JPH0131879B2 - - Google Patents

Description

[Detailed description of the invention]

[Technical Field] The present invention relates to a novel method for producing amylase using Bacillus bacteria. [Prior Art] Glucoamylase, β-amylase, and α-amylase that break down starch into glucose or maltose are known to exist widely in nature, but glucoamylase, β-amylase, and α-amylase, which break down starch into glucose or maltose, are known to exist widely in nature. Maltetraose (G4), Maltopentaose (G5) Maltohexaose (G6)
There are few reports on amylases that cleave at oligosaccharide units such as. These oligosaccharides are attracting attention as fillers, excipients, or sweetness modifiers for foods. especially,
Each oligosaccharide of G4 to G7 is also attracting attention as a substrate for measuring the activity of diagnostic amylase. [Purpose and effect] With the aim of establishing a method for producing these oligosaccharides, the present inventor has extensively searched for microorganisms in the natural world, and as a result, the inventors have found that α-glucans such as amylose, amylopectin, and starch are specifically malted. A novel amylase that hydrolyzes tetraose (G4) has been developed in Bacillus circulans.
circulans). As an amylase that generates maltotetraose from starch, it is known that amylase produced by Pseudomonas stuzeri hydrolyzes maltotetraose units from the non-reducing end of amylose and amylopectin. However, this enzyme is reported to leave high molecular weight limitodextrin from amylopectin and glycogen. [Archive Biochemistry and Biophysics (Archive
Biochemistry and Biophysics) Vol. 145, p. 105 (1971)]. It is reported that the yield of maltotetraose from starch by this enzyme is about 55%, and the yield from amylopectin is 52% [United States Patent USP 3654082 (1972) Amylase
Symposium, vol. 6, p. 31 (1971)]. other than this,
Amylase G4 and 5 are known as amylases that produce maltotetraose from starch (Japanese Patent Publication No. 59-4119), but this enzyme mainly produces both oligosaccharides maltotetraose and maltopentaose from starch. For example, the enzyme is produced as a component of maltotetraose from starch, from page 1, column 2, line 34 to page 2, column 3, line 4.
It is described that it produces glycated products containing 35% and 15-30% maltopentaose. However, the amylase produced by the Bacillus bacterium of the present invention was found to produce maltotetraose from starch at an extremely high yield of about 65% to 75%. However, as is clear from Table 1 and Figure 2, the amount of maltopentaose produced is
The amount is extremely small. The yield of maltotetraose is determined by the type of starch used, the degree of liquefaction (DE),
It depends on the amount of enzyme, etc., but usually glucose 1-5%, maltose 5-15%

〔composition〕

The present invention is directed to amylase G4, which belongs to the genus Bacillus and is an α-amylase with endo-type action that produces maltotetraose as a main component from starch.
This is a method for producing Bacillus amylase G4, which is characterized by culturing a microorganism that produces Bacillus amylase G4 and collecting amylase G4 from the culture. The contents of the present invention will be explained in more detail below. Amylase G4 produced according to the present invention has the following enzymatic properties. (1) Action: Decomposes α-glucans such as amylose, amylopectin, and glycogen into decomposition products whose main component is maltotetraose. This enzyme is a type of α-amylase that has an endo-type decomposition mode, and when it acts on liquefied starch, it has a
Maltotetraose is obtained with a yield of %. (2) Action temperature range and optimum action temperature: When used in the presence of 1% soluble starch and 0.05M phosphate buffer, it works up to about 75°C, and the optimum action temperature is about 50°C. (Figure 1a). (3) Action PH range and optimum action PH: Acts over a wide range of about 4 to about 12. The optimal working pH is 6-8.5 (works in 0.05M acetic acid or phosphate buffer, 1% soluble starch, Figure 1b). (4) Thermal stability: Approximately 40% when heated under 0.1M Tris buffer (PH7.0) at 50℃ for 10 minutes
The activity was inactivated, and approximately 85% of the activity was lost by heating at 55°C for 10 minutes [Figure 1c]. (5) PH stability; under 0.1M buffer at room temperature (25℃)
After standing for 3 hours, residual activity was measured. As a result, it was stable in the pH range of 6 to 9 [Fig. 1 d]. (6) Stabilization; when calcium ions are present,
An increase in thermal stability was observed (dashed line in Figure 1c). (7) Inhibitor: This enzyme is treated with 5×10 ^-3 M HgCl ₂ ,
CuSO ₄ , ZnSO ₄ , AgNO ₃ respectively
About 100%, about 95%, about 50%, about 30% inhibition. (8) Purification method: This enzyme was purified from the centrifuged supernatant of the liquid culture by ammonium sulfate fractionation, DEAE-Sepharose column chromatography, Biogel A0.5m column chromatography, and Biogel A0.5m column chromatography. It can be electrophoretically purified to homogeneity by rechromatography. (9) Molecular weight: The molecular weight measured by gel filtration using Biogel A0.5m was approximately 10,000. (10) Titer measurement method: Add an appropriate amount of enzyme to 0.5 ml of 2% soluble starch dissolved in 0.1 M phosphate buffer (PH7.0), make up to 1 ml with water, and react at 40°C. Under these conditions, the amount of enzyme that produced a reducing power equivalent to 1 μmol of glucose per minute was defined as 1 unit. Regarding the above enzymatic properties, when compared with the maltotetraose-generating amylase of Pseudomonas stutzeri known before the present invention, (1) the optimum action PH is in the wide PH range of PH 6 to 8.5 for amylase G4; The enzyme of Pseudomonas stutzeri has a pH of around 8. (2) When amylase G4 is applied to starch, maltotetraose is obtained with a yield of about 65-75%, whereas the enzyme of Pseudomonas stutzeri has a pH of around 8. (3) The molecular weight of amylase G4 is around 10,000, while the enzyme of Pseudomonas stutzeri has a molecular weight of 48,000 and 58,000.
Biochemistry and Biophysics Acta Volume 566,
88 (1979)], amylase G4 and the Pseudomonas stutzeri enzyme have significant differences in properties such as the yield of maltotetraose from starch, optimal action pH, and molecular weight. It can be called an enzyme.
As an exemplary bacterium that produces amylase G4, Bacillus circulans G-
List 4. The mycological properties of this bacterium are as shown below, and it has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as FAIKEN Article No. 820. (1) Morphology: Bacillus, width 0.5-1 x 5-6μ, non-motile, Gram negative. (2) Spores: Usually one near the end, no swelling of the sporangia. (3) Meat juice; cloudy and sediment. (4) Juicy agar: Good growth, light yellow to light brown, with unevenness around the edges and surface. (5) Glucose juice agar; good growth, pale yellow to pale brown. (6) Glucose nitrate agar; growth is poor. (7) Potato: Good growth, milky white. (8) Glucose-asparagine agar; growth is not very good. Pale yellow to yellowish brown. (9) Tyrosine agar; grows well, slightly brown. (10) Milk; slow coagulation, peptonization. (11) Indole; not generated. (12) Catalase; produced. (13) Acetylmethylcarbinol; not produced. (14) Hydrogen sulfide; not generated. (15) Citric acid; Use. (16) Nitrate reduction; negative. (17) Citric acid; use. (18) Salt broth: Grows well in medium containing 8% salt, and grows slightly in 10% salt. (19) Utilization of carbohydrates; acid is produced from D-glucose, D-fructose, D-mannose, L-arabinose, D-ribose, maltose, sucrose, starch, etc., but no gas is produced. D-xylose, L-rhamnose, L
- Availability of sorbose is not good. (20) Optimal growth temperature: around 26℃. (21) Maximum growth temperature: approximately 60℃ (22) Death temperature: Will not die even if heated at 100℃ for 30 minutes. The above mycological properties are described in Bergey's Manual of Determinative Bacteriology.
7th and 8th editions of Bacteniology (The Williams and Wilkins Company)
(1957 and 1974), this bacterium was identified as a type of Bacillus circulans and named Bacillus circulans G-4. The culture for producing amylase G4 according to the present invention uses organic nitrogen sources commonly used for culturing microorganisms, such as meat extract, peptone, yeast extract, casein, corn steep liquor, and soybean meal. Starch, dextrin, maltose, glucose, sucrose, etc. are used as carbon sources. and,
As a supplementary nutrient source, a medium containing an inorganic nitrogen source, phosphate, magnesium salt, and various metal salts is used. Cultivation is carried out aerobically at pH 5-9 and temperature 20-60°C. Since amylase G4 is an enzyme produced outside the bacterial cells, after the culture is completed, bacteria are removed by filtration or centrifugation, and the supernatant is collected. Concentrate if necessary and salt out with ammonium sulfate or sodium sulfate, or
Alternatively, an organic solvent such as acetone, isopropanol, ethanol, or methanol is added to obtain the enzyme as a precipitate, which is then dried and stored. The reaction of saccharifying starch using amylase G4 is carried out as follows. Starch is acid or α-
It is liquefied by amylase. Since the degree of liquefaction affects the yield of maltotetraose, it is preferable to
Liquefied starch with a DE of 20 or less is used (DE is the percentage of reducing power expressed as glucose in the solid content).
The substrate concentration is usually 5-40%. reaction PH
is usually 5 to 9, and the temperature is 40 to 60°C. This enzyme is significantly thermostabilized by the presence of calcium ions, so during the saccharification reaction, 5×10 ^-4 ~
Approximately 2×10 ⁻² M of calcium salt is added. Starch, amylopectin, by amylase G4
In saccharification reactions using substrates with branched α-1.6-glucosidic bonds, such as glycogen, if the reaction is carried out in the presence of α-1.6-glucidases such as isoamylase and prunalase, amylase G4 is activated to promote the saccharification reaction. Save money or
The yield of maltotetraose can be increased. Next, the details of the present invention will be explained with reference to Examples. Example 1 1% polypeptone, 0.3% dipotassium phosphate, 0.1% magnesium sulfate (heptahydrate), 1% soluble starch
Put 30ml of the medium consisting of into a 200ml Erlenmeyer flask,
After sterilizing at 120℃ for 10 minutes, inoculation with Bacillus circulans G4 (Feikoken Joyori No. 820)
The cells were cultured with shaking (160 rpm) at ℃ for 4 days. As a result of measuring the amylase G4 produced in the supernatant obtained by centrifugation after culturing, it was found that 1 ml of the medium
It was 6.4 units per unit. Example 2 The enzyme solution obtained in Example 1 was used to saccharify starch. As a substrate, liquefied starch with DE4.2
Calcium chloride 5×10 ^-3 M per 100 mg (solid content)
and amylase G4 as shown in Table 2, Substrate 1
Add 0.5 to 4 units per g to make a total volume of 1 ml, 50
The reaction was carried out at ℃ for 44 hours. After the reaction, the produced maltotetraose was quantified by high performance liquid chromatography, and the results were as shown in Table 2.

[Table] With an enzyme amount of 4u/g substrate, the sugar composition of the glycated product at 44 hours is glucose 0.0%, maltose 4.6
%, maltotriose 0.0%, maltotetraose 72.6%, and other sugars 22.8%. Example 3 In Example 2, the reaction was carried out in the presence of amylase G4 and pullulanase belonging to the genus Klebsiella (manufactured by Amano Pharmaceutical Co., Ltd.). The results obtained are shown in Table 3.

[Table] As is clear from the table, when starch was saccharified using amylase G4, when the reaction was carried out in the presence of pullulanase, saccharification was promoted and a saccharified product with a high maltotetraose content was obtained.

[Brief explanation of drawings]

Figure 1 a, b, c and d are amylase
Optimal working temperature, optimal working PH, thermal stability and PH of G4
Indicates stability. FIG. 2 shows the sugar composition of glycated products separated by high performance liquid chromatography. 1: Glucose, 2: Maltose, 3: Maltotriose, 4: Maltotetraose.

Claims (1)

[Claims] 1. A microorganism that belongs to the genus Bacillus and produces amylase G4, which is an endo-type α-amylase that produces maltotetraose as a main component from starch, is cultivated, and amylase G4 is collected from the culture. Bacillus amylase
G4 manufacturing method.