JPH0249584A - Heat-resistant alkaline amylase and production thereof - Google Patents

Abstract

NEW MATERIAL:A heat-resistant alkaline amylase having the following properties. Action and substrate specificity, specifically hydrolyzing alpha-1,4-glycoside bond of starch, amylose, amylopectin and their partial decomposition products to form dextrin and maltooligosaccharide; optimum pH, pH10-10.5; stable pH, stable at pH6-11.5 under heating condition at 60 deg.C for 30min; optimum working temperature, 75 deg.C; thermal stability, stable up to 65 deg.C for 30min in glycine buffer solution of pH10.3 and up to 70 deg.C for 30min in Ca<2+>-added glycine buffer solution of pH9.5. USE:A component of a desizing agent for textile industry or exclusive detergent for automatic dish washer. It has high desizing and cleaning effect. PREPARATION:A microbial strain belonging to genus Bacillus and capable of producing the objective enzyme [preferably Bacillus alcalothermophilus A3-8 (FERM P-10180)] is cultured and the objective enzyme is separated from the culture product.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a novel heat-stable alkaline amylase and a method for producing the same. For more information, see Bacillus
The present invention relates to a heat-stable alkaline amylase obtained from a novel microorganism belonging to the genus P. lus), and a method for producing the same.

(Prior Art) Amylase is an enzyme that specifically hydrolyzes α-1,4-glycosidic bonds in starch, and during hydrolysis, the viscosity of a reaction solution containing starch is significantly reduced. Taking advantage of this action, amylase is used as a desizing agent for the textile industry and as a component of detergents for automatic dishwashers.

The amylase used for the above purpose is preferably an amylase that acts at high temperatures and has excellent heat resistance. Such amylase is mainly produced by Bacillus subtilis.
5ubtilis) + Bacillus amyloliquefaciens (Ba.
slicheniformis).

The optimum reaction temperature of amylase derived from these bacteria is 70° C. or higher, and the optimum pH is around neutrality of 6-7. Therefore, when these amylases are used in the desizing process in the scouring and bleaching process of the textile industry, the reaction is first carried out in a neutral state, and then the pH is adjusted to an alkaline level of 10 or more before the scouring process is carried out. If amylase with an optimum pH of 10 or more, ie, alkaline amylase, can be used in the desizing step, there is no need to adjust the pHtM between at least both steps, and the operation can be simplified. Also, the pH is 9-IO
Conventional amylase added to detergents does not have sufficient effect when washing dishes with the above-mentioned automatic dishwasher detergents at near or higher alkalinity, but if alkaline amylase could be added, it would be extremely effective. It is effective for

Currently, Bacillus A-40 is used as alkaline amylase.
Enzymes produced by -2 strains (Agric, Biol, Che
m, +35+1783, (1971)), an enzyme produced by Bacillus NRRL strain B-3881 (J, B
acteriol, 1 interest, 992. (1972))
, Streptomyces genus KSM-9 (nail B knΩtan S
ρ, enzyme produced by KSM9) strain (Japanese Patent Application Laid-open No. 61-20
9588).

and Bacillus sp.
Enzyme produced by H-167) strain (JP-A-62-20
No. 8278) is known. However, the optimal temperature for action of all enzymes is 55°C or lower, and the stable temperature range is also low at 55°C or lower. Therefore, sufficient effects cannot be expected in the desizing process, which is generally carried out at high temperatures (65° C. or higher), or in dishwashing using automatic dishwashing machines (60° C. or higher).

From the above points of view, it works well at higher temperatures.

Alkaline amylase with excellent heat resistance is desired.

(Problems to be Solved by the Invention) An object of the present invention is to provide an alkaline amylase that has a high optimum temperature for two reactions and has excellent heat resistance. Another object of the present invention is to provide a method for producing alkaline amylase having the above-mentioned valuable properties.

(Means for solving the problem) As a result of searching for various microorganisms that produce alkaline amylase, the inventors found a new bacterial species belonging to the genus Bacillus collected from the soil of Hata City, Omi, Shiga Prefecture.

The present invention was completed based on the discovery that thermostable alkaline amylase can be efficiently produced by aerobic culture.

The thermostable alkaline amylase of the present invention has primary properties.

(a) Action and substrate specificity: starch, amylose,
amylopectin and their partial decomposition products α-1,
It specifically hydrolyzes 4-glycosidic bonds, producing mainly dextrins and maltooligosaccharides.

(C) Optimum pH: 10.0-10.5° (C) Stable p
H: Under heating conditions of 60°C for 930 minutes, p)16.
Stable at 011.5.

(d) Range of suitable temperature for action: The optimum temperature for action is around 75°C.

(e) Thermal stability: Stable up to 65°C when heated for 30 minutes in a glycine buffer at pH 10.3, and stable up to 65°C when heated for 30 minutes in a glycine buffer at pH 9.5 containing calcium ion. It is stable up to 70''C.

The method for producing heat-stable alkaline amylase of the present invention includes Bacillus (
The method includes a step of culturing a microorganism belonging to the genus Bacillus and producing the heat-stable alkaline amylase; and a step of separating the heat-stable alkaline amylase from the culture obtained in the step.

The alkaline amylase of the present invention can be applied to bacteria of the genus Bacillus, particularly Bacillus alcalothermophilus ^3-8 (Bacillus alcalothermophilus ^3-8).
lus alcalothermo hilus
A3-8) strain. This bacterial strain is a new bacterial strain isolated by the 8 inventors from soil collected from Omihachiman City, Shiga Prefecture. Since this strain is an aerobic sporulating bacillus, it is clear that it is a microorganism belonging to the genus Bacillus. Furthermore, this strain has the characteristic of being thermophilic and alkalophilic in that it does not grow in a medium with a pH of 7.0 or below and at a culture temperature of 35° C. or below. Table 1 shows the mycological properties of this strain. In Table 1, the PTT of the medium is 9.0 and the culture temperature is 55° C. unless otherwise specified.

Table 1 This strain having the above mycological properties was used as described in Bergey's Manual of Determinative Bacteriology, 8th edition.
eterminative Bacreriolog
y 18th edition (1974)) and The Genus Bacillus (1974))
Bacillus stearothermophilus (Ba.llus (1973)), which belongs to the genus Bacillus.
cillus stearothermo hilu
s) and the thermoalkalophilic bacterium Bacillus IC (B
acillus No, IC) similar to ( Agri
c, Biol, Chem, +51+ 2429+
(1987)). However, the mycological properties of the Ipponzono strain are somewhat different from these strains. The differences are summarized in Table 2 below.

(The following is a blank space) From Table 2, the inventors identified this strain as belonging to a new bacterial species of the genus Bacillus.
It was named er Ushimochi Katsukawa A3 ▽8). This strain has been deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology, with deposit number 10180.
No. (IIERM P-10180).

The medium for culturing the above-mentioned bacterial strain does not need to be special, and a normal medium can be used. Carbon sources include various starches (soluble starch, liquefied starch, etc.), dextrin, and amylopectin.

Amylose etc. are used. Nitrogen sources include peptone, yeast extract, soybean flour, corn staple liquor, casein, meat extract, and amino acids. In addition to these carbon sources and nitrogen sources, there are various salts such as magnesium salts and potassium salts.

Sodium salts, phosphates, etc. are added as necessary. Furthermore, since this strain is an alkalophilic bacterium, separately sterilized sodium carbonate, sodium hydroxide, etc. are added to adjust the pH of the medium to 7.5▽-11.0. In such a medium, the culture temperature is 36▽-▽72℃, preferably 55℃.
Incubate aerobically for 24▽-▽72 hours at °C with stirring or shaking. The thermostable alkaline amylase of the present invention is mainly secreted and accumulated in the culture solution.

In order 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. Since this enzyme is mainly secreted outside the bacterial cells (into the culture solution), a crude enzyme solution can be easily obtained by removing the bacterial strain, for example, by filtration and centrifugation. This crude enzyme solution was further purified using known methods.

For example, salting out with ammonium sulfate; methanol, ethanol,
Examples of purification methods using precipitation with an organic solvent such as acetone; ultrafiltration; gel filtration chromatography, ion exchange chromatography, and various other chromatography alone or in combination are shown below.

(1) Centrifuge the culture solution to obtain a supernatant. (2) Salting out the supernatant with ammonium sulfate (0-60% saturation).

(3) The obtained precipitate was dialyzed and DEAE-)Yopal (
▽Toyopearl) Perform chromatography using a 650M column (▽pH▽8, ▽O~1.0
M NaC1 concentration gradient) (4) DEAE-▽) Perform a second chromatography using Copal 650M column (▽pl+8.▽0▽~0.5 M NaC] concentration gradient), (5) Sephacryl (Sephacryl)
) Perform gel filtration chromatography using an S-200 column (▽pl+8). Table 3 shows the degree of enzyme purification in each step when purified by such a method.

The activities in Table 3 are values measured by the activity measuring method described below. The value in step (1) is 3.6 I! of the supernatant obtained by centrifuging the bacterial culture. This is the measured value when , is used as a sample. In this way, in step (5), 108 units/m, which is 1,768 times more purified than the original enzyme supernatant! A purified enzyme solution of (the definition of unit will be described later) is obtained. The properties of the enzymes described below are as follows.

This purified enzyme was used for investigation.

(Left space below) Foil 1J1 [063% soluble starch solution dissolved in 100 mM glycine buffer (pH 10,0) is used as a substrate. 100 μl of a sample containing this enzyme of unknown activity is added to 500 μl of the substrate solution and reacted at 60° C. for 10 minutes, and then 1 mft of 0.05N hydrochloric acid-0.2M sulfosalicylic acid solution is added to stop the reaction. 420μN for this
Add 4 mM of iodine-82 mM potassium iodide solution to develop color, and measure the decrease in absorbance at a wavelength of 660 nm.

One unit of enzyme (AAU; alkaline amylase unit) is the amount of enzyme that reduces the absorbance at 660 nm by 1.0.

箆聚傅情况! The physicochemical properties of the enzyme of the present invention are shown below.

■Action and substrate specificity Each enzyme is added to a glycine buffer containing amylose and amylopectin as substrates instead of soluble starch, an enzymatic reaction is carried out according to the activity measurement method, and the reaction solution is subjected to HPLC over time. The product was investigated. As a result, 3 pyroligneous acid acts not only on starch but also on amylose, amylopectin and their partial decomposition products, specifically hydrolyzing α-1,4 glycosidic bonds (endo-type decomposition), and mainly converting dextrin and maltooligosaccharides. It was found that it was generated.

(2) Optimum pH and Stability p)1 Using this enzyme, an enzyme reaction was carried out under pH conditions in the range of pH 3.8 to 12 according to the activity assay method. However, the buffer used was 0.1M in the pH range of 3.8 to 8.
Tris-malate buffer, and 0.1M or 0.2M glycine-NaOH buffer in the pH range 8.5-12. The relative activities are shown in FIG. In Figure 1, the solid line shows the results at 60°C, and the broken line shows the results at 75°C.
The results are shown below. From FIG. 1, it can be seen that the optimum pn is about 10.0 to 10.5 at 75°C.

This enzyme was dissolved in the specified p)I buffer and heated to 60℃ for 30 minutes.
Residual activity was measured after holding for a minute. The result is the second
As shown in the figure. The buffers used were Tris-malate buffer at pH 5.5-8.0 and Tris-malate buffer at pH 8.7-11.
7 is a glycine-NaCl-NaOH buffer. From the second test, it can be seen that the stable pH range is from pH 6.0 to 11.5 under heating conditions of 60° C. for 30 minutes.

(2) Suitable temperature range for action Using this enzyme as a soluble starch substrate, enzyme reactions were carried out under temperature conditions in the range of 40 to 85°C according to the activity assay method. The relative activity is shown by the solid line in FIG. From FIG. 3, it can be seen that the optimum temperature when using soluble starch as a substrate is around 75°C.

2 when measuring activity in the above temperature range (40-85℃)
1 g of 3mM CaC or 3mM ethylenediaminetetraacetic acid (EDT^) was added to the reaction system, and the influence on the optimal temperature of the two enzymes was investigated. The results are shown in FIG. 3 together with the above results. Even if 3+11M CaC1 was added, no effect on the optimum temperature was observed, and almost the same results as those without the addition, shown by the solid line in FIG. 3, were obtained.

On the other hand, 3n+M l! When DTA is added, the results shown by the broken line in FIG. 3 are obtained. From Figure 3, E
Addition of DTA inhibits the activity of this enzyme at temperatures above 65°C.

It can be seen that the optimum temperature is lower than 75°C when no additive is used.

(2) Stability against temperature The present enzyme was dissolved in a specified buffer solution, and the residual activity was measured after holding the solution for 30 minutes at a temperature in the range of 40 to 80°C. The results are shown in FIG. The buffer used was 0.
05M glycine-41 NaOH solution (pHIO, 3).

0.05M glycine-NaOH buffer (pH 9,5) and 0.05M glycine-NaOH buffer (pH 9,5)
) and 0.05 M glycine-NaOH buffer (pH 9
, 5.3mM CaC1z). In FIG. 4, these results are shown by dashed lines, solid lines, and broken lines, respectively. From Figure 4.

This enzyme is stable up to 65°C when heated for 30 minutes in a glycine buffer of pH 3, and up to 70°C when similarly heated in a glycine buffer of pH 9.5 containing calcium ions. It can be seen that it is stable.

■ Stability against surfactants and chelating agents The enzyme was dissolved in 100 mM glycine buffer (pHlo, o) and the residual activity was measured after heating at 60°C for 30 minutes with the following surfactants and chelating agents. .

The results are shown in Table 4. In Table 4, POEAE is polyoxyethylene higher alcohol ether, DBS
represents sodium dodecylbenzenesulfonate, SDS represents sodium dodecyl sulfate, EDTA represents ethylenediaminetetraacetic acid, and STP represents sodium tripolyphosphate.

Table 4 As is clear from Table 4, one enzyme contained 0.1% POEAE, 0.01% DBS, 2
It is stable to 0mM EDT and 20mM STP, but is almost inactivated by 0.1% DBS and 0.1% SO3.

Both 11R3-deficient enzymes were compared with previously reported alkaline amylases.

Table 5 shows the characteristics of this enzyme and various alkaline amylases (optimum p11, optimum temperature, stable pl+, and stable temperature).

(The following is a blank space) From Table 5, it can be seen that the optimum reaction temperature for this enzyme is 75°C, which is higher than the optimum temperature for any other alkaline amylase and is different from these enzymes.

This enzyme is similar to alkaline amylase derived from Bacillus sp. If-167 in terms of optimum pH and stable pH, but the above-mentioned optimum temperature and stable temperature are 10 to 15°C higher. Thus, it is clear that the thermostable alkaline amylase of the present invention is a new enzyme different from any known alkaline amylases, and is the most thermostable alkaline amylase that acts at high temperatures.

(Example) The invention will be explained below with reference to examples.

3-8 strains to Bacillus Arcalothermophilus.

2% soluble starch, 1% polypeptone, 1% meat extract
, KJPO40.1%, MgSO4・7H200,
02%.

Shake flask 5 containing medium (pH 10) 100- containing 10.5% NaC and 0.5% Na2CO3
The cells were inoculated and cultured with reciprocating shaking at 55°C for 3 days. After culturing.

The culture solution was centrifuged at 10.00 rpm for 10 minutes to remove bacterial cells. Ammonium sulfate powder was added to the obtained supernatant until 60% saturation, and then 10. OOOrp
The precipitate was collected by centrifugation at m for 10 minutes. The precipitate thus obtained was air-dried at 40° C. for 1 day and further dried at room temperature for 3 days under reduced pressure to obtain 2.7 g of crude enzyme powder containing 1,080 units/g.

Forty-five shake flasks containing 100 ml of the same medium solution as in Example 1 were prepared, and Bacillus aerulothermophilus strains 3-8 were inoculated. After culturing this in the same manner as in Example 1, it was centrifuged to obtain 3.62 g of supernatant.

The alkaline amylase activity of this supernatant was 4.8 units/
It was rd. Next, ammonium sulfate powder was added to this supernatant solution until it reached 60% saturation, and the mixture was heated to 10.00 Or
The precipitate was collected by centrifugation at pm for 10 minutes. This precipitate was dissolved in 10 mM glycine buffer (pl+9) and dialyzed against a buffer of the same grade for 1 day. After that, the dialysis fluid was
DE equilibrated with mM l-Lis-HCl buffer (pH 8)
AE-) Copal 650M column (φ2.6 X35c
m) and eluted with an O-0, 1M NaCl concentration gradient. The active fraction eluted around 0.2M NaCl was equilibrated with 20mM Tris-HCl buffer (pH 8) using a DEAE-) Copal 650M column (φ2.6
x21cm), and (1-0.5M NaC
1 concentration gradient. Then 0.25M Na
The active fraction eluted around C1 was treated with Sephacryl S-200 equilibrated with 501 Tris-HCl buffer (pH 8).
Gel filtration was performed through a column.

Elution was performed with the same buffer used to equilibrate the column.

By collecting the active fractions, 50 m2 of an enzyme solution containing 108 units/ml was obtained. In this way, thermostable alkaline amylase was obtained from cultured synovial fluid at a yield of 31%, and was purified approximately 1,768 times in terms of specific activity.

(Effects of the Invention) According to the present invention, a novel thermostable alkaline amylase can be obtained from Bacillus arcothermophilus A3-8 strain. This enzyme has a high thermostability of 65 to 70'C and an optimum action temperature of 75C, making it the enzyme with the best thermostability among the alkaline amylases known so far.

By using this enzyme, it is possible to significantly improve the degradation of starch in alkaline solutions than when using conventional amylases. For example, this enzyme can be used in the desizing process during the scouring and bleaching process in the textile industry to simplify the process, and by incorporating it into automatic dishwasher detergent, it can improve the cleaning effect on starch stains. It is possible. In addition, this enzyme is used in an extremely wide range of fields, such as enzyme-containing detergents containing protease, amylase, etc., as a viscosity regulator for alkaline glue used in the two-corrugated board industry, as a waste liquid treatment agent, and in the process of processing starch in alkaline areas. can be done. Furthermore, since the enzyme is secreted outside the bacterial cells, the method of the present invention allows the enzyme to be obtained in large quantities and at low cost through simple steps.

is a graph showing the stable pH range of wood enzymes.

Figure 3 is a tree Graph showing the optimum temperature of enzymes.

And Figure 4 is a book It is a graph showing the stable temperature range of enzymes.

Below Up

Claims (1)

[Claims] 1. A heat-resistant alkaline amylase having the following properties. (a) Action and substrate specificity: starch, amylose,
amylopectin and their partial decomposition products α-1,
It specifically hydrolyzes 4-glycosidic bonds, producing mainly dextrins and maltooligosaccharides. (2) Optimal pH: 10.0-10.5. (c) Stable pH: Under heating conditions at 60°C for 30 minutes, p
Stable at H6.0-11.5. (d) Range of suitable temperature for action: The optimum temperature for action is around 75°C. (e) Thermal stability: 3 in glycine buffer at pH 10.3
It is stable up to 65°C when heated for 0 minutes, and stable up to 70°C when heated for 30 minutes in a pH 9.5 glycine buffer containing calcium ions. 2. The heat-stable alkaline amylase according to claim 1, which is obtained from a bacterium belonging to the genus Bacillus. 3. Bacillus alcalothermophilus A3-8 (￥
Bacillus￥￥alcalothermoph
illus A3-8) strain according to claim 1. 4. A step of culturing a microorganism that belongs to the genus Bacillus and produces the heat-stable alkaline amylase according to claim 1; and a step of separating the heat-stable alkaline amylase from the culture obtained in this step; A method for producing heat-resistant alkaline amylase comprising: 5. The manufacturing method according to claim 4, wherein the microorganism is cultured at 40-70°C. 6. The manufacturing method according to claim 4, wherein the microorganism is cultured at a pH of 7.5 to 11.0. 7. The microorganism is Bacillus alcalothermophilus A.
3-8(￥Bacillus￥ ￥alcalothe
rmophilus\A3-8) strain according to claim 4.