JP2001061490A - Alfa-glucosidase and production thereof and ethyl-alfa- glucoside produced by utilizing the same and the production thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide the subject novel α-glucosidase that is stable to heat and pH, can be used at ambient temperature and is useful for producing ethyl-α- glucoside by culturing a microorganism in Mortierella, for example, Mortierella alliacea YN-15 in a culture medium. SOLUTION: A microorganism in Mortierella, for example, Mortierella alliacea YN-15 (FERN P-16106) is cultured in a culture medium to produce the objective novel α-glucosidase that hydrolyze the α-D-glucoside bond in maltose, is stable to heat and pH, can be used at an ambient temperature and has a sufficient sugar-converting activity and is useful for production of ethyl-α- glucoside. The enzyme is obtained by culturing a microorganism in Mortierella, for example, Mortierella alliacea YN-15 (FERM P-16106) is cultured in the medium at a pH of 7.0 and a culturing temperature of 28 deg.C for 7 days and isolating and purifying the product.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to maltose α-D
The present invention relates to α-glucosidase which hydrolyzes a glucoside bond, a method for producing the same, and ethyl-α-glucoside produced using the α-glucosidase and a method for producing the same. In particular, Mortierella ariasea, a microorganism belonging to the genus Mortierella Α starting from YN-15
-Glucosidase and a method for producing the same, and ethyl-α-glucoside produced using the α-glucosidase and a method for producing the same.

[0002]

2. Description of the Related Art Ethyl-α-glucoside is prepared by using maltose and ethyl alcohol as substrate solutions and adding α-glucosidase as a catalyst thereto. This means that the α-glucoside bond of maltose is hydrolyzed with α-glucosidase, and the obtained α-glucoside group is transferred to the ethyl group of ethyl alcohol to produce ethyl-α-glucoside.

[0003] Ethyl-α-glucoside is known as a taste component of sake and mirin.

[0004] By the way, α-glucosidase exists widely in the living world, and various types of α-glucosidase exist in yeast, filamentous fungi, bacteria, mammals, plant seeds and the like.

Therefore, conventionally, various types of α-glucosidase such as those derived from yeast, those derived from bacteria and those derived from plant seeds have been produced.

[0006]

However, α-glucosidase obtained by the above-mentioned conventional method has the following problems. (1) Those derived from yeast, for example, α-glucosidase derived from Saccharomyces cerevisiae, do not have sufficient transglycosylation activity necessary for producing ethyl-α-glucoside. (2) Those derived from bacteria, for example, α-glucosidase derived from the bacterium Bacillus stearothermophilus do not have the activity of degrading maltooligosaccharides, and are not suitable for producing ethyl-α-glucoside using maltooligosaccharide as a substrate. (3) As for α-glucosidase derived from plant seeds, for example, α-glucosidase derived from sugar beet seeds has an optimal pH on the acidic side and is not suitable for use near neutrality.

Accordingly, the present invention provides an α-glucosidase which is stable against heat, pH, etc., and which has a sufficient transglycosylation activity required for the production of ethyl-α-glucoside, a method for producing the same, and an α-glucosidase. -It is an object of the present invention to provide ethyl-α-glucoside produced using glucosidase and a method for producing the same.

[0008]

Means for Solving the Problems In order to achieve the above object, the present inventors have made various studies and found that Mortierella aliasea YN, a microorganism belonging to the genus Mortierella, is used.
It has been found that α-glucosidase can be produced by culturing -15 in a medium, and the present invention has been completed.

[0009] Mortierella Ariasea YN-15
(Mortierella alliacea YN-1
5. Mortierella sp. SAM2197 strain, FERMP
-16106) is known as a microorganism having resistance to a high concentration of a carbon source, but is also a starch-assimilating bacterium. Therefore, they secrete amylolytic enzymes into the culture.

Therefore, Mortierella Ariasea YN
-15 was confirmed to secrete α-glucosidase, one of the amylolytic enzymes, and the present invention was completed.

[0011] Mortierella ariasea YN-15 is present in soil. The present invention is to extract and culture Mortierella ariasea YN-15 present in this soil to produce α-glucosidase.

Accordingly, the method for producing ethyl-α-glucoside according to the present invention is directed to a method for producing ethyl-α-glucoside by adding α-glucosidase to a solution containing maltose and ethyl alcohol as substrates. Wherein the α-glucosidase is produced by culturing a microorganism belonging to the genus Mortierella in a medium.

The ethyl-α-glucoside according to the present invention is obtained by adding α-glucosidase produced by culturing a microorganism belonging to the genus Mortierella in a medium in a solution containing maltose and ethyl alcohol as substrates. It is characterized by being manufactured.

The method for producing α-glucosidase according to the present invention is characterized in that α-glucosidase is produced by culturing Mortierella ariasea YN-15, which is a microorganism belonging to the genus Mortierella, in a medium. .

The α-glucosidase according to the present invention is produced by culturing Mortierella ariasea YN-15, which is a microorganism belonging to the genus Mortierella, in a medium.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, an embodiment of an α-glucosidase according to the present invention, a method for producing the same, ethyl-α-glucoside produced using the α-glucosidase and a method for producing the same will be described with reference to the drawings. I will explain it.

The microorganism used in the present invention, which belongs to the genus Mortierella, is Mortierella ariasea YN-1.
5 (Mortierella alliacea YN)
-15, Mortierella sp. SAM2197 strain, FERM
P-16106) is Mortierella (Mortiere)
rella), subgenus Mortierella (Subg)
enus Mortierella) and a sub-genus Mortierella of the genus Alpina (Section)
Alpina), and further belongs to the species Ariacea in the Alpina ward.

The Mortierella ariasea YN-15 used in the present invention can be cultured under the following culture conditions and culture methods.

In order to culture the strain used in the present invention, spores, hyphae of the strain or a pre-culture solution obtained by pre-culturing is inoculated into a liquid medium and cultured.

The carbon sources that can be used include glucose,
Maltose, maltitol, sucrose, lactose,
There are isomaltose, trehalose, soluble starch, amylose, amylopectin, glycogen, pullulan, dextran and the like, and particularly preferred is maltitol, soluble starch and amylopectin.

As the nitrogen source, sodium nitrate, ammonium chloride, ammonium sulfate, ammonium hydrogen phosphate, corn steep liquor, casein, polypeptone, yeast extract and the like can be used. In particular, ammonium hydrogen phosphate, casein, polypeptone and yeast extract can be used. preferable.

In addition, if necessary, as a micronutrient source,
Phosphates such as potassium phosphate and potassium dihydrogen phosphate, magnesium sulfate, iron sulfate, copper sulfate, magnesium chloride,
Inorganic salts such as potassium chloride and vitamins can also be used.

The medium components such as the above-mentioned carbon source, nitrogen source, inorganic salts and vitamins can be added not only at the start of the culture but also to the medium before the start of the culture or the culture solution during the culture.

These components may be added all at once,
It may be added continuously or may be added in plural times. These medium components can be added alone or in advance by mixing.

These medium components are not particularly limited as long as they do not impair the growth of microorganisms, but the total amount of the carbon source is 1% to 3%, preferably about 2%.

The total amount of the nitrogen source added is 0.1% to 1%.
And preferably around 0.5%.

The culturing temperature is 5 to 40 ° C., preferably about 28 ° C.

The pH of the medium is 5.5 to 8.5, preferably about 7, and aeration-agitation culture, shaking culture, or static culture is performed.

The culture period is 2 to 12 days, preferably 4 to 12 days.
~ 7 days.

After completion of the culture, α-glucosidase is obtained in the culture supernatant.

When α-glucosidase is thus obtained, α-glucosidase is added to maltose and ethyl alcohol as a substrate solution by a known method to produce ethyl-α-glucoside.

Next, the present invention will be described more specifically based on examples.

[0033]

Embodiment 1 First, Mortierella Ariasea YN-
An example in which α-glucosidase is produced by culturing No. 15 will be described as Example 1.

A platinum loop of Mortierella ariasea YN-15 was inoculated into a medium having a composition of 2% soluble starch, 0.5% yeast extract, 0.5% ammonium hydrogen phosphate, pH 7.0, and inoculated at 28 ° C. For 7 days with shaking.

The culture supernatant obtained from this culture was used as a crude enzyme solution (α-glucosidase), and the PNPG hydrolysis activity was measured.

As a result, 30 mU using PNPG as a substrate
/ Ml of hydrolysis activity.

Here, 1 U (unit) is 1 U at 37 ° C.
The amount of enzyme that releases 1 μmol of p-nitrophenol per minute.

[0038]

Example 2 Next, an example in which ethyl-α-glucoside is produced using α-glucosidase derived from Mortierella ariasea YN-15 obtained in Example 1 will be described as Example 2. .

As a substrate solution, 8 ml of 2.5% maltose and 3.2 ml of 40% ethyl alcohol were added 30 mU (unit) of α-glucosidase, and the mixture was cultured at 45 ° C. for 24 hours.

After the reaction, ethyl-α-glucoside in the reaction solution was quantified. As a result, it was confirmed that 4 mg of ethyl-α-glucoside was produced.

Next, the present invention will be further described based on experimental examples.

[0042]

[Experimental example 1] First, Mortierella Ariasea YN-
The evaluation of the carbon source when α-glucosidase is produced from No. 15 will be described with reference to FIG.

In FIG. 1, when examining the evaluation of the carbon source, p-nitrophenyl-α-glucoside (hereinafter, PN
The evaluation of the carbon source is being investigated from the hydrolysis activity using PG) and the reaction for producing ethyl-α-glucoside.

Here, the hydrolysis activity was measured using PNPG by measuring 100 μl of 20 mM PNPG solution and 0.1 M of PNPG solution.
100 μl of an α-glucosidase solution was added to 200 μl of an acetate buffer (pH 5.0), and reacted at 37 ° C. for 15 minutes.
μl was added, and the absorbance was measured at 400 nm.

As described above, one unit (U) of the enzyme activity is the amount of the enzyme that releases 1 μmol of p-nitrophenol per minute at 37 ° C.

The measurement of the hydrolysis activity using PNPG is based on the fact that maltose and PNPG both have an α-glucoside bond, and PNPG is a substance which develops a yellow color called p-nitrophenol by a hydrolysis reaction. This is because it is easy to measure the enzyme activity.

The ethyl-α-glucoside formation reaction is carried out by adding α-glucosidase using 2.5% maltose and 40% ethyl alcohol as a substrate solution,
The reaction is carried out for 4 hours, and the amount of ethyl-α-glucoside produced is measured.

After the completion of the reaction, a part of the reaction solution was measured by thin layer chromatography (TLC).

As an analysis condition, a thin layer was obtained from Merck s.
Ilica gel 60 was used, and acetonitrile-water (85:15 (volume ratio)) was used as a developing solvent. The detection was performed by heating the thin layer after spraying with sulfuric acid, and the concentration of the obtained spot was measured with a densitometer.

In FIG. 1, the experimental conditions were as follows: the amount of addition of the carbon source was 2%, ammonium hydrogen phosphate was 0.5% as the nitrogen source, pH was 7.0, the culture temperature was 28 ° C., and the culture time was 7 days. And

In the graph of FIG. 1, the upper row of each carbon source shows PNPG hydrolysis activity (mU / ml), and the lower row shows ethyl-α-glucoside formation reaction (mg / ml).
DCW (dry cell weight) is Mortierella ariasea YN- cultured using each carbon source.
15 shows the dry cell weight.

FIG. 1 shows that maltitol, soluble starch, amylopectin and the like are excellent as carbon sources.

[0053]

Experimental Example 2 Next, Mortierella Ariasea YN-
The evaluation of the nitrogen source when producing α-glucosidase from No. 15 will be described with reference to FIG.

The contents of the experiment were the same as in FIG. 1, and the evaluation of the nitrogen source was examined based on the PNPG hydrolysis activity and the ethyl-α-glucoside formation reaction.

The experimental conditions were as follows: soluble starch as a carbon source was 2%, nitrogen sources were all 0.5%, pH was 7.0, the culture temperature was 28 ° C., and the culture time was 7 days.

FIG. 2 shows that ammonium hydrogen phosphate, casein, polypeptone, yeast extract and the like are excellent as nitrogen sources.

[0057]

Experimental Example 3 Next, Mortierella Ariasea YN-
The evaluation of the carbon source concentration when α-glucosidase is produced from No. 15 will be described with reference to FIG.

The experimental conditions were as follows: soluble starch was used as a carbon source, and ammonium hydrogen phosphate was used as a nitrogen source at 0.5%.
%, PH 7.0, the culture temperature was 28 ° C., and the culture time was 7 days.

In FIG. 3, 31 indicates PNPG hydrolysis activity (mU / ml), 32 indicates ethyl-α-glucoside formation reaction (mg / ml), 33 indicates DCW (g / l),
The vertical axis shows each scale.

As understood from FIG. 3, the carbon source concentration of the PNPG hydrolysis activity 31 and the ethyl-α-glucoside formation reaction 32 is preferably 1% to 3%, and more preferably 2%.
There is a peak around%.

The DCW 33 increases as the carbon source concentration increases.

[0062]

Experimental Example 4 Next, Mortierella Ariasea YN-
The evaluation of pH when α-glucosidase is produced from No. 15 will be described with reference to FIG.

In FIG. 4, the experimental conditions were as follows: soluble starch 2% as a carbon source, ammonium hydrogen phosphate 0.5% as a nitrogen source, a culture temperature of 28 ° C., and a culture time of 7 days.

In Experimental Example 4, the evaluation of pH
PG hydrolysis activity, ethyl-α-glucoside formation reaction,
DCW is being investigated.

In FIG. 4, 41 indicates PNPG hydrolysis activity (mU / ml), 42 indicates ethyl-α-glucoside production reaction (mg / ml), 43 indicates DCW (g / l),
The vertical axis shows each scale.

As understood from FIG. 4, the PNPG hydrolyzing activity 41 is preferably pH 7 to 8, the ethyl-α-glucoside production reaction 42 has a peak around pH 7, and the DCW 43 is preferably pH 7 to 8.

[0067]

Experimental Example 5 Next, Mortierella Ariasea YN-
The evaluation of the culture period when α-glucosidase is produced from No. 15 will be described with reference to FIG.

In FIG. 5, the experimental conditions were as follows: soluble starch 2% as a carbon source, ammonium hydrogen phosphate 0.5% as a nitrogen source, pH 7.0, and a culture temperature of 28 ° C.

In Experimental Example 5, regarding the evaluation of the culture time,
PNPG hydrolysis activity and ethyl-α-glucoside formation reaction have been investigated.

In FIG. 5, 51 indicates the PNPG hydrolysis activity (mU / ml), 52 indicates the reaction for producing ethyl-α-glucoside (mg / ml), and the vertical axis indicates the respective scales.

As can be understood from FIG. 5, the PNPG hydrolyzing activity 51 becomes saturated around the fourth day of the culturing period and starts to decrease around the seventh day.

As is clear from the above experimental examples, when α-glucosidase is produced from Mortierella ariasea YN-15, the optimal culture conditions are that 2% of soluble starch is used as a carbon source and phosphate is used as a nitrogen source. It can be seen that 0.5% ammonium hydrogen was used, the pH was 7.0, the culture temperature was 28 ° C., and the culture time was 4 to 7 days.

[0073]

As described above, the present invention relates to a method for producing ethyl-α-glucoside in which α-glucosidase is added to a solution containing maltose and ethyl alcohol as substrates to produce ethyl-α-glucoside. Since the α-glucosidase is produced by culturing a microorganism belonging to the genus Mortierella in a medium, heat, p,
Α-glucosidase which is stable to H and the like, can be used at normal temperature, and further has a sufficient glycosyltransferase activity necessary for producing ethyl-α-glucoside, a method for producing the same, and the α-glucosidase. Ethyl-α prepared by
-Glucoside and its production method can be obtained.

[Brief description of the drawings]

FIG. 1: Mortierella Ariasea YN-15 to α
-The figure explaining the evaluation of the carbon source at the time of producing glucosidase.

FIG. 2: Mortierella Ariasea YN-15 to α
-The figure explaining evaluation of the nitrogen source at the time of producing glucosidase.

FIG. 3. Mortierella Ariasea YN-15 to α
-The figure explaining the evaluation of the carbon source concentration at the time of producing glucosidase.

FIG. 4: Mortierella Ariasea YN-15 to α
-The figure explaining evaluation of pH at the time of producing glucosidase.

FIG. 5: α from Mortierella Ariasea YN-15
-Figure explaining the evaluation of the culture time when producing glucosidase.

[Explanation of symbols]

 31, 41, 51 PNPG hydrolysis activity 32, 42, 52 Ethyl-α-glucoside formation reaction 33, 43 DCW

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification FI FI Theme Court ゛ (Reference) C12R 1: 645) (72) Inventor Tomohiro Aki 24-4-606, Saijocho, Higashihiroshima City, Hiroshima Prefecture (72) Invention Person Shoji Kawamoto 8-6-3-203 Saijo Chuo, Higashihiroshima City, Hiroshima Prefecture (72) Inventor Yoshio Tanaka F-term (reference) 4B050 CC01 DD03 LL05 4B064 AF41 CA21 CC03 CD06 CD09 DA10

Claims (7)

[Claims] An α-glucosidase is added to a solution containing maltose and ethyl alcohol as substrates, and ethyl-α-glucosidase is added to the solution.
-A method for producing ethyl-α-glucoside for producing glucoside, wherein the α-glucosidase is produced by culturing a microorganism belonging to the genus Mortierella in a medium. 2. The method for producing ethyl-α-glucoside according to claim 1, wherein the microorganism belonging to the genus Mortierella is a microorganism belonging to the subgenus Mortierella. 3. The method for producing ethyl-α-glucoside according to claim 2, wherein the microorganism belonging to the subgenus Mortierella is a microorganism belonging to Ariasea species. 4. The method for producing ethyl-α-glucoside according to claim 3, wherein the microorganism belonging to the ariasea species is Mortierella ariasea YN-15. 5. An ethyl-α produced by adding α-glucosidase produced by culturing a microorganism belonging to the genus Mortierella in a medium in a solution containing maltose and ethyl alcohol as substrates. -Glucoside. 6. An α-glucosidase is produced by culturing Mortierella ariasea YN-15, which is a microorganism belonging to the genus Mortierella, in a medium to produce α-glucosidase.
-A method for producing glucosidase. 7. An α-glucosidase produced by culturing Mortierella ariasea YN-15, a microorganism belonging to the genus Mortierella, in a culture medium.