DE2218783B2 - METHOD FOR GENERATING AN ALKALINE AMYLASE - Google Patents

Description

The invention relates to a method for producing an alkaline amylase which a Aktivitätsmaxi- yo maximum at pH 4.5, and which has at pH 7 and at pH 9 due to the besitu Molekulargcwichtsbestimmung by gel filtration, a molecular weight of about 40,000.

Amylases are starch degrading: enzymes that are preferred are active in the acidic or neutral range (see "Advances in Applied Microbiology" I, p. 293 [1965]). Various methods are known for producing these enzymes.

The object of the present invention is to provide a method for producing a novel alkaline Amylase using a novel microorganism.

The invention consists in a process for the production of an alkaline amylase which has a maximum activity at pH 4.5, at pH 7 and at pH 9 and which, due to the molecular weight determination by gel filtration, has a molecular weight of about 40,000 , contains a carbon source, a nitrogen source and an inorganic material, inoculated with the Sitamm Bacillus ATCC 21783 and then after adjusting to a pH of 7 to 11 at a temperature of about 30 to 37 C is incubated aerobically until the culture medium has a considerable activity on the alkaline amylase, whereupon the alkaline amylase produced in the culture medium is separated from the culture medium by the usual physical-intrinsic methods for the recovery of enzymes. The Bacillus ATCC 21783 is made up of one : - ■■. Soil sample has been isolated from the Hiros.iwa district of Wako, Saitama Prefecture, Japan.

The microorganism designated as Baciiius ATCC 2i7S3 is on March 23, 1972 with unrestricted access with American. Type Culture Collection; ■ ·; (ATCC), 12 301 Parklaun Drive. Rockville, Maryland 20852, USA.

The microbiological properties were after

(D Nutrient bouillon 10 (or not added) sodium 5 Repton 3 Beef extract (2) Nutrient broth with Agar Agar 10 (or not added) 'Sodium 5 Peptone 3 Beef extract 15th Agar Agar (3) Bouillon with glucose lü (or not added) sodium 5 Peptone 3 Beef extract 10 glucose (4) Bouillon with glucose and agar-agar 10 (or not added) sodium 5 Peptone 3 Beef extract 10 glucose 15th Agar Agar (5) Gelatin medium 10 (or not added) sodium 5 Peptone 3 Beef extract 5 gelatin Peptone water medium 10 (or not added) sodium 5 Peptone (7) Potato medium 10 (ö'jer .- ■ 1..
i 111 IlI ZügcSciZi) Nuiiiuiiii'aiuuiliii 50 Potato extract IS Agar Agar (Si test 1 i \ sodium 1 Peptone 5 glucose 5 NaCl

(9) Nitrate Reduction

Sodium carbonate 10

Peptoo; 5

Beef extract 3

KNO ₃ 1

10) Catalase reaction

Sodium carbonate 10 peptone 5

Yeast extract 5

Strength 20 K ₂ HPO ₄ 1

Agar-agar 15 MgSO ₄ -7H ₂ O 0.2

(11) hydrolysis of casein

Sodium carbonate 10 Yeast extract 5 Glucose 10

Casein 5

K ₂ HPO ₄ 1

Agar-agar 15

MgSO ₄ -7H ₂ O 0.2

(12) starch hydrolysis

Sodium carbonate! Q

Peptone 5

Yeast extract 5

Strength 20 K ₂ HPO ₄ 1

Agar-agar 15

MgSO ₄ -71I ₂ O 0.2

;; 3) utilization of citrate

Sodium carbonate 10 sodium chloride 1

MgSO ₄ -7 H ₂ O 0.2

(NH ₄ ) ₂ KPO ₄ 1

KH ₂ PO ₄ 0.5

Citrate 2

Agar-agar 15

Table 1

(14) recovery of Ammonium salt

sodium 10 Yeast extract 0.2 glucose 5 (NH ₄ ) ₂ HPO ₄ 1 KCl 0.2 MgSO ₄ 0.2 Saccharide 5 (Glucose, Sucrose and the like) (15) bouillon with Sodium chloride

Sodium carbonate 10

Peptone 5

Beef extract 3

NaCl 70

(16) Dextrose nitrate medium

Sodium carbonate 10 Glucose 10

KH ₂ PO ₄ 1

NaNO ₃ 1

Agar-agar 15

(17) growth below anaerobic conditions

Sodium carbonate 10

Peptone 10

Beef extract 3 Yeast extract 2 Glucose 10

K ₂ HPO ₄ 5

(18) Glucose-Asparagine Medium

sodium glucose

Aspatagin

K ₂ HPO ₄

Agar Agar

The growth of Bacillus ATCC 21 783 on various nutrient media can be seen from Table 1.

10 (or not added) 10

0.5

0.5 15

medium

(D Nutrient broth (2) Bouillon with agar-agar (3) Bouillon with glucose (4) Bouillon with glucose and Agar Agar (5) Gelatin medium 16) Peptone-water medium (7i Potato medium

pH value of the medium
pH 7

pH 10.2 ')

very weak
growth

very weak
growth

weak growth
weak growth

very weak
growth

weak growth weak growth

Turbidity; good growth good growth

Growth; Dissolution

growth

good growth

In table 1, column 1 shows the used Culture media and in columns 2 and 3 the growth at a pH value of 7 and 10.2, respectively.

The microorganism has dimensions from 0.5 to 0.6 x 2.0 to 3.0 _μ πν, the sporangium is clearly swollen, and the spore formed in the end part of the cell is oval and has dimensions of 0.9 to 1.0 x 1 , 2 to 1.5 ^ m.

The microorganism has pertrichous flagella, as can be seen from its electron microscope image. Said Bacillus strain has a positive Gram stainability and negative antacidity. It grows on the nutrient medium described below (soluble starch, yeast extract, peptone, K ₂ HPO ₄ , MgSO ₄ · 7H ₂ O and 1% Na ₂ CO ₃ , adjusted to a pH Value of 10.2) very well in the form of freely moving colonies. This Bacillus strain is distinguished by the fact that it grows well in an alkaline nutrient medium with a pH of 7.5 to 11, preferably about 10, better than in a neutral nutrient medium.

The following physiological properties were noted:

(1) Optimal growing conditions:

pH about 10

Temperature 37 to 40 C - aerobic

(2) Conditions under which the microorganism can grow:

PH value
temperature

7.5 to 11

up to 45 C - aerobic

With the aid of the _{nutrient medium containing 1% Na 2} CO ₃ , the following experiments were carried out and the following results were obtained:

Table 2

(3) Gram dyeability

(4) Voges-Proskauer reaction

(5) Nitrate is reduced

(6) catalase

(7) hydrolysis of gelatin
and casein

(8) hydrolysis of starch

(9) Utilization of citrate

Nutrient medium

(10) recovery of
Ammonium salts

(11) Growth in 7%
Sodium chloride solution

(12) Growth on glucose-nitrate-nutrient
medium

(13) growth below
anaerobic conditions

(14) growth on one
Dextrose asparagine nutrient medium

positive positive (8)

(9)

positive (10) positive (5), (11)

positive (12)

low (13) recovery

positive (14) weak (15) positive (16)

positive (17) positive (18)

In a nutrient medium of the following composition

Sodium carbonate 10 *)

(NH ₄ ) ₂ HPO ₄ 1

KCl 0.2

MgSo ₄ 0.2

Yeast extract 0.2

Saccharide (glucose, sucrose, etc.) 5

*) Figures in grams per liter of water

the saccharides are dextrose, fructose. Xylose, wood sugar, cane sugar, malt sugar, milk sugar and pectin sugar used On the other hand, galactose, ergot sugar and inulin are only used to a small extent recovered. Acid is generated Yeast extract, peptone, corn steep liquor and are used as nitrogen sources like used

The Bacillus ATCC 21783 then became a comparative Subjected to examination according to the classification procedure outlined in said publication "Aerobic Spore-forming Bacteria" and in "Bergey's Manual of Determinative Bacteriology" (1957) (on p.613ff). This investigation showed that the microorganism mentioned in in some respects resembled the known microorganisms of the genus Bacillus, in certain properties however, was completely different from them, and that there were no species in the known genus which agree with the present microorganism in terms of the properties mentioned. This led to that Conclusion that it was a new strain of the genus Bacillus. Since the known Microorganism is an aerobic, spore-forming bacterium, it naturally belongs to the genus Bacillus. The microorganism is characterized in particular by the fact that it is in an alkaline medium grows particularly well, with the optimal pH value being around 10

Known bacterial species were compared with the Bacillus strain ATCC 21783, for example the Bacillus firmus. This is negative in the Voges-Proskauer reaction and has a glucose-containing Nutrient medium an optimal pH value of 6.2. In contrast, the Bacillus strain ATCC 21 783 is at positive for the Voges-Proskauer reaction and shows growth in a nutrient medium containing dextrose at a pH of 7.5 to 11, the optimum pH being around 10.

While the growth of Bacillus firmus is inhibited by grape sugar, this is the case with the Bacillus strain ATCC 21783 does not.

The Bacillus differs in these points ATCC 21783 strain clearly distinguished from Bacillus firmus.

One can also use the Bacillus subtilis for comparison

use with the Bacillus strain ATCC 21783 In Bacillus subtilis, however, there is only a small difference between the main axis of the mycelium:

and the minor axis of the spore present (major axis of the mycelium: 0.7 to 0.8 μm, minor axis of the spore 0.6 to 0.9 μΐη). On the other hand, in the case of Bacillus Strain ATCC 21783 the main axis of the mycelium:

6s 0.5 to 0.6 μΐη and the axis of the spore 0.9 to 1.0 _μ ΐη With regard to the physiological properties be

carries the optimal pH value of the nutrient medium for the Bacillus subtilis about 5.5, while the BaciUus Stamrr

ATCC 21 783 good growth in a nutrient medium adjusted to a pH of 7.5 to 11 shows, the optimum pH being around 10. The Bacillus strain differs in these points ATCC 21 783 clearly from the Bacillus subtilis.

In summary, it should be stated that the Bacillus strain ATCC 21 783 is a microorganism which, as an aerobic, spore-forming bacterium, belongs to the genus Bacillus and which, in view of its microbiological properties, in particular the fact that the optimal pH value for its growth is around 10 , can be regarded as a new strain of this genus. This strain is named Bacillus ATCC 21,783.

In the practice of the invention, fermentation can be carried out by the following procedure will.

The method is characterized by the addition of a carbonate to the culture medium. In this Medium are starch, soluble starch and the like as a carbon source, yeast extract, peptone, corn steep liquor and the like as a nitrogen source and various carbonates, e.g. B. potassium carbonate, Sodium carbonate and sodium bicarbonate, used as inorganic salts. These salts become the solution added to prepare a culture medium adjusted to a pH of about 10.5.

The medium prepared in this way is inoculated with the Bacillus ATCC 21 783, which is then cultured with shaking at a temperature of about 30 to 37 ° C. The activity of the enzyme produced generally peaks after about 24 to 64 hours of culture.

Since the time to reach maximum enzyme activity at a given temperature and in the case of a culture medium consisting of the same components, on the aeration and stirring conditions depends, the cultivation time is expedient in each individual case by measuring the enzyme activity certainly.

The second important parameter for breeding is the pH of the medium. At the beginning you have to use carbonates or bicarbonates set a pH of 7 to 11 will. When using a sugar-covered culture medium, the optimal pH value is between 8 and II.

To obtain the enzyme from the culture broth the usual physico-chemical procedures can be used. For example, you can use the culture broth cool down and if necessary / ur neutralization Add acetic acid. The culture broth is used for the quantitative precipitation of the alkaline amylase then added ethyl alcohol. The precipitate is separated off thoroughly with ethyl alcohol washed and dried. That obtained in this way from the culture of the said microorganism Enzyme, the alkaline amylase, turns out to be an alkaline amylase, whose pH values at optimal Maintaining their activity at pH 4.5, pH 7 and pH 9.

The cultivation must be carried out in an alkaline medium with a high concentration of carbonate. Various carbonates are added to the medium containing a carbon source and a nitrogen source as required for the growth of the microorganism. For example, you have to use a medium that is glucose. K _: Contains HPO ₄ , yeast extract, peptone and MgSO ₄ · _{7H 2} O. Add sodium carbonate or sodium bicarbonate. The concentration of the added carbonate in the medium is expediently 0.5 to 5%.

For the advantageous production of the desired alkaline Amylase, the addition of a carbonate to the medium mentioned is extremely important. This goes from the test results set out in Table 3.

The growth of the microorganism Bacillus strain ATCC 21783 was checked by adding the culture broth after 18 hours placed in a cuvette with a beam path of 1 cm length and its absorption capacity was measured at 660μΐτι. Further the activity of amylase was measured under the conditions described below. The results are given in Table 3, from which it can be seen that the presence of a carbonate in the medium is an important prerequisite for the generation of the alkaline amvlase.

Table 3

Added salt

Initial pH value

Growth slum

Amy läse-Akliviüil (U / ml)

pH 4

pH 9

(1) No added salt (broth contains only soluble starch, K ₂ HPO ₄ , yeast extract, peptone and MgSO ₄ · 7 H ₂ O)

(2) NaCl 1%

(3) KCl 1%

(4) Phosphate buffer solution: 5) NaHCO ₃ 1%

'6) Na ₂ CO ₃ 1.0%

7) K ₂ CO., 1.0%

8) Na ₃ PO ₄ and K ₃ HPO ₄

10.0 0.7 150 100 10.0
(with NaOH 0.5
set) 160 100 10.0
(with NaOH 0.5
set) 160 100 8.0 0.9 350 210 8.8 1.2 2500 1200 10.2 U 3100 2000 10.2 1.2 2600 1400 11.2 0.67 450 300 709 524/353

The next most important factor in breeding is pH. The results of the experiments described below show that the pH value during cultivation must be in the range from 7 to 11 in order to produce the alkaline amylase.

The influence of the pH on the production of alkaline amylase was investigated by using HCl or NaOH, the pH of said culture medium was changed, the I% sodium carbonate contained. According to Table 4 it was found that the best results at a pH of 7 to II, especially from 8 to 11, in attendance of glucose. Table 4 also reveals a prevention of amylase activity recognize.

Table 4

Medium amylase activity (U / ml)

PH value:
7 8 9 OK

1100 2800 3000 800 100 100 100 100

contains 1% 200
Na ₂ CO,

does not contain 100
1% Na ₂ CO,

Thereafter, the influence of pH on dii production of alkaline amylase was determined by the Ver the pH change of the culture medium ₁ ml NaHCO or Na ₂ CO ₁ uniersucht. The result is shown in Table 5.

Table 5

Added SaI /

Initial pH End pH Growth

Amylase Activilate (U / ml)

Without added salt (medium consists of 2%
soluble starch, 0.1% K ₁ HPO ₄ , 0.5% HeTeextrakt, 0.5% peptone. 0.02% MgSO ₄ 7 H ₂ O)

NaHCO, 0.5%
NaIICO., 1.0%
NaHCO-, 2.0%
Na ₂ CO., 0.5%
Na ₂ CO, 1.0%

Under the culture conditions given above, the medium is mixed with that previously described in the same ^ s Culture medium pre-incubated Bacillus ATCC 21783 inoculated and this then in a shaking culture suitable conditions, e.g. B. grown at 30 to 37 C for 24 to 64 hours. When the breeding ends is, the cells are separated and the broth is made with acetic acid or the like Acid neutralized or not neutralized. then will precipitate the generated alkaline amylase a solvent such as ethyl alcohol or acetone is added. The precipitate is then drained and dried to obtain the desired substance.

The alkaline amylase obtained in this way has an activity of about 2000 to 3000 U / ml and an optimal pH of about 9 in the alkaline range.

The enzyme obtained according to the invention, the novel alkaline amylase, has the following physicochemical properties:

(1) molecular weight

When determined with the help of gel filtration - about 40,000.

(2) Optimal pH range and range of stable pH values

The pH was adjusted to 4 to 5 with acetate, 5 to 8.5 with tris-maleate buffer solution, 9 to 11 with glycine sodium hydroxide and 11 to 12 with carbonate. Furthermore, the enzyme used was desalted with the aid of gel filtration. FIG. 1 shows in a graph the optimal pH values of the alkaline amylase, the activity at the

7.0 7.0 0.1 not generated 8.9 9.4 0.8 750 9.0 9.5 0.9 1500 9.2 9.6 1.0 1800 9.7 9.6 0.9 1400 10.3 9.8 9.8 1500

pH 9 is given as 100%. From Figure 1 it can be seen that optimal pH values at pH 4.5, pH 7 and pH 9.

0.01 ml of the enzyme according to the invention were heated to 60 ° C. for 30 minutes together with 0.1 ml of a buffer solution with a given pH value and 1.0 micromol CaCl _{2 as a stabilizer.} 0.2 ml of a buffer solution having a pH of 9.0 was then added to the mixture mentioned, whereupon the residual activity was determined. The result is given in Table 6.

Table 6

PH value residual activity

4th 5

7th 8th

10 11th

Acetate buffer solution Tris-maleate buffer solution

Na ₂ CO, / NaHCO ₃ Na ₂ COj

12th

100

70 10

(K)

(3) Study of enzyme activity

0.01 ml of said alkaline amylase were diluted so that the absorption capacity at 700 nm has been reduced to 10 to 20%. The diluted amylase was added to 0.2 ml of a 0.2% potato starch solution and 0.2 ml of a 0.2 molar glycine-NaCl-NaOH buffer solution with a pH of 9.0 (or to 0.2 ml of an O.lmolar acetate buffer solution with

the pH value 4.0) and incubated for 10 minutes at 40 ° C. After the reaction, 0.5 ml of 0.5 molar acetic acid was added to the mixture. Then 3 ml of a 0.005% iodine solution were added and the absorption capacity of the sample was measured at 700 μm. To correct the readings, the value for a blank sample in which the enzyme solution had been mixed with 0.5 molar acetic acid before the addition of the starch solution was subtracted.

Definition of enzyme activity

A unit of activity of the enzyme is defined as that amount of the enzyme which the absorption \ enable at 700 ηm under the standard conditions described above by 10%.

(4) Use temperature range

The enzyme activity was measured according to the usual method at pH 8 and at various temperatures. The measurement was carried out with a solution that did not ^{contain any Ca ++} as a stabilizer.

The residual activities indicated in Table 7 were obtained in this way.

Table 7 Time Home activity temperature (Min.) (%) (C) 15th 100 50 30th 100 50 15th 80 55 15th 10 60

(5) activation and stabilization

To investigate the activation of the enzyme, Ca * ^{1 was} added. The activation was not observed, but it was recognized an increase in the heat resistance, when 8 Ca ^{+ +} was added at the pH.

This is shown in Table 8. All samples were at the stated temperature for 30 minutes long incubated.

Table 8

temperature

Residual activity (%)
pH 4 pH 9

50
55
60
65
70
75
80

Table 9

100 100 100 100 100 100 100 100 61 65 10 10 0 0

(6) cleaning method

The culture broth, which had been obtained by culturing the Bacillus ATCC 21783, was adjusted _{to pH 7 by adding 5 molar CaCl 2} solution, with precipitates forming which were centrifuged off.

_{V 2} volumes of acetone were added to the filtrate, whereupon precipitates formed again.

These were separated and dissolved in water.

The solution was subjected to dialysis overnight and then concentrated with polyethylene glycol. After gel filtration chromatography, active fractions were separated off and absorbed on a DÄDÄ cellulose column. This was equilibrated with a solution which was 1 millimolar of CaCl, and 10millimolaran Fris-hydroxymethylaminomethane-HCl and had a pH of 8.5. The enzyme was then _{eluted with CaCl 2} solution, which had a concentration of 5-50 millimoles / l, usually 40 millimoles / l.

The active fraction was thus separated and for the recovery of the end product by gel filtration chromatography cleaned.

From curves showing the relationship between pH and enzyme activity before and after Cleaning / own, no difference emerges.

(7) Uniformity of the enzyme

From the results shown below, it can be seen that the enzyme obtained in the present invention is uniform.

1. The sedimentation constant in the analysis with the ultracentrifuge was about 4 S.

2. Gel filtration chromatography gave only a single peak of activity.

3. Disk electrophoretic analysis at pH 8.3 confirmed that the enzyme was monodisperse was.

4. The ratio between the enzyme activities at the pH values between 4 and 9 was not changed even if the enzyme activity was partially lost through heating

In Table 9, the physico-chemical properties of the enzyme are compared with those of known amylases, such as saccharin ornamental and liquefying amylases, the Bacillus subtilis ge gained were prepared from a culture of the microorganism (see "Advances in Applied Microbiology," 7, P. 293 11965]).

The enzyme according to the invention is characterized in that it has three optimal pH values, of pH 4.5, pH 7 and pH 9, ie an activity in a wide range.

These results lead to the conclusion that enzyme since the invention is a novel verfiüssi constricting amylase

40

Amylases obtained from a culture of Bacillus subtilis according to the invention
obtained amylase

Art
Heat resistance

liquefying
65-80 C

saccharifying
55-70 C

liquefying

60-70 C

(in the presence of

Ca ⁺⁺ )

continuation

Amylases obtained from a culture of Bacillus subtilis according to the invention
obtained amylase

Stable pH range 4.8-10.8 4.0-7.8 Optimal pH values 5.4-6.0 4.8-5.2 Stabilization by Ca ^{++ +} ) ") Starch hydrolysis 35% 70% ⁺ ) Stable. ") Not stable.

5-10

4.5, 7.0, 9.0

15%

In summary it can be stated that from the comparison of the physicochemical properties of the enzyme according to the invention, alkaline amylase, with those of the known amylase clearly shows that it is a novel amylase, which is different from known amylases differs

Such amylases are in the German Offenlegungsschriften 2044 513 and 2044 984. To demonstrate the novelty of the invention Bacillus strain ATCC 21783 its activity was compared with that of that in the DT-OS2044513 called bacilli

ATCC 21592 (A-40)
ATCC 21591 (A-59)
ATCC 215% (27-1)
ATCC 21593 (124-1)
ATCC 21595 (135)
ATCC 21594 (169)

and those of the bacilli mentioned in DT-OS 20 44 984

FERM P-374 Bacillus subtilis AJ-3250

FERM P-375 AJ-3252

FERM P-376 Bacillus sp. AJ-3255

FERM P-660 AJ-3298

FERM P-661 AJ-3299

compared.

It shows for the strain ATCC 21783 according to the invention and the known ones listed above Strains Optima at the following pH values: see comparative list.

Comparative compilation

tribe 21783 pH Present invention ATCC 21592 4.5; 7; 9 DT-OS 2044 513 ATCC 21591 10.5 ATCC 21596 10.5 ATCC 21593 10.2 ATCC 21595 10.2 ATCC 21594 4.6; 10.2 ATCC P-374 4.8; 10.2 FERM P-375 4-9 DT-OS 20 44 984 l-HRM P-376 5.5-8.5 IERM P-660 8.5-9.5 FERM P-661 10.5 FERM Il

The Bacillus ATCC 21783 according to the invention is the only Bacillus to have three activity maxima and thus an activity range from almost pH 3 to almost pH 12.

To prove the technical progress

also the stability of the enzyme produced according to the invention with that of the enzymes according to DT-OS 20 44 513 and 2044984 compared.

In terms of heat stability, the invention. B produced enzyme has an activity of 100% at 65 C and pH 4 and pH 9. In contrast, enzymes known from DT-OS 2044 513 have a residual activity of only 50% ^{in the presence of Ca ++ at 55 C.}

The enzymes known from DT-OS 20 44 984 are at a pH 5.5 to 9, or at a pH 7.0 up to 10.0, or at a pH 8.0 to 10.0 and at a pH 6.0 to 11.0 stable. This is the place to be A person skilled in the art has the chance, if work has to be carried out at pH 4, immediately on the enzyme of the invention To be able to fall back on the procedure.

When the enzyme prepared according to the invention is used as an additive to detergents, it becomes in powder form introduced directly into the detergent, for example from sodium dodecylbenzenesulfonate (DBS) can exist. This preparation is not subject to any restrictions e.g. B. with regard to the pH of the finished detergent solution or the mixing ratio of the detergent ingredients and additives. For example, by adding 0.01 to 1.0 percent by weight of the invention produced enzyme has a good effect, d. H. a good amylolysis effect.

The enzyme produced according to the invention was in the presence for a certain period of time a detergent was incubated at 40 ° C. The residual activity of the enzyme was then measured.

Experimental method

At the time of contact with the enzyme of the present invention, the detergent had a concentration of 0.1%. Sodium dodecylbenzenesulfonate (DBS) was used as a detergent. At the time the reaction was carried out with the aid of a glycine-NaCl-

f) o NaOH buffer solution set a pH of 10.5.

As can be seen bcsit / t as cin / iucr Bacillus der Bacillus ATCC 21783 three optima

Reaction solution g

Detergent 0.5%

Glycine-NaCl-NaOH buffer solution
Manufactured according to the invention
Enzyme (2000 U / ml)
Water additive

ml

ml

ml

ml

Results

Residual activity of the produced according to the invention
Enzyme

Reaction temperature: 40 ^{= C}

Name of the microorganism

Residual activity (%)
0 min. 30 min.

60 min.

Bacillus ATCC 21783 100

110

98

Below will be
Invention described:

Soluble starch
K ₂ HPO ₄
Yeast extract
Peptone
MgSO ₄ · _{7H 2} O

Embodiments of the

example 1

20 g

Ig

5g
10 g

0.2 g

The approach given above was carried out in 900 m! Dissolved water and sterilized the solution at 115 C for 15 minutes. A solution of 10% Na ₂ CO ₃ in 100 ml of water was sterilized at 115 C for 15 minutes. By mixing these two solutions, a culture medium was obtained, 250 ml of which was placed in a shake flask with a capacity of 1 liter. The culture medium in the flask was inoculated with the Bacillus ATCC 21783, which had been pre-incubated in the same medium overnight. The medium was then adjusted to pH about 10 and shaken at 37 ° C. for 48 hours. The cells were separated from the culture medium using a centrifuge. At pH 9, ImI of the culture filtrate contained 2500 units of alkaline amylase.

After the culture broth had cooled down thoroughly, 3 volumes of acetone were added to it, whereby the enzyme was precipitated quantitatively. The precipitate was washed thoroughly with acetone and air dried. 1 I became the culture bouillon obtained about 11 g of a brownish powder.

The sample thus obtained had an optimal pH of about 9.0 and was found to be alkaline amylase (specific activity: 200,000 U / g).

Example 2
Composition of the culture medium:

Soluble starch 20 g K ₂ HPO ₄ ig Corn steep liquor 50 ml MgSO ₄ · 7 H ₂ O 0.1 g

The batch given above was dissolved in 900 ml of water, adjusted to a pH of 6 to 6.5 with NaOLI and sterilized at 115 ° C. for 15 minutes. A solution of 10% Na ₂ CO ₁ in 100 ml of water was sterilized at 115 ° C. for 15 minutes. By mixing the two solutions, a culture medium was obtained, 100 ml of which was poured into a

placed shake flasks with a capacity of 11. The one in the piston Culture medium was inoculated with the Bacillus ATCC 21733 in the same medium Had been incubated the night before. The medium was then shaken at 37 ° C. for 72 hours Cells were separated from the culture medium using a centrifuge. Contained at pH 9 1 ml of the culture filtrate 3800 units of alkaline amylase.

After the culture broth had cooled down thoroughly, 3 volumes of acetone were added to it Enzyme was precipitated quantitatively. The precipitate was washed thoroughly with acetone and air dried About 12 g of a brownish powder were obtained from 11 of the culture broth.

The sample thus obtained had an optimal pH of about 9.0 and was found to be as alkaline amylase (specific activity: 350,000 U / g).

Example 3

Composition of the medium:
Approach 1

Soluble starch 20 g

K ₂ HPO ₄ 1 g

Yeast extract 5 g

Peptone 10 g
Solution of the salt mixture B
in 100 ml of water

Salt mixture B

MgSO ₄ x 7 H, 0.4 g

NaCl 0.2 g

FeSO ₄ -7 H, 0.16 g

MnSO ₄ 0.16 g

Approach A was dissolved in 900 ml of water and sterilized at 115 ° C. for 15 minutes. A solution of 1% NaHCO ₃ in 100 ml of water was sterilized at 115 ° C. for 15 minutes. By mixing these two solutions, a culture medium was obtained, 250 ml of which was placed in a shake flask with a capacity of 1 liter. The culture medium in the flask was inoculated with the Bacillus ATCC 21783 which had been pre-incubated in the same medium overnight. The medium was then adjusted to a pH of about 10 and shaken at 37 ° C. for 72 hours. The cells were separated from the culture medium using a centrifuge. At pH 9, ImI of the culture filtrate contained 3500 units of alkaline amylase.

After the culture broth had cooled down thoroughly, 3 volumes of acetone were added to it, whereby the enzyme was precipitated quantitatively. The precipitate was washed thoroughly with acetone and air dried. About 10.5 g of a brownish powder were obtained from i i the Kuiturbouiiion.

The sample thus obtained had an optimal pH of about 9.0 and was found to be alkaline anylase (specific activity: 32,000 U / g).

1 sheet of drawings

709 524/353

Claims (1)

Claim ;: Process for the production of an alkaline amylase which has an activity maximum at pH 4.5 at pH 7 as well as pH 9 and which due to the molecular weight determination by gel filtration has a molecular weight of about 40,000, characterized in that a culture medium, that is a carbonate, a carbon source, ι ο a nitrogen source and an inorganic material contains, inoculated with the strain Bacillus ATCC 21783 and then after adjustment to a A pH of 7 to 11 at a temperature of about 30 to 37 ° C is incubated aerobically until the culture medium has a considerable activity on alkaline amylase, whereupon the in the Culture medium produced alkaline amylase from the culture medium with for the recovery of Enzymes are separated from the usual physico-chemical processes. the procedure tested by Nathan R. Smith, RE. Gordon and F.E. Clark under the title "Aerobic Sporeforming Bacteria" (United States Department of Agriculture, November 1952) and those in "Bergey's Manual of Determinative Bacteriology" (1957). The properties of this microorganism as well as the novel alkaline amylase are shown below explained. The figure shows a graph showing the optimal pH values of the alkaline amylase according to the invention. The nutrient media that are used in the experiments for Tables 1 and 2 were used. The composition is in grams per each Liters of water indicated: