FI58157B - VAERME- OCH SYRASTABILT ALFA-AMYLASENZYM OCH FOERFARANDE FOER DESS FRAMSTAELLNING - Google Patents

Description

ri ADVERTISEMENT rft1c7 JSA m (11) UTLÄGGN INQSSKItl PT 3Ö1 0 / ($ 4) pc .- · - p (51) KyJfcVci.3 σ 12 I 9/28, σ 07 G 7/00 FINLAND —FINLAND (21) hewtdh * mw-Fwwami «wlii | 7712U0 (22) HriMwitipttvt — AitelcwInpAt 19.0U.77 (23) Altopilvi — GiMgh «Csd * t 19.0U.77 (41) Tvihit (from outside — .feIMt offntNg 20 10 77

Patents * and registries (44) Date of filing and registration date. -

Patent and Regulatory Authority AiwCUcan utl ** d ech utl. »Kr * ft * n publkarad 29.08.80 (339 (33) (31) Pyri« «r« CmoHc ·! * - ·· * «prforltat 19.oU.76 02.02.77 USA (US) 678513, 76U923 (71) CPC International Inc., International Plaza, Englewood Cliffs,

New Jersey 07632, USA (72) Masaki Tamura, Yokosuka-shi, Kanagawa Pref., Mutsuo Kanno, Iruma-gun,

Saitama Pref., Yoshiko Ishii, Tokyo, Japan-Japan (JP) (7U) Oy Roister Ab (5U) heat- and acid-resistant alpha-amylase enzyme and method for its preparation - Rim- and ochyrastäbilt alpha-amylase enzyme and for the preparation of framställning This invention relates to a novel heat and acid resistant alpha-amylase enzyme and to a process for its preparation.

Alpha-amylase enzyme preparations are used to hydrolyze, liquefy - and / or convert starchy substances into starch hydrolysates; they are also used in detergent compositions. Alpha-amylase enzymes are used to treat starchy substances in the manufacture of many starch hydrolysates, such as maltodextrins, corn syrup, dextrose, levulose, and maltose. The enzyme alpha-amylase hydrolyzes starch molecules, cleaving them into various medium molecular weight fragments called maltodextrins. The maltodextrins are then treated with one or more other enzyme preparations, e.g. glucoamylase, beta-amylase and / or glucose isomerase, to obtain the desired end product. Alternatively, many such enzyme preparations can be added simultaneously to the starch-raw material slurry to directly obtain the desired starch hydrolyzate.

Alpha-amylase enzymes are obtained from a wide variety of sources. Most alpha-amylase enzymes are produced by bacteria such as Bacillus subtilis, I> 58157

Bacillus licheniformis, Bacillus stearothermophilus, which are cultured in a suitable culture broth, after which the cells produced are lysed and the enzyme preparation is separated from the culture broth and purified.

Many of the currently produced commercial alpha-amylase enzymes are derived from cultures of the microorganism Bacillus subtilis. When these enzymes are used to convert starch to starch hydrolyzate, the optimum temperature is generally about 80 ° -85 ° C, and the optimum pH is about 6.0. For efficient use of the enzyme, these temperature and pH conditions have two drawbacks. First, if the starch is modified by an enzyme at a pH of about 6 and a temperature of about 80 ° -85 ° C, some of the reducing end groups of the starch are isomerized and in the subsequent conversion process maltulose is formed which reduces the desired product, e.g. dextrose, Levuose. yield. Second, the optimum pH of the glucoamylase used in the conversion and sugar conversion process is usually about 4.5 for Aspergillus niger-type enzymes and about 5.0 for Rhizopus-type enzymes. Therefore, at the end of the liquefaction step, when using an alpha-amylase enzyme, the pH must be adjusted to between about 6-4.5 or 5.0. This pH adjustment increases the ion concentration and the use of ion exchanger resins in the purification of the final product increases the purification costs.

In recent years, various heat-resistant alpha-amylase enzymes have been developed. Examples of such heat-resistant alpha-amylase enzymes are those produced with strains of the microorganism Bacillus stearothermophilus / 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) ¾ S.L. Pfueller and W.H. Elliot, J. Biochem, 244 48 (1969) 7 · Thermaraylalfa amylase enzymes, manufactured by Novo Industri A / S and having good heat resistance in neutral or poorly temporal solutions, have recently been marketed under the trademark. They are prepared by culturing Bacillus licheniformis as described in GB Patent No. 1,296,839, Die Stärke, 25, 304, 305 and 308 (1973) and Shigemasa Saito, ABB, 155, 290 (1973). Although alpha-amylase enzymes from Bacillus licheniformis are relatively warm; durable in neutral and poorly temporal solutions, so their stability is not sufficient for economical use under acidic conditions.

The invention thus relates to an alpha-amylase enzyme which has good heat resistance and stability under acidic conditions and which can be used under conditions comparable to those under which other amylases, such as glucoamylase, are used.

The alpha-amylase enzyme of the invention is characterized in that it has been prepared by culturing the microorganism Bacillus stearothermophilus strain ATCC 31 199 and has the following properties: 3,585,157 (1) molecular weight as determined by sodium dodecyl sulfate plate electrophoresis is about 90,000 - 100 000; (2) at least n, 70% of its initial alpha-amylase activity remains after heating for 10 minutes at 90 ° C at pH 6.0 without the addition of calcium ion; (3) at least about 50% of its initial alpha-amylase activity remains after heating for 30 minutes at 85 ° C at pH U.5 in a medium having a calcium ion content of 0.005 m and a starch content of 22.5 wt%; (it) at least about 50% of its initial alpha-amylase activity remains after heating for 60 minutes at 90 ° C at pH 6.0 without the addition of calcium ions; and (5) at least about 50% of its initial alpha-amylase activity remains after heating for 10 minutes at 80 ° C at pH U.5 in a medium having a calcium ion content of 0.005 m

The alpha-amylase enzyme of the invention is also characterized in that it lacks substantial protease activity, for example, its protease / alpha-amylase ratio is less than 3, and essentially less than 1.

Figure 1 shows the optimum pH ranges of the enzyme of the invention and Thermamyl alpha-amylase.

Figure 2 shows the optimum temperatures of the enzyme of the invention and Thermamyl alpha-amylase.

Figure 3 shows the thermal stability of the enzyme of the invention and Thermamyl alpha-amylase at 80 ° C at pH 5.5 in the presence of 5 mmol Ca ++ ions.

Figure 1+ shows the thermal stability of the enzyme of the invention and Thermamyl alpha-amylase at 90 ° C and pH 6.0.

Figure 5 shows the thermal stability of the enzyme of the invention and Thermamyl alpha-amylase at 90 ° C at pH 6.0 in the presence of 1 ninol Ca ++ ions.

Figure 6 shows the thermal stability of the enzyme of the invention and Thermamyl alpha-amylase at 85 ° C and pH + + in the presence of 22.5% starch.

Figure 7 shows the thermal stability of the enzyme of the invention and Thermamyl alpha-amylase at 85 ° C at pH i +, 5 in the presence of 22.5% starch and 5 mmol Ca ++ ions.

Figure 8 shows graphically the determination of the molecular weight of an enzyme according to the invention by the method of sodium sodium decodate sulphate plate electrophoresis.

Figure 9 shows the relationship between the pH and enzyme activity of an enzyme according to the invention and two known alpha-amylases from Bacillus stearothermo-philus.

Figure 10 shows the relationship between the temperature of the enzyme according to the invention and the known alpha-amylase from Bacillus s tearothermo-philus and the activity of the enzyme.

Figure 11 shows the deactivation curves of the enzyme of the present invention and previous alpha-amylases from Bacillus subtilis and Bacillus licheniformis when treated at 80 ° C at pH U in the presence of 5 mmol of calcium ions.

Figure 12 shows the deactivation curves of the enzyme of the present invention and previous alpha-amylases from Bacillus licheniformis and Bacillus stearothermophilus when treated at 90 ° C and pH 6.0 without the addition of calcium ions.

The ability of different microorganisms to produce heat-stable alpha-amylase was studied by culturing them at elevated temperatures (37 ° C, U5 ° C, 55 ° C, and 70 ° C), different pH values (5.0 and 7.0), and broth. Isolated thermophilic radial fungi were found to produce thermolabile alpha-amylase in high yield at 55 ° C and thermophilic fungi at t5 ° C, while thermophilic bacteria produce thermostable alpha-amylase in low yield at 70 ° C. Based on these initial studies, microorganisms were screened for alpha-amylase acid and thermal stability by performing cultures at 55 ° C and 70 ° C.

A total of 550 microbiological samples were collected from different locations, such as soil, hot spring water, compost, etc. samples.

Acid and thermal stability was obtained by determining the alpha-amylase activity of the culture filtrates before treatment for 10 minutes at 80 ° C, pH 5.0 in the presence of Ca ions (10 mmol), and after treatment.

Alpha-amylase activity was determined as follows:

A 0.2% by weight solution of soluble starch was prepared weekly as follows: 200 mg of soluble starch in 50 ml of 0.2 M acetate buffer (pH 0.5) with 0.013 moles of CaCl 2 · 2 ^ 0 was heated to 100 ° C. in boiling water, and the resulting solution was diluted to 100 ml with the same buffer. A tube containing 0.1 ml of enzyme solution and 0.3 ml of 0.2% by weight soluble starch solution was incubated for 5 minutes at 60 ° C. The reaction was quenched by the addition of 1.0 mL of 0.5 N acetic acid. 3.0 ml of 0.015% by weight iodine solution was added, stirred and the optical density was read against HgO at a wavelength of 700. The enzyme-free tube served as a blank and its optical density at 700 pn was denoted OD ^. One unit of enzyme was the amount that catalyzed the $ 10 decrease in blue color per minute under the conditions described above.

'(OD - 0D) x 100! WL units = cörrmö- b

Unless otherwise stated, the reported alpha-amylase activity has been determined by the method described above (in NML units). When alpha-amylase activity is denoted as CPC alpha-amylase units, the CPC unit is approximately 1/10 of the NML unit.

The units of CPC alpha-amylase activity are determined by the following method:

One milliliter of the appropriately diluted enzyme solution is added to 10 ml of a 1% solution of soluble starch in 0.03 molar acetic acid buffer solution (pH 6.0) and the reaction is carried out for 10 minutes at 60 ° C. One milliliter of the reaction solution is placed in a 100 ml graduated flask containing 50 ml of 0.02% hydrochloric acid solution, 3 ml of 0.05% iodine solution is added, and the total volume is adjusted to 100 ml by adding water. Measure the evolving absorbance of the blue color at a wavelength of 620 μm. The amount of enzyme required to degrade 10 mg of starch per minute is determined as 1 CPC unit.

1 crc unit = 1 ° ° s i ioViÖ x (dilution factor)

O

where D = absorbance of the control solution (water has been added instead of the enzyme solution) D = absorbance of the reaction solution s

The conditions and nutrient solutions used in the screening study are summarized in Table 1.

The results of the first and second screening studies are reported in Table 2.

6 58157

table 1

Culture conditions and nutrient solutions

Conditions

Plate culture ^ ^ Oblique surface culture ^ ^ Bottle culture ^^ a) 55 ° C pH 5 55 ° C, pH 5 50 ° C, pH 6 b) 55 ° C, pH T 55 ° C, pH 7 50 ° C, pH 7 c) 70 ° C, pH 5 70 ° C, pH 5 60 ° C, pH 6 d) 70 ° C, pH 7 70 ° C, pH 7 60 ° C, pH 7 (1) Cup growing (2) Sloping (3) Bottle culture ~ · nutrient solution___ culture lysis nutrient (BB) nutrient solution (BD) (bM)

Amylopectin 0.02% Soluble starch 1.0% 3.0% (NH 4) HPO, 0.025% Bacto-tryptone 0.5% $ 0.5 (Difco)

Yeast extract 0.025% Yeast extract 0.5 55 1.0 ί

Na citrate 0.01% CaCl 2 · 2H 2 O 0.05% 0.05%

MgSO 2 · 7H 2 O 0.05% MnCl 2 · i + HgO 0.05% COD 0.15% MgSO 2 • 7H 2 O - 0.05

CaCl2 * 2H2O 0.05% KH2PO0 0.1% 0.1%

Agar 2.0% Agar 2.0% 581 57 7

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8 58157

About 25 Xilla of thermophilic ray fungi isolated at 55 ° C had alpha-amylase production of 100-2,500 units per milliliter of culture broth, but no activity remained after heat treatment (80 ° C, 10 minutes, pH 5.0).

Of the 10βΐ thermophilic bacterial strains isolated at 55 ° C, a total of 35 strains had alpha-amylase production of 100–1000 units, and 10 of these strains produced thermostable alpha-amylase. Almost all of the alpha-amylases produced by thermophilic bacteria isolated at 70 ° C were acid and heat stable; the best yields were obtained from those isolated at pH 5.0.

The relationship between the activity of the 219 strains isolated at 70 ° C at pH 5.0 and the size of the Kirk region was examined. The results are shown in Table 3, which shows that $ 90 of these strains had a clear region diameter of less than 3 cm and produced 0-200 units of alpha-amylase. The highest production was obtained with strains with a clear area greater than 3 cm in diameter, but these accounted for only 10% of the strains tested.

Table 3

Relationship between the activity of the isolated strain and the size of the clear area

The size of the clear area from the insulation board (diameter in centimeters) _

Activity in the bottle (units / ml) <2 2-3 3-k.-K total 0 - 100 153 U0 15 0 208 - 200 32 10 6 - 500 0 0 2 0 2 - 1000 00 10 1 - 1500 00 11 2

Total 156 k2 20 1,219

It can be seen that an excellent screening method for finding microorganisms capable of producing acid- and west-resistant alpha-amylase in high yield involves isolating bacteria in the above experiments from colonies with a clear area diameter greater than 3 cm.

As a result of this screening, strains producing acid- and heat-resistant alpha-amylase enzymes were selected as the best producing.

This stock is deposited in the permanent collection of the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, where it is freely available.

9 58157

Deposited organism NML strain η · ο ATCC n; o

Bacillus stearothermophilus B-968 31 199 The bacteriological characteristics of the strain deposited with the ATCC are as follows: (1) Morphological characteristics: A. Cell shape and size: 0.6 x 2-3 pn; rodent bacteria rarely in chains B. Pleomorphism: negative C. Mobility: mobile and with flagella D. Spore: 0.6 x 1.5 / ml, egg-shaped: tennis racket-shaped spore body E: Gram staining: positive F: Acid resistance: negative (2) Growth in different media: A. Nutritional agar plate: actively spreading colonies with a rough surface and an uneven edge B. Nutrient agar sloping surface: good growth, cloudy white, active spread, comb-like growths C. Broth culture: transparent brown, white opaque surface D. Nutritional gelatin liquefaction of cuttings: E. Nutritional gelatine - agar plate: wide clear area F. Salt-nutrient liquid culture: growth inhibition when salt is 2% G. Milk agar plate: clear area when casein is hydrolysed H. Glucose agar v germination surface: good growth, similar colonies to nutrient agar I. Proteosi-peptone agar sloping surface: No growth (3) Physiological properties: A. Nitrate reduction: positive B. Catalase test: positive C. Voges-Proskauer reaction: positive D. Citric acid utilization: positive E. Hydrogen sulphide formation: positive F. Starch hydrolysis: strong hydrolysis G. Acid and gas formation: glucose, xylose, arabinose, mannitol form acid but no gas 10 581 57 H. Temperature and pH: effect on growth: ATCC No. 31,199 (B-968) _ 50-70 ° C good growth

The preferred pH range for growth is 5 ~ 8

Optimum pH 6-7

The above experiments were performed according to "Laboratory Methods Microbiology", W.F. Harrigan et al., And "Manual, of Microbiological Methods," published by the American Bacteriological Association.

The strain having the above characteristics was identified as a Bacillus stearo thermophilus strain according to Bergey's Manual of Determinative Bacteriology, 0th ed.

This strain was further purified by plate culture. The isolation, culture, and purification conditions for the selected strain are summarized in Table h. As can be seen from Table h, the purified strain ATCC No. 31,199 (B-968) produces 2111 NML units of alpha-amylase activity units per milliliter of culture broth (about 15 CFC points).

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The alpha-amylase enzyme of the invention is produced by culturing Bacillus stearothermophilus strain ATCC No. 31,199 in a culture medium suitable for its cultivation, which contains an assimilable source of carbon and nitrogen and other necessary nutrients.

Suitable assimilable carbon sources include carbohydrates such as starches, corn flour, wheat flour. The amount of carbohydrate used in the culture medium can vary within wide limits, for example from about 1% (w / v) to 25% (w / v), and preferably from about 10% (w / v) to 20% ( w / v) (percentages are calculated as dextrose). Preferred assimilable carbohydrates are starch and partially hydrolyzed starch (in a weight percentage of 1 to 5% by weight). "

The nitrogen source in the culture medium may be inorganic and / or organic. Suitable inorganic nitrogen sources include ammonium salts and inorganic nitrates.

Suitable organic nitrogen sources include peptone, meat extract, enzyme extract, casein, corn steep liquor, malt extract, soybean meal, skim milk.

In addition, the medium should contain conventional trace elements such as inorganic salts such as calcium chloride, magnesium sulfate, phosphates, sodium chloride, potassium chloride.

These carbon sources, nitrogen sources and inorganic salts can be used alone or in suitable mixtures. In addition, small amounts of metal salts, vitamins, amino acids can be used to increase growth and bacterial productivity.

The culture conditions used in the preparation of the alpha-amylase enzyme of the present invention are the same as those normally used for culturing thermophilic bacteria. Mainly the strain is cultured submerged in culture broth with stirring and aeration for 1-5 days at 50 ° C-70 ° C with a pH of 5 ~ 9. The enzyme accumulates in the cultured nutrient solution.

After preparation of the alpha-amylase enzyme according to the invention, the microbial cells are removed by conventional means, such as, for example, by centrifugation. Inorganic salts such as ammonium sulfate, sodium sulfate or magnesium sulfate, and / or water-miscible organic solvents such as acetone, ethanol, 2-propanol are then added to precipitate and concentrate the enzyme from the filtrate. Alpha-amylase can also be recovered by adding starch to the filtrate so that the alpha-amylase is absorbed into the starch.

To purify the enzyme from the enzyme-containing filtrate, the filtrate is treated with cold acetone, which is used in twice the volume of the filtrate, whereupon the enzyme precipitates. The precipitated enzyme is dissolved in 0.05 molar tris-hydrochloric acid buffer solution (pH 8.5) and the solution is allowed to run through a DEAE-cellulose column equilibrated with the same buffer solution (pH 8.5).

Most of the alpha-amylase remains in solution. Other proteins, pigments, etc. are absorbed into DEAE cellulose. The filtrate containing the alpha-amylase enzyme is referred to herein as the "partially purified enzyme". The partially purified enzyme can be further purified by dialysis against 0.01 tris-hydrochloric acid buffer solution (pH 7.9) and then passed through a hydroxyapatite column equilibrated with the same buffer solution. The enzyme is absorbed into the column at this stage. The absorbed enzyme is eluted by increasing the concentration of ammonium sulfate linearly from 0 molar to 0.5 molar.

After concentration, the partially purified enzyme thus obtained is passed through a Sephadex G-150 column. The enzyme is poorly absorbed into Sephadex and is extracted into a fraction with a molecular weight of less than 100,000. The activity of the purified enzyme thus obtained has increased 100-fold compared to the original filtrate, but in plate electrophoresis, among other alpha-amylase enzyme bands, other strips, and the enzyme is not yet crystalline.

The following example is intended to more fully illustrate the method described above and to present the preferred mode for carrying out the various objects of the invention.

Example

In a culture medium containing 3% corn starch, 0.5% peptone, 1% corn steep liquor, 0.05% calcium chloride, 0.05% manganese chloride, 0.05% magnesium chloride and 0.05% potassium chloride, the pH was adjusted to 7.5 . A 50 ml sample of this culture medium was placed in a 500 ml batch flask and sterilized for 15 minutes at 121 ° C. The sterilized broth was inoculated with Bacillus s.tearother-mophilus strain ATCC No. 31,199 (B-968) and stirred for U days at 55 ° C. After culturing, the microbial cells were removed by centrifugation. The alpha-amylase activity of the filtrate was 1U CPC units / ml. Alpha-amylase was precipitated by adding twice the volume of 2-propanol relative to the amount of filtrate. The precipitate was lyophilized to a dry powder. The crude enzyme powder was dissolved in 0.05 M Tris-HCl buffer (pH 8.5), and the solution was passed through a DEAE-cellulose column equilibrated with the same buffer solution. The alpha-amylase enzyme was not absorbed into DEAE-cellulose, while most other proteins, pigments, etc. were absorbed into DEAE-cellulose and were thus removed. The partially purified enzyme thus obtained was concentrated and then passed through a Sephadex-G-150 column. The enzyme was poorly absorbed into Sephadex and eluted therefrom as a fraction having a molecular weight of less than 100,000. The purified enzyme thus obtained was lyophilized and had a relative activity of 230 CPC units / mg protein.

The effect of temperature and pH on alpha-amylase formation was tested with the isolated strain. Alpha-amylase activity was reported as the average of the peak activities of the three experiments. The composition of the nutrient solution used in both the pre-culture and the main culture was the same as shown in Table 1 (B-M nutrient solution). The results of these experiments are summarized in Table 5.

Table 5 111 58157 Effect of temperature and pH on δ-amylase formation.

Temperature__Activity units / ml culture time_ ATCC (° C) pH 5.5 pH 6.0 pH 6.5 pH 7.0 pH7.5 pH 8.0 No. 31 199 1 + 5 1U3 (139) 133 (139) 113 (139) 115 (139) 86 (139) 50 686 (139) 663 (139) 5 ^ 3 (139) ^ 93 (139) * + 91 (139) 55 1 608 (U3) 1 365 (1 + 3 ) 1 267 (1 + 3) 1 025 (1 + 3) 9 * + 6 (1 + 3) 60 1 k85 (1 + 3) 1 1 + 57 (1 + 3) 1 295 (1 + 3) 1 15l + (1 * 3) 1 117 (1 + 3) 65 0 (65) o (65) o (65) o (65) o (65)

It can be seen from Table 5 that in the culture of strain ATCC No. 31,199, the optimum pH of the nutrient solution is 5.5 at a temperature of 55-60 ° C. -

Table 6 summarizes alpha-amylase production in the isolated strain.

Experimental slides were first used in the experiments, and the strain was transferred 9-H times to two types of sloping slides, followed by culture in liquid.

Thus, a suitable sloping nutrient solution was found that was able to maintain continuous alpha-amylase production. Table 7 defines the effect of nutrient solution volume on alpha-amylase formation. The effect of aeration on alpha-amylase formation was studied by varying the volume of the nutrient solution in 500 ml flasks.

Table 6 Formation of C 1-4 amylase using multiple inocula on sloping surfaces.

ATCC

Nutritional solution Pullovil.i life - 'no. and number of transplants Activity, Conditions units / ml temperature „_ (v il.i lifetime. t) (° C) ___ 31 199 BD * 1 2 1 163 (W +) 55 5.5 BD, 9 1 013 ( 68) 55 5.5 NA * 2 9 775 (68) 55 5.5 * 1 Sloping surface nutrient solution * 2 Nutrient agar sloping surface for screening Bacto-peptone (Difco) 0.5%

Soluble starch 1.0% Bacto beef extract

Bacto-tryptone (Difco) 0.5% (Difco) 0.3%

Yeast Extract 0.5% Agar 2.0?

CaCl 2 · 2 H 2 O 0.05% pH 7.2%

MnCl 2 + H 2 O 0.05% KHgPO ^ 0.1%

Agar 2.0% PH 5.0

Table 7 15 58157

Effect of nutrient solution volume on amylase formation

Pullovil.j elmä ATCC Oblique Surface- Conditions Nutrient Activity, No. Transitions T_. . pH volume ml / 500 ml units / ml number f ° C) & flask (culture time, t) 30 1 131 (Ui1) ho 1 2ko (hh) 31 199 BD-2 55 5.5 50 1 163 (¾¾ ) 60 1 05U (68) TO 1 22¾ (68) The strain of this invention (ATCC No. 31 199) was purified and the properties of the alpha-amylase produced by it were compared with those of purified Thermamyl Liquid 60, Batch AN 1005 · η by the method described below.

The alpha-amylase enzyme was precipitated from the culture filtrate of ATCC 31,199 or Thermamyl-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. Soluble starch was added to the solution to give a 20% starch suspension. This suspension was heated for 30 minutes at 85 ° C and the precipitate was removed by centrifugation. The solution was then dialyzed against 0.05 M Tris-HCl buffer (pH 8.5) containing 10 mmol Ca ++ ions by changing the buffer twice. The dialysate was treated on a DEAE-cellulose column equilibrated with dialysis buffer. Alpha-amylase was not absorbed onto the column and eluted with the same buffer. Fractions with alpha-amylase activity were pooled and the alpha-amylase enzyme was concentrated by precipitation with acetone. Alpha-amylase activity was determined by the CPC method described above. The cleaning method is summarized in Table 8.

Table 8 16 5 81 5 7 Purification of oC amylase

Purification Broth Acetone Thermal DEAE Process or Crude Precipitation Treatment Cellulose Q 1 -amylase S i ent Enzyme - Finished st e ATCC No. 31 199

Total activity (CPC units) 9,910 8,210 7,214 2,310

Total protin (mg) 4914 1969 1206 33

Cmi female active mouth (units / mg) 2.0 4, 2 6.0 70

Yield (%) 100 82.8 72.8 23.3 _ fr

Thermamyl ^ (manufactured by Novo Industri A / S)

Total activity (CPC units) 12,100 9,400 9,540 2,410

Total protein content (mg) 684 286 I83 24

Specific activity (units / mg) 17.7 32.9 52.1 100.4

Yield (%) 100 77.7 78.8 19.9

The acid and heat resistance of the partially purified alpha-amylase preparation and Thermanyl-alpha-amylase (containing 7 units / ml) produced by strain ATCC No. 31,199 was determined by incubation for 60 minutes under the following conditions: - (a) 90 ° C, pH 6.0, Ca ++ 0 or 1 mmol »(b) 80 ° C, pH 4.5 Ca ++ 5 mmol} and (c) 85 ° C, pH 4.5, Ca ++ 1 mmol or 5 nmol, 22.5% soluble starch. 15 ml of alpha-amylase preparation was dialyzed in a Visking 8/32 cellulose tube against 0.05 M calcium acetate buffer (pH 4.5-6.0) containing 0-5 nmol calcium acetate for 3 hours at 4 ° In C, changing the buffer twice. 4 ml of dialysate was then placed in small test 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-amylase activity was determined. idc. .

0.3 ml of dialyzed alpha-amylase enzyme solution, 0.5 ml of 1 mol. calcium acetate buffer, 0.2 ml 0.5 ~ mol. CaCl 2 and 3.0 ml of a $ 30-soluble starch slurry were pipetted into a test tube. The mixture was then incubated at 85 ° C for 30 minutes with constant agitation. The test tubes were then rapidly cooled in an ice water bath and the remaining alpha-amylase activity was determined.

Residual alpha-amylase activities were determined 5, 10, 20, 30, and 60 minutes after incubation.

The pH optimum and optimum temperature of ATCC 31 199 alpha-amylase and Thermamyl alpha-amylase were otherwise determined under the CPC experimental conditions described above except that the pH or temperature of the reaction mixture was changed. The results of the experiments are shown in Figures 1 and 2. The pH optimum of ATCC No. 31 199 alpha-amylase was between H, 0-5.2 and that of Thermamyl alpha-amylase U.5 · Thermamyl alpha-amylase was found maintaining high enzymatic activity in the neutral and alkaline pH range.

The optimum temperature for ATCC No. 31,199 alpha-amylase was 75 ° C and for Thermamyl alpha-amylase 85 ° C. The ratio of enzymatic activity to reaction temperature was very different for ATCC No. 31,199 alpha-amylase and Thermamyl alpha-amylase. It was found that the alpha-amylase activity of Thermamyl alpha-amylase increased by 20-30 ° C when incubated at 85 ° C and pH 6.0 in the presence of starch and Ca ++ ions. This fact may explain the difference in enzymatic activity and reaction temperature between ATCC No. 31 199 alpha-amylase and Thermamyl alpha-amylase.

Figure 3 depicts the inactivation curves of ATCC No. 31,199 alpha-amylase and Thermamyl alpha-amylase when incubated at 80 ° C at pH k.5 and in the presence of 5 mmol CaCl 2. The thermal stability of ATCC No. 31 199ϊη alpha-amylase was greater than the thermal stability of Thermamyl alpha-amylase under these conditions.

Figure h depicts alpha-amylase of ATCC No. 31,199, alpha-amylase of Thermamyl, and alpha-amylase produced by Bacillus stearothermophilus (described by Ogasawara et al., J. Biochem., 67, 65, 77, 83 (1970)). )) inactivation curves when incubated at 90 ° C at pH 6.0 in the absence of Ca ++ ions (described by Ogasawara et al.) incubation conditions were similar to those used in this experiment).

As can be seen from Figure U, the thermal stability of the alpha-amylase of this invention was much higher than that of both Thermamyl alpha-amylase and Ogasawara et al. Oh. alpha-amylase (although the alpha-amylase of the invention is also produced by Bacillus stearothermophilus).

Figure 5 depicts ATCC No. 31 199ίη alpha-amylase and Thermamyl alpha-amylase inactivation curves under otherwise the same conditions as described above for Figure 1+ (e.g. 90 ° C and pH 6.0), but the nutrient solution contained 1 nmole Ca ++ ions. The thermal stability of the alpha-amylase of this invention was still higher than that of the alpha-amylase of Thermamyl, but the difference was not as great as when incubated without Ca ++ ions. These facts suggest that ATCC No. 31 199 alpha-amylase binds Ca ++ ions more strongly than Thermamyl alpha-amylase and therefore require fewer Ca ++ ions to stabilize the protein molecule than Thermamyl alpha-amylase.

18 _ Λ ^ r- n 58157

Figures 6 and 7 depict the inactivation curves of ATCC No. 31 199 alpha-amylase and Thermamyl alpha-amylase when incubated at 85 ° C at pH 1.5 + soluble starch (22.5% dry matter). and in the presence of Ca ++ ions (1 mmol Ca ++ ions as described in Figure 6 and 5 mmol Ca ions as described in Figure 7). The thermal stability of alpha-amylase of ATCC No. 31,199 was better than that of Thermamyl1 alpha-amylase under both Figure 6 and Figure 7 conditions. Since the conditions of the above experiments described in Figures 6 and 7 are similar to many industrial starch liquefaction conditions, it is expected that the thermal stability of the alpha-amylase of this invention will prove to be better in the acidic pH range for industrial starch liquefaction and conversion.

The molecular weights of alpha-amylase of ATCC No. 31,199 were determined to be 96,000 as measured by the method of Weber and Osborn, J. Biol. Chem., 2kb, i + Uo6 (1969) by sodium dodecyl sulfate plate electrophoresis. The marker proteins were albumin (mp. 67,000), ovalbumin (mp. 1 * 5,000), chymotrypsin (mp. 25,000) and cytochrome c (mp. 12,500). The position of the alpha-amylase of this invention on a polyacrylamide gel was determined by placing the gel on an amylose-Azure agar plate and incubating it at 37 ° C.

The results of this experiment are described in Figure 8.

The molecular weight value of 96,000 for the alpha-amylase of this invention is much higher than the molecular weight values for the alpha-amylases produced by Bacillus stearothermophilus reported by Ogasawara et al., J. Biochem., 67, 65, 77, 83 (1970) ( reported molecular weight U8,000) and Manning et al., J. Biol. Chem., 236, 2952, 2958, 2962 (1961) (reported molecular weight 15,600).

The protease activity of the isolated strain was studied because, in the presence of a protease or a proteolytic enzyme among alpha-amylase, the enzymes tend to react with various proteins in many starch raw materials to form water-soluble protein hydrolysates such as amino acids. Therefore, the presence of the protease enzyme as an impurity among the alpha-amylase enzyme is detrimental to the efficient hydrolysis of starch raw materials. The protease activity of the alpha-amylase enzyme produced by the isolated strain was determined from the acetone precipitates of the culture filtrates at the peak of alpha-amylase formation using a modified Anson-Kagi reserve method.

The results obtained were compared with the enzyme preparations of Thermamyl alpha-amylase and CFC-bacterial alpha-amylase. As shown below, the culture filtrates of Thermamyl contained large amounts of protease. On the other hand, the thermostable alpha-amylase-producing strains of this invention did not generate significant amounts of protease.

Alpha-amylase protease activity

Enzyme Protease / alpha-amylase _ ratio_ ATCC No. 31,199 0.06 CPC-ELA 6.2

Thermamyl 60 36.5 19 58157

As can be seen from the above results, the alpha-amylase of this invention lacks significant amounts of protease activity. This is a significant advantage when using an enzyme to hydrolyze starch raw materials.

Use of an alpha-amylase according to the invention

The following use experiment describes the use of the alpha-amylase enzyme of the invention for liquefying starch.

Thirty grams of potato starch was suspended in 70 ml of water, 75 mg of calcium chloride dihydrate was added and the pH was adjusted to 1.5. To this slurry was added 50 CFC units of purified, ground enzyme obtained in the example to liquefy the starch at 85 ° C over 30 minutes. The hydrolysis rate (D.E.) of the liquefied solution was about 21 and the pH was U.3. After lowering the temperature to 60 ° C, the glucoamylase enzyme produced by Aspergillus niger * was added and the solution was saccharified and converted at 60 ° C for 18 hours. D.E. of the sugared and converted solution. was 97.5 and had a dextrose content of 96.0%.

As shown above, the alpha-amylase enzyme of the present invention hydrolyzes and liquefies starch. It can be used to convert soluble starch, amylose, amylopectin, glycogen, etc., to rapidly lower the viscosity of these substrates. When the enzyme of this invention reacts with soluble starch at a pH of 1.5 at 60 ° C, the starch-iodine reaction disappears at a rate of hydrolysis (D.E.) of about 15 and a final D.E. rate of 32-36. The sugars of the hydrolyzed product were analyzed and contained maltose, maltotriose, maltotetrose, and other malto-oligosaccharides with a small amount of glucose. The mutation of the reduced sugar product has been found to be negative. Accordingly, the enzyme of the present invention is a type of liquefying alpha-amylase enzyme and hydrolyzes the alpha-1,1 bonds of starch without hindrance.

The relationship between liquefaction (relative value) using the enzyme of the present invention and the pH used in the operation is shown in Figure 9 and w is compared to the corresponding ratio for known alpha-amylase enzymes produced by Bacillus stearothermophilus.

As shown in Figure 9, the optimum pH for the enzyme of the present invention is 1.2-5.2. The enzyme of the present invention does not lose its activity even if left for 2 hours at room temperature at a pH between 3 and 11.

The relationship between the liquefaction (relative value) using the enzyme of the present invention and the pH used in the operation is shown in Figure 10 and compared to the corresponding ratio for alpha-amylase produced by Bacillus stearothermophilus * as described by Ogasavara et al. for the enzyme of the invention, the preferred operating temperature is about 80 ° C.

As can be seen from the above, the alpha-amylase enzyme of the present invention can be used for the liquefaction and conversion of starch from the starch to sugar converting industry, the textile industry for glue removal and as additives in detergent compositions in the same manner as bacterial alpha-amylase.

The alpha-amylase enzyme of the present invention is particularly suitable for the liquefaction and conversion of starch in the production of maltodextrins for subsequent dextrose preparation using glucoamylase, as end-group isomerization can be avoided because the enzyme can be used efficiently at pH 4. , 5-5.0), thus increasing the yield of dextrose. The use of this new enzyme also reduces the ion exchange load of the purification process, as the pH does not need to be adjusted before conversion to sugar and conversion to glucose by the enzyme glucoamylase.

In a preferred use of the alpha-amylase enzyme of this invention, the enzyme is used to convert starch to starch hydrolyzate, leaving the remaining unconverted starch in its granular form. These processes are described in U.S. Patents 3,992,196 to 3,922,197; 3,922,198} 3,922,199 »3,922,200 and 3,922,201, all published November 25, 1975. In these methods using granular starch, the starch is treated at relatively low temperatures, i. at a temperature below the onset of gelatinization of the starch to the actual gelatinization temperature of the starch. In these processes, the glucoamylase enzyme is preferably used in combination with the alpha-amylase enzyme. The enzyme of the present invention is particularly suitable for use in this method because it has the same optimal pH range as glucoamylase.

The alpha-amylase enzyme of the invention can also be advantageously used in the process disclosed in U.S. Patent No. 3,912,590 to liquefy a starch slurry containing at least 25 weight starches, such as corn starch slurry, by treating at about 100 ° C to 115 ° C; at preferably 105 ° -110 ° C for 1-60 minutes, preferably 5-10 minutes, and then at 80-100 ° C, preferably 90-100 ° C, wherein the viscosity of the diluted solution is less than 300 cP . measured at 95 ° C. An incomparable advantage of using the alpha-amylase enzyme of the invention in this method is that a lower pH can be used, so that little or no pH change is required in the subsequent saccharification with glucoamylase.

In the preferred use of the alpha-amylase enzyme of the present invention, the starch slurry is treated to liquefy the starch with the enzyme at a pH in the range of about 3.5-6.5, preferably 4-5, at a temperature in the range of about 50 ° C to 100 ° C, preferably 60 ° C. 95 ° C.

In the case of the above-described hydrolysis methods for granular starch, the temperature range extends from the normal onset of gelatinization to the actual gelatinization temperature of the starch at about 60 ° C for corn starch. In the case of a direct liquefaction procedure, the enzyme-containing starch slurry 21 58157 is heated to liquefy the starch, e.g., with a jet heater, at a temperature in the range of about 85 ° C to about 95 ° C, and preferably about 90 ° C to 92 ° C. After initial leaching (such as in the hydrolysis of granular starch) or liquefaction, the slurry is preferably subjected to a "heat shock" treatment at a temperature above 100 ° C, preferably about 110 ° C to about 150 ° C, to liquefy any remaining starch granules. The liquefied starch is then cooled and treated by the addition of additional alpha-amylase, alone or with other enzymes such as glucoamylase, beta-amylase, pullulanase, glucose isomerase sequentially or simultaneously. If alpha-amylase is used alone, the temperature in the second enzyme treatment step is preferably between about 80 ° C and about 90 ° C, and preferably about 85 ° C, which is the optimum temperature for the enzymes of this invention.

If other enzymes such as glucoamylase and / or glucose isomerase are present, the temperature is slightly lower, i.e. 55 ~ 75 ° C and mostly about 60 ° C.

Conclusion The alpha-amylase enzyme of the present invention can be clearly distinguished from previously known alpha-amylases obtained from animals, plants, yeasts,

Fungi from imperfect fungi and skirts because the thermal stability of these previously known enzymes is so low that they completely lose their activity when treated for 5 minutes at 70 ° C at pH 6.0. Figure 12 compares the thermal stability of the alpha-amylase according to the invention (values used are essentially the same as in Figure 6) with the thermal stability of known alpha-amylases. It can be seen that the alpha-amylase enzyme of the invention has much better acid and heat resistance than previous alpha-Amylase enzymes. It is also shown in Figure 12 that the alpha-amylase of this invention retains at least 90% of its original activity when heated at 90 ° C, with a pH of 6.0, for 10 minutes without Ca ions and at least about 50% of its original alpha-amylase activity. when heated at 90 ° C, pH 6.0, for 60 minutes. It can be seen from Figure 11 that the alpha-amylase of the invention retains at least about 50% of its original alpha-amylase activity when heated for 10 minutes at 80 ° C at pH 1.5 in the presence of 5 mmol of calcium ions.

Table 9 shows the pH dependence of the alpha-amylase of this invention and the alpha-amylase enzymes produced by industrially used Bacillus subtilis and Bacillus licheniformis, as well as the alpha-amylase enzymes produced by Bacillus stearothermophilus known from the literature.

22 581 57

Table 9

Tested Optimal Suitable working Molecular enzymes working temperature weight _ PH_ ATCC No. 31 199 1 +, 0-5.2 80 ° C 96 000 Produced by B. subtilis _ alpha-amylase 1 1 +, 5-6.5 l + 5-60 ° C 1 + 9,000 Alpha-amylase produced by B. licheniformis c 5.0-9.0 76-78 C 22,500 - alpha-amylase produced by B. stearothermophilus a) Ogasawara et al. Δ 5.0-6.0 65-70 ° C 1 + 8,000 b) Campbell et al., Λ 55-70 ° C 15,600 ° C Ogasavara et al., J. Biochem., 67. (1970) » ^ Shigemasa Saito ABB, 155. 290 (1973).

^ Campbell et al., J. Biol. Chan. 236. 2952 (1961).

^ Campbell et al., J ^. Biol. Chem., 236, 2958 (1996).

GB Patent No. 1,296,839 discloses an alpha-amylase produced by B. licheniformis having a molecular weight of 18,000 to 20,000 and an alpha-amylase produced by B. subtilis having a molecular weight of 96,000.

Weaving some other known alpha-amylase enzymes, the enzyme of this invention is inhibited by mercury and EDTA, but is stabilized by calcium.

Claims (2)

23 58157 Pat entt iva suction set: A heat- and acid-resistant alpha-amylase enzyme, characterized in that it is prepared by culturing the microorganism Bacillus stearothermophilus strain ATCC 31 199 and has the following properties: (1) the molecular weight determined by sodium dodecyl sulfate plate electrophoresis is about 90,000-100,000; (2) at least about 70% of its initial alpha-amylase activity remains after heating for 10 minutes at 90 ° C at pH 6.0 without the addition of calcium ion; (3) at least about 50% of its initial alpha-amylase activity remains after heating for 30 minutes at 85 ° C at pH 1.5 in a medium having a calcium ion content of 0.005 μm and a starch content of 22.5% by weight; (* 0 at least about 50% of its initial alpha-amylase activity remains after heating for 60 minutes at 90 ° C at pH 6.0 without the addition of calcium ion; and (5) at least about 50% of its initial alpha-amylase activity remains about 50% after heating for 10 minutes at 80 ° C at pH 1.5 in a medium with a calcium ion content of 0.005 m. A process for producing a heat- and acid-resistant alpha-amylase enzyme according to claim 1, characterized in that the strain of the microorganism Bacillus stearothermophilus ATCC 31 199 is cultured in a culture broth containing an assimilable carbon and nitrogen source at a temperature of 5 to 9 in the range of about 50-70 ° C for 1-5 days.