JP2000041689A - Production of inositol - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain inositol important as a kind of vitamin useful for a nutritive food, a feed additive, a medicine, etc., in a high yield by using a fungus having resistance to a halogenated pyruvic acid, capable of secreting and producing inositol. SOLUTION: A fungus [e.g. Candida boidinii BP22 (FERM P-15,743), etc.], having resistance to a halogenated pyruvic acid, capable of secreting and producing inositol is cultured in a medium, inositol is accumulated in a culture solution and collected from the culture solution to give the objective inositol important as a kind of vitamin in higher animals useful for a nutritive food, a feed additive, a medicine, etc., in a high yield economically. Candida boidinii BP22 strain is obtained by treating Candida boidinii δ-65 (FERM P-15, 743) (FERM P-15,379) with a mutagenetic agent and culturing the treated strain under conditions in which no parent strain can grow.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION Inositol is an important substance as a type of vitamin in higher animals,
Used for feed additives and pharmaceuticals.

[0002]

2. Description of the Related Art Conventionally, inositol has been extracted from rice bran, corn steep liquor and the like (JP-A-61-56142).
And a method of culturing and producing baker's yeast (European Patent Publication No. 506289). Further, a method of culturing and producing a microorganism belonging to the genus Candida by the present inventors (JP-A-8-258, JP-A-8-8)
9266, JP-A-8-38188).

[0003]

The method of extracting rice bran, corn steep liquor and the like contains many impurities other than inositol, is difficult to purify, and is economically problematic. In addition, the method of culturing and producing baker's yeast has low productivity, is still economically problematic, and has no industrial record. In addition, the method of culturing and producing a microorganism belonging to the genus Candida has not been industrially satisfactory in terms of yield and yield.

[0004]

Means for Solving the Problems In order to solve the above problems, the present inventors have found that a mutant strain of a microorganism belonging to the genus Candida accumulates inositol in good yield and yield. The present inventors have conducted intensive studies on a method for producing inositol with higher productivity, and as a result, by imparting resistance to halogenated pyruvate to a microorganism capable of producing inositol, the accumulated concentration of inositol and the production yield were reduced. The present inventors have found that the temperature is remarkably improved, and arrived at the present invention.

That is, the present invention comprises culturing a microorganism which is resistant to halogenated pyruvate and has the property of secreting inositol, accumulates inositol in the culture solution, and collects inositol from the culture solution. And culturing a microorganism having tolerance to pyruvate halide and capable of producing inositol, and performing a reaction to produce and accumulate inositol using the obtained cells or a processed product thereof, and the reaction solution A method for producing inositol, which comprises collecting more inositol.

[0006] The halogenated pyruvic acid as used herein is a pyruvate antimetabolite. That is, by adding a substance that inhibits the growth of microorganisms, it exhibits an inhibitory or inhibitory effect on enzymes involved in pyruvate biosynthesis or decomposition,
It is a substance whose inhibition or inhibition is restored by the addition of pyruvic acid. Examples of the halogenated pyruvic acid include bromopyruvic acid, fluoropyruvic acid, chloropyruvic acid, and the like, and the respective substances can be used alone or in combination.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The microorganism used in the present invention may be any microorganism that secretes inositol, and the parent strain is Candida, a hexachlorocyclohexane-resistant mutant obtained by the present inventors.・ Voidinii (Candida boidinii)
It is preferable to use δ-65 (FERM P-15739). As long as it has the property of secreting inositol, it may also have properties such as resistance to drugs and auxotrophy, and any microorganism that secretes inositol is included in the present invention.

[0008] The induction of the mutant strain may be carried out by irradiating the parent strain with ultraviolet light,
Alternatively, after treatment with a mutagenic agent (for example, N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonic acid, etc.), growth is possible on a solid medium containing pyruvate halide at such a concentration that the parent strain cannot grow. What is necessary is just to collect various strains.

[0009] A halogenated pyruvate-resistant mutant is a strain that is more resistant to halogenated pyruvate than the parent strain. In the present invention, the relative growth rate of the parent strain is 30%.
In the concentration range of halogenated pyruvic acid which shows:
It is preferable to obtain a mutant showing a relative growth degree of 0% or more. Here, the relative growth was measured by measuring the absorbance of the culture at 660 nm, and expressed as a relative value when the absorbance of the culture without the addition of halogenated pyruvic acid was taken as 100%. Commercially available halogenated pyruvic acid may be used for assaying the resistance.

The culturing method of the present invention will be described. The medium for producing inositol is an ordinary medium containing a carbon source, a nitrogen source, inorganic ions and, if necessary, other organic trace components.

Examples of the carbon source include glucose, fructose, hydrolysates of starch and cellulose, sugars such as molasses, organic acids such as fumaric acid, citric acid and succinic acid, and alcohols such as methanol, ethanol and glycerol. 0.1 to 4.0% of an organic ammonium salt such as ammonium acetate, an inorganic ammonium salt such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium nitrate, ammonia gas, aqueous ammonia, urea, etc. as a nitrogen source. As the organic trace component, the required substance such as biotin is 0.00%.
A medium containing 0001% to 0.1%, and if necessary, 0 to 5% of corn steep liquor, peptone, yeast extract and the like, respectively, is appropriately used. In addition to these,
Potassium phosphate, magnesium sulfate, calcium chloride,
Sodium chloride, zinc sulfate, copper sulfate, ferrous sulfate, etc. are added as trace components. Preferably, an antifoaming agent is also added to stabilize the culture conditions.

The culture is usually performed under aerobic conditions. During the culture,
The pH of the medium is adjusted to 3-8, the temperature is adjusted to 20-35 ° C,
Preferable results can be obtained by shaking or aeration and stirring culture for 4 to 96 hours.

Next, a method for producing inositol by the enzymatic method according to the present invention will be described. The cells are cultured in the same manner as in the medium in the fermentation method to obtain cells. This cell may be used for the reaction as it is, but preferably, it is subjected to a protoplasmic separation treatment by a known method. The reaction is carried out in the presence of nicotinamide adenine dinucleotide and ammonia at a pH of 3 to 8 and a temperature of 20 to 35 ° C. with shaking for 10 to 72 hours.

The inositol secreted and accumulated in the culture solution can be used as a feed without isolation and collection as it is. In addition, inositol can be collected from a culture solution or a reaction solution by a known method. For example, crystals can be obtained by removing cells by centrifugation or the like, removing ionic substances with a cation and anion exchange resin, and concentrating.

[0015]

The present invention will be described below in detail with reference to examples. Example 1 (Isolation of a bromopyruvic acid resistant mutant)
Cells of the Voidinii δ-65 strain were treated with N-methyl-N′-nitro-N-nitrosoguanidine (300
(g / ml, 30 ° C. for 10 minutes), the cells were diluted appropriately, applied to a plate medium containing bromopyruvic acid (BP) at a concentration of 5 mg / l in the medium shown in Table 1, and then plated at 30 ° C. At 4
Cultured for days. The grown Candida voidinii colonies that have undergone mutagenesis are isolated as pure mutants, and Candida voidinii BP22 (FERM) is isolated.
P-16743).

[0016]

[Table 1] (Degree of Resistance of Bromopyruvic Acid-Resistant Mutants) Each strain of Candida voidinii BP22 and Candida voidinii δ-65 was shake-cultured at 30 ° C. for 24 hours using a medium excluding the agar medium shown in Table 1, The grown cells were collected and washed with physiological saline. Each of these cell suspensions was inoculated into 5 ml of the medium shown in Table 1 to which bromopyruvic acid at the concentration shown in Table 2 was added, and cultured at 30 ° C. Examined. The results are as shown in Table 2. The mutant strain resistant to bromopyruvate used in the present invention shows that growth was not inhibited by a high concentration of bromopyruvic acid and that the mutant strain acquired strong bromopyruvic acid resistance as compared with the parent strain.

[0017]

[Table 2] Example 2 (Culture of bromopyruvic acid-resistant mutant strain and production of inositol by fermentation method) Candida voidinii B
P22 and Candida voidinii δ-65 were each inoculated in a loop of 5 ml of the medium shown in Table 3 at 30 ° C.
Preculture was performed by shaking for 24 hours. This culture solution was previously
50 ml of a medium having the composition shown in Table 4 and steam sterilized at 5 ° C. for 10 minutes.
And transferred to a 500 ml Erlenmeyer flask containing
After culturing for 72 hours under the conditions of m 2 and amplitude 30 cm, glucose previously sterilized was added at 50 g / l, and culturing was performed for a total of 120 hours.

After completion of the culture, the concentration of inositol in the filtrate from which the cells and calcium carbonate had been removed was quantified by high performance liquid chromatography, and the results shown in Table 4 were obtained. Compared to the parent strain, the mutant strain showed a significant increase in inositol production.

[0019]

[Table 3]

[0020]

[Table 4] Example 3 The supernatant of 1 L of the culture solution obtained in Example 2 was passed through a cation exchange resin DIAION SK1B (manufactured by Mitsubishi Chemical Corporation) to collect a flow-through fraction, and further anion exchange resin DIAION PA316 (manufactured by Mitsubishi Chemical Corporation). ), The flow-through fraction was collected, concentrated and crystallized to obtain 14.2 g of inositol crystals having a purity of 98% or more.

[0021]

According to the present invention, when inositol is produced in a culture solution by a fermentation method using the yeast of the present invention, compared with the existing method,
More economical production of inositol becomes possible.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (reference) C12R 1:72)

Claims (7)

[Claims] 1. A method for producing inositol, comprising using a microorganism which is resistant to halogenated pyruvate and has the ability to produce inositol secretion. 2. The production of inositol according to claim 1, wherein a microorganism having tolerance to halogenated pyruvate and capable of producing inositol secretion is cultured to accumulate inositol in the culture solution. Method. 3. A method of culturing a microorganism having resistance to halogenated pyruvate and capable of producing inositol, accumulating inositol in the culture solution, and collecting inositol from the culture solution. Item 2. The method for producing inositol according to Item 2. 4. A method of culturing a microorganism having pyruvate halide resistance and capable of producing inositol secretion, and performing a reaction for producing and accumulating inositol using the obtained cells or a processed product thereof. The method for producing inositol according to claim 1, wherein 5. A method of culturing a microorganism which is resistant to halogenated pyruvate and has an ability to produce and secrete inositol, and performing a reaction for producing and accumulating inositol using the obtained cells or a processed product thereof. The method for producing inositol according to claim 4, wherein inositol is collected from the reaction solution. 6. The method for producing inositol according to any one of claims 1 to 5, wherein the microorganism capable of producing and secreting inositol is a microorganism belonging to the genus Candida. 7. The microorganism belonging to the genus Candida is Candida voidinii.
The method for producing inositol according to the above.