JPS62118885A - Heat-resistant alpha-amylase capable of producing maltotriose - Google Patents

Abstract

PURPOSE:To obtain an enzyme capable of effective hydrolyzing a starch based substrate into maltotriose, by obtaining an alpha-amylase, having 5.6 optimum pH at 60 deg.C and capable of hydrolyzing alpha-1,4 bonds of alpha-glucan to produce maltotriose. CONSTITUTION:A strain belonging to the genus Bacillus is cultivated in a culture medium containing a carbon source, nitrogen source, inorganic salt, etc., and the resultant culture fluid is filtered or centrifuged to remove microbial cells. The resultant fluid is then concentrated, ultrafiltered or dialyzed to afford the aimed heat-resistant alpha-amylase having the following properties; (1) Hydrolyzing alpha-1,4 bonds in alpha-glucan and producing maltotriose. (2) Optimum pH; 5.6 at 60 deg.C and stable pH; 5.9-8.1 at 55 deg.C and 4.0-11.9 at 25 deg.C. (3) Optimum temperature within the acting optimum temperature range; 65-75 deg.C, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to α-amylase that degrades starch mainly into maltotriose.

(Prior Art) Conventionally, known amylases include glucoamylase, which decomposes starch from its non-reducing end into glucose 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 glucose and maltose. In contrast, few enzymes are known that effectively degrade starch-based substrates into maltotriose and maltotetraose units.

As an enzyme that can hydrolyze starch-based substrates to maltotriose, Streptomyces gricius (Strep
amylase produced by strain N-A468 (Japanese Patent Publication No. 57-6915, Japanese Patent Publication No. 57-6915);
α-Amylase (US) produced by strain YT-1004 (
Japanese Patent Publication No. 60-15315) is only known.

Maltotriose is a component of low-sweetness foods because it has a lower sweetness than glucose and maltose.

It has high utility value as an infusion ingredient instead of glucose or 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) α of the present invention
-Amylase has the following properties.

(2) Action and substrate specificity: Hydrolyzes the α-1,4 bond of α-glucan to produce maltotriose.

■Optimal pH: 5.6 at 60°C.

■ Stable pH: 5.9-8.1.25℃ at 55℃
is 4.0 to 11.9.

■ Range of suitable temperature for action: The optimum temperature is 65 to 75°C.

■Thermal stability: In the presence of 51 calcium ions.

It is stable up to 75°C when maintained at pH 6.8 for 10 minutes.

■Molecular weight: The molecular weight determined by sodium dodecyl sulfate electrophoresis is 78,000.

■Isoelectric point: 4.6 ■Michaelis constant Km value for amylopectin: 1
．． Omg/m7! ■Amino acid analysis: The α-amylase of the present invention is
s) Bacteria of the genus, especially the facultative thermophile Bacillus thermoamy
loliquefaciens) sp, nov KP1
Strain 071 (Feikoken Bibori No. 8477; I'ERM P
-8479). This strain is a new strain isolated and collected from soil by the present inventors.

Its mycological properties are shown in Table 1. In Table 1, unless otherwise specified, the culture temperature was 55°C.

Table 1 Bacterial strains that produce the enzyme of the present invention are based on the above-mentioned mycological properties as described in Purge Aids Manual of Determinative Bacteriology, 8th edition (1974) and P.D. Walker (P. , D.Walke
r) ・Spore Research (J, Wolf) (19
74) From pages 247 to 264, Bacillus (Bacillus)
It was identified as one bacterial species belonging to the genus P. llus). This strain is classified as Bacillus according to the above-mentioned classification by Alker and Wolf.
It approximates the second group of stearothermophilus (
1) Sporangia are non-swellable, <Q powder is hydrolyzed, (3) acid is not produced from cellobiose, rhamnose and xylose, (4) minimum growth temperatures are different.

f5) different G C contents, and (6) Bacillus stearothermophilus ATCC12980 (ty
pe 5train) has 29% homology to DNA.
Bacillus (B), which does not belong to this group because of its low
It turned out to be a new strain of the genus Acillus.

The culture medium for the above-mentioned strain does not need to be special, and a normal culture 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, soy flour, corn distiller's solubles, etc. are used. As inorganic salts.

K211PO4+ KII2PO41CaC1z+ M
g5o4. Na2HPOa+ (Nl14)zl, etc. are used. For example, a primary medium is preferably used.

Culture p11 is 6.5-8.5. Preferably 7.5. The culture temperature is 45-60°C, preferably 55°C, and about 16°C.
Carry out the culture with aerobic agitation or shaking for an hour. The α-amylase of the present invention is secreted outside the bacterial cell. For example 21
According to the activity measurement method described below, the amount of enzyme produced when 200 ml of culture medium was placed in an Erlenmeyer flask and cultured with rotational shaking at 190 revolutions/min with a shaking width of 3.4 cm was 0.04 units 7 m (1 ( medium).

Collection and purification of the enzyme of the present invention from the above-mentioned culture solution can be accomplished using known purification methods 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, f&, DEAE Sephadex column, phenyl Sepharose column, and hydroxyapatite column were subjected to salting out with ammonium sulfate, etc.

Purification is performed using a DEAE cellulose column, etc. An example of a purification method is shown below.

(1) After removing and concentrating the bacterial cells from the culture solution, 5mM sodium maleate/5mM CaC1z/0.0
2% NaN.

Dialyze against a buffer containing (pH 6, 8). (2
) Salting out with ammonium sulfate (saturation level 40-70%)
).

This was subjected to (3) chromatography on DEAE Sephadex 8-5 and 30mM glycine sodium 1
Buffer containing 5mMCaC1z10.02% NaNz (
Elution is performed using a concentration gradient elution method of 0 to 1M NaCl (pH 8.0) and NaCl. Next, (4) Phenyl Sepharose CL-48 chromatography was carried out, and the above buffer solution (p
H8,0) as a solvent and ethylene glycol as 0 to 50
% and ammonium sulfate from 1 to 0 M using a gradient elution method). Furthermore, (5) hydroxyapatite IITP chromatography was applied to phosphate buffer (
Elution is performed using a concentration gradient elution method of 5 to 100 mM using pif 6.8) as a solvent. This was followed by f61 DEAE cellulose chromatography with 10mM sodium citrate, 15mM CaClz, 10.02% NaNt.
NaC1 by buffer solution (pH 6, 8) containing NaC1
Elution is performed using a concentration gradient elution method from O to 0.5M. Table 2 shows the degree of enzyme purification in each step when purified by such a method. The activities in Table 2 are values measured by the activity measuring method described below. The value in step 1 is a value measured when a culture solution from which bacterial cells have been removed is concentrated and used as a sample. In this way, in step 6, 11.8
units/g of protein (85 compared to the original enzyme concentrate)
A purified enzyme (purified twice) is obtained. The properties of the enzyme described below were investigated using this purified enzyme.

(Left below) CaC1z and oyster glycogen as a substrate were added to a maleate buffer (pH 6,8) containing 40mM sodium maleate.
gen) were added to 5mM and 1%, respectively.

To this is added 0.1 mj2 of an enzyme solution whose activity is unknown. 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.

Enzyme properties The physicochemical properties of the enzyme of the present invention are shown below.

(2) Effect and Substrate Specificity The present enzyme was added to a 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.

In Table 3, α-limited dextrin (1) was used with human salivary gland α-amylase, and α-limited dextrin (2) was used with Bacillus subtilis.
using α-amylase produced by S. btilis).

Each was prepared. This enzyme does not hydrolyze substrates other than those listed in Table 3, such as dextran, isomaltose, maltose, panose, sucrose, trehalose, and melezitose.

(Left below) 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 varies from 1 to 100 depending on each substrate.
After 24 hours, the reaction solution was subjected to paper chromatography to examine the main product. The results are shown in Table 4.

D Two enzymes from Tables 3 and 4 are soluble starch.

It can be seen that it acts on glucans such as amylopectin, amylose, glycogen, maltotetraose, and maltopentaose to produce maltotriose. Since all of the maltotriose produced is in the alpha form, one enzyme acts on alpha-glucan.

It is clear that it is α-amylase that hydrolyzes the 1-4 bond to produce maltotriose.

■Optimal pH and stable pH range Using this enzyme, pHi3. 14 in the range t~10.4
The enzymatic reaction was carried out under seed pt+ conditions according to the method for measuring activity. However, the buffer used was pH 3,
40mM acetate buffer in the range of 1 to 4.0;
20mM succinate buffer in the range of pH 4.5 to 5.6, 20mM maleate buffer in the range of pH 6.2 to 7.1, and 20mM borate buffer in the range of pH 8.2 to 10.4. The relative activities are shown in FIG. From FIG. 1, it can be seen that the optimum pH is about 5.6 at 60°C.

This enzyme was contained in a predetermined pH bag containing 5mM CaCl.

After dissolving in Sofa and maintaining at 25°C for 15 hours, residual activity was measured. pH is 3.0.3.5.4.0.5.9
．． It was set at 8.1°, 10.8 and 11.8. next,
Temperature is 55℃, pH 14, 2, 4, 7, 5, 2, 5,
Residual activity was measured in the same manner for 9, 8, 1, 8, 3, 8, 8, 9, 0 and 9.5. The residual activity of each is shown in Figure 2. The buffer used had a pH of 3.0 to 3.6.
Then, 20mM acetate buffer, pH 4,1-5.
5 with 20mM succinate buffer, pus, o
~6.5 20mM maleate buffer, and p
H7.5-11.5 is 20mM borate buffer. From Figure 2, the stable pl+ range is 4.0 to 11 at 25℃.
．． It can be seen that the temperature is 5.9 to 8.1 at 9.55°C.

(2) 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 Figure 2, this enzyme is pl! at 55°C. It is clear that the activity is inactivated in 15 hours at a pH of 4.1 or lower or a pH of 9.5 or higher.

After dissolving the product in 40mM maleic acid buffer with pH 6 and 8 and maintaining it at a predetermined temperature for 10 minutes, the residual activity was measured. The holding temperature is 4℃, 55℃, 6
0℃, 70℃, 72.5℃, 75℃ and 77.5℃
was set to . The respective residual activities are shown in FIG. 4 by two-dot chain lines. FIG. 4 shows that this enzyme is stable up to 70°C under the above conditions. Next, 5mM CaCIz was added, 4°C, 55°C, 260°C, 70°C, 72.5
°C175”c, 77.5°C, 80°C and 82.5
Residual activity was measured in the same manner at °C. Also for the system in which EDTA (ethylenediaminetetraacetic acid) was added instead of CaCl2, the temperature was 4'C, 55°C, 160°C, 62.5°C.
Similar experiments were conducted at , 65°C and 67.5°C.

The respective residual activities are shown in FIG. 4 by solid lines and dashed-dotted lines.

From FIG. 4, it can be seen that when calcium ions are added, this enzyme is stabilized and no deactivation occurs up to 75°C.

When EDTA is added, the heat resistance decreases and the stable range is 60
It can be seen that the temperature is up to ℃.

■Inhibition, Activation, and Stabilization Using this enzyme, enzymatic reactions were carried out in a solution containing various metal ions and reagents instead of Ca ions according to the activity measurement method. The relative activities are shown in Table 4. In Table 4, ρ-chlormercuric linkhenzoe) (pCMB)
and 5,5”-dithio-bis(2-nitrobenzoate)
For (DTBNB), these reagents and enzyme were incubated in advance at 55°C for 20 minutes, and then the substrate was added and allowed to react.

Table 4 From Table 4, this enzyme is 11g2+, pb2+, and u2
+, 7. n2+.

It can be seen that the activity is inhibited by heavy metals such as Mn''+.

Next, we investigated the effects of amines (including ammonium salts and amino acids) and sugars. Enzyme reactions were carried out in a solution containing the following amines at a ratio of 5mM or the following sugars at a ratio of 10mM according to the activity measurement method. Table 6 shows the degree of inhibition (%). In Table 6, the Ki value of maltotriitol is the value at 50% inhibition.

Table 6 Table 6 shows that the two enzymes are strongly inhibited by histidine, benzylamine and maltotriitol. The enzyme is inhibited non-reactively by histidine and retardantly by benzylamine.

(2) Molecular Weight The molecular weight of sodium dodecyl sulfate (SDS) is approximately 78,000 as determined by pneumophoresis. The value obtained by the above-mentioned SDS electrophoresis method agrees well with the value calculated from the measured value of sedimentation coefficient (S!.-, 5.48).

■Mobility (by disk electrophoresis) 7.5% polyacrylamide gel tube (0.5φ
Electrophoresis of nine enzymes was performed using a 5 cm x 5 cm).

Using Tris-Glycine Puffer (pH 8.3).

Electrify the tube at 40°C with a current of 2 mA/tube for 2 hours.
After electrophoresis, it was stained with amide black and decolorized with 7% acetic acid. The mobility is a 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. The distance traveled by BPB is 4.6 c
m, so the mobility of one enzyme is 0.31. SD
A single band was also obtained by S electrophoresis, indicating that the two enzymes were a single substance.

■ Isoelectric point Wrigley's method (Wrigley, C, W, ; J,
Chromatogr.

The isoelectric point measured by the electrophoresis method according to Japanese Journal 362-365 (1968) is 4.6.

■Km value Km value, maximum reaction rate (V) when soluble starch, amylopectin, amylose and glycogen are used as substrates
and V/Km are shown in Table 7. The enzyme reaction was carried out according to the activity measurement method.

From Table 7, the most suitable substrate for the two enzymes is amylopectin, and its Km value is 1.0■/+4! It can be seen that it is.

[Phase] 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 one enzyme had a molecular weight of 78,000 and had 14 methionine residues. Trp was measured optically.

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.

This enzyme is produced by the Japanese Patent Publication No. 60-15315 listed in the prior art section.
It differs from the α-amylase derived from Bacillus YT-1004 strain described in the publication in various respects. For example, the range of temperature suitable for one action is 65 to 75°C, which corresponds to YT-1004 strain α-
Much higher than 50°C for amylase. YT-10
While α-amylase of the 04 strain is inactivated by about 70% when stored in a buffer at 50°C for 10 minutes, the enzyme of the present invention exists stably 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 is
As mentioned above, the enzyme is characterized by its excellent heat resistance. Furthermore, the production rate of maltotriose is high, and when the substrate is amylose, it is completely decomposed into maltotriose. in this way.

Judging from its properties and molecular characteristics, the enzyme of the present invention was found to be a new maltotriose-producing α-amylase.

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 1, food improver (moisturizer, etc.), and as an infusion ingredient, so effective use of the 9 enzymes is desired.

Culture of Bacillus KP1071 strain: soluble starch 1.6%, peptone 0.8%, yeast extract 0.
3%.

Buffer y-(pH 7.5) containing KH□PO40.3% and KHzPO40.1% was used as a medium, and Ba
Cillus KP1071 strain was cultured. Cultivation was carried out at 55° C. for about 16 hours using a 9-revolution shaking culture method.

(B) Collection and purification of enzyme: The culture solution 49.41 obtained in section (A) was centrifuged to remove bacterial cells.

After concentrating this, 5mM sodium maleate 15mM
It was dialyzed against a buffer containing CaCIz/0.02% NaN3 (pl+6.8) and salted out with ammonium sulfate (saturation 40-70%). DEAE this
- Sephadex A-50 chromatography (column 4.5φ x 42.5cm) and 30mM glycine sodium/5m MCaClzlo, 02% NaN
, with a buffer containing (pH 8,0) and NaCl.
Elution was performed by concentration gradient elution method of IMNaC1. Next, it was subjected to phenyl Sepharose CL-4B chromatography (column 3.0 φ x 24.5 Cm), and the buffer solution containing ethylene glycol-ammonium sulfate (p
H8,0) as a solvent and ethylene glycol as 0 to 50
% and ammonium sulfate from 1 to OM.

Furthermore, it was subjected to hydroxyapacite HTP chromatography (column 3.0 φ x 28.0 cm), using phosphate buffer (pH 6, 8) as a solvent, to a concentration of 5 to 100 mM.
Elution was performed using a concentration gradient elution method. Then DEAE
Cellulose chromatography (column 2.0 φX2
8.4 cm) and 10 mM sodium citrate.
0 to 0.5 M with a buffer (pH 6,8) containing mMCaCIglo, 02% NaN, and NaCl.
Elution was performed using the NaC+ concentration gradient elution method to obtain purified enzyme.

(C) Production of maltotriose = Using the enzyme obtained in section (B), an enzymatic reaction was carried out according to the activity assay method using amylose as a substrate. The concentration of amylose was 0.2%, and the enzyme was used at position 0.04541.

The reaction liquid volume was 0.25 nl. After starting the enzyme reaction, 10
minutes, 20 minutes, 40 minutes, and 60 minutes later, paper chromatography (developing solvent: n-butanol/pyridine/
Water = 6:4:3 (v/v)) was added to examine the production status of maltotriose. The results are shown in FIG. In FIG. 5, 01 to G9 indicate the glucose numbers of 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: Activity measurement method by adding the enzyme solution obtained in section (B) (containing 0.45 units of enzyme) to 2.5 ma of buffer containing 1% soluble starch. Enzyme reaction was carried out for 4 hours according to . This reaction solution was kept at 95° C. for 5 minutes to inactivate the enzyme. Next, desalting was carried out using monovent of an ion exchange resin, and the mixture was 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 Bacillus thermoamyloliquefaciens strain P4O10. 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, as a food improver (moisturizing agent, etc.), as an infusion acid, and so effective use of these enzymes 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 are paper chromatography showing the production status of maltotriose when an enzyme reaction is carried out using this enzyme and using amylose as a substrate. Attorney Shusaku Yamamoto, Patent Attorney Figure 1 H Figure 2 H Figure 4 Temperature (0C) Figure 5

Claims (1)

[Claims] 1. α-amylase having the following properties. (1) Action and substrate specificity: α-1 and α-glucan
4 bonds are hydrolyzed to produce maltotriose. (2) Optimal pH: 5.6 at 60°C. (3) Stable pH: 5.9-8.1, 25 at 55°C
It is 4.0-11.9 at °C. (4) Range of suitable temperature for action: The optimum temperature is 65 to 75°C. (5) Thermal stability: In the presence of 5mM calcium ions, p
It is stable up to 75°C when held for 10 minutes at h6.8. (6) Molecular weight: The molecular weight determined by sodium dodecyl sulfate electrophoresis is 78,000. (7) Isoelectric point: 4.6 (8) Michaelis constant Km value for amylopectin:
1.0mg/ml (9) Amino acid analysis: ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ *1 Asp and Asn *2 Glu and Gln 2, obtained from Bacillus genus bacteria and described in claim 1 α-Amylase. 3. Bacillus thermoa amyloliquefaciens (Bacillus)
llus thermoamyloliquefaci
Claim 1 or 2 derived from
α-Amylase as described in Section. 4. 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 as described.