JPH0253035B2 - - Google Patents

Description

[Detailed description of the invention]

[Technical Field] The present invention relates to a method for producing maltose using a novel amylase G2 produced by Bacillus bacteria. [Prior Art] Various enzymes are known for producing maltose from starch, such as β-amylase produced by plants and microorganisms, and α-amylase produced by filamentous fungi such as Aspergillus oryzae. β-Amylase is an enzyme that regularly releases β-maltose from the non-reducing end of amylose and amylopectin.
When used in combination with enzymes that hydrolyze 1,6-glucosidic bonds), maltose can be produced in high yields. On the other hand, amylase produced by filamentous fungi such as Aspergillus degrades amylose and amylopectin in the endo-type.
In addition to maltose, glucose, maltotriose, and other oligosaccharides are also produced in large quantities. Recently, H. Outtrup and BENorman reported that Bacillus stearothermophilus (Bacilus stearothermophilus)
We reported that a novel maltogenic enzyme produced by A. stearothermophilus decomposes the non-reducible end of starch into the xo form, producing α-maltose. This enzyme does not strictly hydrolyze maltose units like β-amylase, but in the early stage of the reaction, in addition to maltotetraose (G4), maltotriose (G3), and maltose (G2),
Small amounts of maltopentaose (G5) and maltohexaose (G6) are also produced. It also breaks down Shardinger dextrin into maltose and glucose, and breaks down maltotriose into maltose and glucose. Therefore, the starch decomposition product produced by this enzyme contains 6 to 8% glucose. [Purpose and effect] The present inventor has conducted a wide search for microorganisms in search of amylase-producing bacteria that specifically produce maltose from starch, and as a result, isolated from soil and identified Bacillus megaterium. The identified bacterium was found to produce a novel and heat-resistant maltogenic enzyme. The inventor named this enzyme amylase G2. This enzyme is thought to hydrolyze starch into maltose units from its non-reducing end, but it was confirmed by metarotation and gas chromatographic analysis of the product that the sugar produced was the α-type. This enzyme cannot break down the α-1,6-glucoside bond of amylopectin, so it leaves limit dextrin, but the rate of decomposition of amylopectin to maltose is 2-3% compared to that of β-amylase. It is considered that the degree of decomposition is high and that it can be decomposed closer to branch bonds. The present invention has been made based on this knowledge. [Structure] That is, the present invention relates to a method for producing maltose using amylase G2, which is characterized in that a novel amylase G2 produced by a bacterium of the genus Bacillus is allowed to act on amylose, amylopectin, starch, or a partial decomposition product thereof. It is. The present invention will be explained in more detail below. Details of the properties of the enzyme of the present invention are as shown below. (1) Action: Hydrolyzes α-1,4-glucoside bonds of amylose, amylopectin, and glycogen into maltose units from their non-reducing ends. Since the sugar produced is α-maltose, α
- Although it is considered to be a type of amylase, it does not exhibit endo-type degrading action and leaves limit dextrin from amylopectin and glycogen. It does not decompose cyclodextrin, and its ability to decompose maltotriose is weak. It is similar to β-amylase from plants and bacteria, except that the sugar produced is α-type, but its decomposition limit for amylopectin and starches containing it is 2 to 3% higher. (2) Action temperature range and optimum temperature: When allowed to act under 1% soluble starch and 0.05M phosphate buffer, it acts up to about 75°C, and the optimum temperature is about 60°C (Figure 1a). . (3) Effective PH range and optimum PH; wide PH from about 3 to about 11
Acts on a range. The optimum pH is about 7-7.5 (works under 0.05M phosphate buffer, 1% soluble starch, (Figure 1b). (4) Thermal stability: 0.05M Tris buffer (PH7.0) When heated at 50℃ for 10 minutes, no deactivation is observed.
10% was inactivated, approximately 70% was inactivated by heating at 65°C for 10 minutes, and more than 90% was inactivated by heating at 60°C for 10 minutes (Fig. 1c). (5) PH stability: under 0.1M buffer, room temperature (30℃)
The mixture was left for 3 hours and the remaining activity was measured. As a result, it was stable in the pH range of 4.5 to 8 (Fig. 1d). (6) Stabilization: No increase in thermal stability due to calcium ions was observed. (7) Inhibitor: This enzyme is treated with 1×10 ^-3 M HgCl ₂ ,
AgNO, CuSO ₄ , NiSO ₄ each about 85
%, approximately 75%, approximately 70%, approximately 60% inhibited. Furthermore, it was inhibited by about 70% by 1×10 ⁻³ M p-chloromercury benzoate. (8) Purification method: This enzyme was purified from the supernatant of the liquid culture by ammonium sulfate fractionation, DEAE-Sepharose column chromatography, rechromatography using the same column, and column chromatography using Biogel A0.5m. It can be electrophoretically purified to homogeneity. (9) Molecular weight: The molecular weight measured by Cephadex G-200 was about 60,000. (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, make up to 1 ml with water, and react at 40°C. In this article, the amount of enzyme that generates a reducing power equivalent to 1 μmol of glucose per minute is defined as 1 unit. The above enzymatic properties were known prior to the present invention. Table 1 shows the results of a comparison with β-amylase of plant origin, β-amylase of bacteria of the genus Bacillus, and maltose-producing amylase of Bacillus stearothermophilus. That is, while plant β-amylase and Bacillus β-amylase produce only β-maltose, the enzyme of the present invention produces only α-maltose. Bacillus stearothermophilus amylase, like the enzyme of the present invention, produces α-maltose, but it has been reported that in the early stage of the reaction, it produces maltotetraose, maltotriose, and trace amounts of maltohexaose and maltopentaose. has been done. Therefore, these enzymes are essentially different from the enzymes of the present invention. Also, the optimal pH,
Since differences are also observed in enzymatic properties such as optimal temperature and molecular weight, the enzyme of the present invention can be said to be a novel enzyme. An example of a bacterium that produces the enzyme of the present invention is Bacillus megaterium. The mycological properties of this bacterium are as shown below.
It has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microbiological Research Institute No. 7978. (1) Form: Width: 0.7-1.3 x 2.4-5.0μ, usually 2 or more pieces connected in short chain form. Gram negative. (2) Spores; 2 near each end. Swelling of cells containing spores is hardly observed. (3) Growth; aerobically grown. Almost no growth is observed under anaerobic conditions. (4) Meat juice; cloudy and sediment. (5) Meat juice agar: Good growth, smooth surface, light brown color, but no pigment formation. (6) Glucose broth agar; grows better than broth medium. (7) Glucose nitrate agar; grows well. (8) Tyrosine agar; produces brown pigment.

[Table] (9) Glucose-asparagine agar; grows quite well. (10) Potato: Good growth, smooth surface, light brown color. Produces a brown pigment in the medium. (11) Use of citric acid; positive. (12) Starch hydrolysis; positive. (13) Acetylmethylcarbinol; not produced. (14) Gelatin; decomposes. (15) Milk; coagulates and peptonizes. (16) Nitrate reduction; positive. (17) Catalase; positive. (18) Utilization of carbohydrates: D-glucose, D-fructose, D-galactose, D-mannose, D-ribose, L-rhamnose, L-arabinose, D-xylose, D-mannitol, D-sorbitol, maltose, sucrose These methods are used to produce gas without producing any gas. (19) Optimal growth temperature: around 40℃. (20) Maximum growth temperature: approximately 50℃. (21) Killing temperature: Will not die even after heating at 100℃ for 20 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
(1974), this bacterium was named Bacillus megaterium G-2. A general culture for producing amylase G2 by culturing Bacillus megaterium G2 is performed as follows. Cultivation is usually carried out by aeration and stirring culture using a liquid medium. Nitrogen sources include meat extract, peptone, yeast extract, casein, corn staple, liquor, and soybean meal, which are commonly used as organic nitrogen sources when culturing microorganisms, or supplementary nitrogen sources such as ammonium chloride, Inorganic nitrogen sources such as ammonium phosphate and nitrates are used. As the carbon source, starch, dextrin, maltose, glucose, sucrose, etc. are used. Cultivation is carried out aerobically at pH 5-9 and temperature 20-60°C. Since amylase G2 is an enzyme produced outside the bacterial cells, after completion of the culture, bacteria are removed by filtration or heart separation, and the supernatant liquid is collected. The culture supernatant is concentrated if necessary, and the enzyme is collected as a precipitate by salting out with ammonium sulfate, sodium sulfate, etc., or by adding an organic solvent such as acetone, isopropanol, ethanol, or methanol, and then dried. save. The reaction of saccharifying starch using amylase G2 is carried out as follows. Starch is first liquefied with acid or the starch liquefaction enzyme α-amylase. Starch liquefaction degree (DE)
significantly affects the yield of maltose, and maltose can be obtained in higher yields by using liquefied starch with a lower degree of liquefaction. (DE is the percentage of reducing power expressed as glucose in the solid content). The reference concentration is usually
5-40%, the reaction pH is 5-8, and the temperature is 50-60°C. This enzyme cannot break down α-1,6-glucoside bonds in amylopectin, so when using amylopectin, starch containing it, or its derivatives as a substrate, it is necessary to use When the reaction is carried out in the presence of α-1,6-glucosidase such as amylase or pullulase, the saccharification reaction can be promoted and maltose can be produced in good yield. Next, the details of the present invention will be explained with reference to Examples. Example 1 Soy protein 2%, rice bran 2%, K ₂ HPO ₄ 0.3
%, MgSO ₄ 7H ₂ O 0.1%, soluble starch 2%,
CaCl ₂ 1 × 10 ^-3 M, MnCl ₂ × 1 × 10 ^-4 M,
^{A culture} _medium ⁽ _PH
7.0) Pour 30ml into a 200ml Erlenmeyer flask and heat to 120°C.
After sterilizing it for 15 minutes, it was inoculated with Bacillus megaterium (Feikoken Bacteria No. 7978) and cultured with shaking (160 rpm) at 30°C for 3 days. After culturing, the amylase G2 activity produced in the supernatant obtained by centrifugation was measured and found to be 9.0 units per ml of culture medium. Example 2 Ammonium sulfate was added to the culture supernatant obtained in Example 1 to a saturation of 80%, and the resulting precipitate was collected using a centrifuge and dissolved in water.
The mixture was dialyzed overnight against distilled water containing 10 ^-3 M L-cysteine and used as an enzyme solution for starch saccharification. The enzyme recovery rate was about 28%. Liquefied starch with DE1.83, 5.81, 8.05 and 12.6 100 each
1 unit of amylase G2 was added to make the total volume 1 ml, and the reaction was carried out at 55°C for 3 days. After the reaction, the sugar composition of the saccharified solution was analyzed by liquid chromatography, and the results were as shown in Table 2.

[Table] Example 3 Ammonium sulfate was added to the culture supernatant obtained in Example 4 to a saturation of 8%, and the resulting precipitate was collected using a centrifuge and dissolved in water to give a concentration of 1×10 ^{- 3} M L-
Distilled water containing cysteine was dialyzed overnight and the enzyme recovery rate for starch saccharification enzyme solution was approximately 82%.
It was %. ) Add 6 x 10 ^- units of amylase to 20 mg each of soluble starch, short-chain amylose (DP17), amylopectin (made from corn), and glycogen (made from oysters),
The total volume was 2 ml, and the reaction was carried out at pH 7 and 40°C for 6 days. After the reaction, the sugar composition of the saccharified solution was measured by liquid chromatography, and the results were as shown in Table 3.

[Table] Example 4 Add 2G of amylase to 1g of liquefied starch with DE1.43.
Units and Klebsiella pneumonia
1, 2 or 3 units of prilanase (manufactured by Nakaze Biochemical Co., Ltd.) produced by Pneumoniae) were added, and the PH
7. The reaction was carried out at 55°C for 3 days. After the reaction, the sugar composition of the saccharified solution was analyzed and the results were as shown in Table 4.

【table】 [Brief explanation of the drawing]

Figure 1 a, b, c and d are amylases, respectively.
It shows the optimum temperature, optimum PH, thermal stability and PH stability of G2.

Claims (1)

[Claims] 1 Amylase G2 produced by Bacillus bacteria,
A method for producing maltose using amylase G2, which is characterized by acting on amylose, amylopectin, starch, or a partial decomposition product thereof.