JP3190537B2 - Method for producing novel maltose 1-epimerase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The present invention relates to β-maltose and α-maltose.
The present invention relates to a method for producing a novel maltose 1-epimerase which catalyzes an interconversion reaction with maltose.

[0002]

BACKGROUND OF THE INVENTION Maltose is added to starch, for example, β-
It can be obtained by the action of amylase, but recently, industrially pure products have been mass-produced and their uses have been expanding. The sweetness of this maltose is said to be 30 to 40% of sugar, but the mild rounded sweetness is considered to be the most delicious sweetness among sugars. Maltose is widely used in the field of foods, in addition to being used to reduce the sweetness of sugar, using the physical properties of maltose itself, especially for improving taste, maintaining color, and improving food properties. It is used for the purpose of. In addition, in medicine, maltose is absorbed and digested without the need for insulin, and isotonic solution having twice the concentration of glucose can be prepared, so that it is used for diabetic patients and for intravenous injection of patients during and after surgery. It is also used as a sugar replenisher. For this reason, quantifying the maltose content in food has a very important industrial significance as well as the sensory test in evaluating food. It is also strongly desired by those skilled in the art to easily measure the maltose content in the blood of diabetic patients and the like.

[0003] Conventionally, as a method of quantifying maltose in a maltose-containing sample, for example, a method of reacting α-glucosidase on maltose in the sample and quantifying the produced glucose [Method of Enzymatic Analysis (Massod of Enzymatic Analysis)] Methods of Enzym
atic Analysis), Vol. 3, No. 1185
P. 1974], a method of reacting maltose phosphorylase on maltose in the sample in the presence of arsenate to determine the amount of glucose produced [Analytical Biochemistry (Analytical Biochemistry)].
misry), vol.57, p.303, 1974]
Etc. are known. However, the above method
Since α-glucosidase acts on maltotriose to maltopentaose in addition to maltose, maltose cannot be quantified accurately. On the other hand, the above method is known to be excellent in maltose quantification because maltose phosphorylase specifically acts on maltose and does not act on other oligosaccharides.

However, maltose phosphorylase has α
-Acts very specifically on maltose [Archives
Of chemistry and biophysics (Archives of Biochemistry)
y and Biophysics), Vol. 281, p. 58, 1990], since it does not act on β-maltose, the amount of maltose in a normal maltose-containing sample containing both α-maltose and β-maltose anomers is determined. The disadvantage is that it takes a very long time,
It is not suitable for quantifying maltose in maltose-containing samples such as food and blood.

[0005] Therefore, as a result of various studies, the present inventors have previously found that a novel maltose 1-epimerase having the following physicochemical properties and a microorganism belonging to the genus Lactobacillus, which is suitably used for the quantification of maltose, use the enzyme. It was found to be produced (see Japanese Patent Application No. 6-301706).

[0006]

SUMMARY OF THE INVENTION The object of the present invention is to provide a method for producing the above-mentioned maltose 1-epimerase using other microorganisms.

[0007]

Means for Solving the Problems The present inventors have conducted intensive studies to achieve the above object, and as a result, the strain belonging to the genus Streptococcus, Leuconostoc, Enterococcus or Pediococcus has been transformed into the maltose 1 strain.
-It was found that epimerase was produced, and the present invention was completed based on this finding.

That is, the present invention relates to maltose 1-epimerase which belongs to the genus Streptococcus, Leuconostoc, Enterococcus or Pediococcus and has an action of catalyzing an interconversion reaction between β-maltose and α-maltose. A method for producing a novel maltose 1-epimerase, which comprises culturing a strain having the ability to produce in a medium and collecting the maltose 1-epimerase from the culture, and the present invention further provides Streptococcus sp. A strain belonging to the genus Stock, Enterococcus or Pediococcus and capable of producing maltose 1-epimerase having the following physicochemical properties is cultured in a medium, and the maltose 1-epimerase is collected from the culture. Novel maltose 1-epimera This is a method for producing ose. (1) Action: catalyzes the interconversion reaction between β-maltose and α-maltose. (2) Substrate specificity: specifically acts on maltose and does not substantially act on cellobiose or lactose. (3) Optimum pH and stable pH range: Optimum pH is 5.5
The stable pH range is pH 5.0 to 8.5 after treatment at 30 ° C. for 30 minutes. (4) Molecular weight: about 43,000 (gel filtration method).

Hereinafter, the present invention will be described in detail. First, a novel enzyme, maltose 1-epimerase (hereinafter sometimes referred to as "the present enzyme"), which the present inventors have previously found.
The physicochemical properties of are as follows. (1) Action: Using 10 ml of 50 mM Hepes-sodium hydroxide buffer (pH 7.0) containing 1.0% β-maltose and 60 units (U) of the present enzyme as a reaction system,
The reaction was carried out at 25 ° C. for 10 minutes, and the specific rotation [α] _D after the reaction was measured using a polarimeter by the method described in [Measurement Method 1] described below. As a result, the specific rotation of the reaction solution is +1
It was 30 ゜. The specific rotation before the enzymatic reaction is + 112 °, β-maltose is converted to α-maltose,
Both anomers have reached equilibrium, which means that the enzyme

[0010]

Embedded image

As shown by the chemical formula 1, it has been found that it catalyzes an interconversion reaction between β-maltose and α-maltose. In addition, it was confirmed by high performance liquid chromatography that maltose did not undergo decomposition or the like after the enzymatic reaction.

(2) Substrate specificity: As a result of examining the substrate specificity by measuring the change in optical rotation of each substrate using a polarimeter according to [Measurement method 1] described below, the enzyme was found to be specific for maltose. It acts on cellobiose or lactose. Table 1 shows an example of examining the relative activity of the present enzyme for various substrates.

[0013]

[Table 1]

(3) Optimal pH and stable pH range: optimal p
H is a 50 mM acetic acid-sodium acetate buffer solution (pH 4.
2-5.5), 50 mM female-sodium hydroxide buffer (pH 5.5-6.5), 50 mM Hepes-sodium hydroxide buffer (pH 6.5-8.5) and 50 mM bistrispropane-hydrochloride buffer. Using the solution (pH 8.5 to 9.0), the activity of the present enzyme at each pH was measured according to [Measurement Method 1] described below. The results are as shown in FIG. 1, and the optimum pH of the present enzyme is 5.5 to 7.5, particularly pH 6.5 to 7.0. In the marks shown in FIG. 1, □ indicates a 50 mM acetic acid-sodium acetate buffer solution (pH
4.2 to 5.5), ■ indicates a 50 mM female-sodium hydroxide buffer (pH 5.5 to 6.5), and ● indicates a 50 mM hepes-sodium hydroxide buffer (pH 6.5 to 8.5).
Indicates the results when 50 mM bistrispropane-HCl buffer (pH 8.5 to 9.0) was used. The stable pH range is 50 mM acetic acid-sodium acetate buffer (pH 4.0 to 5.0), 50 mM
mM female-sodium hydroxide buffer (pH 5.5-6.
0), 50 mM Hepes-sodium hydroxide buffer (pH
7.0-8.5) and 50 mM bistrispropane-HCl buffer (pH 9.0-10.0), pH 4.0.
After treatment at 30 ° C. for 30 minutes at １10.0, the residual activity of the present enzyme was measured and determined (using assay method 2 described below). The results are as shown in FIG. 2, and the stable pH range of the present enzyme is 5.0 to 8.5. In the marks shown in FIG. 2, □ indicates a 50 mM acetic acid-sodium acetate buffer (pH 4.0 to 5.0), and Δ indicates a 50 mM female-sodium hydroxide buffer (pH 5.5 to 6.0). ,
● indicates 50 mM Hepes-sodium hydroxide buffer (pH
7.0 to 8.5), and ○ shows the results when using 50 mM bistrispropane-HCl buffer (pH 9.0 to 10.0), respectively.

(4) Molecular weight: TSKgel G3000
The molecular weight is about 43,000 by a gel filtration method using SW _XL (manufactured by Tosoh Corporation). In addition, since the molecular weight by SDS-polyacrylamide gel electrophoresis is about 45,000, this enzyme is considered as a monomer.

(5) Measuring method of titer: The titer of the enzyme was measured by the following two methods, and these measuring methods were appropriately used. The amount of the enzyme that converts 1 μmol of β-maltose into α-maltose per minute is defined as 1 unit (U). [Measurement method 1] Weighed maltose monohydrate 105.3
A 50 mM Hepes-sodium hydroxide buffer solution (pH 7.
0) Add 10 ml, stir immediately, dissolve, and use a polarimeter equipped with a measurement cell kept at 25 ° C., at 589 nm.
(Sodium lamp) The amount of optical rotation change (millidegree / min) of the enzyme reaction solution is measured. In addition,
The measurement of the optical rotation change of the control was carried out with 50%
mM Hepes-sodium hydroxide buffer (pH 7.0) 1
The measurement is performed in the same manner as described above except that 0 ml is added. Next, the converted α-maltose is quantified based on the difference in the amount of optical rotation between the enzyme reaction solution and the control.

[Measurement Method 2] <Preparation of Coloring Reagent> Maltose phosphorylase 110
U, mutarotase 1000 U, glucose oxidase 900 U, peroxidase 100 U, monopotassium phosphate 400 μmol, phenol 100 μmol and 4
Prepared by dissolving 5.0 μmol of aminoantipyrine in 8 ml of 50 mM Hepes-sodium hydroxide buffer (pH 7.0). <Preparation of Substrate Solution> Immediately before the enzymatic reaction, 21.6 mg of maltose monohydrate was added to 20 ml of ice-cooled 50 mM Hepes-sodium hydroxide buffer (pH 7.0).
Prepare by dissolving in <Measurement method> 0.8 ml of the above-mentioned coloring reagent and 0.1 ml of the enzyme solution appropriately diluted were placed in a micro cuvette and placed at 25 ° C.
After preliminarily heating for 2 minutes, the reaction was started by adding 0.1 ml of the above substrate solution, and while reacting at 25 ° C. for 3 minutes,
The amount of change in absorbance at 505 nm is measured with a spectrophotometer. The control was 50 instead of 0.1 ml of the enzyme solution.
The measurement is performed in the same manner as described above except that 0.1 ml of a mM Hepes-sodium hydroxide buffer (pH 7.0) is added. Next, the converted α-maltose is quantified based on the difference in the amount of increase in absorbance between the enzyme reaction solution and the control per minute during 1 to 2 minutes of the reaction. This [Measurement method 2]
The increase in absorbance at 505 nm per minute of 0.3 determined in the above step was 1 of the titer of the enzyme determined in [Measurement method 1].
Equivalent to unit.

Next, examples of other physicochemical properties of the present enzyme will be described. (6) Range of suitable temperature for operation: various temperatures (20 ° C to 45 ° C)
The activity of the present enzyme was measured according to the above [Measurement method 2]. The results are as shown in FIG. 3, and the suitable temperature range for the action of the present enzyme is 37 ° C. to 43 ° C., particularly around 40 ° C.

(7) Deactivation conditions due to pH, temperature, etc .:
When treated with this enzyme at 30 ° C. for 30 minutes, p
It is stable at H5.0 to 8.5 and, as can be seen from FIG. 2, rapidly deactivates on the acidic side and alkaline side, and deactivates by 50% or more at pH 4 or lower and pH 10 or higher. In addition, this enzyme was heated at each temperature (30 ° C. to 7 ° C.) using a 50 mM Hepes-sodium hydroxide buffer solution (pH 7.0).
(5 ° C.) for 10 minutes, and the thermal stability was examined according to the above [Measurement Method 2]. The results are as shown in FIG. 4. The enzyme is stable up to 45 ° C., beyond which it starts to be inactivated, and at 65 ° C. or more, there is 50% or more inactivation.

(8) Inhibition, activation and stabilization: 50 mM
This enzyme and various additives (metal salt and metal chelating agent) were dissolved in a Hepes-sodium hydroxide buffer (pH 7.0) to a concentration of 2 mM each, and treated at 30 ° C. for 30 minutes. The enzyme activity of the treatment solution was measured according to the above [Measurement method 2]. The results are as shown in Table 2,
Although the enzyme is strongly inhibited by HgCl ₂ and CuSO ₄ , no additives are found that contribute specifically to activation and stabilization.

[0021]

[Table 2]

(9) Purification method: The isolation and purification of the present enzyme can be carried out according to a conventional method, for example, ammonium sulfate precipitation, organic solvent precipitation, adsorption treatment using an ion exchanger, ion exchange chromatography, and the like. Hydrophobic chromatography, gel filtration chromatography, adsorption chromatography,
Affinity chromatography, electrophoresis and the like are used alone or in appropriate combination.

(10) Km value: When the initial rate of the present enzyme is measured by changing the substrate concentration at 25 ° C. according to a conventional method, a sharp increase in the substrate blank value due to spontaneous rotation of light is observed particularly at a high substrate concentration. Since the initial rate of the enzyme could not be measured, the Km value was measured at 5 ° C. (using [measurement method 2]). From the Lineweaver-Burk plot, Km
The value is 2.2 × 10 ⁻³ M (relative to β-maltose).

(11) SDS-polyacrylamide gel electrophoresis: SDS-polyacrylamide gel electrophoresis was carried out by a conventional method using a polyacrylamide gel having a concentration gradient of acrylamide of 10 to 20% (W / V). As shown in FIG. 5, a single band of the present enzyme was observed. Relative mobility (Rf) after energization at 60 mA for 60 minutes
Is 0.34.

Next, a method for producing the present enzyme will be described. First, the microorganism used is a strain belonging to the genus Streptococcus, the genus Leuconostoc, the genus Enterococcus or the genus Pediococcus and having the maltose 1-epimerase-producing ability, and specific examples thereof include:
In the genus Streptococcus, Streptococcus sp.
In the genus Leuconostoc, such as 12546, Leuconostoc mesenteroides (Leuconostoc)
enterococcus hirae (Enterococcus hirae) IFOs in the genus Enterococcus, such as AT.
3181, and in the genus Pediococcus, Pediococcus pentosaceus (Pediococcus p.
entosaceus) IFO 3894, etc., and variants or mutants of these strains can also be used. The novel maltose 1-epimerase in the present invention may have any of the above-described functions and major physicochemical properties such as substrate specificity, and other physicochemical properties may exhibit some differences. Are also included as the present enzyme. Each of the aforementioned microorganisms is an example of a bacterium used to obtain such maltose 1-epimerase, and in the present invention belongs to the genus Streptococcus, Leuconostoc, Enterococcus or Pediococcus, and maltose 1-epimerase. Any strain capable of producing can be used.

Next, in order to produce maltose 1-epimerase using a microorganism capable of producing maltose 1-epimerase, an ordinary solid culture method may be used, but a liquid culture method is preferred. As the medium, either a synthetic medium or a natural medium can be used as long as it contains a carbon source, a nitrogen source, an inorganic substance, and other nutrients appropriately. As the carbon source, any assimilable carbon compound may be used, and for example, maltose, glucose, starch hydrolyzate, glycerin, fructose, molasses, and the like are used. As the nitrogen source, any available nitrogen compound may be used. For example, yeast extract, peptone, meat extract, corn steep liquor, soybean flour, amino acids, ammonium sulfate, ammonium nitrate and the like are used. In addition, salt, potassium chloride, magnesium sulfate, manganese chloride, ferrous sulfate,
Various salts, vitamins, such as potassium monophosphate, potassium diphosphate, sodium carbonate, sodium acetate,
An antifoaming agent is used. Each of these nutrients can be used alone or in combination.

In order to produce the present enzyme using the liquid medium thus prepared, aerobically culturing may be carried out by aeration and stirring submerged culture or shaking culture. Is preferred. At that time, the initial pH of the medium
Is adjusted to about 5 to 9, and 20 to 37 ° C., preferably 30
10 to 50 hours at a temperature of about 50 ° C., preferably 15 to 25 hours.
Incubate for hours. By doing so, the present enzyme is produced and accumulated in the culture. In order to collect the present enzyme from this culture, ordinary enzyme collecting means can be used.

Since the present enzyme is mainly present in the cells, it is preferable to separate the cells from the culture by operations such as filtration and centrifugation, and to collect the cells from the cells. . In this case, a conventional method, for example, a method of destroying cells using various disrupting means such as an ultrasonic crusher, a French press, and a dynamyl, a method of lysing cell walls using a cell wall lysing enzyme such as lysozyme, Triton X-1
For example, a method of extracting an enzyme from cells using a surfactant such as 00 can be used alone or in combination. Next, insoluble substances are removed by filtration or centrifugation to obtain a crude enzyme solution of the present enzyme. The above-mentioned purification method can be applied to isolate the present enzyme from the crude enzyme solution thus obtained, if necessary.

[0029]

According to the present invention, a novel maltose 1-epimerase which acts specifically on maltose and is capable of performing an interconversion between β-maltose and α-maltose in a very short time can be obtained with high efficiency. Can be manufactured well.

[0030]

Next, the present invention will be described in more detail by way of examples, but the present invention is not limited to these examples.

Example 1 Maltose monohydrate 1.0% (W / V), polypeptone 2.0% (W / V), yeast extract 1.0% (W / V), sodium acetate 1.0 % (W / V), MgSO 4 · 7H 2 O0.0
2% (W / V), MnCl ₂ .4H ₂ O 0.0002% (W
/ V) and 20 liters of medium (pH 7.0) consisting of tap water
The mixture was sterilized in a 0-liter jar at 120 ° C. for 10 minutes.
This medium contains Leuconostoc mesenteroides (L
euconostocmesenteroides) A
From a stock slant (three) of TCC 12291, a cell suspension prepared using physiological saline was inoculated, and this was inoculated with 3 cells.
A seed culture solution was obtained by stirring and culturing at 0 ° C. for about 24 hours at a rotation speed of 100 rpm without aeration. Then, the seed culture 1 was placed in a 500-liter tank containing 400 l of the same medium as described above.
0l, inoculated at 30 ° C for about 24 hours, rotation speed 50rpm
And agitated culture without aeration. After completion of the culture, the culture solution 40
0l to Microza (PW-303) manufactured by Asahi Kasei Corporation
The cells were collected using a 10 mM phosphate buffer (pH 6.
5) After washing the cells with (buffer A), the cells were suspended in the same buffer to 15,000 ml. Purification of this enzyme was carried out at 1500 m
1 was performed by the following operation.

Step 1 (Preparation of Crude Enzyme Solution): 7 ml of Triton X-100 was added to 1500 ml of the cell suspension.
And 1.5 g of lysozyme were added, mixed, heated at 35 ° C. for 2 hours, and left at 5 ° C. overnight. Next, after adding and mixing 40 g of ammonium sulfate powder, 30 ml of a 10% (V / V) aqueous solution of polyethyleneimine (pH 7.0) was added dropwise with stirring to perform nucleic acid removal treatment. This supernatant was dialyzed against the buffer A containing 0.2 M potassium chloride using an ultrafiltration membrane. Step 2 (First QAE-Sephadex A-5
0. Chromatography): dialysate (1400ml)
Using a column of QAE-Sephadex A-50 (13 ×
After the adsorption, the column was washed with buffer A containing 0.2 M potassium chloride and then eluted with buffer A containing 0.6 M potassium chloride to limit the active fraction. Externally concentrated and dialyzed against buffer A containing 0.2 M potassium chloride. Step 3 (2nd QAE-Sephadex A-5
0. chromatography): dialysate (150 ml)
Column of QAE-Sephadex A-50 (6.0 × 2
0 cm), the column was washed with buffer A containing 0.2 M potassium chloride, and then washed with 0.2 M
The buffer A containing 0.6 M potassium chloride was eluted by the linear gradient method, and the active fraction was ultra-concentrated. Step 4 (Phenyl-Sepharose CL-4B / Chromatography): After adding ammonium sulfate powder to the concentrated liquid (40 ml) so as to be 20% saturated and mixing, a phenyl-Sepharose CL-4B column (2.5 × 30c
m), the column was washed with buffer A containing 20% saturated ammonium sulfate, and then eluted by reverse linear gradient method of buffer A containing 20% to 0% saturated ammonium sulfate. The active fraction was ultra-concentrated and concentrated in 2 mM phosphate buffer (pH
6.8) (hereinafter referred to as buffer B). Step 5 (hydroxyl apatite chromatography): After adsorbing dialysate (20 ml) to a column of hydroxyl apatite (2.5 × 10 cm), washing the column with buffer B, then buffer B And a buffer B in which only the concentration of the phosphate buffer was changed to 50 mM, and eluted by the linear concentration gradient method, and the active fraction was ultra-concentrated. Step 6 (Sephadex G-200 chromatography): Concentrate (about 2 ml)
After passing through a -200 column (2.5 x 95 cm), gel filtration was performed using buffer A containing 0.1 M sodium chloride, and 45 ml of the eluted active fraction was collected. The fraction was a purified sample of the enzyme determined to be homogeneous by SDS-polyacrylamide gel electrophoresis (FIG. 5), and had a total protein amount of 16.1 mg and a total activity of 26.
100 U, specific activity was 1620 U / mg.

Example 2 Using 10 ml of 50 mM Hepes-sodium hydroxide buffer (pH 7.0) containing 1.0% β-maltose, first, the course of spontaneous polarized light to be a control, that is, specific rotation [α] _D was measured with a polarimeter at 25 ° C. for 10 minutes. Next, 5% containing 1.0% β-maltose and 15 U, 30 U or 60 U of the present enzyme obtained in Example 1.
0 mM Hepes-sodium hydroxide buffer (pH 7.0)
Using 10 ml as a reaction system, the reaction was carried out at 25 ° C. for 10 minutes, and these specific rotations [α] _D were similarly measured using a polarimeter.
Was measured. In addition, based on the 12th revision Japanese Pharmacopoeia, in order to quickly perform the rotation, the time course of the rotation when the reaction solution was made alkaline (pH 10 or more) by adding ammonia water to the above control was also measured. did. FIG. 6 shows the result. In FIG. 6, (a) is a control, and (b), (c) and (d) are each the enzyme 15
Shows the course of specific rotation [α] _D when U, 30U, and 60U are used in the reaction system, and (e) shows the course of polarized light rotation when ammonia water is added to the control to make the reaction solution alkaline. Is shown. As is clear from FIG. 6, the specific rotation of the reaction solution is rapidly shifted from + 112 ° before the reaction to + 130 ° after the enzyme reaction by using this enzyme, and β-
It was observed that maltose was converted to α-maltose and both anomers had reached equilibrium, which was completely consistent with the specific rotation of the alkaline control. That is, the present enzyme
Because it rapidly catalyzes the interconversion reaction between β-maltose and α-maltose, for example, by using it in combination with maltose phosphorylase that acts very specifically on α-maltose, Maltose was recognized as a suitable enzyme that can be quantified in a short time.

Example 3 A 30 ml liquid medium prepared in the same manner as in Example 1 was placed in a large test tube and sterilized at 120 ° C. for 10 minutes. In this medium, Streptococcus sp.
occussp. ) From a stock slant of IFO 12546, inoculate one loopful of the bacterial mass and inoculate it at 30 ° C. for about 22
The culture was allowed to stand for a period of time. The growth amount of the strain during the culture was determined by measuring the absorbance of the culture at 660 nm. Next, cells were collected from 30 ml of the culture by centrifugation, washed with 10 mM phosphate buffer (pH 7.0) (hereinafter referred to as buffer C), and then washed at -20 ° C.
And stored frozen overnight. After thawing the frozen cells at room temperature, 2.0 ml of a buffer C containing 0.5% Triton X-100 and 0.2% lysozyme was added, mixed, and then mixed at 35 ° C.
For 2 hours. A supernatant was collected from the treated solution by centrifugation and used as an enzyme solution. According to the above [Measurement method 2], maltose 1 in the enzyme solution derived from the present strain was used.
-Measure the epimerase activity (U) and determine the growth amount (660 n
The activity value (U / growth amount) per OD value (m) was determined. As a result, the present strain belonging to the genus Streptococcus suitably produces maltose 1-epimerase (0.1
43U / growth amount).

Example 4 In a large test tube containing 30 ml of a liquid medium prepared in the same manner as in Example 1, Enterococcus hirae (Entero) was placed.
One loopful of the bacterial mass was inoculated from a stock slant of Coccus hirae IFO 3181, and this was incubated at 37 ° C. for about 22 hours. Then, this culture 30 m
An enzyme solution was prepared from Example 1 in the same manner as in Example 3. The maltose 1-epimerase activity (U) in the enzyme solution derived from the present strain was measured according to the above [Measurement method 2], and the growth amount (6
Activity value (U / growth amount) per OD value at 60 nm)
As a result, the strain belonging to Enterococcus produced maltose 1-epimerase appropriately (0.139 U
/ Growth amount).

Example 5 A Pedococcus pentosaceus (Ped) was placed in a large test tube containing 30 ml of a liquid medium prepared in the same manner as in Example 1.
iococcus pentosaceus) IFO
From 3894 stock slants, one loopful of bacterial mass was inoculated, and this was statically cultured at 30 ° C. for about 26 hours. Next, an enzyme solution was prepared from 30 ml of the culture in the same manner as in Example 3. According to the above [Measurement method 2], maltose 1-epimerase activity (U) in the enzyme solution derived from this strain was measured.
Was measured, and the activity value (U / growth amount) per growth amount (OD value at 660 nm) was determined. As a result, it was confirmed that the present strain belonging to Pediococcus preferably produced maltose 1-epimerase (0.028 U / growth amount).

[Brief description of the drawings]

FIG. 1 is a graph showing the optimum pH of the present enzyme.

FIG. 2 is a graph showing a stable pH range of the present enzyme.

FIG. 3 is a graph showing a suitable temperature range for the action of the present enzyme.

FIG. 4 is a graph showing the thermostability of the present enzyme.

FIG. 5 is an electrophoretogram of the present enzyme.

FIG. 6 is a graph showing the course of the rotation of light from β-maltose to α-maltose when the present enzyme is used.

Claims (1)

(57) [Claims] 1. A genus of the genus Streptococcus, Leuconostoc, Enterococcus or Pediococcus,
A method for producing a novel maltose 1-epimerase, comprising culturing a strain capable of producing maltose 1-epimerase having the following physicochemical properties in a medium, and collecting the maltose 1-epimerase from the culture. (1) Action: catalyzes the interconversion reaction between β-maltose and α-maltose. (2) Substrate specificity: specifically acts on maltose and does not substantially act on cellobiose or lactose. (3) Optimum pH and stable pH range: Optimum pH is 5.5 to
7.5, and the stable pH range is 30 ° C. for 30 minutes.
H is 5.0 to 8.5. (4) Molecular weight: about 43,000 (gel filtration method).