JPS6314955B2 - - Google Patents

Description

[Detailed description of the invention]

[Technical Field] The present invention relates to a method for producing maltotetraose using a novel amylase produced by bacteria belonging to the genus Bacillus. [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. There are few reports on amylases that cleave oligosaccharide units such as maltotetraose (G4), maltopentaose (G5), and maltohexaose (G6). Attention has been paid. In particular, each oligosaccharide of G4 to G7 is 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 Sseudomonas 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 approximately 55%, and the yield from amylopectin is 52% [United States Patent USP 3654082 (1972), amylase
Symposium, vol. 6, p. 31 (1971)]. However, the amylase produced by the Bacillus bacteria of the present invention was found to produce maltotetraose from starch at an extremely high yield of about 65 to 75%. The yield of maltotetraose is affected by the type of starch used, degree of liquefaction (DE), amount of enzyme, etc., but usually glucose is 1-5% and maltose is 5-15%.

〔composition〕

The present invention is directed to the production of maltotetraose by allowing amylase G4, which produces maltotetraose as a main component from starch, to act on starch, amylopectin, glycogen, or a partial decomposition product thereof. The present invention relates to a method for producing maltotetraose, which is characterized by saccharification. The contents of the present invention will be explained in more detail below. The enzyme 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 75%. (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
About 85% of the activity was inactivated 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 is 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 oxygen that produced a reducing power equivalent to 1 μmol of glucose per minute was defined as 1 unit. Regarding the enzymatic properties mentioned above, when compared with the maltotetraose-generating amylase of Pseudomonas stutzeri, which was known before the present invention, (1) the enzyme of the present invention has a wide pH range of 6 to 8.5;
(2) When the enzyme of the present invention is applied to starch, maltotetraose can be obtained with a yield of approximately 65-75%; (3) The molecular weight of the enzyme of the present invention is around 10,000, whereas the molecular weight of the enzyme of Pseudomonas stutzeri is about 48,000 and 58,000 [Biochemistry
Biochemistry and Biophysics Acta Volume 566,
88 (1979)], the enzyme of the present invention and the enzyme of Pseudomonas stutzeri have significant differences in properties such as the yield of maltotetraose from starch, optimal action pH, and molecular weight, and are novel. It can be called an enzyme. The α-1.6-glucosidase used in the enzyme-based saccharification reaction of the present invention is an enzyme that decomposes α-1.6-glucosidic bonds present in amylopectin, glycogen, pullulan, etc., and is used in various bacteria, actinomycetes, Produced by yeast etc. Depending on the difference in substrate specificity, it is sometimes called isoamylase, pullulanase, etc.
Collectively called debranching enzymes,
It is also called α-1.6-glucosidase. Amylase G4 of the present invention can efficiently increase the yield of maltotetraose using any α-1.6-glucosidase. For example, when starch is used as a substrate and amylase G4 is used alone, the yield of maltotetraose is about 65%, but when the saccharification reaction is carried out in the coexistence of pullulanase, the yield is about 65%.
I was able to get it up to 74%. As an exemplary bacterium that produces the enzyme of the present invention, Bacillus circulans G-4
List G-4. The bacteriological 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.
Bacteriology, 7th and 8th editions (The Williams and Wilkins Company);
(1957 and 1974), this bacterium was identified as a species 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 completion of the culture, bacteria are removed by filtration or centrifugation, and the supernatant liquid is collected. Concentrate if necessary,
Salting out with ammonium sulfate, sodium sulfate, or acetone, inpropanol, ethanol,
An organic solvent such as 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 to 2
Calcium salt of approximately ×10 ⁻² M is added. Next, the details of the present invention will be explained with reference to examples. Example 1 Polypeptone 1%, dipotassium phosphate 0.3%, magnesium sulfate (heptahydrate) 0.1%, soluble starch 1%
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 amylase G4 produced in the supernatant obtained by centrifugation after culturing, it was found that 1 ml of culture 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, 100 mg (solid content) of liquefied starch with DE4.2, calcium chloride 5 x 10 ^-3 M and amylase were added.
Add 2 units of G4 per 1 g of substrate, and add 2 units or 5 units of Klebsiella pullulanase (manufactured by Nagase Biochemical Co., Ltd.) per 1 g of substrate, make the total volume 1 ml with water, and react at 50°C. I let it happen. A certain amount was taken at 20 hours and 44 hours after the start of the reaction, and the produced maltotetraose was quantified by high performance liquid chromatography. The results are shown in Table 2.

[Table] As is clear from the table, in the reaction of starch saccharification using amylase G4, when the saccharification reaction is performed in the coexistence of pullulanase, the saccharification reaction is accelerated and the malt content is higher than when amylase G4 is used alone. A saccharified product with high tetraose content was obtained.

[Brief explanation of the drawing]

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. When amylase G4, which produces maltotetraose as a main component from starch, acts on starch, amylopectin, glycogen, or their partial decomposition products to produce maltotetraose, in the presence of α-1.6-glucosidase. A method for producing maltotetraose characterized by saccharification.