JPH0822237B2 - Method for producing trehalose - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel method for producing trehalose, which is expected to be applied to the fields of medicine, food and agriculture.

[0002]

BACKGROUND OF THE INVENTION Trehalose is ubiquitous in organisms and is known as an energy storage substance in microorganisms and plants and as a protective substance against environmental changes such as heat, drying, freezing and osmotic pressure. Also in yeast, for example, the relationship between trehalose accumulation and environmental resistance in baker's yeast has been clarified (Chemistry and organism 31, 6, 3).
74-381, 1993). It is also known that insects are used as an energy source for flight.

On the other hand, as a method for producing trehalose,
1) A method of culturing a strain such as baker's yeast that accumulates a large amount of trehalose and extracting it with hot water, alcohol, etc.
-360692, Japanese Patent Laid-Open No. 5-91890), 2) Method of producing in culture by using bacteria such as Brevibacterium (Japanese Patent Laid-Open No. 5-212882), 3) Maltose phosphorylase and trehalose phosphorylase treatment. Method (JP-A-58-216)
No. 695) is known.

In baker's yeast and the like, attempts have been made to suppress the activity of trehalose-degrading enzyme (trehalase) in cells in order to increase the amount of trehalose accumulated (eg, trehalase activity deficiency due to mutation, addition of enzyme inhibitor, etc.). However, since various trehalases having different properties are present in various places in cells and they are induced by various causes, it is considered difficult to suppress the activity.

[0005]

SUMMARY OF THE INVENTION The present invention provides a method for efficiently producing trehalose.

[0006]

The present inventors have found that yeast belonging to the genus Candida, particularly Candida fermentii, constitutively accumulates trehalose in cells, and The inventors have found that trehalase activity can be easily deleted by mutation treatment, and that these mutant strains produce a significantly higher amount of trehalose than the yeast before mutation, and completed the present invention.

That is, the present invention is based on the candida (Ca
The present invention provides a method for producing trehalose, which comprises culturing a mutant strain of trehalose-producing yeast belonging to the genus Ndida) and collecting trehalose from the culture.

The yeast used in the present invention is a strain belonging to the genus Candida and having a trehalose-producing ability, which has been deficient in trehalose assimilation by mutation treatment. Candida), which has the ability to produce trehalose, can be used in any strain as long as it is a trehalose-assimilating revertant strain obtained from a strain in which trehalose assimilation and accumulation are simultaneously deleted by mutation treatment. it can.

The parent strain of yeast used in the present invention is, for example, Candida ferme isolated from soil collected in Taichung City, Republic of China.
ntii) No. 3110 strain (FERM P-1234
8) can be mentioned, but any strain other than this strain can be used as a parent strain of the mutant strain used in the present invention as long as it belongs to the genus Candida and has the ability to produce trehalose. it can.
Further, in order to isolate a mutant strain suitable for the purpose of the present invention,
The above-mentioned parent strain having the ability to produce trehalose is subjected to a mutation method known per se, for example, UV treatment, NTG.
(N-methyl-N′-nitro-N-nitrosoguanidine) treatment or the like may be performed and appropriately selected.

Candida Fairmenty (C
andida fermentii) No. The 3110 strain has the following mycological properties. 1. Morphological properties It has an oval or spherical shape with a size of 3 to 7 μm, and has no mycelium-like structure. No spores are formed in any of the gypsum, Klein medium and malt extract medium.

2. Physiological Properties Yeast by JA Barnet et al .: Characterization and identification.
classification and identification) according to the second edition. The various physiological properties of this strain are as follows.

(1) Fermentability test D-glucose + lactose-D-galactose-cellobiose-maltose-meletitose-methyl α-D-glucoside-raffinose + sucrose + inulin + α, α-trehalose-starch-melibiose- D-xylose-

(2) Growth test D-glucose + D-galactose-L-sorbose + D-glucosamine-D-ribose-D-xylose-L-arabinose-L-rhamnose-sucrose + maltose-α, α-trehalose + Methyl α-D-glucoside-cellobiose-salicin-arbutin-melibiose-lactose-raffinose + meletitose-inulin + starch-glycerol + erythritol-ribitol-xylitol + L-arabitonyl-D-glucitol-D-mannitol. Myo-inositol-D-glucono-1,5-lactone + 2-keto-D-gluconic acid + 5-keto-D-gluconic acid-D-gluconic acid-D-glucuronic acid + D-galacto Turonic acid-DL-lactic acid + succinic acid-citric acid-methanol-ethanol-propane-1,2-diol-butane-1,2-diol-potassium nitrate-potassium nitrite-ethylamine-L-lysine + gadaverine D creatinine-glucosamine + Vitamin deficiency + D: Growth is delayed

(3) Tolerance test 0.01% cycloheximide-0.1% cycloheximide-1% acetic acid-50% D-glucose + 60% D-glucose +

(4) Growth temperature 25 ° C. + 30 ° C. + 35 ° C.-37 ° C.-40 ° C .- (5) Additional test DBB reaction-

3. Other Properties (1) Number of Ubiquinone Side Chains Agricultural and Biological Chemistry (Agricultural Biological)
1 Chemistry) Vol. 37, No. 3, p. 621 (1973), the number of ubiquinone side chains was 6 units as measured by reverse phase thin layer chromatography.

(2) GC content, published by Japan Society for Publishing, edited by Kazuo Komagata, experimental method for chemical classification of microorganisms, page 227-242, using Shimadzu spectrophotometer UV-2110 and temperature controller SRP-8. , A100 change of chromosomal DNA in 1 × SSC buffer during a 40-minute linear temperature gradient of 60-100 ° C. was followed, and from the measured Tm value, GC (%) = (TM-6
The GC content (mol%) of the nuclear DNA of this strain was 44.9% as determined by the formula 9.3) /0.41.

The trehalose assimilation-deficient strain used in the method of the present invention is derived from the above-mentioned yeast belonging to the genus Candida and is a mutation that lacks trehalose assimilation. , All strains that produce trehalose without trehalose assimilation, but are preferred
The Candida fairendi (Candida)
fermentii) No. Candida fermentii No. 3110 strain obtained by mutating the strain 3110 with ultraviolet rays. 311
The 0tn strain can be mentioned.

To isolate a trehalose-producing strain lacking trehalose assimilation, which is suitable for the purpose of the present invention, a strain belonging to the genus Candida and capable of producing trehalose is known per se. It can be obtained by performing the mutation treatment of. Less than,
An example is shown below.

Candida Fairmenty
ida fermentii) No. One platinum loop of strain 3110 was placed in MY liquid medium (yeast extract 0.3%, malt extract 0.3%, polypeptone 0.5%, glucose 1%, p.
H5.5), cultured at 28 ° C. for 16 hours, and washed twice with sterile water. Put this in a petri dish, irradiate from a distance of 30 cm with an ultraviolet lamp (15 W) for 2 minutes, and then 5 m of yeast nitrogen medium (Difco) lacking carbon source
The cells were suspended in 1 and cultured overnight. The cells were collected by centrifugation, suspended in a yeast nitrogen medium containing 0.5% trehalose, cultured for 5 hours, and then nystatin was added at 10 μg / m 2.
1 was added and the culture was continued for another 2 hours. This was washed twice with sterilized water, and glycerin MY plate (yeast extract 0.3%, malt extract 0.3%, polypeptone 0.5%) was added.
%, Glycerin 1%, agar 2%, pH 5.5),
Among the grown colonies, strains that could not grow in a medium containing trehalose as a carbon source were selected.

The isolated strain was cultured in the MY liquid medium in the same manner as above, the bacterial cells were collected by centrifugation, and washed twice with cooled acetone. Acetone was removed by suction to obtain a powder. 10 mg of the obtained powder was suspended in 180 μl of 25 mM potassium phosphate buffer (pH 7.5), further 20 μl of 250 mM trehalose aqueous solution was added, and trehalose remaining with time was measured by liquid chromatography. A strain deficient in degrading activity was obtained. The column used was CLC-NH2 (Shimadzu), the mobile phase was 70% acetonitrile, and the detector used was a differential refractometer RID-7A (Shimadzu).

Next, the intracellular trehalose amount of these trehalose-degrading activity-deficient strains was measured by the following method, and a strain having a significantly increased trehalose accumulation amount as compared with the parent strain was selected. That is, 30 ml of MY liquid medium (yeast extract 0.3%, malt extract 0.3%, polypeptone 0.
250 ml containing 5%, glucose 1%, pH 5.5)
Inoculate the flask, incubate at 25 ° C for 1-2 days, collect the cells by centrifugation, wash well with water, and then add 30 ml.
Suspended in water, heat-treated at 120 ° C. for 15 minutes, intracellular trehalose was extracted, and trehalose was quantified by liquid chromatography to select a strain having a significantly increased accumulated amount as compared with the parent strain. .

By the above method, the mutant strain suitable for the purpose of the present invention was obtained at a ratio of about 1 strain per 100 colonies growing on the glycerin MY plate. One of the strains thus obtained is Candida fermentii No. Three
The bacteriological properties of the 110 tn strain were the same as those of the parent strain No. 110 except that the trehalose assimilation was defective. It showed the same properties as the 3110 strain.

This strain was sent to the Institute of Biotechnology, Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology, on January 1, 1994, at the Candida fermentii N
o. It has been deposited as 3110tn (FERM P-14187).

Further, as a trehalose-assimilating revertant mutant obtained from a strain in which utilisability and accumulation of trehalose, which is another mutant used in the method of the present invention, are simultaneously deficient, the above-mentioned Candida (Candida) is used. ) It is derived from a yeast belonging to the genus, and includes all strains that produce trehalose, which are obtained by a reversion mutation in which trehalose-utilizing ability is recovered from a strain that lacks trehalose-utilizing ability and accumulating ability at the same time. Among them, the Candida ferme (Candidaferme)
ntii) No. 3110 strain was isolated from a strain deficient in utilizability and accumulation of trehalose mutated by ultraviolet rays, and a trehalose assimilation-reverting mutant strain Candida ferment.
ii) No. The 3110tr strain can be mentioned.

A method known per se can be used to isolate a revertant strain that produces trehalose with restored trehalose assimilation from a strain that is deficient in trehalose assimilation and accumulation at the same time, which is suitable for the purpose of the present invention. Thus, a trehalose-producing strain belonging to the genus Candida can be treated and obtained. An example thereof will be shown below.

Candida Fairmenty
ida fermentii) No. Mutagenizing treatment and measurement of intracellular trehalose amount were carried out in the same procedure as the above-mentioned procedure for obtaining strain 3110 having a trehalose-degrading activity deficiency, and a strain deficient in trehalose assimilation and accumulation was obtained. By the above-mentioned method, the mutant strain that was deficient in assimilation and accumulation at the same time was obtained at a ratio of about 1 strain per 100 colonies growing on the glycerin MY plate. These 10 ⁸ to 10 ⁹ cells were applied to a yeast nitrogen medium (trehalose 0.5%) containing trehalose as a carbon source, and cultured at 28 ° C. for 3 days to utilize trehalose at a frequency of about 10 ^-8. A revertant strain that can be obtained can be obtained.

[0028] One of the strains thus obtained is Candida fe
rmentii) No. The mycological property of the 3110tr strain was No. 3110tr. It showed the same properties as the 3110 strain.

This strain was sent to the Institute of Biotechnology, Institute of Biotechnology, Institute of Industrial Science, on January 1, 1994, at Candida fermentii N
o. It has been deposited as 3110 tr (FERM P-14188).

The production of trehalose according to the present invention is derived from yeast belonging to the genus Candida,
Strains producing trehalose lacking trehalose assimilation, such as the above-mentioned Candida fairendi (C
andida fermentii) No. 3110t
n strain, or a trehalose assimilation-reverting mutant strain obtained from a yeast strain belonging to the genus Candida, which is deficient in trehalose assimilation and accumulation at the same time, for example, Candida fairendi (Candida)
da fermentii) No. It is produced by inoculating the nutrient-containing medium with the 3110tr strain and allowing it to grow aerobically.

As the nutrient source, those usually used as yeast nutrient sources can be used, for example, carbon source can be glucose, glycerin, sucrose and the like, and nitrogen source can be, for example, meat extract, yeast extract, peptone. ,
Organic substances such as corn steep liquor and inorganic nitrogen such as ammonium sulfate, ammonium chloride, ammonium nitrate and ammonium phosphate can be used. In addition, inorganic salts such as sodium chloride, potassium chloride, phosphate, and other heavy metal salts and vitamins are added as necessary, and in order to suppress foaming during culture, for example, silicone (manufactured by Shin-Etsu Chemical Co., Ltd.-KM75 Defoaming agents such as; trademark) can be added as appropriate.

Culturing conditions such as temperature, pH, aeration and incubation time are selected so that the strain used accumulates the maximum amount of trehalose. For example, when the MY liquid medium is used, it is advantageous to carry out the treatment at a temperature of 20 to 37 ° C., preferably 28 to 33 ° C. at a pH of 4 to 7, preferably 5 to 6 for 1 to 2 days.

To isolate trehalose from the culture,
It is possible to perform it by appropriately combining known separation means. Since trehalose accumulates in yeast cells, for example, the cells are suspended in an organic solvent that is miscible with water, such as methanol, ethanol, and acetone, and trehalose is extracted from the cells, and the cells are separated by centrifugation or filtration to separate the supernatant. The trehalose crystals can be obtained by applying the purification operations such as concentration under reduced pressure, ion exchange resin treatment, crystallization and the like individually or in appropriate combination and, if necessary, repeatedly.

When cultivated by the above culturing method, trehalose accumulates in the yeast cells as described above, but 2 to 6% of an organic solvent miscible with water, such as methanol, ethanol or acetone, during the culturing, or Methanol, ethanol, which is obtained by adding a surfactant such as Digitonin to a medium of 0.1 to 1%, or culturing yeast cells cooled.
By washing with an organic solvent that is miscible with water, such as acetone, trehalose produced inside the cells can be converted into permeabilized cells. By these treatments, the cell growth ability is lost, but the ability to produce trehalose from the nutrients in the medium is not lost.

The permeabilized cells prepared by washing with an organic solvent may be immediately used for the production of trehalose, but can be stored at 0 to 10 ° C, preferably 4 ° C. Furthermore, the stability is improved by forming an immobilized microbial cell by using a gelling carrier such as κ-carrageenan and calcium alginate. When the cells treated in this way are used, trehalose is produced in the culture solution, which is advantageous because the extraction operation from the cells can be omitted during purification.

[0036]

The present invention will be described in more detail below with reference to examples.

Example 1 M consisting of 0.3% yeast extract, 0.3% malt extract, 0.5% polypeptone and 1% glucose (pH 5.5)
100 ml of Y liquid medium was put into a 500 ml shake flask and sterilized by heating at 120 ° C. for 20 minutes. Candida ferm (Candida ferm)
entii) No. Inoculate 1 platinum loop of 3110tn strain,
It was shake-cultured at 28 ° C. for 16 hours. 5 of the obtained culture solution
Collect cells by centrifugation at 000 xg for 5 minutes, and
l of water was added and stirred, and the centrifugation was repeated in the same manner to give a total of 3
Washed twice. 1 g of the obtained wet bacterial cells was added to 60% ethanol 2
It was suspended in 5 ml and heat-treated at 80 ° C. for 1 hour. After cooling to room temperature and centrifugation at 5000 xg for 5 minutes, the bacterial cells were removed to obtain a trehalose-containing extract. This was treated with activated carbon and freeze-dried to obtain 90.5 mg of trehalose powder having a purity of 90.5%.

Example 2 100 ml of MY liquid medium in a 500 ml shake flask.
, And sterilized by heating at 120 ° C. for 20 minutes. Candida fermenti (Candidaferm)
entii) No. Inoculate 1 platinum loop of 3110tr strain,
It was shake-cultured at 28 ° C. for 16 hours. 5 of the obtained culture solution
Collect cells by centrifugation at 000 xg for 5 minutes, and
l of water was added and stirred, and the centrifugation was repeated in the same manner to give a total of
Washed twice. 1 g of the obtained wet bacterial cells was added to 60% ethanol 2
It was suspended in 5 ml and heat-treated at 80 ° C. for 1 hour. After cooling to room temperature and centrifugation at 5000 xg for 5 minutes, the bacterial cells were removed to obtain a trehalose-containing extract. This was treated with activated carbon and freeze-dried to obtain 89.5 mg of trehalose powder having a purity of 91.2%.

Example 3 100 ml of MY liquid medium was added to a 500 ml shake flask.
, And sterilized by heating at 120 ° C. for 20 minutes. Candida fermenti (Candidaferm)
entii) No. Inoculate 1 platinum loop of 3110tn strain,
It was shake-cultured at 28 ° C. for 16 hours. 5 of the obtained culture solution
The cells were collected by centrifugation at 000 × g for 5 minutes, and 100 ml of a new MY liquid medium was added thereto to suspend. 2% more
When acetone was added so that the amount of trehalose was 1.40 g / l (liquid chromatography method), trehalose was accumulated in the medium.

Example 4 100 ml of MY liquid medium was added to a 500 ml shake flask.
, And sterilized by heating at 120 ° C. for 20 minutes. Candida fermenti
i) No. Inoculate 1 platinum loop of 3110tn strain, 28 ℃,
It was shake-cultured for 16 hours. 5000 × the obtained culture solution
The cells were collected by centrifugation for 5 minutes for 5 minutes, 50 ml of 25 mM potassium phosphate buffer (pH 8.0) was added thereto, the mixture was stirred, and again centrifuged under the above conditions to obtain 1 g of washed cells.
Furthermore, this is washed twice with 50 ml of cooled acetone,
It was sucked with a vacuum pump and dried. 0.1 g of the powder thus obtained was placed in a 250 ml flask, and a 25 mM potassium phosphate buffer solution (pH 8.
0) 20 ml was added and suspended, and it was shaken at 28 ° C.
The trehalose concentration in the buffer was measured by liquid chromatography.

In the same manner, Candida fermenti No. 31
The 10 tr strain was also measured. Table 1 shows the results.

[0042]

[Table 1]

Example 5 5 ml of 25 mM potassium phosphate buffer (pH 8.0) containing 1% of κ-carrageenan was heated and dissolved, and then 4
Cooled to 2 ° C. In addition to this, the No. 311
0.1 mg of dry powder of 0tn strain was suspended, further cooled to room temperature and solidified. This was cut into about 0.5 cm square pieces, suspended in a 25 mM potassium phosphate buffer solution (pH 8.0) containing 1% glucose, and shaken at 28 ° C. The trehalose concentration in the buffer was measured by liquid chromatography.

In the same manner, Candida fermenti No. 31
The 10 tr strain was also measured. Table 2 shows the results.

[0045]

[Table 2]

Claims (5)

[Claims] 1. A method for producing trehalose, which comprises culturing a trehalose-producing yeast belonging to the genus Candida and collecting trehalose from the culture. 2. The method for producing trehalose according to claim 1, wherein the trehalose-producing yeast belonging to the genus Candida is a strain lacking trehalose-assimilating ability. 3. The trehalose-utilizing revertant strain of claim 1, wherein the trehalose-producing yeast belonging to the genus Candida is a trehalose assimilation-reverting mutant strain obtained from a strain deficient in trehalose assimilation and accumulation at the same time. Production method 4. A trehalose-producing yeast belonging to the genus Candida is Candida fermentii No.
The method for producing trehalose according to claim 1, which is 3110tn strain (FERM P-14187). 5. A trehalose-producing yeast belonging to the genus Candida is Candida fermentii No.
The method for producing trehalose according to claim 1, which is 3110tr strain (FERM P-14188).