75 mg of calcium chloride dihydrate, and the pH was adjusted to k; 5. The refined, powdery enzyme obtained according to Example 2 was added to this suspension in an amount corresponding to 50 CPC units, whereupon the mixture was allowed to ^{react at 85 °} C. for 30 minutes to liquefy the starch. The DE value of the liquefied solution, which had a pH of 4.3, was about 21. After the temperature of the reaction mixture had dropped to about 60 ° C., a glucamylase enzyme derived from Aspergillus niger was added and the solution was kept at 60 for 48 hours ⁰ C saccharified and converted. The DE value of the saccharified and converted solution was 97-5 and its dextrose content was $ 96.0.

Example 3 above shows that the Of-amylase enzymes according to the invention hydrolyze and liquefy starch. They can be used to convert soluble starch, amylose, amylopectin, glycogen, etc. to abruptly reduce the viscosity of these substrates. When allowed to react, the enzymes of the invention at pH 4.5 and 6O ⁰ C and the soluble starch, the starch-iodine reaction disappears at a degree of hydrolysis (DE) of about 15, after which the hydrolysis, however, to a highest degree of hydrolysis or DE value continues from 32 to 36 ... The sugars contained in the hydrolyzed product were analytically identified as maltose, maltotriose, maltotetraose and other maltooligosaocharides mixed with a small amount of glucose. The mutarotation of the reducing sugar product has been found to be negative. Accordingly, the enzymes of the invention are α-amylase enzymes of the liquefying type which smoothly hydrolyze the α-1,4 bonds in starch.

The relationship between the liquefaction and the use of an enzyme according to the invention (relative value) and the working pH is shown graphically in FIG. 9, the results obtained with the known λ-amylase enzymes derived from Bacillus stearothermophilus being shown for comparison. As can be seen from Figure 9, the optimal

709845/0831

Male pH for the enzymes of the invention in a range of 4.2 to 5.2. The preferred enzymes according to the invention do not lose their activity even if they are left to stand at room temperature for Zh hours at pH values in the range from 3 to.

The relationship between the liquefaction by the enzymes according to the invention (relative value) and the working temperature is shown graphically in FIG. 10, the corresponding curve for the iC-amylase from Bacillus stearothermophilus described by Ogasawara and Mitarb being shown for comparison.As from FIG. 10 can be seen, the preferred working temperature for the enzymes according to the invention is about 80 ^° C.

From the above it can be seen that the invention Oi-amylase enzymes for liquefying and converting starch in the starch saccharification industry, in the desizing process in the textile industry and as additives in detergent mixtures, or in the usual areas of application bacterial OC amylase enzymes can be used.

The ö (-amyl as e enzymes according to the invention are particularly suitable for the liquefaction and conversion of starch in the production of mal todextrins, in particular with the subsequent production of dextrose using glucamylase, since the isomerization of the end groups of the molecule can be avoided because / that enzyme efficiently in an acidic pH range, ie at a pH of 4.5 his 5 | 0 can, use, thereby increasing Dextroseausbeute the use of these new enzymes also reduces the Ionenaustauscherbelastung the raffinate ions moved because before the saccharification and. Conversion with glucamylase does not require any pH adjustment.

A preferred application for the inventive oC-amylase enzymes is their use in processes for con—

709845/0838

- ι * -Vi

verting of strength, in which the remaining, non-converting tated starch is retained in granular form. Such methods are described in U.S. Patents 3,922,196, 3,922,197, 3 922 198, 3 922 199, 3 922 200 and 3 922 201 and which is additionally referred to here. In these methods for converting starch, in which the If unconverted starch remains in granular form, the solubilization of the starch material is at least in the The initial phase is carried out at relatively low temperatures, i.e. below the temperature at which the starch begins to gelatinize up to the actual initial gelatinization temperature of the starch. According to a preferred Embodiment of this method is carried out simultaneously with the ck amylase used in the initial stage of solubilization glucamylase enzyme. The enzymes according to the invention are particularly suitable for this process because of their optimal pH range is compatible with glucamylase.

A preferred ajidere possible use for the invention of invention 0 ^ amylase enzymes is the use thereof, which is incorporated in addition reference is made in the method of the U.S. Patents 3,853,706 and 3 8 9 19 ^ ² * known type.

According to a further preferred embodiment of the invention the tf-amylase enzymes according to the invention can be with a Use methods of the type known from US Pat. No. 3,912,590, to which reference is also made here as a supplement. In the Use of the <X -amylase enzymes according to the invention after this known method is a slurry of starch, e.g. Corn starch containing at least 25 wt .- # starch material with the Ct amylase enzyme at a temperature of about 100 to 115 and preferably about 105 to 110 C for about 1 to 60 and preferably 5 to 10 minutes in order to increase the strength liquefy, whereupon the temperature is reduced to 80 to 100 and preferably 90 to 100 ° C when the viscosity of the diluted solution measured at 95 ° C less than 300 amounts to. The special advantage that one achieves in that

jre advantage den

709845/0838

in a process in which the temperature profile mentioned above is used, the process according to the invention ct-amylase enzymes used can be seen in that a lower pH can be used so that no or in a subsequent saccharification process with glucamylase at most a slight adjustment of the pH value is necessary.

Very particularly advantageous and therefore preferred to use the inventive PC-amylase enzymes in a process for converting starch in which a Stärkeauf- slurry to the action of the enzyme at a pH in the range of about 3.5 to 6.5 and preferably about h to 5 »is exposed to a temperature in the range of about 50 to 100 and preferably about 60 to 95 C in order to liquefy the starch. In the case of the above-described processes for the hydrolysis of granular starch, the temperature will be in the range between the normal gelatinization start temperature and the actual gelatinization temperature of the starch, that is to say about 60 ° C. in the case of corn starch. In the case of the method for direct liquefaction, the starch slurry containing the enzyme is preferably heated to a temperature in the range from about 85 to 95 and in particular about 90 to 92 ° C., for example by means of a nozzle heater, in order to liquefy the starch. After the initial solubilization, as in the hydrolysis of granular starch, or liquefaction, the slurry is preferably subjected to a "heat shock treatment" at a temperature above 100 and preferably at a temperature in the range of about 110 to 150 C to liquefy any starch granules that may still be present . The liquefied starch slurry is then cooled and preferably treated with further rf-amylase alone or in combination with other enzymes, such as glucamylase, β-amylase, pullulanase, glucose isomerase, one after the other or in combination. When Of-Amylase is used alone, the temperature in the second enzyme treatment step becomes preferred

709845/0838

in a range from about 80 to 90 and especially at about 85 C, the optimal temperature for the invention Enzymes, kept. If there are other enzymes, like glucamylase and / or glucose isomerase are present, the temperature will be somewhat lower, i.e. about 55 to 75 and especially about 60 C.

The Oi-amylase enzymes according to the invention can be clearly distinguished from the - ~ X-amylases obtained from animals, plants, yeasts, fungi imperfecti and molds according to the prior art in that these known enzymes have such a low heat resistance that they lose their activity completely after a 5-minute treatment at 70 C and pH 6. If one compares the heat resistance of the ot-amylases according to the invention, which is shown in FIG. 12 (the working conditions in the experiments, the results of which are shown in FIG ), it can be clearly seen that the ot-amylase enzymes according to the invention are clearly superior to the compared <X -amylase enzymes according to the prior art in terms of acid and heat resistance. It can also be clearly seen from FIG. 12 that the X-Amyl as enzymes according to the invention are distinguished by the fact that they are able to maintain at least about 70 and preferably at least about 90% of their initial activity when they are used in the absence of added calcium ions 10 minutes, kept at 90 C and a pH of 6.0, and at least about 50 $> zubehalten its initial Λ-Amyläseaktivität, when held for 60 minutes at 90 ° C and a pH of 6.0. As can be seen from FIG. 11, the <* - amylase enzymes according to the invention are further distinguished by the fact that they are able to retain at least about 50 io of their initial Ot-amylase activity when they are used in the presence of 5 mmol of calcium ions for 10 minutes Maintains temperature of 8O ⁰ C and a pH of 4.55.

709845/0838

-H-

Table IX gives a comparative overview of the essential performance characteristics of the 4-amylase enzymes according to the invention and the α-amylase enzymes currently used on a large scale from Bacillus subtilis and Bacillus 1i c heni formis on the one hand and the iX-amylase enzymes described in the literature and derived from Bacillus stearothermophilus on the other again. In particular, the enzymes mentioned are compared in Table IX with regard to the optimum working conditions for them in terms of pH and temperature and with regard to their respective molecular weights. Figures 11 and 12 show a comparative comparison of the properties of these enzymes with regard to heat and acid resistance in graphical representation.

70984 5/0838

Tested enzymes

Table IX

Optimal working pH

Suitable molecular working temp. weight

C ainylase enzymes der
invention

01 — Amylaso from
B. subtilis ¹ '

Ot-amylase from
B.licheniforrais

2)

h _% O - 5.2

^, 5 - 6.5 5.0 - 9.0

«X-Araylase from
B. stearothermophilus

a) Ogasawara and Mit. '5.0 - 6.0

b) Campbell and Mit.

3)

4.8

80 ⁰ C

- 60 ° C

76-78 ° C

65 - 70 ° C
55 - 70 ° C

96,000 hs ooo

22 500

k8 000 15 600

Ogasavara and Mit., J ^ Biochem. , 67 > 65 (197O).

² 'Shigemasa Saito, FIG. 155 , 290 (1973).

^ 'Campbell and Mit., J ^ Biol. Chem. , 236 , 2958 (1961). ^ Campbell and Mit., J ^ Biol ·. Chem. _T 236 . 2958 (1961).

In the British patent, the molecular weight of the X-amylase of B ^ licheniformis is 18,000-20,000 and that of the CK. Amylase from B ^ subtilia given as 96,000 .

If the OC-amylase enzymes according to the invention are compared with the known ct-amylase enzymes from Bacillus subtilis . it is noticeable that, as can be seen from FIGS. 9 and 11, these differ considerably with regard to their optimal operating pH, the suitable operating temperature, the molecular weight and their heat and acid resistance. This shows that the enzymes of the invention are fundamentally different from the known C (amylase from Bacillus subtilis.

709845/0838

- 4f -

If one compares the oC-amylase enzymes of the invention with the o (-Ainyl as e enzymes from Bacillus licheniformis , it can also be found here that the enzymes differ considerably in terms of their optimal working pH value, their molecular weight and their heat and acid resistance as can be seen from Table IX and Figures 11 and 12.

It will be understood by those skilled in the art that the strains used to generate the new (K-Arnyl aseenzyme of the invention can be subjected to the action of known antigenic agents, such as UV light, a chemical treatment and the like according to known methods the invention from the strains ATCC No. 31 195, 31 196, 31 197t, 31 19 ^{8 and} 31 199t as well as their variants and mutants and / or '3-amylase enzymes obtained from submutants of the said variants and mutants or produced by the microorganisms in question.

Like some of the well-known Ö (amylase enzymes, the enzymes of the invention inhibited by mercury and ethylenediaminetetraacetic acid, but stabilized by calcium.

It goes without saying for the person skilled in the art that the invention explained by the above examples can be modified in various ways within the scope of the inventive concept. Numerous variations and modifications of the invention are readily apparent to the person skilled in the art and can be carried out or used by him without departing from the inventive concept described and disclosed above.

As already mentioned, can be the new CC Amyläse enzyme of the invention in an advantageous manner in the hydrolyzing and / or liquefaction of starch used and, because of their stability at low pH values in combination with other acid-resistant amylases such as are used glucoamylase, both in soluble and in immobilized form. 709845/0838

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Claims (1)

Claims _, "" "" Heat and acid-resistant Ot-amyl as e enzyme, characterized in that it is able 1. at least about 70% of its initial ° t-amylase activity to keep if you do it in the absence of added calcium ions for 10 minutes at 90 C and one maintains pH of 6.0, 2. at least about 50 # of its initial oC amylase activity if you keep it in the absence of added calcium ions for 60 minutes at 90 C and maintains a pH of 6.0, 3. at least about 50 $ of its initial Λ-amylase activity if you keep it in the presence of 5 mmol calcium ions for 10 minutes at 80 C and a pH of ^ .55 holds. 2. ot-amylase enzyme according to claim 1, characterized in that that it has a molecular weight as determined by SDS disk electrophoresis of at least about 90,000. 3 · Λ-amylase enzyme according to claim 1 or 2, characterized in that it originates from a microorganism of the genus Bacillus. k. Λ-amylase enzyme according to claim 3 »characterized in that it comes from a microorganism of the species Bacillus stearothermophilus . 5. Λ-amylase enzyme in particular according to claim k _f, characterized in that it is derived from a microorganism of the species Bacillus stearothermophilus ATCC No. 31 195 »31 196, 31 197, 31 198 and / or 31 199 including the variants 709845/0838 ORIGINAL INSPECTED SUBSCRIBED Mutants and submutants of these strains originated. 6. «cC-amylase enzyme according to claim 5, characterized in that it is derived from Bacillus stearothermophilus strain ATCC No. 31 or a mutant of this strain. 7. <&> -amylase enzyme according to any one of claims 1 to 6, characterized in that it is capable of at least about 50% of its initial activity at a temperature of 85 C and a pH of 4.55 in the presence of 5 mmol To retain calcium ions and 22.5 percent by weight starch, dry matter, for a period of 30 minutes. 8. A process for the preparation of a heat- and acid-resistant o £ - amylase enzyme, in particular according to one of claims to 7, characterized in that a microorganism of the species Bacillus stearothermophilus strain ATCC No. 31 195, 31 196, 31 197, 31 198 and / or 31 199, including the variants, mutants and submutants of these strains, are cultivated in a nutrient medium and the enzyme produced in this way is recovered. 9. The method according to claim 8, characterized in that a nutrient medium is used which has an assimilable carbon and nitrogen source. 10. The method according to claim 8 or 9, characterized in that the microorganism is 1 to 5 day (s) at a pH cultivated in the range of 5 to 9 and at a temperature of about 50 to 70 ° C. 11. Use of an oC amylase enzyme according to one of the claims 1 to 7 and in particular an oC-amylase enzyme prepared by the process according to one of claims 8 to 10 to convert starch into a starch hydrolyzate 709845/0838 a) Treating an aqueous starch slurry with the C (amylase enzyme at a pH of 3-5 to 6.5 and preferably at a temperature of about 50 to 100 ° C. and a pH of about 5 to 5 hours to liquefy and convert the starch b) recovering a starch hydrolyzate from the conversion step whereby or after which the starch hydrolyzate is preferably treated with a glueamylase enzyme, heated to over 100 ° C and then cooled and treated with additional ct-amylase
and or
is treated with a glucose isoraerase enzyme. 709845/083 «