JPH0369509B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel amylase, a method for producing the same, and a method for producing maltopentaose using the same. Conventionally, maltopentaose has been produced by hydrolysis of starch or amylose using various amylases. However, amylases used in this conventional method are known to produce approximately 33.3% maltopentaose from amylose [Archives of Biochemistry
and Biophysics, 155 290-298 (1973)], but the decomposition products obtained using this amylase also contain maltooligosaccharides other than maltopentaose, making it unsatisfactory as a maltopentaose-producing amylase. . Maltopentaose has recently been used as a substrate for blood amylase measurement, and its range of use has expanded, and mass production is expected. However, as mentioned above, conventional methods cannot fully meet this demand. As a result of repeated research to search for amylase with excellent maltopentaose production ability, the present inventors aerobically produced Bacillus cereus NY-14, which the present inventors isolated from soil. We discovered that amylase, which has the ability to produce maltopentaose, is produced and accumulated outside the bacterial cells by culturing, and that when this amylase is applied to starch, etc., maltopentaose is produced at a high rate of over 60%. Based on this knowledge, the inventor of the present invention was completed. The present invention first relates to a novel amylase having the following properties. (1) When 0.25ml of this enzyme is applied to 0.5ml of 1% soluble starch and 0.25ml of 50mM acetate buffer (PH6.0, containing 10mM _CaCl2 ), 38% of the starch is reduced after 5 minutes.
of maltopentaose, 67% after 2 hours.
of maltopentaose is produced. (2) This enzyme acts on soluble starch, amylopectin, amylose, dextrin, and maltohexaose, but does not act on bullulan, dextran, β-cyclodextrin, and oligosaccharides from maltopentaose to maltose. (3) The optimal pH for this enzyme is 6 to 7 at 30℃,
It is stable between 6 and 10. (4) The optimum temperature for this enzyme is 50-55℃ at pH 6.0. (5) This enzyme has a pH of 5 or less at 30°C for 1 hour or at 4°C.
If left for 24 hours, it will become inactive. Also, at 70℃, 30
It is inactivated by heating for 1 minute. (6) This enzyme was prepared in 50mM acetate buffer (PH6.0) at 4℃.
The enzyme is completely inactivated by dialysis against CaCl2 for 24 hours, and the inactivated enzyme is not activated even when _CaCl2 is added. Furthermore, there is no deactivation when dialyzing against a solution containing 10mM CaCl ₂ in the same buffer. (7) The molecular weight of this enzyme is approximately 90,000 (according to SDS-acrylamide gel electrophoresis). A second aspect of the present invention is to produce a novel amylase, which is characterized by culturing a microorganism that belongs to the genus Bacillus and has the ability to produce a novel amylase having the above-mentioned properties, accumulating the amylase in the culture, and collecting the amylase. It is a manufacturing method. The third aspect of the present invention relates to a method for producing maltopentaose, which is characterized by allowing a novel amylase having the above-mentioned properties to act on a polysaccharide or oligosaccharide containing an α-1,4-glucosidic bond. The microorganism used as the novel amylase-producing microorganism of the present invention belongs to the genus Bacillus and may be any microorganism that has the ability to produce amylase having the above-mentioned properties.For example, Bacillus cereus isolated from soil by the present inventors NY-14 is mentioned. The mycological properties of this bacterium are as follows. Morphological properties Cell shape and size In 0.5% sodium chloride broth medium at 30°C, 24
Cells cultured aerobically for 1 μ x 3.0-4.0 μ
5 to 5 when there are one or two or more long bacilli.
There are 6 of them in a chain. Presence or absence of interlocking There is no motility. There are no flagella either. Spore formation present. It has an oval shape of 0.7-0.8μ and is located at or near the center. Sporangial cells do not swell. Gram staining is positive. Growth status on various media Broth agar plate culture (30℃, 48 hours) Colonies are expansive, the surface is flat and rough, and the periphery is ear-shaped and grayish white. Juicy agar slant culture (30℃, 48 hours) Growth is abundant, coarse, opaque, expansive, and lobed at the margin. There is no stickiness. Flesh puncture culture (30℃, 5 days) Growth is thick over the entire surface and grows only along the puncture line. Meat juice liquid culture (30℃, 5 days) Growth is good, the liquid is transparent and can be precipitated, does not produce a bacterial film, and forms a bacterial ring that easily collapses. No pigment is produced. 0.5% Sodium Chloride Broth Agar Plate Culture (30℃, 48 hours) The shape of the colony is irregular, the surface is flat and rough, and the periphery is ear-shaped and grayish white. There are no comma-shaped villages. 0.5% sodium chloride broth agar slant culture (30°C, 48 hours) Growth is abundant, coarse, opaque, expansive, and lobed at the margin. There is no stickiness. 0.5% sodium chloride broth puncture culture (30℃,
(5 days) The growth is thick over the entire surface and grows only along the puncture line. 0.5% sodium chloride broth liquid culture (30℃,
(5 days) Growth is good, but the liquid becomes cloudy and sediment is formed. Does not produce bacterial membrane or pigment. Milk agar plate culture (30℃, 24 hours) Casein has a wide hydrolysis zone. Meat juice gelatin puncture culture (20℃, 7 days) Liquefies rapidly in eruptive or layered forms. Physiological properties Reduction of nitrate: Reduces VP test: Produces acetylmethylcarbinol MR test: Positive Utilization of citric acid: Utilizes Indole production: Does not produce Hydrogen sulfide production: Produces Ammonia production: Produces For milk Action: Coagulates Catalase: Positive Growth range: PH5.2-10.2, temperature 7-37°C Attitude towards oxygen: Grows on glucose under aerobic and anaerobic conditions. O-F test: Anaerobically decomposes sugar (glucose) to generate acid. Growth in Sabouraud dextrose culture solution/agar slant culture: Grows Presence or absence of acid and gas production from sugars: D-
It does not grow on xylose, L-arabinose, L-rhamnose, mannitol, D-raffinose, glycerose, D-galactose, D-mannose, lactose, or sucrose. It grows on soluble starch, D-glucose, D-fructose, trehalose, salicin, and maltose and produces acid but does not produce gas. Growth in 0.001% lysozyme: Growth Growth in 0.02% azide: Growth Growth in 7% salt: Growth 1974) (Bergey's Manual of
According to Deerminative Bacteriology 8th ed.1974), this bacterium belongs to Bacillus cereus, and the present inventors identified this bacterium as Bacillus cereus NY.
It was named -14. This bacterium was designated FERM P-6648 (FERM BP-329) by the Institute of Microbial Technology, Agency of Industrial Science and Technology.
It has been deposited as. As mentioned above, the microorganism used in the present invention may belong to the genus Bacillus and have the ability to produce amylase having the above-mentioned properties.
It is not limited to NY-14 and its variants and variants. Cultivation of microorganisms for producing the novel amylase of the present invention is carried out in a medium containing nutrients that can be utilized by common microorganisms. Specifically, for the production of novel amylase, polysaccharides or oligosaccharides containing α-1,4-glucosidic bonds, such as various starches, amylopectin, amylose, dextrin, maltohexaose, etc. are required. . Therefore, Bacillus cereus
The NY-14 medium contains one or more of these polysaccharides or oligosaccharides and other ingredients necessary for the growth of the bacteria, such as organic and inorganic nitrogen sources; organic and inorganic salts; vitamins, etc. Contains are used. Regarding the culture conditions for the microorganisms used in the present invention,
Although it varies depending on the bacteria used, it should be selected to maximize the production amount of the target new amylase. Specifically, Bacillus cereus NY-
For 14 strains, the pH of the medium was 8.0, the culture temperature was 30℃,
Appropriate culture time is about 24 hours, and aerobic culture is performed. For example, in the case of soluble starch, an effective amount of polysaccharide or oligosaccharide added to the medium is in the range of 0.5 to 2%. After completion of the culture, the novel amylase can be collected and purified from the culture solution by a combination of known appropriate methods. For example, solid matter is removed from the culture by appropriate means such as filtration or centrifugation to obtain a supernatant liquid, and the supernatant liquid is subjected to chromatography such as ammonium sulfate fractionation, gel filtration, or ion exchange to remove amylase. Refined products can be obtained. The amylase thus obtained has the following properties. (1) Action Add this enzyme to 0.5ml of 1% soluble starch and 0.25ml of 50mM acetate buffer (PH6.0, containing _{10mM CaCl2} ).
When 0.25ml (1.87U, activity was measured according to the titer measurement method shown in (4) below), 38% of the starch was absorbed after 5 minutes of reaction, and 67% after 2 hours.
of maltopentaose is produced. (2) Substrate specificity This enzyme acts on soluble starch, amylopectin, amylose, dextrin, and maltohexaose, but does not act on bullulan, dextran, β-cyclodextrin, and oligosaccharides from maltopentaose to maltose. (3) Optimal PH and stable PH The optimum pH for this enzyme is 6 to 7 at 30℃, and the PH
It is stable between 6 and 10. The relationship between PH value and amylase activity is as shown in FIG. 1, and the measurement method was according to the titer measurement method shown in (4) below under the following conditions. Measurement conditions: Temperature 30℃, 0.5M acetate buffer for PH4.0-6.0, 0.5M phosphate buffer for PH6.0-8.0,
0.5M Tris buffer for PH8.0~9.0, PH9.0~
10.0 uses 0.5M glycine buffer. (4) Titer measurement method Add 0.25 ml of enzyme solution to 0.25 ml of 50 mM acetate buffer (PH 6.0, containing 10 mM CaCl ₂ ) to 0.5 ml of 1% soluble starch, react at 30°C for an appropriate time, and then incubate. Add 0.2 ml of the solution to 0.4 ml of dinitrosalicylic acid (DNS reagent) solution, heat in a boiling water bath for 5 minutes, then add 1.8 ml of water and compare the colors at 540 μm. The amount of enzyme that generates a reducing power equivalent to 1 μmol of glucose per minute was defined as 1 unit (U). (5) Range of optimal temperature for action The optimal temperature for this enzyme is PH6.0 as shown in Figure 2.
at 50-55℃. The relationship between temperature and amylase activity was measured according to the measurement method described in (4) above, except that the reaction temperature was varied from 30 to 80°C. (6) Conditions for inactivation such as pH and temperature
If left for 24 hours, it will become inactive. Also, at 70℃, 30
It is inactivated by heating for 1 minute. (7) Inhibition, activation and stabilization This enzyme was prepared in 50mM acetate buffer (PH6.0) at 4°C.
The enzyme is completely inactivated by dialysis against CaCl2 for 24 hours, and the inactivated enzyme is not activated even when _CaCl2 is added. Furthermore, there is no deactivation when dialyzing against a solution containing 10mM CaCl ₂ in the same buffer. Various metal ions of this enzyme (final concentration 1×
10 ^-3 M) (treatment at 30°C for 10 minutes) is shown in Table 1. As is clear from the table, iron,
Inhibitory effects were observed in copper, mercury, silver, zinc, and cadmium. Table 1 Relative activity of metal compounds (%) - 100 MgCl ₂ 90 FeSO ₄ 2 CoCl ₂ 114 CuCl ₂ 0 HgCl ₂ 1 LiCl 107 BaCO ₃ 95 ZnSO ₄ 63 CaCl ₂ 109 NiCl ₂ 102 CdCl ₂ 57 AgNO ₃ 7 (8) Purification method The culture solution obtained by liquid culture using an Erlenmeyer flask in step 5 was filtered, and this filtrate was used as a crude enzyme solution. 950 ml of a crude enzyme solution with an activity of 0.533 units per ml is cooled with water, ammonium sulfate is added while keeping the temperature at 0 to 5°C, and a precipitated fraction is collected between 0.6 and 0.7 saturation. The obtained precipitate was added to a small amount of 50mM.
After dissolving in acetate buffer (PH6.0, containing 10mM CaCl ₂ ), dialysis is performed with the same buffer at a temperature of 0 to 5°C for one day and night using a dialysis membrane. Insoluble matter generated in the dialyzed enzyme solution is further removed by centrifugation or the like. Next, the enzyme was dissolved in 50mM acetate buffer (PH
Sephadex G- ₁₀₀ column (column volume 1.5 ×
27.5cm) and drain with the same buffer. The active fraction thus obtained is powdered by lyophilization. The enzyme purified by the above method was found to be single by polyacrylamide gel electrophoresis. In addition, its specific activity is 525.6 (units/mg protein)
It was hot. (9) Molecular weight SDS-acrylamide gel electrophoresis (Methods in Enzymology, Vol. 26, 3-27
(Page, 1972), this enzyme has a molecular weight of approximately
It is 90000. (10) Polyacrylamide disk electrophoresis Davis' original method [BJDavis, Ann.New
Tris-glycine buffer (PH8.3) with reference to York Acad.Sci., 121, Art 2, 404 (1964)]
When electrophoresed on a 7.5% medium standard gel for 90 minutes at 3 mA/unit and then stained with Coomassie blue, this enzyme showed one band as shown in Figure 3. The ratio of maltopentaose was determined by high performance liquid chromatography, and the protein was measured by the Lowry method using bovine serum albumin as a standard. The amylase obtained by the method of the present invention can produce maltopentaose in a high proportion from starch and the like. That is, polysaccharides or oligosaccharides containing α-1,4-glucoside bonds, such as various starches, amylopectin,
When the amylase of the present invention is applied to amylose, dextrin, maltohexaose, etc.,
Maltopentaose is generated and accumulated in the reaction solution. The reaction should be carried out under conditions that maximize the amount of maltopentaose produced, and may be appropriately determined in consideration of the properties of the amylase. After the reaction is completed, the reaction is stopped by heating to inactivate amylase, and the reaction solution is subjected to chromatography such as gel filtration or ion exchange to obtain a purified product of maltopentaose. According to the present invention, it is possible to efficiently produce maltopentaose in large quantities, and it is expected that the fields of application of maltopentaose, including the above-mentioned applications, will be expanded. Moreover, unlike conventional methods, the method of the present invention is characterized in that the purification operation is easy because there are few by-products of substances other than maltopentaose. Next, the present invention will be explained in detail with reference to examples. In addition, the amount of carbohydrates in the examples is expressed in terms of glucose using the phenol/sulfuric acid method, and sugar analysis was performed using high performance liquid chromatography and paper chromatography using the following method.
A certain amount of the sugar-containing solution was spotted on Toyo Paper No. 5 (19 x 19 cm) and developed twice in a closed container by the ascending method at 70°C using 65% n-propyl alcohol as a developing agent. After development, each oligosaccharide was hydrolyzed by treatment with glucoamylase at 40°C for 30 minutes, and color was developed by an alkaline acetone-silver nitrate immersion method. To determine the amount of maltopentaose produced, the culture solution or reaction solution was developed using paper chromatography using the above method, and then treated with glucoamylase, and the portion corresponding to maltopentaose was cut out.
The product extracted with hot water at 100°C for 15 minutes was measured in terms of glucose using the phenol-sulfuric acid method, and the weight was determined as maltopentaose weight. In addition, high performance liquid chromatography was also used as necessary. Amylase activity was determined by adding 0.25 ml of enzyme solution to 0.5 ml of 1% soluble starch and 0.25 ml of 50 mM acetate buffer (PH 6.0, containing ₁₀ mMCaCl2), incubating at 30°C for an appropriate time, and then adding 0.25 ml of enzyme solution to 0.5 ml of 1% soluble starch. ml to 0.4 ml of dinitrosalicylic acid solution and heated in a boiling water bath for 5 minutes, then add 1.8 ml of water.
ml was added and the color was compared at 540 μm, and the amount of enzyme that produced a reducing power equivalent to 1 μmol of glucose per minute was defined as 1 unit. Example 1 Various carbohydrates 0.5%, peptone 1%, salt 0.5%
Put 10 ml of medium (PH8.0) into a test tube (21 x 210 mm)
After dispensing and sterilizing Bacillus cereus NY-14
(FERM BP-329) was inoculated with one platinum loop and heated at 30℃.
Culture was performed with shaking (250 rpm, amplitude 20 mm) for 24 hours. After the culture was completed, the culture was filtered to remove bacterial cells, and the amylase activity in the supernatant was measured. Second result
Shown in the table.

[Table] The supernatant was sufficiently cooled (3 to 5°C), ammonium sulfate was added to give a saturation of 0.9, and the precipitate fraction was collected. The obtained precipitate was dissolved in a minimum amount of 50mM acetate buffer (PH6.0, containing _{10mM CaCl2} ), and then dialyzed against the same buffer using a dialysis membrane for one day and night. Insoluble matter generated in the dialyzed enzyme solution was further removed by centrifugation. A powdered enzyme preparation was obtained by freeze-drying this enzyme solution (yield: 90~
100%). Example 2 500 ml of a medium (PH8.0) containing 0.5% soluble starch, 1% peptone, and 0.5% salt was dispensed into a 5000 ml Erlenmeyer flask with a hemlock and sterilized. One method is to use Bacillus cereus NY-14 in a medium with the same composition as this medium.
(FERM BP-329) was cultured with shaking at 30°C for 24 hours, 10ml of this preculture was inoculated into the medium, and incubated at 30°C.
Shaking culture was performed at 120 revolutions per minute for 24 hours. After completion of the culture, the culture was centrifuged to obtain a supernatant. The amylase titer of this supernatant was measured and found to be 0.533 units/ml, and its specific activity was
It was 0.294 units/mg protein. Next, 950 ml of the supernatant liquid was sufficiently cooled (3 to 5°C).
Then ammonium sulfate was added and the precipitate fraction occurring between 0.6 and 0.7 saturation was collected. The obtained precipitate was added to a small amount of 50mM acetate buffer (PH6.0, 10mM
After dissolving in _CaCl2- containing), dialysis was performed for 24 hours against the same buffer using a dialysis membrane. Insoluble matter generated in the dialyzed enzyme solution was further removed by centrifugation. Next, this enzyme was equilibrated with 50mM acetate buffer (PH6.0, containing _10mM CaCl2) on a Sephadex G-100 column (column volume: 15x
27.5cm) and eluted with the same buffer. The resulting active fraction was single when electrophoresed on a polyacrylamide gel. In addition, the specific activity of this enzyme is
It was 525.6 units/mg protein and purified 1788 times.
A purified enzyme powder was obtained by freeze-drying this enzyme solution. Example 3 10 pieces of a medium (PH8.0) containing 0.5% soluble starch, 1% peptone, and 0.5% salt were placed in a 30-volume jar fermenter and sterilized at 121°C for 10 minutes. Bacillus cereus precultured in a medium with the same composition as this
Inoculate 200 ml of NY-14 (FERM BP-329) bacterial suspension and keep at 30℃ for 24 hours at aeration rate of 16/min and shaking rate.
Shaking culture was performed at 150 rpm. After the cultivation was completed, the culture was collected and centrifuged at 4°C to remove the bacterial cells. The amylase titer of the supernatant obtained was measured and found to be 0.513 units/ml, and the specific activity was 0.290 units/mg protein. Next, purified enzyme powder was obtained from this supernatant in the same manner as in Example 2. Example 4 0.5ml of 1% soluble starch, 50mM acetate buffer (PH
0.25 ml (1.87 U) of the amylase solution of the present invention was added to 0.25 ml (6.0, containing 10 mM CaCl ₂ ) and reacted at 30°C, and after 2 hours, the reaction was stopped by heating. Maltopentaose in the reaction solution was 3.17 mg/ml, and the starch ratio was 67%. The reaction solution was passed through an activated carbon column and a liquid chromatography column, and the resulting solution was concentrated and freeze-dried to obtain 3 mg of white powder of maltopentaose.

[Brief explanation of drawings]

FIG. 1 is a graph showing the relationship between PH and activity of the amylase of the present invention, and FIG. 2 is a graph showing the relationship between temperature and activity. FIG. 3 is an acrylamide gel disc electropherogram of purified amylase.

Claims (1)

[Claims] 1. A novel amylase having the following properties. (1) When 0.25ml of this enzyme is applied to 0.5ml of 1% soluble starch and 0.25ml of 50mM acetate buffer (PH6.0, containing 10mM _CaCl2 ), 38% of the starch is reduced after 5 minutes.
of maltopentaose, 67% after 2 hours.
of maltopentaose is produced. (2) This enzyme acts on soluble starch, amylopectin, amylose, dextrin, and maltohexaose, but does not act on bullulan, dextran, β-cyclodextrin, and oligosaccharides from maltopentaose to maltose. (3) The optimal pH for this enzyme is 6 to 7 at 30℃,
It is stable between 6 and 10. (4) The optimum temperature for this enzyme is 50-55℃ at pH 6.0. (5) This enzyme has a pH of 5 or less at 30°C for 1 hour or at 4°C.
If left for 24 hours, it will become inactive. Also, at 70℃, 30
It is inactivated by heating for 1 minute. (6) This enzyme was prepared in 50mM acetate buffer (PH6.0) at 4℃.
The enzyme is completely inactivated by dialysis against CaCl2 for 24 hours, and the inactivated enzyme is not activated even when _CaCl2 is added. Furthermore, there is no deactivation when dialyzing against a solution containing 10mM CaCl ₂ in the same buffer. (7) The molecular weight of this enzyme is approximately 90,000 (according to SDS-acrylamide gel electrophoresis). 2. A method for producing a novel amylase, which comprises culturing a microorganism that belongs to the genus Bacillus and has the ability to produce a novel amylase having the following properties, accumulating the amylase in the culture, and collecting the amylase. (1) When 0.25ml of this enzyme is applied to 0.5ml of 1% soluble starch and 0.25ml of 50mM acetate buffer (PH6.0, containing 10mM _CaCl2 ), 38% of the starch is reduced after 5 minutes.
of maltopentaose, 67% after 2 hours.
of maltopentaose is produced. (2) This enzyme acts on soluble starch, amylopectin, amylose, dextrin, and maltohexaose, but does not act on pullulan, dextran, β-cyclodextrin, and oligosaccharides from maltopentaose to maltose. (3) The optimal pH for this enzyme is 6 to 7 at 30℃,
It is stable between 6 and 10. (4) The optimum temperature for this enzyme is 50-55℃ at pH 6.0. (5) This enzyme has a pH of 5 or less at 30°C for 1 hour or at 4°C.
If left for 24 hours, it will become inactive. Also, at 70℃, 30
It is inactivated by heating for 1 minute. (6) This enzyme was prepared in 50mM acetate buffer (PH6.0) at 4℃.
The enzyme is completely inactivated by dialysis against CaCl2 for 24 hours, and the inactivated enzyme is not activated even when _CaCl2 is added. Furthermore, there is no deactivation when dialyzing against a solution containing 10mM CaCl ₂ in the same buffer. (7) The molecular weight of this enzyme is approximately 90,000 (according to SDS-acrylamide gel electrophoresis). 3. A new amylase-producing bacterium belonging to the genus Bacillus is Bacillus cereus NY-14 (FERM BP-329)
The manufacturing method according to claim 2. 4. A method for producing maltopentaose, which comprises reacting a polysaccharide or oligosaccharide containing an α-1,4-glucosidic bond with a novel amylase having the following properties. (1) When 0.25ml of this enzyme is applied to 0.5ml of 1% soluble starch and 0.25ml of 50mM acetate buffer (PH6.0, containing 10mM _CaCl2 ), 38% of the starch is reduced after 5 minutes.
of maltopentaose, 67% after 2 hours.
of maltopentaose is produced. (2) This enzyme acts on soluble starch, amylopectin, amylose, dextrin, and maltohexaose, but does not act on bullulan, dextran, β-cyclodextrin, and oligosaccharides from maltopentaose to maltose. (3) The optimal pH for this enzyme is 6 to 7 at 30℃,
It is stable between 6 and 10. (4) The optimum temperature for this enzyme is 50-55℃ at pH 6.0. (5) This enzyme has a pH of 5 or less at 30°C for 1 hour or at 4°C.
If left for 24 hours, it will become inactive. Also, at 70℃, 30
It is inactivated by heating for 1 minute. (6) This enzyme was prepared in 50mM acetate buffer (PH6.0) at 4℃.
The enzyme is completely inactivated by dialysis against CaCl2 for 24 hours, and the inactivated enzyme is not activated even when _CaCl2 is added. Furthermore, there is no deactivation when dialyzing against a solution containing 10mM CaCl ₂ in the same buffer. (7) The molecular weight of this enzyme is approximately 90,000 (according to SDS-acrylamide gel electrophoresis). 5. The production method according to claim 4, wherein the polysaccharide or oligosaccharide containing an α-1,4-glucoside bond is at least one substance selected from starch, amylopectin, amylose, and dextrin. .