JPH1189566A - Production of disaccharide phosphorylase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain disaccharide phosphorylase necessary in enzymatically producing trehalose in high yield by culturing a microorganism having disaccharide phosphorylase producing ability in a culture medium containing validamycin. SOLUTION: Validamycin (also containing validamycylamine), normally in an amount of 0.0001-0.50% (w/v) is added to a culture medium and a microorganism having disaccharide phosphorylase-producing ability, e.g. Plesiomonas SH-35 strain (FERM P-5144) is cultured in the culture medium. Thereby, disaccharide phosphorylase such as trehalose phosphorylase maltose phosphorylase is obtained in good productivity.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a disaccharide phosphorylase. In particular, the present invention efficiently produces trehalose phosphorylase and maltose phosphorylase, which are disaccharide phosphorylases required when enzymatically producing trehalose useful as a food property improver or a pharmaceutical or enzyme stabilizer. On how to do it.

[0002]

2. Description of the Related Art Disaccharide phosphorylase decomposes a disaccharide in the presence of inorganic phosphoric acid in the presence of inorganic phosphoric acid to form glucose monophosphate and glucose, and forms a disaccharide from glucose monophosphate and glucose. An enzyme that catalyzes the reversible reaction of the synthesis reaction, for example, trehalose phosphorylase, maltose phosphorylase, cellobiose phosphorylase and the like are known. Trehalose phosphorylase is an enzyme that catalyzes a reversible reaction represented by the following (1). In addition, maltose phosphorylase is as follows
It is an enzyme that catalyzes the reversible reaction shown in (2). However, in each formula, a rightward arrow indicates a forward reaction, and a leftward arrow indicates a reverse reaction.

[0003]

Embedded image

[0004] Trehalose phosphorylase is, for example,
Euglena gracilus [Biochim. Biophys. Acta., Vol. 198, 151-
154 (1970), Journal of Biological Chemistry (J. Biol. Chem.), Volume 247,
3223-3228 (1972)], Flammulina velutipes [FEMS Microbiol. Le
tt.), Vol. 55, pp. 147-150 (1988)] and Bradyhizobium japonicam.
ponicum) [Plant Physio
l.), Vol. 81, pp. 538-541 (1986)], and the like.

[0005] Maltose phosphorylase can be obtained from Neisseria meningitidis [Journal of Biological Chemistry (J. Biol.
Chem.) 199, 153, 1952], Lactobacillus brevis IFO3345.
[Agricultural and Biological Chemistry (Agr. Biol. Chem), vol. 37, 2813]
, 1973], Lactobacillus brevis (Lactob
acillus brevis) DSM 20054, NCIB 8836, 8561, 8526,
Lactobacillus plantarum
um) DSM 20174 and Microtechnical Laboratories No. 4628, Lactobacillus reuteri (DSM20016), Lactobacillus fermentum (Lactobacillus fermentum)
tum) DSM 20052, Streptococcus bacterium (Streptoco
ccus sp.)
No. 25, No. 4626, No. 462
No. 7, etc. (Japanese Patent Publication No. 60-54036): Lactobacillus sanfrancis
It has been known that microorganisms such as co) (JP-A-1-91778) have an intracellular enzyme. Furthermore, Plesiomonas strain SH35 belonging to the genus Plesiomonas (Nippon Life Science Co., Ltd., No. 5144) is known to be a useful cell that simultaneously produces both maltose phosphorylase and trehalose phosphorylase. Kaihei 8-1311157).

According to the above formulas (1) and (2), trehalose can be produced enzymatically using maltose and inorganic phosphoric acid as raw materials and using maltose phosphorylase and trehalose phosphorylase. Trehalose is a compound useful as an agent for improving the properties of foods or as a stabilizer for pharmaceuticals and enzymes, and its application range is expanding. Furthermore, both enzymes have been used for the quantification of maltose and trehalose (JP-A-8-280399).

[0007]

The above disaccharide phosphorylases such as maltose phosphorylase and trehalose phosphorylase can be obtained as intracellular enzymes by culturing various microorganisms as described above. Due to the growing need for trehalose, efficient production of maltose phosphorylase and trehalose phosphorylase, which are essential enzymes for the production of trehalose at low cost, has been desired. Little has been done to improve sex. Therefore, an object of the present invention is to provide a method capable of producing a disaccharide phosphorylase such as trehalose phosphorylase or maltose phosphorylase in a higher yield. In order to achieve the above object, the present inventors have conducted various studies.As a result, when a microorganism having a disaccharide phosphorylase-producing ability such as trehalose phosphorylase or maltose phosphorylase is cultured in a medium containing validamycin, the amount of enzyme production is remarkably increased. The inventors have found that the present invention is improved and completed the present invention.

[0008] Validamycin is widely used as an agricultural antibiotic and has a structure consisting of validoxylamine and D-glucose. It has been reported that validamycin and its basic skeleton, validoxylamine, strongly inhibit the forward phosphorolytic reaction of trehalose phosphorylase shown in the above (1) [Biosc
i. Biotech. Biochem., Vol. 59, 1908-1912
P. (1995)]. It is also known that when culturing a microorganism having the ability to produce trehalose, the addition of validamycin or a derivative thereof significantly increases the amount of trehalose produced (Japanese Patent Publication No. 3-130008).
4). However, it has not been known at all that when a microorganism having a disaccharide phosphorylase-producing ability such as trehalose phosphorylase or maltose phosphorylase is cultured in a medium containing validamycin, the production amount of the enzyme is remarkably improved.

[0009]

That is, the present invention provides a disaccharide phosphorylase characterized in that a microorganism having a disaccharide phosphorylase producing ability is cultured in a medium containing validamycin, and the produced disaccharide phosphorylase is recovered. It relates to a manufacturing method.

[0010]

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a disaccharide phosphorylase. Examples of the disaccharide phosphorylase include trehalose phosphorylase and / or maltose phosphorylase. Further, in the production method of the present invention, a medium containing validamycin is used. Validamycin is widely used as an agricultural antibiotic, and it has six structural isomers A to F, and a commercially available product is usually available as a mixture thereof. In the present invention, validamycin amine, which is a basic skeleton of validamycin, can also be used. In the present invention, validamycin also includes validamycin amine. The amount of validamycin to be added to the medium may be selected within a range that does not inhibit the growth of the microorganism used, and is usually about 0.0001 to 0.50% (w /
v), preferably in the range of 0.02 to 0.10% (w / v) is effective.

The microorganism capable of producing disaccharide phosphorylase can be, for example, a microorganism capable of producing trehalose phosphorylase, a microorganism capable of producing maltose phosphorylase, or a microorganism capable of producing trehalose phosphorylase and maltose phosphorylase. Specifically, as a microorganism having trehalose phosphorylase and maltose phosphorylase producing ability, for example, a microorganism belonging to the genus Plesiomonas can be mentioned, and Plesiomonas (Plesiomonas) can be exemplified.
monas) microorganisms belonging to the genus Plesiomonas SH-
35 strains (Shiken Kenjo No. 5144) can be mentioned. However, it is not limited to this.

[0012] Examples of microorganisms having the ability to produce trehalose phosphorylase include Euglen (Euglen).
a) Genus, genus Flammulina, genus Bradyhizobium, Actinomadura (Ati)
nomadura), Amycolata (Amycola)
ta) and Kineosporia (Kineospori)
a) Microorganisms belonging to the genus can be mentioned. The microorganisms belonging to the genus Euglena and the genus Flammulina are as described above. Actino Madura (A
ctinomadura, Amycolata (Amyc)
olata) and Kineosporia (Kineospo)
More specifically, microorganisms belonging to the genus ria) are, specifically, Actinomadura luteofleoressens (Actinom).
adra lutefluoresens, ATC
C25469), Actinomadura verrucososp
ora, ATCC 27299), Amycolata autotrophica
lica, ATCC 19727), Kineosporia auranti (Kineosporia auranti)
aca, ATCC 29727).
However, it is not limited to these.

[0013] Further, as a microorganism capable of producing maltose phosphorylase, Neisserria
Microorganisms belonging to the genus, Streptococcus, Enterococcus, Lactobacillus and Leuconostoc can be mentioned. The genus Neiserria,
Examples of microorganisms belonging to Streptococcus are as described above. Entrococcus (E
lactobacillus (Lac)
genus and Leuconostoc (Le)
More specifically, microorganisms belonging to the genus Uconostoc are Enterococcus (Enterococcus).
sp. , ATCC 10541), Lactobacillus mari, ATCC2
7053), Lactobacillus brevis (Lactobacillu
s brevis IFO3345), Leuconostoc mesenterer
oides ATCC 10830). However, it is not limited to these.

Preferably, validamycin is added to the medium in advance. In addition, as a nutrient source of the medium used for the culture, a carbon source, a nitrogen source, inorganic salts, organic acid salts, and trace nutrients that can be used by the strain can be used.
For example, as a carbon source, maltose, trehalose,
Glucose, sucrose, galactose, lactose, starch and the like are used, and are not limited as long as the carbon source can be used by the cells. As a nitrogen source, for example, various ammonium salts (ammonium sulfate, ammonium nitrate, salt ammonium, phosphorus ammonium),
Inorganic or organic nitrogen compounds such as corn steep liquor, peptone, meat extract, yeast extract, dried yeast, soy flour, and urea. As the inorganic salts, salts of potassium, sodium, calcium, magnesium, iron, manganese, cobalt, zinc and phosphoric acid are used.

As a means for culturing, static cultivation, shaking cultivation, aeration stirring cultivation and the like may be used. Of course, it depends on the type of strain and the like, and in short, it may be selected according to individual cases so that the target product is produced most efficiently.

Each enzyme can be isolated by a conventional method by ultrasonic treatment or lytic enzyme treatment. Further, the crude enzyme can be recovered by extracting the enzyme from the cells. Further, the enzyme is also contained in the culture supernatant outside the cells, and the remaining culture solution obtained by separating the cells can be used as a crude enzyme-containing solution. Furthermore, these crude enzymes can be purified by a conventional method such as a solvent precipitation method using ethanol, acetone, isopropanol or the like, an ammonium sulfate fractionation method, ion exchange chromatography, gel chromatography and the like.

[0017] Enzyme activity assay decomposition reaction: the addition of enzyme solution 0.01ml in 20mM maltose or trehalose solution 0.5ml dissolved in phosphate buffer (pH 7.0) of 50 mM, 1 at 50 ° C.
After reacting for 5 minutes, heat in a boiling water bath for 3 minutes to stop the enzymatic reaction. Next, after cooling in running water, the generated glucose is measured by a glucose oxidase method (Glucose C-II Test Wako, manufactured by Wako Pure Chemical Industries, Ltd.).
Here, 1 unit of enzyme activity is 1 μmo per minute under the same conditions.
It is the amount of enzyme that produces 1 (micromol) glucose.

[0018]

The present invention will be specifically described below with reference to examples. Example 1 Intracellular and extracellular trehalose phosphorylase and / or
Refers to the production of maltose phosphorylase Plesiomonas SH35 strain (Shiken Kenjo No. 5144)
Trehalose 1.65% (w / v), soy peptide (Hinute SMS, Fuji Oil) 2.0%, yeast extract (RB-2, Sapporo Beer) 1.5%, urea 0.3
%, Ammonium nitrate 0.2%, dipotassium phosphate 0.3
%, Magnesium sulfate 0.02%, manganese chloride 30m
The cells were inoculated into a liquid medium of pH 8.0 containing g / l. next,
Each cell was inoculated into 25 ml of this liquid medium, and
The cells were shaken at 180 rpm for 4 hours and cultured aerobically. A culture solution was prepared by adding various concentrations of validamycin to the culture solution from the beginning, and a comparative example was prepared by similarly culturing without adding validamycin. The culture solution obtained by culturing in this manner was treated with an ultrasonic cell disrupter to destroy the cells, and then the enzyme activity per 1 ml of the disrupted cell suspension was measured. As a result, as shown in Table 1, the enzyme productivity was improved by adding validamycin.

[0019]

[Table 1]

Example 2 Preparation of trehalose phosphorylase Actinomadura luteofuloresence (Acti)
nomadura luteofluoresens,
ATCC 25469), Actinomadura verrucoso
spora, ATCC27299), Amycolataautophilica (Amycolataautotrophic)
ilica, ATCC 19727), Quineosporia
Aulanca Red, Kineosporia auran
tiaca), trehalose 1.0% (w / v), peptone 0.2%, meat extract 0.1%, yeast extract 0.1
% Of a liquid medium containing pH 7.2.

Next, each cell was inoculated into 25 ml of the liquid medium, shaken at 28 ° C. for 4 days at 180 rpm, and cultured aerobically. A culture solution prepared by adding 0.02% of validamycin to the culture solution from the beginning and a culture solution prepared by adding no validamycin in the same manner as a comparative example were prepared. The culture solution obtained by culturing in this manner was treated with an ultrasonic cell disrupter to destroy the cells, and then the enzyme activity per 1 ml of the disrupted cell suspension was measured. As a result, as shown in Table 2, the enzyme productivity was improved by adding validamycin.

[0022]

[Table 2]

Example 3 Preparation of Maltose Phosphorylase Enterococcus s
p. , ATCC 10541), Lactobacillus mari, ATCC2
7053), Lactobacillus brevis (Lactobacillu
s brevis) IFO3345, Leuconostoc mesenteroides, sucrose 2.0% (w / v), tryptone 1.0%, yeasts
extract 1.0%, dipotassium phosphate 0.5
%, Magnesium sulfate 0.04%, iron sulfide 0.001
%, Manganese sulfide 0.001%, vitamin C 0.005%
% Of the liquid medium at pH 7.5.

Next, each cell was inoculated into 25 ml of the liquid medium and anaerobically cultured at 30 ° C. for 1 to 2 days. A culture solution prepared by adding 0.02% of validamycin to the culture solution from the beginning and a culture solution prepared by adding no validamycin in the same manner as a comparative example were prepared. The culture solution obtained by culturing in this manner was treated with an ultrasonic cell disrupter to destroy the cells, and then the enzyme activity per 1 ml of the disrupted cell suspension was measured. As a result, as shown in Table 3, the enzyme productivity was improved by adding validamycin.

[0025]

[Table 3]

[0026]

According to the present invention, a method for easily producing a culture solution containing a large amount of trehalose phosphorylase or maltose phosphorylase and producing a disaccharide phosphorylase such as trehalose phosphorylase or maltose phosphorylase in a higher yield. Can be provided.

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Claims (9)

[Claims] 1. A method for producing a disaccharide phosphorylase, which comprises culturing a microorganism capable of producing disaccharide phosphorylase in a medium containing validamycin and collecting the produced disaccharide phosphorylase. 2. The method according to claim 1, wherein the disaccharide phosphorylase is at least one selected from the group consisting of trehalose phosphorylase and maltose phosphorylase. 3. The microorganism according to claim 1, wherein the microorganism capable of producing disaccharide phosphorylase is a microorganism capable of producing trehalose phosphorylase, a microorganism capable of producing maltose phosphorylase, or a microorganism capable of producing trehalose phosphorylase and maltose phosphorylase. The manufacturing method as described. 4. The method according to claim 1, wherein the microorganism having the ability to produce trehalose phosphorylase and maltose phosphorylase is a microorganism belonging to the genus Plesiomonas. 5. A microorganism belonging to the genus Plesiomonas, wherein the microorganism belonging to the genus Plesiomonas is a strain of Plesiomonas SH-35.
No. 144). 6. The microorganism having trehalose phosphorylase-producing ability may be selected from the group consisting of Euglena, Flammulina, and Bradyhizobiu.
m), Actinomadura, Amycolata, and Kineosporia
ia) a microorganism belonging to the genus;
The production method according to the paragraph. 7. A microorganism capable of producing trehalose phosphorylase, wherein the microorganism has the genus Actinomadura,
Amycolata and Kineosporia
The method according to claim 6, which is a microorganism belonging to the genus eosporia). 8. The microorganism capable of producing maltose phosphorylase may be selected from the group consisting of Neisseria, Streptococcus, and Entrococcus.
The method according to any one of claims 1 to 3, wherein the microorganism is a microorganism belonging to the genus rococcus, the genus Lactobacillus, or the genus Leuconostoc. 9. The production method according to claim 8, wherein the microorganism having maltose phosphorylase producing ability is a microorganism belonging to the genus Enterococcus, the genus Lactobacillus, and the genus Leuconostoc.