JPH09201194A - New alpha-glucosidase and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce a new α-glucosidase, capable of hydrolyzing an α-glucoside bond and providing α-D-glucose and useful for measurement, etc., of α-amylase activities by culturing a strain, belonging to the genus Bacillus and having the ability to produce the specific α-glucosidase. SOLUTION: This new α-glucosidase has functions to hydrolyze an α-glucoside bond and produce α-glucose, is capable of acting on maltooligosaccharide having the polymerization degree ranging from maltose to maltooctaose, a p- nitrophenyl-α-D-glucopyranoside, a p-nitrophenyl-α-D-maltoside, a p-nitrophenyl-α- D-maltopentaoside and nigerose without acting on isomaltose and kojibiose, has 6.5-7.0 optimum pH, 50-55 deg.C optimum temperature and about 42,000±5000 (measured by a gel filtration method) and is useful for measurement, etc., of α-amylase activities. The enzyme is obtained from a cultured product of a strain belonging to the genus Bacillus and having the ability to produce the α-glucosidase.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to maltooligosaccharides having a degree of polymerization of maltose to maltooctaose, paranitrophenyl-α-D-glucopyranoside (hereinafter referred to as PNPG), and paranitrophenyl-.
α-D-maltoside (hereinafter referred to as PNPG2), para-nitrophenyl-α-D-maltopentaoside (hereinafter, referred to as
PNPG5), acting on nigerose etc., α-D
-It hydrolyzes a glucoside bond to produce α-D-glucose, but has a substrate specificity that does not act on isomaltose and kojibiose, and has excellent heat resistance and pH stability,
Further, the present invention relates to a novel α-glucosidase useful as a conjugate enzyme in the α-amylase activity measurement and the quantification of chloride ion, which has a suitable temperature range for practical use, and a method for producing the same.

[0002]

2. Description of the Related Art It is known that α-glucosidase is widely present in the living world and is present in yeast, filamentous fungi, bacteria, mammals, plant seeds and the like. Further, these α-glucosidases have various enzymatic properties such as heat resistance, optimum pH and substrate specificity. Examples of conventionally known α-glucosidases that hydrolyze maltose and PNPG include bioscience, biotechnology and biochemistry (Biosci. Biotech. Bioc.
hem. ) Vol. 57, No. 11, 1902-1905
Yeast-Derived, Starch, pp. 1993
Kamen (Denpun Kagaku) Volume 36, Volume 3
Pp. 181-187, 189-195, 19
1989, derived from rice, Biochemica Biophysica Actor (Biochimica et Bi
Ophysica Acta) Volume 787, Volume 281-
289, 1984, Bacillus stearothermophilus.
Those derived from rmophilus) are known.
In addition, examples of commercially available enzymes include those derived from yeast (Sigma Co., Toyobo Co., Ltd.), those derived from rice (Sigma Co.), and microorganisms (Toyobo Co., Ltd.).

However, among the above-mentioned α-glucosidases, α-glucosidase derived from yeast is heat,
It lacks stability against H, inhibitors, etc. and causes problems in enzyme production and use at room temperature. The rice-derived enzyme is
Since it is a plant seed-derived enzyme, it takes time to produce it, and the nature of the enzyme is not suitable for use in the vicinity of neutrality because the optimum pH is on the acidic side. Furthermore, the Bacillus stearothermophilus-derived enzyme has excellent heat resistance, but since the optimum temperature is around 70 ° C, the reaction at around 37 ° C, which is a general enzyme reaction, It is necessary to use a large amount of enzyme.

[0004]

The present invention overcomes the drawbacks of the above-mentioned conventional α-glucosidases, and, for example, as a conjugated enzyme used in the measurement of α-amylase activity, quantification of chloride ion, etc. Α-Glucosidase that can be used as an enzyme for measuring and eliminating maltooligosaccharides such as maltose and as an enzyme for elimination, or in industrial production such as production of glucose from oligosaccharides, and monosaccharide sugars such as fructose, and this enzyme at low cost, and The purpose of the invention is to provide a method for easily manufacturing.

[0005]

Means for Solving the Problems As a result of a search for microorganisms capable of producing α-glucosidase widely from nature in order to achieve the above-mentioned object, the present inventors have found that it belongs to the genus Bacillus isolated from soil. One strain belonging to is maltooligosaccharide, PNPG, PNPG2, PN having a polymerization degree from maltose to maltooctaose.
Based on this finding, it was found that a novel α-glucosidase that acts on PG5 and nigerose, but does not act on isomaltose and cordiose, has excellent stability and has a suitable action temperature range in the vicinity of practical measurement is produced. The present invention has been completed.

That is, the present invention has the following physicochemical properties: (a) Action: α-glucoside bond is hydrolyzed to give α-glucoside bond.
It produces D-glucose. (B) Substrate specificity: maltooligosaccharides having a degree of polymerization from maltose to maltooctaose, paranitrophenyl-α-D-glucopyranoside, paranitrophenyl-α-D-maltoside, paranitrophenyl-
It acts on α-D-maltopentaoside and nigerose, but does not act on isomaltose and cordiose. (C) Optimum pH and stable pH range: The optimum pH is 6.5.
It is around 7.0 and the stable pH range is pH 6.0 when treated at 25 ° C. for 20 hours in the presence of 0.2% bovine serum albumin.
１10.5. (D) Optimum temperature range of action: The optimum temperature range of action is 50-55.
It is around ℃. (E) Conditions of inactivation due to pH, temperature, etc .: pH at 20 ° C. for 20 hours at 25 ° C. in the presence of 0.2% bovine serum albumin
It is stable in the range of 6.0 to 10.5 and deactivates at pH 4.5 or lower or pH 11 or higher. Also, the thermal stability is pH
It is stable up to 55 ° C after treatment for 7.0 minutes, and completely deactivates at 65 ° C or higher. (F) Molecular weight: about 42,000 ± 5,000 (gel filtration method). (G) Effect of inhibitor treatment: In the presence of 0.2% bovine serum albumin, pH 7.0, 25 ° C, 1 hour inhibitor treatment, copper, mercury and silver heavy metal ions and sodium dodecyl sulfate, p- Deactivated by chloromercuribenzoic acid. (H) Km value: Km at pH 7.0 and 37 ° C. using para-nitrophenyl-α-D-glucopyranoside as a substrate.
The value is 2.8 × 10 ⁻³ M. It is a novel α-glucosidase having, also belonging to the genus Bacillus, culturing a strain having an α-glucosidase-producing ability having the physicochemical properties in a medium, and collecting the α-glucosidase from the culture. A method for producing the above-mentioned α-glucosidase, which is characterized. Hereinafter, the present invention will be described in detail.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION First, a novel enzyme of the present invention, α-
Details of physicochemical properties of glucosidase (hereinafter referred to as the present enzyme) are as follows.

(1) Action: This enzyme hydrolyzes a glycoside or an oligosaccharide having an α-glucoside bond from its non-reducing terminal side to produce α-D-glucose.

(2) Substrate specificity: acts on maltooligosaccharides having a degree of polymerization of maltose to maltooctaose, PNPG, PNPG2, PNPG5 and nigerose, but does not act on isomaltose and kojibiose. Table 1 shows an example of the results of examining the relative activity of the enzyme with respect to various substrates when the glucose production amount of PNPG is 100.

[0010]

[Table 1]

The conditions and method for measuring the relative activity are as follows. 0.11 ml of each substrate (20 mM), 0.
Contains 1% bovine serum albumin (hereinafter referred to as BSA) 10
0 mM potassium phosphate buffer (pH 7.0) 0.49 m
1 and 0.39 ml of distilled water were mixed and preincubated at 37 ° C. for 5 minutes. Then BSA 0.1
% Potassium phosphate buffer (pH 7.
This enzyme solution (0.0826 mg / ml) diluted with 0) was added to 0.01 ml of the reaction solution, and the hydrolysis reaction was accurately performed at 37 ° C for 15 minutes. After completion of the reaction time, a test tube containing the reaction solution was put into boiling water to stop the enzymatic reaction, and the amount of glucose produced was measured by the glucose oxidase method using Glucose CII Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). .

(3) Optimum pH and stable pH range: optimum p
H is 100 mM acetic acid-sodium acetate buffer (pH 3.5 to 5.5), 100 mM MES as a buffer.
-Sodium hydroxide buffer (pH 5.5-7.0),
100 mM HEPES-sodium hydroxide buffer (p
H 7.0-8.0), 100 mM TAPS-sodium hydroxide buffer (pH 8.0-9.0), 100 m
M CHES-sodium hydroxide buffer (pH 9.0)
.About.10.0), and 100 mM CAPS-sodium hydroxide buffer solution (pH 10.0 to 11.0) were used. Activity is measured by 0.7 ml of each buffer, 20 mM PNPG solution
After adding 0.25 ml, preincubation was carried out at 37 ° C. for 5 minutes, and then the enzyme solution (0.000033) was added.
Adjust the concentration to mg / ml and use 0.2% as the dilution buffer.
% BSA-containing 10 mM potassium phosphate buffer pH 7.0
0.05 ml was added, and the reaction was carried out exactly at 37 ° C. for 15 minutes. After the lapse of the reaction time, 1 ml of 0.2 M sodium carbonate solution was added to stop the reaction, and the produced para-nitrophenol was colored, and the produced amount was measured using a Hitachi spectrophotometer (U-2000A manufactured by Hitachi Ltd.). 400n
It was determined by measuring the absorbance at m. The results are shown in Fig. 1, and the optimum pH of this enzyme is
It is around 6.5 to 7.0.

The stable pH range of the buffer solution is 1
00 mM acetic acid-sodium acetate buffer (pH 3.5-
5.5), 100 mM MES-sodium hydroxide buffer (pH 5.5 to 7.0), 100 mM HEPES
-Sodium hydroxide buffer (pH 7.0-8.0),
100 mM TAPS-sodium hydroxide buffer (pH
8.0-9.0), 100 mM CHES-sodium hydroxide buffer (pH 9.0-10.0), 100 mM
CAPS-sodium hydroxide buffer (pH 10.0-
11.0), 100 mM potassium phosphate buffer (pH
6.5-8.0) and 100 mM Tris-HCl buffer (pH 7.5-9.0) were used. Each buffer 0.05m
0.01 ml of 0.34 mg / ml enzyme, 0.5
% BSA (0.04 ml) was added, and the mixture was treated at 25 ° C. for 20 hours. After the treatment, it was diluted 500-fold with a 100 mM potassium phosphate buffer solution pH 7.0 containing 0.2% BSA, allowed to stand for 10 minutes, and its enzyme activity was measured by a conventional method to determine the residual enzyme activity. The results are shown in Fig. 2, and the stable pH range of the present enzyme is pH 6.0 to 10.5.

(4) Optimum temperature range of action: The titer of this enzyme was measured at various temperatures using a mixture of substrate and enzyme in the titer determination method described later. The results are as shown in FIG. 3. The optimum temperature range for the action of the enzyme is around 50-55 ° C., and it shows a value of about 80% of the maximum activity even at around 40 ° C.

(5) Conditions for inactivation due to pH, temperature, etc .: This enzyme is stable in the range of pH 6.0 to 10.5 when treated at 25 ° C. for 20 hours in the presence of 0.2% BSA. As shown in 2, 20% at pH 4.5 or below and pH 11 or above
The above is deactivated. Regarding the thermostability, 100 mM potassium phosphate buffer (pH 7.0) was used as a buffer and the enzyme concentration was adjusted to 0.0782 mg / ml, followed by incubation at each temperature for 30 minutes.
After the incubation, the enzyme activity was measured 500 times with a 10 mM potassium phosphate buffer (pH 7.0) containing 0.2% BSA, and the enzyme activity was measured by a standard method to determine the residual enzyme activity. The results are shown in FIG. 4, and the present enzyme is stable under these conditions up to 55 ° C. and has a pH of 7.0, 30.
It is completely deactivated at a temperature of 65 ° C or higher by minute treatment.

(6) Molecular weight: TSKgel G3000
The molecular weight was calculated to be about 42,000 ± 5,000 by the gel filtration method using SWXL (manufactured by Tosoh Corporation). Also, SD
As a result of electrophoresis by S-polyacrylamide gel electrophoresis (using gel: Multi-gel 10-20, Daiichi Kagaku Co., Ltd. acrylamide gradient 10-20% gradient gel), a single band was obtained as shown in FIG. It was confirmed that it was obtained and sufficiently purified. Its molecular weight was calculated to be 57,500, and together with the results of the gel filtration method, it was determined that this enzyme has a monomer structure.

(7) Effect of inhibitor treatment: The influence of various inhibitor treatments was carried out by the following method. First, 0.01 mM of 22 mM of each inhibitor (however, 1.1% in the case of a surfactant), 0.2 mM BSA-containing 100 mM HEPES-sodium hydroxide buffer pH 7.0 containing 0.017 mg / ml of enzyme solution was added. 0.1 ml was added,
The treatment was carried out for 1 hour. Then, after the above treatment, 0.2% B
SA-containing 10 mM potassium phosphate buffer (pH 7.0)
Was diluted 200 times and the residual enzyme activity was determined by the activity measuring method described below. The results are shown in Table 2, and the enzyme is inactivated by heavy metal ions of copper, mercury, silver, sodium dodecyl sulfate (SDS), and p-chloromercuribenzoic acid (PCMB).

[0018]

[Table 2]

(8) Km value From the line Weber-Burk plot, the Km value is P
1. NPG as a substrate at pH 7.0 and 37 ° C.
It is 8 × 10 ⁻³ M.

(9) Method for measuring titer: The titer of this enzyme was measured by the following method, and 1 μmo / min was measured from PNPG.
The amount of enzyme that liberates 1 para-nitrophenol was defined as 1 unit (U).

1 solution: Substrate solution 20 mM PNPG solution 0.603 g of PNPG was dissolved in distilled water to obtain 100 ml.
And 2 solution; buffer solution 0.1M potassium phosphate buffer solution pH 7.0 1M-potassium phosphate solution and 1M-dipotassium phosphate solution were mixed to adjust to pH 7.0. 3 solution: Reaction stop solution 0.2 M sodium carbonate solution 21.2 g of anhydrous sodium carbonate was dissolved in distilled water and 10
00 ml. Enzyme diluent A 0.01 M potassium phosphate buffer (pH 7.0) containing 0.2% BSA was prepared.

(Measurement procedure) First, the above-mentioned 1 liquid 0.2
5 ml, 2 solutions 0.5 ml were mixed, pre-incubated at 37 ° C. for 5 minutes, and then the pre-incubated solution was mixed with 0.25 ml of enzyme solution adjusted to about 0.005-0.02 U / ml, 15 at 37 ° C
Allowed to react accurately for minutes. Then, after the reaction time is over, 1 ml of the 3rd solution is added to stop the reaction, and the para-nitrophenol is allowed to develop color.
(A, manufactured by Hitachi Ltd.), the absorbance of the sample at 400 nm was measured. In addition, the blank value is measured by mixing the substrate solution and the buffer solution, pre-incubating, and then incubating for 15 minutes exactly without adding the enzyme solution.
ml was added, and then the enzyme solution was added to measure the blank absorbance.

(Calculation of titer) From the respective values of the sample absorbance and the blank absorbance determined by the above-mentioned measuring method, the titer was determined by the following formula 1.

[0024]

[Equation 1]

(10) Purification method: The present enzyme can be isolated and purified by a conventional method, for example, ammonium sulfate salting-out method, organic solvent precipitation method, ion exchange chromatography method, gel filtration chromatography method, adsorption method. The chromatographic method, the affinity chromatographic method, the electrophoretic method, the hydrophobic chromatographic method and the like can be used alone or in an appropriate combination.

The main physicochemical properties of the enzyme of the present invention are as described above, and the reason why this enzyme is novel is shown below. As mentioned above, various αs derived from microorganisms have been used.
-Glucosidases have been found, where the major physicochemical properties of this enzyme and those known enzymes are shown in Table 3 for comparison.

[0027]

[Table 3]

In Table 3, the data on the yeast-derived α-glucosidase (Note 1) is shown in Bioscience Biotechnology Biochemistry (Biosci. Bi).
otech. Biochem. ) Volume 57, Issue 11,
Pp. 1902-1905, 1993, and (Note 2) Bacillus stearothermophilus (Bacil)
The data of α-glucosidase derived from L. stearothermophilus is shown in Biochemica Biophysica Actor (Biochimica et Bi).
Ophysica Acta) Volume 787, Volume 281-
289, 1984, and data for α-glucosidase derived from rice in (Note 3) are further described in Starch Kagaku (Denpun Kagaku) Vol. 36, No. 3, 181-187, 189-195. , 1989 respectively.

As can be seen from Table 3, in terms of its substrate specificity, this enzyme acts well on maltooligosaccharides having a degree of polymerization of maltopentaose to maltooctaose and also on soluble starch. Further, since it is excellent in heat stability and pH stability, it is different from the yeast-derived enzyme. Since the present enzyme is stable in this way, it is extremely advantageous in the use and production of the enzyme. Is. Further, unlike the rice-derived enzyme, this enzyme has an optimum pH in the vicinity of neutrality, and thus is suitable for use in the vicinity of neutrality. This enzyme is also used in Bacillus stearothermophilus.
Unlike the enzyme derived from lus), the optimum temperature is 50-55 ° C.
Since the activity at around 40 ° C shows about 80% of its maximum activity, it is generally performed at 30 ° C or 3 ° C.
It is useful as an enzyme for conjugation, measurement, elimination, etc. used for enzyme reaction at 7 ° C or the like. From these facts, the enzyme of the present invention is a novel α-glucosidase having a useful property that has not been heretofore available.

Next, the method for producing the present enzyme will be described. First, as a bacterium to be used, any bacterium belonging to the genus Bacillus may be used as long as it is a strain having the ability to produce this enzyme,
Further, it may be a variant or mutant strain of these bacteria. And as a specific example of the microorganism, Bacillus sp.
cillus sp. ) KS-41B, and variants or mutants of the strain can also be used. This Bacillus sp. KS-41B is a strain obtained by the present inventors from soil in Kumamoto prefecture, and its mycological properties are as shown below. The experiments for identifying the mycological properties were conducted mainly by Takeshi Hasegawa, “Classification and Identification of Microorganisms, The University of Tokyo Press (1975). As a criterion for classification and identification, the Vergiz Manual of Determinative Bacteriology (No. 1) was used. 8th edition (1974
Year).

Bacteriological properties of Bacillus sp. KS-41B (A) Morphological properties Microscopic observation (50 on broth agar medium (pH 7.0))
C, 5-7 hours culture). (1) Cell shape and size: 0.1 to 0.5 × 1 to 5 micron bacilli. (2) Mobility: Mobility. (3) Spore presence: Yes. Formed at the end. (4) Gram staining: positive.

(B) Growth state in each medium (pH 7.0) (1) Meat broth agar plate culture A light brown circular colony in static culture at 50 ° C. for 16 hours.
To form No pigment production is observed. (2) Slope culture of broth agar It shows filamentous growth when cultured at 50 ° C. for 16 hours. (3) Liquid culture of broth Grows on the whole medium (turbidity) by static culture at 50 ° C for 16 hours.
Is recognized. Precipitation occurs at the bottom. (4) Meat juice gelatin stab culture Gelatin is liquefied by static culture at 50 ° C for 3 days. (5) Litmus milk culture After standing at 50 ° C. for 48 hours, no coagulation or liquefaction of litmus milk is observed, and no production of acid or alkali is observed.

(C) Physiological properties The test was carried out mainly using a medium adjusted to pH 7.0. (1) Reduction of nitrate: No reduction. (2) Denitrification reaction: None. (3) MR test: negative. (4) VP test: negative. (5) Indole generation: No generation. (6) Generation of hydrogen sulfide: No generation. (7) Hydrolysis of starch: It is hydrolyzed. (8) Use of citric acid: Do not use. (9) Use of inorganic nitrogen source: Use both nitrate and ammonium salt. (10) Formation of dye: No formation. (11) Urease: negative. (12) Oxidase: weak but positive. (13) Catalase: Positive. (14) Growth range: temperature; 33 to 65 ° C., pH;
1 to 8.8 (15) Attitude toward oxygen: aerobic. (16) OF test (Hugh-Leifson
Method): Oxidation (adding 0.5% glucose as a carbon source) (17) Generation of acid and gas from saccharides: As shown in Table 4, D-glucose, D-fructose, D-xylose, D-arabinose, Generation of acid is observed from D-mannitol, D-sorbitol, and dextrin, but no gas generation is observed from any saccharide.

[0034]

[Table 4]

This strain, which has the above-mentioned mycological properties and has a novel α-glucosidase-producing ability, is positive in Gram staining, has sporulation ability, has motility, and OF test. Is determined to belong to the genus Bacillus. Therefore, this strain was designated as Bacillus sp. KS-41B.
It was named and identified. This strain has been deposited as FERMP-15346 at the Institute of Biotechnology, Institute of Biotechnology, AIST. As the α-glucosidase in the present invention, the above-mentioned action, as long as it has major physicochemical properties such as substrate specificity, even if other physicochemical properties show some differences, Included as an enzyme. The above-mentioned microorganism is an example of a bacterium used for obtaining such α-glucosidase, and in the present invention, any microorganism can be used as long as it belongs to the genus Bacillus and has the α-glucosidase-producing ability.

In order to produce the present enzyme using the above-mentioned strain used in the present invention, any medium can be used as long as it contains a carbon source, a nitrogen source, an inorganic substance and other nutrient sources in a proper amount. The carbon source may be any carbon compound capable of inducing the present enzyme, and for example, a medium containing maltose, soluble starch, powdered starch syrup (manufactured by Showa Sangyo Co., Ltd.) or the like is preferably used. The nitrogen source may be any available nitrogen compound, and examples thereof include yeast extract, peptone, meat extract, corn steep liquor, soybean powder, amino acid, ammonium sulfate, ammonium nitrate and the like. In addition, salt, potassium chloride, magnesium sulfate, manganese chloride,
Various salts such as ferrous sulfate, potassium dihydrogenphosphate, potassium dihydrogenphosphate and sodium carbonate, vitamins, antifoaming agents and the like are used. These nutrient sources can be used alone or in combination.

In order to produce the present enzyme using the liquid medium prepared as described above, aerobically culturing is preferably carried out by aeration-agitation deep culture or shaking culture. At that time, the initial pH of the medium is adjusted to about 6.5 to 7.0,
Culturing is performed at a temperature of 35 to 55 ° C., preferably about 50 ° C. for 7 hours or more. After completion of the culture, the enzyme can be collected from the culture using conventional enzyme collecting means.

Since the present enzyme is an enzyme mainly present in the microbial cells, it is preferable to separate the microbial cells from the culture by an operation such as filtration and centrifugation, and collect α-glucosidase from the microbial cells. . In this case, although the bacterial cells can be used as they are, an ultrasonic crusher, a French press,
A method of destroying bacterial cells using various destroying means such as dynamyl, a method of lysing bacterial cell wall using a cell wall lysing enzyme such as lysozyme, an enzyme from bacterial cells using a surfactant such as Triton X-100 It is preferable to employ a method of extracting the above or the like, and these can be used alone or in appropriate combination. Then, impurities are removed by filtration or centrifugation to obtain a crude enzyme solution of the present enzyme.
In order to isolate the present enzyme from the crude enzyme solution thus obtained, the method used for the usual enzyme purification as described above can be used.

The present enzyme obtained as described above is used, for example, as a coupling enzyme for measuring α-amylase activity and quantifying chlorine ion, as an enzyme for measuring and eliminating maltooligosaccharides such as maltose, or from oligosaccharides. It can be used as an industrially produced enzyme for producing glucose as well as monosaccharide sugars such as fructose.

[0040]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples. (Example 1) Powdered starch syrup (manufactured by Showa Sangyo Co., Ltd.) 2.0%, polypeptone 2.0%, yeast extract 1.0%, 1 potassium phosphate 0.01%, 2 potassium phosphate 0.01%,
100 ml of a medium (pH 7.0) consisting of 0.01% magnesium sulfate heptahydrate and tap water was placed in Sakaguchi Korben and sterilized at 121 ° C. for 7 minutes. Bacillus sp. KS-41B was added to this medium.
(1) from the preservation slant of (FERM P-15346)
A platinum loop was inoculated, and this was cultured with shaking on a shaker at 40 ° C. for about 16 hours to obtain a seed culture solution. Then, another antifoaming agent (Nissan Dishome CC-48
5 ml of 5) was added, and 100 ml of the seed culture solution (for one Sakaguchi Korben) was inoculated into a 30 L jar fermenter containing 20 L of medium prepared by sterilizing at 121 ° C. for 7 minutes, and the rotation speed was 300 rpm and the internal pressure was 0. 0.5L, ventilation 2
The culture was performed at 0 L / min and a temperature of 50 ° C. for about 7 hours. After completion of the culturing, from 20 L of the culture solution, Microza PW-303
The cells were collected using (Asahi Kasei), washed with tap water, and then concentrated to about 10 L.

Purification of this enzyme was performed by the following procedure. Step 1 (preparation of crude enzyme solution):
0.55M EDTA disodium salt (pH 8.0)
1 L was added and mixed, and the mixture was allowed to stand at 20 ° C. overnight, then 11 g of egg white lysozyme was further added and allowed to stand overnight to lyse the cells.
After lysis, 5% aqueous protamine solution (pH 8.0) 2
Nucleic acid removal treatment was performed by dropping 00 ml with stirring.
This supernatant was added to a 0.01 M potassium phosphate buffer (pH 7.0) containing 0.1 M potassium chloride using an ultrafiltration membrane.
It was dialyzed against (hereinafter referred to as buffer solution A).

Step 2 (DEAE-Cellulose treatment): The above dialysate (about 10 L) was wet weighted at about 9 K.
After adding and mixing g DEAE-cellulose to adsorb the present enzyme, the DEAE-cellulose was washed with 0.1 M potassium chloride-containing buffer A, and then 0.3 M potassium chloride-containing buffer A was added. The present enzyme was eluted with the filter, the eluate was concentrated using a follofiber type ultrafiltration device, and dialyzed by substituting the buffer A with 0.05 M potassium chloride.

Step 3 (QAE-Sephadex A-
50 column chromatography): the above dialysate (1
000 ml), 1000 ml of QAE-Sephadex A-50 was added and mixed to adsorb the present enzyme.
The QAE-Sephadex A-50 was washed with the buffer A containing 0.07M potassium chloride, and then 0.07M-
Elution was performed with the buffer solution A containing 0.2 potassium chloride by the linear concentration gradient method. The eluate was concentrated using a follofiber type ultrafiltration device, and dialyzed by replacing with 0.04 M potassium chloride-containing buffer solution A.

Step 4 (DEAE Toyopearl 650
C column chromatography): The above dialysate (500
ml) was adsorbed on a column of DEAE-Toyopearl 650C (4.2 x 30 cm), washed with buffer A containing 0.04 M potassium chloride, and then 0.04 M to 0.
Elution was performed with the buffer A containing 1 M potassium chloride by the linear concentration gradient method. The eluted active fraction was concentrated using a follofiber type ultrafiltration device, and dialyzed for substitution with buffer solution A.

Step 5 (Butyl Toyopearl 650C column chromatography): The dialysate (37 ml)
To the butyltoyopearl 650C column (2.7 × 20c), an equal amount of 2M ammonium sulfate-containing buffer solution A was added.
m) and washed with 1 M ammonium sulfate-containing buffer A, and then eluted with 1 M to 0.5 M ammonium sulfate-containing buffer A by the linear concentration gradient method. The eluted active fraction was concentrated using a follofiber type and a flat membrane type ultrafiltration device to obtain 0.01M potassium phosphate buffer solution pH 7.0.
Dialysis was performed to replace

Step 6 (Hydroxyapatite column chromatography): The above dialysate (40 ml) was adsorbed on a hydroxyapatite column (2.7 × 10 cm), and 0.01 M potassium phosphate buffer pH 7.0.
And then eluted with a linear concentration gradient method using 0.01 M to 0.2 M potassium phosphate buffer (pH 7.0). The eluted active fraction was concentrated using a fiber optic type and flat membrane type ultrafiltration equipment to
Dialysis was performed by replacing with M potassium phosphate buffer.

Step 7 Gel filtration (Bio-Gel
A1.5m200-400mesh): The above dialysate (about 2 ml) was added to Bio-Gel A1.5m200-
Column filled with 400 mesh (2.5 x 95c
buffer solution A containing 0.1 mM potassium chloride.
Gel filtration was carried out with and the eluted active fraction was collected.
By the above purification operation, a purified enzyme preparation (about 106.7
U, 127.8 U / mg protein).

[0048]

INDUSTRIAL APPLICABILITY The present enzyme has malt oligosaccharides having a degree of polymerization ranging from maltose to maltooctaose, paranitrophenyl-α-D-glucopyranoside, paranitrophenyl-α-D-maltoside, and paranitro in substrate specificity. It acts on phenyl-α-D-maltopentaoside and nigerose, but it does not act on isomaltose and kojibiose, and it has excellent heat resistance and pH stability, and its optimum pH is near neutral. The α-amylase activity, which has been widely performed by yeast-derived enzymes having poor stability, is characterized by the fact that the optimum temperature range is about 80% of the maximum activity even at a practical measurement temperature, for example, 40 ° C. As a coupling enzyme for measurement and quantification of chloride ion, and for malto-oligosaccharides such as maltose. As the amount and erasing enzyme, a very high utility value as a good stability enzymes. The enzyme can also be used in industrial production such as production of glucose and monosaccharides such as fructose from oligosaccharides, and enzymatic synthesis of rare hetero-oligosaccharides utilizing the glycosyl transfer activity. Furthermore, according to the method of the present invention, a large amount of the present enzyme can be obtained in a single hour by culturing a microorganism. Moreover, since the present enzyme has excellent stability against heat and pH, heat treatment up to 55 ° C., for example. , PH
Since it can be purified in a wide range of 6.0 to 10.5 and is stable to the treatment with a surfactant, the enzyme can be easily produced at low cost and is extremely useful in industry. is there.

[Brief description of drawings]

FIG. 1 is a graph showing the optimum pH of this enzyme at 37 ° C.

FIG. 2 In the presence of 0.2% BSA of this enzyme at 25 ° C.
The graph which shows the stable pH range in a 20-hour process.

FIG. 3 is a graph showing the optimum temperature range for action of the present enzyme at pH 7.0.

FIG. 4 is a graph showing the thermal stability of this enzyme at pH 7.0.

[Fig. 5] Electropherogram of this enzyme.

Claims (2)

[Claims] 1. A novel α-glucosidase having the following physicochemical properties. (A) Action: α-glucoside bond is hydrolyzed to produce α-
It produces D-glucose. (B) Substrate specificity: maltooligosaccharides having a degree of polymerization from maltose to maltooctaose, paranitrophenyl-α-D-glucopyranoside, paranitrophenyl-α-D-maltoside, paranitrophenyl-
It acts on α-D-maltopentaoside and nigerose, but does not act on isomaltose and cordiose. (C) Optimum pH and stable pH range: The optimum pH is 6.5.
It is around 7.0 and the stable pH range is pH 6.0 when treated at 25 ° C. for 20 hours in the presence of 0.2% bovine serum albumin.
１10.5. (D) Optimum temperature range of action: The optimum temperature range of action is 50-55.
It is around ℃. (E) Conditions of inactivation due to pH, temperature, etc .: pH at 20 ° C. for 20 hours at 25 ° C. in the presence of 0.2% bovine serum albumin
It is stable in the range of 6.0 to 10.5 and deactivates at pH 4.5 or lower or pH 11 or higher. Also, the thermal stability is pH
It is stable up to 55 ° C after treatment for 7.0 minutes, and completely deactivates at 65 ° C or higher. (F) Molecular weight: about 42,000 ± 5,000 (gel filtration method). (G) Effect of inhibitor treatment: In the presence of 0.2% bovine serum albumin, pH 7.0, 25 ° C, 1 hour inhibitor treatment, copper, mercury and silver heavy metal ions and sodium dodecyl sulfate, p- Deactivated by chloromercuribenzoic acid. (H) Km value: Km at pH 7.0 and 37 ° C. using para-nitrophenyl-α-D-glucopyranoside as a substrate.
The value is 2.8 × 10 ⁻³ M. 2. A strain which belongs to the genus Bacillus and has the α-glucosidase-producing ability having the physicochemical properties according to claim 1, is cultured in a medium, and the α-glucosidase is collected from the culture. The method for producing α-glucosidase according to claim 1.