SI7710998A8 - Process for obtaining the heat and acid resistant alpha-amylase enzyme - Google Patents

Description

Technical field

The invention relates to the field of enzyme production, in particular the alpha-amylase enzyme resistant to heat and acid, which can be used for the hydrolysis, cutting and / or conversion of starch-containing material into starch hydrolysates.

According to the International Patent Classification, the invention is classified in C 07G 7/02; A 61K 37/48.

Tehnlčkl problem

Alpha-amylase enzyme preparations are used for the hydrolysis, acquisition and / or conversion of various starch-containing materials into starch hydrolysates and are also used in detergent formulations. When the alpha-amylase enzyme is used to treat starch-containing materials, they are used as an initial stage in the production of a number of synthetic starch hydrolyzate-based materials such as malt-dextrins, corn syrups, dextrose, levulose, maltose, etc.

The alpha-amylase enzyme hydrolyzes starch molecules by breaking them into various fragments of molecular weight intermediates known as malt-dextrins. Malto-dextrins are subsequently treated with one or more supplementary enzyme preparations including gluco-amylase, beta-amylase and glucose isomerase in order to produce the desired one! the final product. Alternatively, a number of these enzyme preparations may be introduced into the suspension of the starch material so that the desired starch hydrolyzate is directly produced. Alpha-amylase enzymes are available from a variety of sources. Many alpha-amylase enzymes are produced from bacterial sources such as Caillus subtillis, Bacillus licheniformis, Bacillus stearothermpohilus, and others that are cultured in suitable culture media, and the cells that are produced are then destroyed and the enzyme pepper is separated from the broth. and purifying.

Many commercially available alpha-amylase enzymes that are produced today are derived from Bacillus subtillis microorganisms. When these enzymes are used to convert starch to hydrolyzate of starch, they will typically have an optimum temperature interval of about 80 ° C to about 85 ° C, and an optimum pH of about 6.0. The temperature conditions and the optimum pH interval for efficient use of the enzyme have two disadvantages. First, the starch is translated with the enzyme to a pH of about 6 and a temperature of about 80 ° C to about 85 ° C, and under these conditions some of the reducing end groups of the starch are isomerized, so that in the subsequent conversion process, maltulose is produced which reduces the degree of isolation of the desired product for example, dextrose, levulose, or maltose. Second, the optimum pH value of glucoamlase used for conversion in the saccharification process is approximately 4.5 in the case of Aspergillus nlger enzyme and pH is about 5.0 in the case of Rhizopus type enzymes. Therefore, after completion of the acquisition phase using alpha-amylase enzyme, it was found necessary to adjust the pH from about 6 to 4.5 or 5.0. This pH adjustment increases the ion concentration and therefore increases the loading and subsequent refining costs by using the ion-exchange resins used in the purification of the final product.

Given the above issues, it was necessary to produce an alpha-amylase enzyme that would be resistant to heat and acids.

The invention solves the problem of producing alpha-amylase enzyme resistant to heat and acids by culturing a microorganism of a strain selected from the group consisting in particular of Bacillus stearothetmophilus ATCC numbers: 31, 195, 31, 196, 31, 197, 31, 198 and 31, 199 , as well as their variants, mutants and sub-mutants of these mutants by culturing in a clutting medium containing carbonation sources and nitrogen and including corn starch, corn yeast, farmamedia, and inorganic salts such as calcium chloride, magnesium sulfate, manganese chloride and sodium chloride, at pH 5 to 9 over a period of one to five days.

The state of the art

Many different heat-stable afla-amylase enzymes have been found in recent years. Examples of such heat stable alphaamylase enzymes include those produced 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 recently, alpha-amylase enzymes have become available that have good thermal stability in neutral and weakly alkaline solutions. These alpha-amylase enzymes, which are heat and alkaline stable, are marketed under the name Thermamyl. ” They are produced by the cultivation of Bacillus licheniformis microorganisms, as described in British Patent Application no. 1,296, 839, published November 22, 1972, Madesen et al., Die Starke, 25, 304, 305, and 308 (1973) and Narimasa Saito, ABB, 155, 290 (1973).

While the alpha-amylase enzymes produced by Bacillus licheniformis have relatively good sealing stability in neutral and slightly alkaline solutions, they do not have the appropriate stability under acidic conditions to make their use economically commercially viable.

Description of a solution to a technical problem with Exemplary examples

As already mentioned, the invention relates to a process for the preparation of an alpha-amylase enzyme resistant to heat and acids, or an alpha-amylase enzyme in which the disadvantages of the currently available enzymes have been eliminated.

In particular, the alpha-amylase enzymes produced by the process of the present invention are characterized by (1) being able to retain at least 70% of their initial alpha-amylase activity when kept at 90 ° C and at a pH of about 10 minutes in the absence of additional calcium ion; (2) capable of retaining at least about 50% of their initial alpha-amylase activity when kept at 90 ° C. at pH 6.0 for 60 minutes in the absence of added calcium ion and (3) capable of holding at least about 50% of their initial alpha-amylase activity at 80 ° C and at pH 4.55 in the presence of 5 mM calcium ion for 10 minutes. The enzymes of the invention are also capable of retaining at least about 50% of their initial activity at a temperature of 85 ° C and a pH of 4.55 in the presence of 5 mM calcium ion and 22.5 wt. percentage of starch, d.s. The enzymes of the invention are further characterized by being able to retain at least about 95% of their initial alpha-amylase activity at a temperature of 80 ° C. and to a pH in the range of about 4 to about 6.0. These enzymes are preferably derived from the Baclllus microorganism and more preferably from the Baclllus stearothermophilus microorganism.

The alpha-amylase enzymes of the invention have a molecular weight of at least 90,000 as determined by SDS disk electrophoresis and are characterized by being virtually free of protease activity, e.g., they generally have a protease / alpha-amylase ratio less! of 3, and preferably less than 1.

The new alpha-amylase enzymes of the invention are produced by culturing in a suitable medium the microorganism Bacillus stearothermophilus, preferably by cultivating a strain selected from the group comprising Bicillus stearothermpohilus ATCC No. 31,195 31,196, 31,197, 31,198, 31,199, their variants, mutants and sub-mutants, and subsequently isolating the alphaamylase enzyme produced.

FIG. 1 illustrates the ratio of the optimum pH of the two enzymes of the invention to Thermamyl alpha-amylase.

Si. 2 illustrates the optimal temperature ratio of the two enzymes of the invention with Thermamy1 alpha-amylase.

FIG. 3 illustrates the thermostability ratio of the two enzymes of the invention with Thermamyl alpha-amylase at 80 ° C, pH 4.55 with 5 mM Ca ++.

FIG. 4 illustrates the thermostability ratio of the two enzymes of the invention with Thermymyl alpha-amylase at 90 ° C and at pH 6.0.

FIG. 5 compares the thermostability of the two enzymes of the invention with Thermamyl alpha-amylase at 90 ° C, at pH 6.0 and in the presence of 1mM Ca ++.

FIG. 6 illustrates the thermostability ratio of the two enzymes of the present invention with Thermamyl at 85 ° C and at pH 4.55 in the presence of 22.5% starch, d.s.

FIG. 7 illustrates the thermostability ratio of the two enzymes of the present invention with Thermamy1 alpha-amylase at 85 ° C, at pH 4.55 in the presence of 22.5% starch and 5 mM cA + +.

FIG. 8 illustrates graphically the molecular weight determination of an enzyme of the invention by means of SDS disk eletrophoresis.

FIG. 9. illustrates the relationship between the pH and the enzymatic activity of one of the enzymes of the invention with previous alpha-amylases derived from Bacillus stearothermophilus microorganisms.

FIG. 10 illustrates the relationship between the temperature of the enzyme of the present invention with alpha-amylase from the foreground derived from Bacillus stearothermophilus.

FIG. 11 illustrates deactivation curve enzymes of the present invention with alpha-amylase From prior art derived from Bacillus subtilis I with alpha-amylase derived from Bacillus licheniformis when treated at 80 ° C and at pH 4.5 in the presence of 5

MM of calcium ion.

FIG. 12 illustrates the decivation curves of enzymes of the present invention with alpha-amylases from prior art derived from Bacillus licheniformis and Bacillus stearothermophilus when treated at 90 ° C. and at pH 6.o without calcium ion.

The production and thermal stability of alpha-amylase from various types of microorganisms have been tested under various conditions such as growth temperature (37 ° C, 45 ° C, 55 ° C and 70 ° C), pH (5.0 and 7.0) and substrates. Thermophilic airborne fungi isolated at 55 ° C and thermophilic fungi isolated at 45 ° C produce thermolabile alpha-amylase in high yields, whereas thermophilic bacteria at 70 ° C produce thermostable alpha-amylase in low yields. Based on these initial studies, microbial testing for thermostable and acid-resistant alpha-amylase was performed by culturing at 55 ° C and 70 ° C. A total of 550 samples of microbial sources were collected from various locations including soil, hot streams, rubble, etc. Thermostability and acid resistance were determined by testing the alpha-amylase activity before j after heat treatment of the Clutrate filtrate at 80 ° C and at pH 5.0 for 10 minutes in the presence of 10 mM calcium ion.

The alpha-amylase activity of the determination is as follows:

Make a 0.2% solution of starch once a week as follows: 200 mg of starch solution in approximately 50 ml. 0.2M acetate buffer (pH 4.5) containing 0.013M CaCl ₂ .2H ₂ O was heated to 100 ° C in boiling water and the resulting solution was made up to 100 ml with the same buffer. Test tube containing 0.1 ml. of enzymatic solution and 0.3 ml of 0.2% solution of starch solution are incubated for 5 minutes at 60 ° C. The reaction was stopped by adding 1.0 ml. 0.5N acetic acid. As 3.0 ml is added. 0.015% of the iodine solution is mixed, the apparent optical density at 700 m versus H ₂ O. The enzyme-free tube serves as a control and its optical density at 700 m is denoted OD ^. One unit of enzyme was the amount that catalyzed a 10% reduction in true value per minute under the conditions described above.

NMLunit = (OD ^ - OD) x 100 / OD ^ x 5x 10

Unless stated otherwise, said alpha-amylase activity was determined by the above procedure (NMLs). When alpha-amylase activity is indicated in CPC alpha-amylase units, CPC units are approximately 1/140 part of NML units. The CPCs of alpha-amlase activity boats are determined by the following procedure:

One milliliter of properly diluted enzyme solution was added to 10 ml of 1% starch solution - 0.03 M acetic acid buffer solution (pH 6.0) and the reaction was carried out for 10 minutes at 60 ° C. One milliliter of the reaction solution is placed in a 100 ml graduated balloon. containing 50 ml of 0.02N hydrochloric acid, and since 3 milliliters of 0.05% iodine solution is added to this, the total volume is made up to 100 ml. by adding water. The evolving blue color measures absorbance at 620 mu. Amount of enzyme required to decompose 10 mg. starch in one minute is defined as - CPC unit.

CPCJedlnlca = θ ₀ 'Οθ / θ ₀ x50 / 10x10x (dilution factor) where jo

D _q = apeorbance of the control solution (water is added instead of the anime solution)

Dg = apeorbance of the reaction solution

The conditioning conditions and the substrates used in the testing study are summarized in Table 1.

The results of the first and second tests are listed in Table 2.

Tablloa 1

F

Ualovl kultlvlnanja 1 pofllorco

Unlovl Culture on the plateA ² ' Culture / a ¹ ) in a balloon '' Culture on the tile ^ ¹ ^ and?) * · - 55 ° C pH 5 55 ° O, ρΠ 5 50 ° C, pil 6 Λ) 55% pH 7 55 ° C, drunk? 50 ° 0, pH 7 c) 70 ° C, pH 5 70% pH 5 60 ° C, pil 6 d) 7Q ° q, pH 7 70 ° G, pH 7 60 ° 0, pil 7

(2) Lining it (3) Pocllop; »zn (1) Lining it

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About 25% of thermophilic airborne fungi isolated at 55 ° C showed 100-2,500 units of alpha-amlase production per milliliter of culture broth, but did not retard activity after heat treatment (80 ° C, 10 minutes, pH 5.0).

A total of 35 strains of 1,061 thermophilic bacterial strains isolated at 55 ° C showed 100-1,000 alpha-amylase production units, and 10 of these strains produced thermostable alpha * amylase. Almost all alpha-amllases From thermophilic bacteria Isolovan at 70 ° C showed thermostability and acid stability; and the highest producers were obtained from pH 5.0 isolates.

The relationship between the activity and size of the clear zone in at least 219 Isolated strains at 70 ° C and at pH 5.0 was examined. The results of this study are shown in Table 3, where it can be seen that 90% of these strains yielded a clear zone less than 3 cm in diameter and produced 0-200 alpha-amylase units. The highest production was obtained from strains that gave a clear zone with a diameter greater than 3 cm, but only 10% of the tested strains behaved like this.

Table 3.

Relationship between activity and size of clear zone Isolated strain

Size of biotroone on aaaol board (cm in sandbox)

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219

On the basis of the above tests, isolation of bacterial colonies having a clear zone greater than 3 cm in diameter, on aa substrate plate used in the assays, has been found to be a great method for testing microorganisms capable of producing high yielding alpha-amylase enzymes resistant to acids and temperature.

Based on this testing, five strains were selected to be the highest producers of alpha-amylase enzymes that are thermostable and acid resistant.

Each of these five strains in purified form, as described below, is deposited permanently in the American Type Culture Collecetion (ATCC) Collection, 12301 Praklawn Drive, Rockville, Maryland 20852. ATCC maintains these strains under contract between ATCC and CPC International Inc. ., the owner of this patent application.

The agreement between ATCC and CPC International Inc., ensures the permanent accessibility of cultures or subcultures to all users without restriction after (1) the U.S. release. a patent describing and identifying the deposits in question and describing the ATCC numbers assigned to them; ill (2) after publication or public disclosure of any U.S. ill A foreign patent application that describes and identifies the deposits in question and describes the ATCC number assigned to them. CPC International Inc. has agreed that, if any of these deposited cultures die or be destroyed, during the effective validity of the patent, it shall replace the live cultures of the same organism. Further, CPC International Inc. has authorized ATCC to make available to the United States Patent and Trademark Office and the West German Patent Office free of charge access to cultures or sub-cultures at any time at the request of the authorized persons of those offices.

Deposited organisms

NML Soy 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

Further, the NML strains B-501 and B-781 were deposited with the Fermentation Research Institue, Industrial Technology Agency, MITI, as FRI No. 3389 and 3390.

(1) Morphological characteristics:

A. Cell size and shape: 0.6 x 2 - 3u; single stick rarely in strings (all strains)

B. Pleomorphism: Negative (all strains)

C. Mobility: mobile and have flagella (all strains)

D. Spora; 0.6x1.0 - 1.5 in, oval shaped; rocket spore housing (all strains!)

E Gram Color: Positive (All Shows)

F. Acid Consumption: Negative (All Soybeans!) (2) Growth on Various Substrates:

A Fruit with nutrient agar: Active colonies spreading with a rough surface and a rough edge (all strains)

B. Nutrient agar plate hair: Good growth, white-opal, active propagation, outer comb-shaped growth (all strains)

C. Nutritious culture broth for weeks: Translucent eclipse, white matte surface (eve strains)

D. Nutrient Gelatin Culture Broth: Uquakefaction (All Strains)

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

F. Co-nutrient culture fluid: Inhibition of growth in 2% salt (all strains)

G Milk agar plate: Trimming the clear zone by hydrolysis of casein (all strains)

H. Glucose agar hair plate: Good growth, similar colonies to those on nutrient agar (all strains)

I. Proteosis peptone agar tile hair: 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. citric acid consumption: Positive (all strains)

E. Hydrogen sulfide cropping: Positive (all strains)

F. Starch hydrolysis: Strong hydrolysis (all strains)

G. Acid and gas trimming: Positive for acid but gas not trimmed Glucose, xylose, arablose, mannitol (all strains)

H. Temperature and pH for growth:

ATCC No. 51,195 ATCC No. 31,197 (n-501) Ο '/ οι) 37 ^? C no raat slight rnct 41 ° C unknown raat uinoron rnab 50 -70 ° C good rcat get 'mrit pil interval zn m s l; The score is now 5-0 - 0 OptlHolan pil G - 7 G - 7

The above tastes were performed according to Labaratory Methods In Microbiology by W.F. Harrigan et al. and the Manual of Microbiological Methods published by the American Bacteriological Assoclation.

Based on the previous characteristics, five (5) selected strains were identified as Badllus stearothermophilus according to Bergey's Manual of Determinatl Bacteriology, 8th Edition.

These five strains were further purified using a plate lubrication process. The isolation, cultivation and purification conditions of the five selected strains are summarized in Table 4. As can be seen from Table 4, ATCC No. purified strain. 31,199 (B-968) was the most! of the five strains selected, 2,111 NML units of alpha-amylase activity per milliliter of culture broth (about 15 CPC units) were produced.

Testing_Other ~ Testing_Purification

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In the manufacture of the alpha-amylase enzymes of the present invention, a strain of a microorganism capable of producing an acid and heat-stable alpha-amylase enzyme such as that which satisfies the tastes in Table 3 (e.g., Bacillus stearothermophilus strains ATCC No. 31,195. 31,196, 31,197, 31,198 or 31,199) are cultured in a nutrient medium known to cultivate thermophilic bacteria. Such culture media should contain a source of assimilating carbon and nitrogen along with other essential nutrients.

Suitable sources of assimilating carbon include carbohydrates such as starches, hydrolysed starches, corn meal, wheat flour, etc. The concentration of carbohydrates to be used in the substrate may vary over wide granules, e.g., it can vary from about 1% w / v to about 25% w / v, and is best varied from about 10% w / v to about 20% w / v, where percentages are calculated as dextrose. A preferred assimilating carbohydrate is starch or partially hydrolysed starch (and when present on a weight basis they are present in an amount ranging from 1 to 5% by weight).

The source of nitrogen in the nutrient medium may be inorganic and / or organic in nature. Suitable inorganic nitrogen sources include ammonium salts and inorganic nitrates, etc. Suitable organic nitrogen sources include peptone, meat extract, enzyme extract, casein, corn liquid, malt extract, soy flour, skim milk, etc.

Further, the nutrient medium should contain common trace substances such as inorganic salts including calcium chloride, magnesium sulfate, phosphates, sodium chloride, potassium chloride, etc.

These sources of carbon, nitrogen and inorganic salts may be used individually or in suitable combinations. Further, a small amount of metal salts, vitamins, amino-xelines, etc. can be used. to promote bacterial growth and productivity.

The cultivation conditions used to produce the alpha-amylase enzymes of the present invention are the same as those normally used for the cultivation of thermophilic bacteria. It is best cultivated immersed in a liquid culture medium under stirring and aeration for 2 to 5 days at 50 ° C to 70 ° C at pH 5 to 9. The enzyme accumulates in the culture medium.

After production of the alpha-amylase enzyme According to the present invention, microbial cells are removed by conventional means such as centrifugation. The filtrate is then preferably subjected to salinization by the addition of inorganic salts such as ammonium sulfate, sodium sulfate or magnesium sulfate and / or the use of water-miscible organic solvents such as acetone, ethanol, 2-propanol, etc. to precipitate the enzyme so that it can be concentrated. It is also possible to isolate the alphaamylase by adding starch to the filtrate so that the alphaamylase will be adsorbed to the starch.

A preferred enzyme purification agent from the enzyme-containing filtrate includes the steps of treating the filtrate with cold acetone in double volume from the filtrate to precipitate the enzyme. The sediment enzyme was then dissolved in 0.05 M tris-hydrochloric acid in buffer solution (pH 8.5) and then passed through a DEAEcellulose column equilibrated with the same buffer solution, At pH 8.5, non-alpha-amylase enzymes, proteins, pigments, etc. are adsorbed on DEAE cellulose while most of the alpha-amylase remains in solution. The filtrate containing the alpha-amylase enzyme is called a 'partially refined enzyme' here. The partially refined enzyme can be further purified by dialysis versus 0.01 tris-hydrochloric acid buffer solution (pH 7.0) and then passed through a hydroxyapatite column equilibrated with the same buffer solution. At this stage, the enzyme is sorbed onto the column. The sorbed enzyme is eluted by linearly increasing the concentration of ammonium sulfate from 0 to 0.5 molar.

The partially refined enzyme thus obtained is passed through a Sephadex G-150 column after concentration. The enzyme is poorly adsorbed on Sephadex and elutes at a molecular weight fraction below 10,000. The activity of the refined enzyme obtained in this way was increased by 100 times that of the original filtrate, but bands of proteins other than the alpha-amylase enzyme were also observed in disc electrophoresis - and crystallization of the enzyme had not yet been achieved.

The following examples serve to describe more fully how to make and use the invention described above, as well as to illustrate the best ways to implement various aspects of the invention. It should be clear that these examples do not limit the true scope of the invention but are given merely to illustrate it. All relationships are in parts by weight, unless otherwise indicated.

Example 1

The culture medium containing 3.0% (wt) soluble starch, 0.5% bactotryptone, 1.0% enzyme extract, 0.05% calcium chloride, 0.05% magnesium sulfate and 0.1% potassium biophosphate was adjusted to pH 7.0. An aliquot of 50 ml. of this medium was poured into a 500 ml conical flask and sterilized for 15 minutes at 121 ° C. The sterilized medium is inoculated with ATCC No. 31,195 (B-501) by Bacillus stearothermophilus strain and boiled under stirring for 4 days at 60 ° C. After cultivation, the microbial cells are removed by centrifugation. The enzyme activity of the filtrate per One milliliter was 10 CPC alpha-amylase Units (determined by the CPC procedure described above). Two volumes of acetone were added to this filtrate to settle the enzyme which was subsequently dissolved in 0.05 molar Trls-HCl buffer (pK 8.5). The solution is then passed through a DEAE-cellulose column which is equilibrated with the same buffer solution. The alpha-amylase enzyme is not adsorbed to DEAE cellulose while many other proteins, pigments, etc., are adsorbed to DEAE cellulose and thus separated. The partially refined enzyme thus obtained is first concentrated and then passed through a Sephadex G-150 column. The enzyme is poorly sorbed on Sephadex and elutes into a molecular weight fraction below 10,000. The refined enzyme thus obtained is converted into a powdered, refined enzyme by freezing and its relative activity was about 200 CPC units / mg of protein.

Example 2

Culture media containing 3% corn starch, 0.5% peptone, 1% corn liquid, 0.05% calcium chloride and 0.05% calcium chloride set M to pH 7.5. 50 ml allquot. of this culture medium was placed in a 500 ml conical flask and sterilized for 15 minutes at 121 ° C. Sterlized substrate Inoculated with ATCC No. 31,196 (B-781) by Badllus stearothermophilus strain and stirred for 4 days at 55 ° C. After cultivation, the microbial cells are removed by centrifugation. The alphaamylase activity in the filtrate was 14 CPC units / ml. AKaamylase was quenched by adding twice the volume of 2-propanol filtrate. The precipitate is ground into dry powder by freezing dry. The activity of the powdered crude enzyme was 3 CPC units / mg. Raflnisani, a sputtered enzyme having an activity of

230 units / mg was obtained from this crude powder enzyme by refining according to the procedure of Example 1.

Five isolated and selected strains (i.e., J., ATCC No. 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. Alpha-amylase activity in the experiments was expressed as the cross-sectional value of the highest activity of triplicate balloons. The substrate composition used for both pre-culture and main culture is the same as shown in Tablid 1 (B-M substrate). The results of these experiments are summarized in Tablid 5.

Effect of temperature and pH on α-amllasa production

Activity in U / ml (Cultivation Boils)

ATCC No. CC) pil 5.5 pil 6.0 PH 6.5 PH 7.0 pa 7.5 pil 8.0 31,195 50 1,055 th most common (90) 514 (90) 100 (90) 55 1,355 th most common (90) 1,334 th most common (90) 120 (90, 60 942 (48) 1,490 th most common (90) 1,040 th most common (90) 65 0 (90) 100 (90) 30 (90) 31,196 45 418 (89) 368 (89) 50 477 (65) 681 (41) 55 608 (41) 916 (41) 60 932 (68) 975 (68) 1,168 th most common (43) 1,051 th most common (43) 65 403 (43) 356 (20) 262 (20) 191 (68) 70 24 (43) 224 (20) 155 (20) 59 (43) V, 197 50 751 (44) 720 (44) 679 (44) 1,001 (44) 55 1,057 th most common (44) 1,155 th most common (27) 1,012 th most common (44) 1,330 th most common (27) ' 60 1,085 th most common (44) 1,166 th most common (27) 1,084 th most common (27) 1,139 th most common (27) 65 1,687 th most common (44) 615 (27) 815 (27) 848 (27) 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 th most common (44) 65 1,340 th most common (68) 782 (44) 776 (44) 698 (44, 661 (44, 31,199 45 143 1139) 133 (139) 113 (139) 115 (139) 86 (139) 50 686 (139) 663 (139) 54 3 (139) 493 (139) 491 (139) 55 1,608 ' (43) 1,365 th most common (43) 1,267 th most common (43) 1,025 th most common (43) 946 (43) ' 00 1,485 th most common (43) 1,457 th most common (43) 1,295 th most common (43) 1,154 th most common (43) .1,117 (43) 65 0 (65) 0 (65) 0 (65) 0 (65) ' 0 (65)

From Table 5 it can be seen that the optimal conditions for ATCC No.

31,195 and 31,196 were 60 ° C, pH 7.0 and 60 ° C, respectively, and pH 7.5. ATCC No. 31,197 and 31,198 produced significant amounts of alpha-amylase when cultured at 65 ° C, but this high cultivation temperature was not suitable for producing good alpha-amylase activity for other isolated strains. At 55 ° C to 60 ° C, pH 8.0 was found to be optimal for the production of alphaamllases for ATCC No. 31,198, and the optimum pH of the substrate for ATCC No. 31,199 was 5.5.

TABLET

Tables 6 and 7 (1) summarize the production of alpha-amylase of the five selected strains using repeated transfers on oblique plates (each strain was treated by making 9-11 transfers on two types of hairpin substrates, and then cultured in liquid so to find a suitable substrate for a hair pad that maintains stable production of alpha-amylase), and (2) determine the effect of the volume of the substrate on the production of alpha-amylase (changing the volume of the substrate in 500 ml balloons) by examining the effect of aeration on production alpha-amylase with the help of each strain.

Production of? -Amllaz using repeated transfers to coaltif plocicsjia ¹¹

Balloon culture

iio. number tr anafor a Activity, U / ml Conditions pH (Warm Cultivls ., ΙΠΙ) 91,195 BD *, 1 056 (90) 60 7.o BD, 10 910 (4-3) II It I1A ', 10 140 (72) II II 51, 1% BD, 2 976 (26) 60 7.5 BD, 10 1,503 (26) It II HA, 10 1,550 (26) 11 n 51.19? BD, 1 1,330 (27) 55 7.5 BD, 10 1,127 (26) II It 11Λ, 10 1,420 (26) n 11 > 1,190 BD, · 4- 736 (44) 1 60 8.0 BD, 11 712 (44) It tl HA, 11 1,117 (44) It 11 31,199 BD, 2 1,163 (44) ⁵⁵ 5.5 ' BD, 9 1,013 (60) II. II HA, 9 775 (60) II Hair pad for x Hair floppy with defensible • treatment agcrom * nestvore! starch 1.09 Bacto-pspton (Difoo) '0.59 Bcto-Tryptone (Difco) 0.59 Extract Bakto-i ', 0- Yeast extract 0.59 most (Difco) 0.37- CeCl ,,. 2H _n 0 0.059 Agar 2.0,9 KnClg.fHIgO 0.059 pil 7.2 kii ₂ by ₄ O.l9 Agor 2.0tf pil 5.0

Table 7

Kfokat gapremlno substrate on amllase production

Balloon culture

Number of tran- ATCC affair at ΤΐΌ. I mow the board. Conditions Substrate al / 500 ul. balloon Activity, U / ml (Time kulbiv., E.) Temp « (° 0) pil 30 150 (65) 40 18? (G5) 31,195 BD-11 60 7.0 50 1,192 (41) 60 790 (41) • 146 (t.5) 30 954 (26) 40 966 (in,;) 31,196 BD-2 GO 7.5 50 976 (; \.) GO 1,236 (86) 70 651 (4J) 30 1,435 (41) 40 1,590 (41) 31,197 BD-3 55 7.5 50 1,510 (41) 60 1,300 (41) 70 640 (41) 30 '600 (44) 1 40 651 (44), 31,198 BD-4 60 8.0 50. 736 (44) 60 939 (44) 7 ° '777 (44) 30. ·, 1,131 (44) 40 1,240 (44) 31,199 BD-2 55 5.5 50 1,163 (44) 60 1,054 (GO) 70 1,224 (60)

The process of purification6 a * ni j; - "Anllas to her

ΛΤΟΟ wantedo. 51,195

Street Effect (Cx'J jedlnloe)

Total protein (n » _G )

Specific activity (iodine / mg)

Prince (',!)

ATCC lio. 51 »199

Home activity (oi'C joflinico)

Total protein (mg)

Specific activity (iodine / iag)

Yield ('O

Thnr. ') Wyl

Ul.unnn activity (CPC iodine)

Total protein (mg)

Specific activity (jodinioe / mg) lrinos (; «)

Table 8

Read alfa-anllass

Broth culture or raw onsink prop. Aoetone precipitation Heat treats- n.1e DEAE- colulose 9,870 th most common 8,640 th most common 8,420 th most common 2,400 th most common 7,990 th most common 2,016 th most common 1,450 th most common 15.7 1.2 4.5 5.8 152.9 100 87.5 85.3 24.3 9,910 th most common .8,210 7,214 th most common 2,310 th most common 4914 1969 1206 33 · 2.0 4.2 6.0 70 100 82.0 7.2.0 23.3 12,100 9,400 th most common '\ 9,540' '2,410 ;> · 684 286 · 183 24 '.. 17.7 32.9 52.1 100.4 loo 77.7 78.8 19.9

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 Thermamy Uquid 60, Batch AN 1005 according to the procedure described below.

The AJfa-amllaznyl enzyme was precipitated from ATCC No. culture filtrate.

31,195 and 31,199 ill from Thermamyi alpha-amylase (diluted twice) by adding two volumes of cold acetone. The precipitate was collected by centrifugation and dissolved in 0.025 M calcium acetate solution. D solution was added to the dissolved starch to make a 20% starch suspension. This suspension was heated to 85 ° C for 30 minutes and the precipitate was removed by centrifugation. The solution was then palatable against 0.05 M Trls-HCl buffer (pH 8.5) containing 10 mM Ca + +, and the buffer was replaced twice. The dialysate is applied to a DEAE-oluous column that is equilibrated with the dyslysis buffer. Alpha-amylase is not adsorbed on the column and eluted with the same buffer. Fractions having alpha-amylase activity are collected and the alpha-amylase enzyme is concentrated by precipitation with acetone. Alpha-amylase activity is determined by CPC using the procedure described above. The purification process is summarized in Table 8.

Stability against acids and thermostability of partially purified alpha-amlacazine preparations from ATCC No. 31,195 i

31,199 I Thermamyl alpha-amylase (having 7 Units / ml) was determined by incubation for 60 minutes under the following conditions:

(a) 90 ° C, pH 6.0, Ca + + O or 1 mM;

(b) 80 ° C, pH 4.55, Ca + + 5 mM, and (c) 85 ° C pH 4.55, Ca ++ 1 mM or 5 mM, 22.5% soluble starch.

* 5 ml. alpha-amylase preparation is dialyzed in a Visking 8/32 cellulose tube against 0.05 M calcium acetate buffer (pH 4.5-6.0) containing 0-5 mM calcium acetate for 3 hours at 4 ° C, with the buffer twice changes. Then, 4 ml of dialysate was placed in small tubes and incubated at the indicated temperature in a water bath. The tubes were rapidly cooled in an ice-water bath and the residual alpha-amlase activity determined.

** 0.3 ml. of dialysed enzymatic solution of alpha-milase, 0.5 ml. 1 M calcium acetate buffer, 0.2 ml 0.5 M CaCl2 and 0.3 ml. 30% of the suspension of soluble starch is entangled in a test tube. The tubes were then rapidly cooled in an ice-water bath and the residual alpha-amylase activity determined.

The residual alpha-amlase activity was determined after 5, 10, 20, 30 and 60 minutes of Incubation.

Optimal temperature and pH of ATCC No. 31,195 l ATCC No.

31,199 alpha-amylase and Thermamyl aHa-amllaze were determined using the CPC test conditions described above, except that the reaction pH and temperature were altered. The results of the tastes are shown in Figures 1 and 2. Optimum pH for ATCC No. 31,195 and ATCC No. 31,199 alpha-amylase was 4.0 - 5.2 and for Thermamyl alpha-amylase 4.5. Thermamyl alphaamylase was found to retain high enzymatic activity at neutral and alkaline pH.

The optimum temperatures were 75 ° C for ATCC No. 31,199 alphaamylase, 80 ° C for ATCC No. 31,195 alpha-amylase and 85 ° C for Thermamyl alpha-amylase. The relationship between enzyme activity and reaction temperature was very similar for ATCC NO. 31,195 and ATCC No. 31,199 alpha-amylase, but for Thermamyl alpha-amylase was very diverse. The alpha-amylase activity of Thermamyi alpha-amylase was found to increase by 20-30% when incubated at 85 ° C and at pH 6.0 in the presence of starch and Ca ++. This fact may explain the difference in the enzyme activityreacon temperature between ATCC No. 31,195 and ATCC No.

31,199 Alpha-amylase I Thermamyl alpha-amylase.

FIG. 3 illustrates the ATCC No. Inactivation curves. 31,195 and ATCC No.

31,199 alpha-amylase and Thermamyl alpha-amylase when incubated at 80 ° C and at pH 4.55 in the presence of 5 mM CaCl2 · ATCC No. 31,199 alpha-amylase shows the highest thermostability after ATCC No. 31,195 alpha-amylase. Thermamyl alpha-amylase is significantly more elabic than ATCC No. 1. 31,195 and ATCC No 31,199 alphaamylase in terms of thermostability under these conditions.

FIG. 4 illustrates the inactivation curves of ATCC No. 31,195 and ATCC No.

31,199 alpha-amylase, Thermamyl alpha-amylase and Badllus oldthermophllus alpha-amylase described by Ogasawara et, al. (J. Biochem. 67, 65, 77, 83 (1970)) when incubated at 90 ° C and at pH 6.0 in the absence of Ca + + (incubation conditions were described by Ogasawara et al. And were the same as in this test).

As can be seen from FIG. 4, the alpha-amylase of the present invention showed much higher thermostability than either Thermamyl alpha-amylase or Ogasawara et. al. alphaamylase (although the alpha-amylase tested in the present invention also belong to the Bacillus stearotehermophilus strain).

FIG. 5 illustrates the ATCC No. inactivation curves. 31,195 I ATCC no:

31,199 alpha-amylase and Thermamyl alfa-1amllaze under the same conditions as described above for FIG. 4 (e.g., 90 ° C and p H 6.0) except that the substrate contained 1 mM Ca + +. The alpha-amylase of the present invention still showed a higher thermostability than the Thermamyl alpha-amylase, but the difference was not as large as in the case without Ca + + added. These facts appear to indicate that ATCC No. 31,195 and ATCC No. 31,199 alphaamylase binds Ca + + more tightly than Thermamyl alpha-amylase, and therefore its needs for CA + + to stabilize the protein molecule are less than Thermamyl alpha-amylase.

Figures 6 and 7 illustrate the ATCC No. inactivation curves. 31,195 and ATCC No. 31,199 alpha-amylase and Thermamyl alpha-amylase when incubated at 85 ° C and at pH 4.55 in the presence of soluble starch (22.5%, ds) iCa + + (1 mMCa + + as illustrated in Fig. 6 and 5 mM Ca ++ as is illustrated in Fig. 7). ATCC No.

31,195 and ATCC No. 31,199 alpha-amylase showed higher thermostability than Thermamyl alpha-amylase under the conditions of Figure 6 I 7. Since the above test conditions illustrated in figures 6 and 7 are similar to many Industry Liquidation conditions, it is expected that alpha-amylase from the present of the invention demonstrate greater thermostability at acidic pH values in industrial starch liquefaction. Molecular weights and ATCC No. ATCC No. 31,1951 31,199 alpha-amylase 96,00 by the procedure of Weber and Osboma, J. Biol. Chem., 244, 1406 (1969) using SDS disk electrophoresis. Marker proteins were albumin (M. wt. 67,000), ovalbumin (M. wt. 45,000), chymotrypsin (M. wt. 25,000) and cltrochrome C (M. wt. 12,500). Location of the alpha-amlase From the present invention, the polycrylamide gel was determined by placing the gel on an amylose-azure agar plate and incubating at 37 ° C. The results of this test are illustrated in FIG. 8.

The value of 96,000 for the molecular weight of alpha-amylase of the present invention is much higher than that for Bacillus stearothermophilus alpha-amylase given by Ogasawara et al., J. Biochem., 67, 65, 77, 83 (1970) (M.W. (given as 48,000) and Manning et al. J. Biol. Chem., 236, 2952, 2958, 2962 (1961) (M. weighted as 15,600).

Five isolated strains have been tested for protease activity because the presence of protease or proteolytic enzyme in alphaamylase enzymes tends to react with the various proteinaceous materials present in many starchy materials to produce protein hydrolysates such as amlno-kisslins. For this reason, the presence of a protease enzyme contaminant in alpha-amylase enzymes is detrimental to the efficient hydrolysis of starch materials. Protease activities in alta-amylase enzymes produced from five Isolated strains were determined from acetone thioga culture filtrate at the maximum production stage of alpha-amylase using a modified Anson-Hagiwara procedure.

The results obtained were compared with the enzyme preparations Thermamyl alpha-amylase and CPC bacterial alpha-amylase. As shown below, Thermamyl and the culture filtrates of isolated thermophilic airborne fungi contained large amounts of protease. On the other hand, the thermostable alpha-amylase production strains of the present invention did not produce a significant amount of protease.

Protease activity of Alpha-amylase

Enzyme Protease / alpha-amylase 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-172 109.0

1 / Thermophilic airborne fungus isolated at 55 ° C and at pH 7.0.

As can be seen From the above results, the alpha-amylase of the present invention does not contain any significant amount of protease activity, i.e., the prosthesis / amylase ratio is less than 3 and generally less than 1. This is a significant advantage when the enzyme is used for hydrolysis starchy materials.

Example 3

The following example illustrates the use of alpha-amylase enzymes of the present invention and the starch liquefaction in the production of starch hydrolysates with high D.E.

Thirty grams of potato starch is suspended in 70 ml. of water, 75 mg of calcium chloride dihydrate was added and the pH was adjusted to 4.5. To this suspension was added 50 CPC units of the refined, powdered enzyme obtained in Example 2 so that the starch was treated at 85 ° C for 30 minutes. D.E. the stock solution was about 21 and the pH was 4.3. When the temperature was lowered to 60 ° C, a glucamlase enzyme derived from Aspergillus niger was added and the solution was saccharified and translated to 60 ° C for 48 hours. D.E. of saccharified and converted solution was 97.5 and dextrose content was 96.0%.

As shown in the previous example (Example 3), the alphaamylase enzyme of the present invention hydrolyzes and attaches starch. They can be used to solubilize starch, amylases, amylopectin, glycogen, etc., to reduce the viscosity of these substrates. When the enzymes of the present invention react with soluble starch to pH 4.5 and 60 ° C, the starch iodide reaction disappears at a hydrolysis rate (D.E.) of about 15 and the required rate is D.E. 32 to 36. The sugars of the hydrolyzed product were analyzed as maltose, maltotriose, maltotetrose and other maltooligosaccharides together with the matte glucose col. Mutarotation of the reducing sugar product is negative. According to the invention, the enzymes of the present invention are alpha-amylase enzymes of the type for lactation and freely hydrolyse alpha-1,4 starch bonds. The relationship between liquefaction using the enzyme of the present invention (relative value) and operative pH is shown in FIG. 9 and is opposed by known alpha-amylase enzymes derived from Bacillus starothermophilus. As shown on Si. 9, the optimum pH for the enzymes of the present invention is pH 4.2 to pH 5.2. Preferred enzymes of the present invention do not lose their activity even if left for 24 hours at room temperature at a pH in the range of 3 to 11.

The relationship between the enzyme liquefaction of the present invention (relative value) and the operating temperature is shown in Si. 10 and is opposed by an alpha-aylase derived from Bacillus stearothermophilus described by Ogasawara st al. As shown in FIG. 10, the preferred operating temperature for the enzymes of the present invention is about 80 ° C.

As can be seen From the foregoing, alpha-amylase enzymes of the present invention can be used in the liquefaction and conversion of starch in the starch saccharification industry, then in the textile industry) and as additives in detergent formulations similar to conventional applications of bacterial alpha-amylase enzymes.

The alpha-amylase enzymes of the present invention are particularly suited for the lcvefaction and conversion of starch in the production of maltodextrin, and the subsequent production of dextrose by using glucoamylase since isomerization of the breeding group of molecules can occur because the enzyme can be effectively used at acidic pH (i.e. pH 4.5-5.0 ), thus increasing the dextrose yield. The use of these new enzymes also reduces the burden of ion exchange in the refining process because no pH adjustment is required before saccharification and conversion with the glucoamylase enzyme.

In one preferred method of using the alpha-amylase enzymes of the present invention, the enzyme is used to convert starch to starch hydrolyzate where the residual unconverted starch remains in granular form. These processes are described and protected in U.S. Pat. Patents No. 3,922,196; 3,922,197; 3,922,198; 3,922,199;

No. 3,922,200 1 3,922,201 All of which were published on November 25, 1975, and their entire contents are incorporated herein by reference. In these granular starch processes, at least the initial dissolution of starch materials is carried out at relatively low temperatures, i.e. Below the initial starch gelatinization temperature. In the most preferred method for performing these procedures, the glucoamylase enzyme is used competitively with the alpha-amylase enzyme in the initial phase of dissolution. The enzymes of the present method are particularly suitable for this process since their pH interval is optimum compatible with glucoamyase.

II another preferred method for the use of alpha-amlase enzymes The present invention may use the methods described in U.S. Pat. No. No. 3,853,706, which was published December 10, 1974 and U.S. Pat. No. No. 3,849, 194, published Nov. 19, 1974, and descriptions thereof are incorporated herein by reference.

In still another preferred embodiment of the alpha-amylazine enzyme of the present invention, the method described in U.S. Pat. No. No. 3,192,590 which was published October 14, 1975 by Slott et. al. a belongs to Novo Industri A / S, so this description is included here as a reference. Using the alpha-amlase enzymes of the present invention by the procedure of Slott et. al., a starch suspension, such as corn starch, which has at least 25% by weight. the starch material is treated with an alpha-amylase enzyme at a temperature in the range of about 100 ° C to about 115 ° C, preferably from 105 ° C to about 110 ° C for 1 to 60 minutes, and preferably 5-10 minutes so that the starch is liquified and thereafter the temperature is reduced to 80-100 ° C and preferably 90 ° C to 100 ° C when the viscosity of the concentrated solution is less than 300 cps melted at 95 ° C. The unique advantage of using the process with this temperature profile with the alpha-amlazine enzymes of the present invention is that a lower pH can be used so that there is no or only a small pH adjustment is required in the later saccharification process with glucoamyase.

The most preferred use of the alpha-amylase enzyme of the present invention for the conversion of starch involves subjecting the starch to euphase action of the enzyme at pH in the range of from about 3.5 to about 6.5, preferably from about 4 to about 5, at a temperature that varies from about 50 ° C to about 100 ° C, and preferably from 60 [deg.] C. to about 95 [deg.] C. so that the starch is quenched. In the case of granulyar starch hydrolysis procedures described above, the temperature will vary from the normal initial gelatinization temperature to the actual initial gelatinization temperature of the starch, i. of about 60 ° C for corn starch. In the case of a direct liquefaction process, the starch suspension containing the enzyme is preferably heated (e.g., with a jet heater) to a temperature ranging from about 85 ° C to about 95 ° C and more preferably from about 90 ° C to about 92 ° C so that the starch is liquefied. After the initial dissolution (as in the hydrolysis of granular starch) or liquefaction, the suspension is initially subjected to heat shock treatment at a temperature above 100 ° C, and preferably varies from about 11 ° C to about 15 ° C so as to identify residual starch in the granules. Subsequently, the liquidated starch suspension is cooled and preferably treated with more alpha-amylase, either alone or in combination with other enzymes such as glucoamylase, beta-amylase, puluanase, glucose isomerase, sequentially or in combination. If alpha-amylase is used alone in the second enzyme phase, the temperature will preferably vary from about 80 ° C to about 90 ° C and most preferably from about 85 ° C, which is the optimum temperature for the enzymes of the present invention. If another enzyme such as glucoamylase and / or glucose isomerase is present, the temperature will be slightly lower, e.g., 5575 ° C and preferably about 60 ° C.

Conclusion

The alpha-amylase enzymes of the present invention can be clearly distinguished from the alpha-amylase from previous science derived from animals, plants, yeasts, imperfect fungi, and algae, since these enzymes from previous science have such a low thermal stability that they completely lose their activity after 5 -minute treatment at 70 ° C and pH 6.0. By comparing the thermal stability of the alphaamylase From the present invention as shown in FIG. 12 (the data for making Fig. 12 are practically the same as those used for El. 6) it is clear that the alpha-amylase enzymes of the present invention are far better in acid and heat stability when compared with the alpha-amylase enzymes of the preceding of science. It is also with FIG. 12 it is clear that the alpha-amylase enzymes of the present pornography are characterized by being capable of containing at least about 70% and preferably at least about 90% of their initial activity when kept at 90 ° C and at PH 6.0 for 10 minutes in the absence of added calcium ion, and are also capable of retaining at least about 50% of their initial activity when held at 90 ° C and at pH 6.0 for 60 minutes. As can be seen from FIG. 11 it is clear that the alpha-amylase enzyme of the present invention is further characterized by being able to retain at least about 50% of its initial α-amylase activity at a temperature of 80 ° C and at a pH of 4.55 in the presence of 5 mM calcium ion during 10 minutes.

Table 9 shows the relationship between alpha-amlase enzymes of the present invention and alpha-amylase enzymes of Bacillus subtills and Bacillus llcheniformis used industrially, as well as those of Bacillus stearothermophilus described in the literature. They were compared in terms of optimal operating pH, correct operating temperature and molecular weight. Figures 11 and 12 contrast their properties with respect to acid and heat stability.

Table 9

Optimal opeTeetiraniz aasim rative nH Aifa-anffifaani cnžTmi / iaovog Findings 4.0 - 5.2 Correct operational the temperature 80 ° 0 Molecular weight 96,000 Alpha-amilssa ·, 'izŠ. subtilis · ^1, 4.5 - 6.5 45 - 60 ° 0 49,000 ⁵ AlfR-nmiasea from _? liTTCioheniformls * 5.0 - 9.0 76 - 78 ° 0 22,500 ⁵ Alpha emilase from h. stearothermopbilus a) Ogasavara et. al. ¹ 5.0-6.0 65 - 70 ° 0 48,000 b) Oampbell et. al. ⁵ 4.8 55 - 70 ° 0 15, (S00 ⁴

^ Ogaeauara et. al., J. Blosotiem,. 62, 65 (1970). ² 3higeinasa Salto, ABB, 155. 290 (1973).

^ Campbell et. al. J. Biol. Cbom .. 236. 2958 (I96I).

4 "

Campbell et. al ,, ibid., 236. 2958 (1961).

^ The British Patent Application lists the molecular weight of alphaamylase from B. licheniformis 18,000-20,000 and from B. aubtilis 96,000. "-When alpha-amylase enzymes From the present invention are compared with the alpha-amylase enzymes from Bacillus subtilis, it can be seen that these are significantly different, as is clear from Figures 9 and 11, in terms of their optimum operative pH, the correct operating temperature , molecular weight and stability against acids and heat. This shows that this enzyme is quite different from the alphaamllazes from Badlus subtilis.

When the alpha-amylase enzymes of the present invention and the alpha-amylase enzymes of Badlus licheniformis are compared, they differ significantly in their optimal operating pH, in molecular weight and in their stability against acids and heat, as shown in Tablid 9 and Figures 11 and 12.

It will be appreciated by the art that the strains used to produce the alpha-amlase enzymes of the present invention may be subjected to mutagenic agents using known techniques such as ultraviolet light, chemical treatment, and the like. According to the invention, the present invention encompasses Ifa-amylase enzymes produced from ATCC No. 1 strains. 31,195, 31,196,31,197, 31,198, 31,199, both variant I mutants of these strains, as well as sub-mutants of said variants and mutants.

Like other known alpha-amylase enzymes, the enzymes of the present invention are Inhibited by mercury and EDTA dl are stabilized by cob.

It will be appreciated by the skilled artisan that various modifications of the present invention can be used, as described in the preceding examples, without departing from the scope of the invention. Many of its variations and modifications will be clear in the art and can be performed without departing from the spirit and spirit of the find described herein.

The strain corresponding to ATCC No. 31,199 (8-968) was mutated to develop strains with higher alpha-amylase activity. The cultivars used in the mean are given below in the tablid.

colony B-968 was cultured at 55 ° C for 16 h with 130 rpm shaking. The cells were washed and codependent in sterile water. Ethyleneimine was then added so that the final concentration was 1 / Ul / 1ml 47% of the colonies gave 30-40 CPC of alpha-amylase units per milliliter of culture medium medium.

One strain, identified as M-1041, was isolated after the above procedure and is still being treated. Cultivation in the middle for planting in the above manner was then washed and suspended in sterile water. It was then treated with UV radiation for 20 sec with stirring on a magnetic stirrer in a Petri dish placed 28 cm below

UV-Jampe. 3% of the accumulated colonies had an activity of 80 to 100 CPC aHa-amllazine Units per millilftar of the culture medium medium.

A strain was identified Identified as M-1327 and cultured for 5 h at

55 ° C in the head, the cells were washed with sterile water and suspended and stirred for 30 min. in 5 ml of phosphate buffer containing 100 [mu] g / ml of N-methyl-N'-nitro-N-nitroso-guanidine at pH 6-0 and room temperature. Mutant strain From this procedure it was identified as M-1717.

'-TOBEICA.

Composition of the mean (% of us / occ.)

Hair Agar Flat For now

Separate starch Corn starch 1.0 3.0 6.0 Up-to-date amylose - 0.1 - - CSL * - - - 1.0 Bacto-trypton 0.5 0.5 - - Yeast extract 0.5 0.5 1.0 0.5 Farmamedia - - - 4.0 CaCl_.2H.O 0.05 0.05 0.05 0.1 MgSO ₄ .7H «O - 0.05 0.05 0.05 MnCl2.4H2O 0.05 0.0001 0.0001 0.001 kh ₂ by ₄ 0.1 0.1 0.1 - NaCI - - - 0.005 Agar 2.0 2.0 - - pH 6.0 6.0 .6.0 6.25.

M-1717 was further subdivided into three types A, B and C. Type B showed the most stable enzyme production and the best aHa-amlase activity. (190-280 CPC-aHa-amylazine equivalents per ml of cultivadone filtrate). This strain, M-1717 B, was cultured in the main medium in the same mouths as M-1327, and then washed and erupted in the same manner. The mutant strain, identified as N-21, had an activity of 330 to 490 CPC-αa-amylase units per ml. of cultivadone filtrate.

For practical Euros, positive mutants were collected by diluting mutagen-treated cells, plating the diluted cells in a flat medium, and culturing overnight at 70 ° C. The clear zones around the colonies were formed by the hydrolysis of amlose-azure in the straight] middle.

The sloping agars were made in the sloping middle of the colonies that had the widest clear zones and were cultivated! are overnight at 70 ° C. Soybeans! with the highest filth they were collected and purified.

Claims (2)

PATENT REQUIREMENTS A process for the production of an alpha-amylase enzyme resistant to heat and acids, comprising cultivating a microorganism of a strain selected from the group consisting of Badllus stearothermophilus ATCC Nos. 31,195, 31,196, 31,197, 31,198 and 31,199 in cultivar containing assimiladone Carbon and nitrogen sources and including 6% corn starch, 1% corn yeast liquids, 0.5% cottonseed flour, 01,% CaCl ^ h ^ O. 0.05% Mg SO ₄ .7H ₂ O, 0.001% MnCIg ^ HgO and 0.05% NaCI, all percentages being w / v, and the cultivation is carried out at a pH of 5 to 9, preferably about 6.25-6.30, during the period from one to five days, at a temperature of about 50-70 ° C, preferably about 55-60 ° C, and the alpha-amylase thus obtained! isolates the enzyme from the fermenadone medium. 2. The method of claim 1, wherein the fermentadone medium still contains up to 2%, 50% solution of acid-hydrolyzed soy protein containing 20% NaCl.