JPH0116157B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to α-amylase that decomposes starch mainly into maltotriose. (Prior art) Conventionally, known amylases include glucoamylase, which decomposes starch from its non-reducing end into glycose units, β-amylase, which decomposes starch into maltose units, and α-amylase, which cleaves starch from its internal chains. , used in the industrial production of glycose and maltose. In contrast, few enzymes are known that effectively degrade starch-based substrates into maltotriose and maltotetraose units.
As an enzyme capable of hydrolyzing starch-based substrates into maltotriose, amylase produced by Streptomyces griseus strain N-A468 (Japanese Patent Publication No. 6915, No. 1983, Japanese Patent Publication No. 1983-1989)
6915) and α-amylase produced by Bacillus strain YT-1004 (Japanese Patent Publication No. 60-15315). The above maltotriose has a lower sweetness than glycose and maltose, so it has high utility value as an ingredient in low-sweetness foods and as an infusion ingredient in place of glycose and maltose. Therefore, it is desired to obtain an enzyme that effectively decomposes starch-based substrates into maltotriose. (Problems to be Solved by the Invention) An object of the present invention is to provide an enzyme that can effectively decompose starch-based substrates into maltotriose. (Means and effects for solving the problems) The α-amylase of the present invention has the following properties. Action and substrate specificity: α-glucan α-
Hydrolyzes the 1,4 bond to produce maltotriose. Optimal pH: 5.6 at 60℃. Stable pH: 5.9-8.1 at 55°C, 4.0-11.9 at 25°C. Range of suitable temperature for action: The optimum temperature is 65-75°C. Thermal stability: in the presence of 5mM calcium ions,
Stable up to 75°C when kept at pH 6.8 for 10 minutes. Molecular weight: Molecular weight determined by sodium dodecyl sulfate electrophoresis is 78,000. Isoelectric point: 4.6 Michaelis constant Km for amylopectin
Value: 1.0mg/ml Amino acid analysis:

【table】

[Table] The α-amylase of the present invention is produced by bacteria belonging to the genus Bacillus, particularly the facultative thermophilic bacterium Bacillus thermoamyloliquefaciens.
thermoamyloliquefaciens) sp.nov KP1071 strain (FERM P-8479). This strain is a new strain isolated and collected from soil by the present inventors, and its mycological properties are shown in Table 1. In Table 1, unless otherwise specified, the culture temperature was 55°C.

【table】

[Table] Identification of bacterial strains The bacterial strains that produce the enzyme of the present invention were identified based on the mycological properties mentioned above.
Dataminative Bacteriology 8th edition (1974), and P. Day. Walker (P.
D.Walker)・J. J.Wolf, Spore Research (1974) 247-264
From the page, it was identified as one bacterial species belonging to the genus Bacillus. This strain is similar to Walker and
According to Wolf's classification, Bacillus stearothermophilus is similar to the second group, but (1) the sporangium is non-swellable, (2) it hydrolyzes starch, and (3) it produces acid from cellobiose, rhamnose, and xylose. (4) the minimum growth temperature is different;
(5) different GC contents, and (6) Bacillus stearothermophilus ATCC 12980 (type
It was determined that this strain is a new strain of the genus Bacillus, which does not belong to this group, as it has a low homology of 29% to the DNA of Bacillus strain. Culture Conditions The medium for the above bacterial strain does not need to be special, and a normal medium can be used. As the carbon source, soluble starch, amylopectin, amylose, glycogen, etc. are used. Corn, potatoes, sweet potatoes, etc. can also be used. Peptone is a nitrogen source,
Yeast extract, soybean flour, corn dairy flour, etc. are used. As inorganic salts,
K ₂ HPO ₄ , KH ₂ PO ₄ , CaCl ₂ , MgSO ₄ ,
_{Na2HPO4 ,} ( _NH4 ) _{2HPO4 ,} etc. are used. For example, the following media are preferably used.

[Table] Culture pH is 6.5 to 8.5, preferably 7.5, culture temperature is
Culture is carried out at 45-60°C, preferably 55°C, for about 16 hours with aerobic stirring or shaking. The α-amylase of the present invention is secreted outside the bacterial cell. For example, when 200 ml of the medium is placed in the Erlenmeyer flask described in 2 and cultured with rotational shaking at 190 revolutions/min with a shaking width of 3.4 cm, the enzyme production amount is 0.04 units/ml (medium) according to the activity measurement method described below. be. Enzyme Collection Method To collect and purify the enzyme of the present invention from the above-mentioned culture solution, known purification methods can be used alone or in combination. For example, the culture solution is filtered or centrifuged to remove bacterial cells and concentrated, and then subjected to ultrafiltration or dialysis. Furthermore, after salting out using ammonium sulfate or the like, purification is performed using a DEAE sephadex column, phenyl sepharose column, droxyapatite column, DEAE cellulose column, etc.
An example of a purification method is shown below. (1) After removing and concentrating bacterial cells from the culture solution, 5mM
Sodium maleate/5mM _CaCl2 /0.02%
Dialyze into a buffer containing _NaN3 (PH6.8). (2) Salting out with ammonium sulfate (saturation degree 40
~70%). This (3) DEAE SEFF A-50
Chromatography is performed, and elution is performed using a buffer solution (PH8.0) containing 30 mM sodium glycinate/5 mM CaCl ₂ /0.02% NaN ₃ and NaCl using a concentration gradient elution method from 0 to 1 M NaCl. Next, (4) Phenylsepharose CL-4B chromatography was performed using the above buffer (PH8.0) containing ethylene glycol-ammonium sulfate as a solvent, and ethylene glycol was 0-50% and ammonium sulfate was 1-0M.
Elution is performed using the concentration gradient elution method. Furthermore, (5) hydroxyapatite was subjected to HTP chromatography using phosphate buffer (PH6.8) as a solvent.
Elution is performed using a 100mM concentration gradient elution method. continue,
(6) DEAE cellulose chromatography, 10mM sodium citrate/5mM _CaCl2 /
Elution is performed using a buffer solution (PH6.8) containing 0.02% NaN ₃ and NaCl using a concentration gradient elution method from 0 to 0.5 M NaCl. Table 2 shows the degree of enzyme purification in each step when purified by this method. The activities in Table 2 are values measured by the activity measuring method described below. The value in step 1 is the value measured when the culture solution from which the bacterial cells have been removed is concentrated and used as a sample. In this way, in step 6, 11.8 units/
mg protein (85 times purified compared to the original enzyme concentrate) of purified enzyme is obtained. The properties of the enzyme described below were investigated using this purified enzyme.

[Table] Activity measurement method Maleic acid buffer (PH6.8) containing 40mM sodium maleate with CaCl ₂ and oyster glycogen as a substrate
were added to 5mM and 1%, respectively.
Add 0.1 ml of an enzyme solution of unknown activity to this. This is incubated at 60°C for 10 minutes, and the increase in reducing power is measured by the Somogyi-Nelson method. One unit of enzyme is the amount of enzyme that produces 1 μmol of formyl group per minute. Properties of Enzyme The physicochemical properties of the enzyme of the present invention are shown below. Action and Substrate Specificity The present enzyme was added to maleic acid buffer containing 10 mM or 1% of the following substrates, an enzymatic reaction was carried out according to the activity measurement method, and the number of moles of formed formyl groups was measured. Table 3 shows the results of experiments conducted using various substrates.

[Table] In Table 3, α-limited dextrin (1) was determined using human salivary gland α-amylase, and α-limited dextrin (2) was determined using human salivary gland α-amylase.
Using α-amylase produced by A. subtilis),
Each was adjusted. This enzyme does not hydrolyze substrates other than those listed in Table 3, such as dextran, isomaltose, maltose, panose, sucrose, trehalose, and melezitose. Five types of substrate solutions each containing 1% of soluble starch, amylopectin, amylose, glycogen, and pullulan were prepared, and enzymatic reactions were performed according to the activity measurement method described above. The reaction time was 1 to 24 hours depending on each substrate, and the reaction solution was subjected to paper chromatography to examine the main product components. The results are shown in Table 4.

【table】

[Table] From Tables 3 and 4, this enzyme can be found in soluble starch,
It is found that it acts on glucans such as amylopectin, amylose, glycogen, maltotetraose, and matotopentaose to produce maltotriose. Since all of the maltotriose produced is in the α-type, it is clear that this enzyme is an α-amylase that acts on α-glucan and hydrolyzes its 1-4 bonds to produce maltotriose. Optimal PH and stable PH range Using this enzyme, 14 types of PH in the range of PH3.1 to 10.4
The enzymatic reaction was carried out under the following conditions according to the method for measuring activity. However, the buffer used is PH3.1 ~
40mM acetate buffer in the 4.0 range, PH
20mM succinate buffer for the 4.5-5.6 range, 20mM maleate buffer for the PH range of 6.2-7.1, and 20mM borate buffer for the PH range of 8.2-10.4. The relative activities are shown in FIG. From Figure 1, it can be seen that the optimum pH is approximately 5.6 at 60°C. This enzyme was dissolved in a buffer of a predetermined pH containing 5 mM CaCl ₂ and maintained at 25° C. for 15 hours, and then the residual activity was measured. PH is 3.0, 3.5, 4.0, 5.9, 8.1, 10.8
and set it to 11.8. Next, the temperature was set to 55℃ and the pH
4.2, 4.7, 5.2, 5.9, 8.1, 8.3, 8.8, 9.0 and 9.5
The residual activity was measured in the same manner. The residual activity of each is shown in Figure 2. The buffer used is PH
20mM acetate buffer for 3.0-3.6, PH4.1
20mM succinate buffer at ~5.5, PH6.0
20mM maleate buffer at ~6.5, and
20mM boric acid buffer for pH7.5-11.5.
From Figure 2, the stable PH range is 4.0 to 11.9 at 25℃, 55℃
You can see that it is 5.9 to 8.1. Range of suitable temperature for action Using this enzyme, enzymatic reactions were carried out under 17 different temperature conditions in the range of 30 to 95°C according to the activity measurement method. The relative activities are shown in FIG. It can be seen from FIG. 3 that the optimum temperature is 65 to 75°C. Conditions for inactivation due to PH, temperature, etc. From FIG. 2, it is clear that this enzyme is inactivated in 15 hours at 55° C. at PH of 4.1 or lower or PH of 9.5 or higher. The enzyme was dissolved in 40mM maleic acid buffer at pH 6.8 and maintained at a predetermined temperature for 10 minutes, and then its residual activity was measured. Holding temperature is 4℃, 55℃, 60℃
℃, 70℃, 72.5℃, 75℃ and 77.5℃. The respective residual activities are shown in FIG. 4 by two-dot chain lines. Figure 4 shows that this enzyme is stable up to 70°C under the above conditions. Next, add 5mM _CaCl2 , 4°C, 55°C, 60°C, 70°C, 72.5°C, 75°C.
The residual activity was measured in the same manner at 77.5°C, 80°C and 82.5°C. 4 for the system in which EDTA (ethylenediaminetetraacetic acid) was added instead of _CaCl2.
Similar experiments were conducted at 55°C, 60°C, 62.5°C, 65°C and 67.5°C. The respective residual activities are shown in FIG. 4 by solid lines and dashed-dotted lines. From Figure 4, when calcium ions are added, this enzyme is stabilized and does not deactivate up to 75℃, but
It can be seen that when EDTA is added, the heat resistance decreases and the stable range becomes up to 60°C. Inhibition, Activation, and Stabilization Using this enzyme, enzymatic reactions were performed in a solution containing various metal ions and reagents instead of Ca ions according to the activity measurement method. Table 4 shows its relative activity.
Shown below. In Table 4, for p-chlormark benzoate (pCMB) and 5,5'-dithio-bis(2-nitrobenzoate) (DTNBB), these reagents and enzymes were pre-mixed at 55°C for 20
After incubating for a minute, the substrate was added and allowed to react.

[Table] From Table 4, this enzyme contains Hg ²⁺ , Pb ²⁺ , Cu ²⁺ ,
It can be seen that the activity is inhibited by heavy metals such as Zn ²⁺ and Mn ²⁺ . Next, we investigated the effects of amines (including ammonium salts and amino acids) and sugars. The following amines at a ratio of 5mM or the following sugars at a ratio of 10mM
An enzymatic reaction was carried out in a solution containing a proportion of . Table 6 shows the degree of inhibition (%)
Shown below. In Table 6, Ki of maltotriitol
Values are at 50% inhibition.

Table 6 shows that this enzyme is strongly inhibited by histidine, benzylamine and maltotriitol. The enzyme is inhibited non-reactively by histidine and retardantly by benzylamine. Molecular weight The molecular weight determined by sodium dodecyl sulfate (SDS) electrophoresis is approximately 78,000. The values obtained by SDS electrophoresis above are the measured values of sedimentation coefficient (S ₂₀ w)
It agrees well with the value calculated from 5.4S. Mobility (by disk electrophoresis) Electrophoresis of this enzyme was performed using a 7.5% polyacrylamide gel tube (0.5φ x 5cm). Using Tris-glycine buffer (PH8.3), conduct electricity for 2 hours at 40°C with a current of 2 mA/tube. After electrophoresis, stain with amido black.
Decolorization was performed with % acetic acid. The mobility is the value when the mobility of bromophenol blue (BPB) added at the same time as the sample is 1.0. This enzyme formed a single band, and the migration distance toward the anode was 1.37 cm. Since the migration distance of BPB is 4.6 cm, the mobility of this enzyme is 0.31. A single band was also obtained by SDS electrophoresis, confirming that the enzyme was a single substance. Isoelectric point Wrigley's method (Wrigley, CW; J.
The isoelectric point measured by electrophoresis according to Chromatogr. 36 362-365 (1968) is 4.6. Km value Table 7 shows the Km value, maximum reaction rate (V), and V/Km when soluble starch, amylopectin, amylose, and glycogen were used as substrates. The enzyme reaction was carried out according to the activity assay method.

[Table] Table 7 shows that the most suitable substrate for this enzyme is amylopectin, and its Km value is 1.0 mg/ml. Amino acid analysis The results of amino acid analysis are shown in Table 8. In Table 8, the number of amino acid residues was calculated assuming that the enzyme has a molecular weight of 78,000 and has 14 methionine residues. Trp was measured optically.

[Table] This enzyme is a simple protein that does not contain sugar, cysteine, cystine, etc., and its N-terminus is threonine. Table 9 summarizes the physical constants of this enzyme.

[Table] This enzyme was introduced in the 1980s Special Publication listed in the prior art section.
It differs from the α-amylase derived from Bacillus YT-1004 strain described in Publication No. 15315 in various respects. For example, the optimal temperature range for action is 65-75℃, which is
It is much higher than the 50℃ of α-amylase of YT-1004 strain. While the α-amylase of YT-1004 strain is inactivated by about 70% when stored in buffer at 50°C for 10 minutes, the enzyme of the present invention remains stable up to 70°C and up to 75°C in the presence of calcium ions. . The molecular weight also differs. The enzyme of the present invention, as mentioned above,
It is characterized by being an enzyme with excellent heat resistance. Furthermore, the production rate of maltotriose is high, and when the substrate is amylose, it is completely decomposed into maltotriose. Thus, the enzyme of the present invention was found to be a new maltotriose-producing α-amylase, judging from its properties and various molecular characteristics. Using this enzyme, maltotriose can be effectively produced from various glucans (starch-based substrates). Maltotriose can be used in many ways, including as a low-sweetness food ingredient, a food improver (such as a humectant), and an infusion ingredient, so effective use of this enzyme is desired. Example (A) Cultivation of Bacillus KP1071 strain: Soluble starch 1.6
%, peptone 0.8%, yeast extract 0.3%,
_{Bacillus _ _ _}
KP1071 strain was cultured. Culture at 55℃ for approx.
The culture was carried out for 16 hours using a rotary shaking culture method. (B) Collection and purification of enzyme: Culture solution 49.4 obtained in section (A) was centrifuged to remove bacterial cells. After concentrating this, 5mM sodium maleate/
It was dialyzed against a buffer (PH6.8) containing 5mMCaCl2 _/ 0.02% _NaN3 , and salted out with ammonium sulfate (saturation degree 40-70%). this
Applied to DEAE-Sephadex A-50 chromatography (column 4.5φ x 42.5cm), 30mM sodium glycinate / 5mMCaCl ₂ / 0.02% NaN ₃
0 to 0 with a buffer containing (PH8.0) and NaCl
Elution was performed using a 1M NaCl concentration gradient elution method.
Next, it was subjected to phenylsepharose CL-4B chromatography (column 3.0φ x 24.5cm),
Elution was carried out using the aforementioned buffer (PH8.0) containing ethylene glycol-ammonium sulfate as a solvent, using a concentration gradient elution method with 0 to 50% ethylene glycol and 1 to 0 M ammonium sulfate.
Further, the mixture was subjected to hydroxyapatite HTP chromatography (column 3.0 φ x 28.0 cm), and elution was performed using a phosphate buffer (PH 6.8) as a solvent using a concentration gradient elution method of 5 to 100 mM. Then,
DEAE cellulose chromatography (column)
_{2.0 φ} Obtained. (C) Production of maltotriose: Using the enzyme obtained in section (B), an enzymatic reaction was carried out using amylose as a substrate according to the activity assay method. The concentration of amylose was 0.2%, 0.045 units of enzyme was used, and the reaction volume was 0.25 ml. 10 minutes after starting the enzyme reaction,
After 20, 40, and 60 minutes, the mixture was subjected to paper chromatography (developing solvent: n-butanol/pyridine/water = 6:4:3 (v/v)) to examine the production status of maltotriose. The results are shown in FIG. In FIG. 5, G1 to G9 indicate the number of glucose in oligosaccharides. For example, G3 indicates maltotriose with 3 glucose numbers, and G6 indicates maltohexaose with 6 glucose numbers. From FIG. 5, it can be seen that when amylose is the substrate, it is completely hydrolyzed to maltotriose in about 60 minutes. (D) Production of maltotriose: 1% soluble starch
The enzyme solution obtained in section (B) (containing 0.45 units of enzyme) was added to 2.5 ml of buffer containing a ratio of 2.5 ml, and an enzyme reaction was carried out for 4 hours according to the activity measurement method. This reaction solution was kept at 95°C for 5 minutes to inactivate the enzyme. Next, the mixture was desalted using an ion exchange resin monobet and concentrated to obtain a colorless and transparent starch sugar solution. When the product composition of this starch sugar solution was confirmed by liquid chromatography, the maltotriose content was 84%. (Effects of the Invention) According to the present invention, a novel α-amylase can be obtained from the Bacillus thermoamyloliquifaciens strain KP1071. This enzyme hydrolyzes a starch-based substrate (α-glucan) to produce maltotriose. This enzyme is characterized by having excellent heat resistance and producing maltotriose in high yield. Maltotriose can be used in many ways, including as a low-sweetness food ingredient, food improver (moisturizer, etc.), and infusion ingredient, so effective use of this enzyme is desired.

[Brief explanation of drawings]

Figure 1 is a graph showing the optimum pH of the enzyme of the present invention, Figure 2 is a graph showing the stable pH range of the enzyme, Figure 3 is a graph showing the optimum temperature of the enzyme, and Figure 4 is a graph of the enzyme of the present invention. A graph showing the stable temperature range, and FIG. 5 is a paper chromatography showing the production status of maltotriose when an enzyme reaction is carried out using this enzyme and using amylose as a substrate.

Claims (1)

[Claims] An α-amylase having the following properties. Action and substrate specificity: α-glucan α-
Hydrolyzes the 1,4 bond to produce maltotriose. Optimal pH: 5.6 at 60℃. Stable pH: 5.9-8.1 at 55°C, 4.0-11.9 at 25°C. Range of suitable temperature for action: The optimum temperature is 65-75°C. Thermal stability: in the presence of 5mM calcium ions,
Stable up to 75°C when kept at pH 6.8 for 10 minutes. Molecular weight: Molecular weight determined by sodium dodecyl sulfate electrophoresis is 78,000. Isoelectric point: 4.6 Michaelis constant Km for amylopectin
Value: 1.0 mg/ml Amino acid analysis: [Table] [Table] 2. α-amylase according to claim 1 obtained from a bacterium of the genus Bacillus. 3. α-amylase according to claim 1 or 2, which is obtained from Bacillus thermoamyloliquefaciens. 4. The α-amylase according to claim 1, which is thermostabilized by calcium ions. 5. Claim 1, wherein the substrate is at least one selected from the group consisting of maltotetraose, maltopentaose, α-limited dextrin, β-limited dextrin, soluble starch, amylopectin, amylose, and glycogen. α-Amylase.