JP4356992B2 - Method for producing L-talitol using reduction reaction of yeast - Google Patents

Description

  The present invention relates to a method for producing L-talitol, and more particularly relates to a method for producing L-talitol from L-psicose using a yeast capable of producing L-talitol from L-psicose. is there.

  L-talitol is a monosaccharide classified as a sugar alcohol and is a rare sugar alcohol that hardly exists in nature. In principle, it can be produced by reducing monosaccharides such as L-talose and L-altrose using hydrogen at high temperature and pressure in the presence of a metal catalyst using organic chemical techniques. It is. However, although this method can produce L-talitol in high yield, it requires a high-pressure autoclave, which not only consumes a large amount of hydrogen and energy, but also requires a high level of safety facilities and management for disaster prevention. There is. In practice, it is extremely difficult to obtain L-talose, L-altrose, etc. as raw materials.

The second method is obtained by an enzyme reaction using ketose 3-epimerase and the like described in Patent Document 1, Patent Document 2, Patent Document 3 and the like, and an enzyme reaction using a polyol dehydrogenase. L-psicose can be produced by the organic chemistry method described above. However, when L-psicose is reduced using hydrogen, the resulting sugar alcohol becomes a mixture of L-talitol and allitol, which has the disadvantages of reduced yield and separation.
Japanese Patent Laid-Open No. 6-125776 JP-A-8-154696 Japanese Patent Laid-Open No. 11-056583

  As in the second method, the third method produces L-talitol in a high yield from L-tagatose obtained by an enzyme reaction using ketose 3-epimerase or the like and an enzyme reaction using polyol dehydrogenase. It is possible. However, it has improvements such as the preparation of the enzyme used, the polyol dehydrogenase, and the formate dehydrogenase conjugated with the enzyme are expensive.

  In recent years, the biochemical industry has developed rapidly, and in the field of carbohydrate chemistry, the demand for many kinds of sugar alcohols that have been hardly required has been rising, and new sugar alcohols are easily and stably produced. Establishment of a method is desired. The present invention provides a method for producing a target L-talitol with high purity and high yield by an environmentally friendly biochemical method.

  In order to solve the above-mentioned problems, the present inventors diligently studied for the purpose of producing L-talitol in a large amount at a low cost by a biochemical method. As a result, it was found that yeast belonging to the genus Methicobia and having L-psicose producing ability from L-psicose easily converts L-psicose in an aqueous solution to L-talitol, and by collecting this, L- The present invention was completed after confirming that tallitol was produced in high yield. The present invention relates to a method for producing L-talitol, and more specifically, L-talitol is produced from L-psicose using a yeast belonging to the genus Methicovia and capable of producing L-talitol from L-psicose. It is about how to do.

That is, the gist of the present invention is the following microorganism (1).
(1) Metschnikowia koreensis LA1 (NITE P-19) having the ability to produce L-talitol from L-psicose.

Moreover, this invention makes the summary the manufacturing method of L-talitol of the following (2)-(7).
(2) In the method for producing L-talitol, which converts L-psicose, which is a raw material, into L-talitol by bringing the microorganism into contact with the microorganism, yeast belonging to the genus Methynicobia having the ability to convert L-psicose into L-talitol as the microorganism A process for producing L-talitol, characterized in that
(3) The method for producing L-talitol according to (2), wherein the contact with the microorganism is carried out by adding L-psicose as a raw material to the cell culture system of the microorganism and culturing.
(4) The method for producing L-talitol according to (2), wherein the contact with the microorganism is carried out by adding L-psicose as a raw material to the cells obtained by culturing the microorganism and incubating.
(5) The method for producing L-talitol according to (3) or (4), wherein a microorganism is brought into contact with L-psicose as a raw material in the form of an aqueous solution containing L-psicose.
(6) The method for producing L-talitol according to any one of (2) to (5), wherein the yeast belonging to the genus Methynicobia is Metschnikowia koreensis .
(7) The method for producing L-talitol according to any one of (2) to (6), wherein the produced L-talitol is recovered.

  The present invention establishes a method for easily producing L-talitol which has been extremely difficult to obtain by a biochemical method. It is possible to provide a method for producing L-talitol, which is advantageous for an industrial production method, capable of producing L-talitol using L-psicose as a raw material using yeast belonging to the genus Methicobia.

In the present invention, the yeast used to produce L-talitol from L-psicose belongs to the genus Methicovia and has the ability to produce L-talitol from L-psicose.
In the present invention, yeast belonging to the genus Methicobia and having L-psicose-producing ability can be used as long as it is a microorganism that can contact L-psicose-containing aqueous solution and produce L-talitol from L-psicose. Well, for example, yeasts such as Methicobia chorensis or mutants thereof are suitable, and these yeasts are usually cultured in a nutrient medium, and preferably cultured under favorable conditions such as shaking tow and aeration stirring. The L-psalitol in the aqueous solution may be converted into L-talitol and the produced L-talitol may be collected using the obtained living microbial cells.
The microorganism used in the examples is Metschnikowia koreensis LA1 having the ability to produce L-talitol from L-psicose.

Metschenikowia koreensis LA1 is a vegetative cell in YM liquid medium with an oval or cylindrical shape (3-6 × 4-7μm) and no film formation.
The color of the colony on YM agar medium is white to cream, and the property is buttery. The colony surface is rough and gloss is dull.
No pseudohyphae formation is observed in Dalumau plate culture using cornmeal medium.
The number of spores in an ascomb formed on a 1 / 10V8 agar medium is 2.
Fermentability is D-glucose: +, D-galactose, sucrose, maltose, raffinose, trehalose:-
The assimilation is D-glucose: +, galactose: +, L-sorbose: +, sucrose: +, maltose: +, cellobiose: plus, trehalose: +, lactose:-, melibiose:-, raffinose: -, Melezitose: +, inulin:-, soluble starch: +, D-xylose: +, L-arabinose:-, D-arabinose: +, D-ribose: +, L-rhamnose:-, ethanol: +, glycerol : +, Erythritol:-, ribitol: +, mannitol: +, sorbitol: +, salicin: +, lactic acid:-, citric acid:-, inositol:-
Nitrate assimilation:-, growth on vitamin-free medium:-, growth at 37 ° C:-, DBB reaction:-, urease test: +

The above strains are Cletus P. Kurtman and Christie J. Robnett, Identification and
phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S)
ribosomal DNA partial sequences. Antonie van Leeuwenhoek 73,
Measure the partial base sequence of the D1 / D2 domain of 26S rDNA according to 331-371, 1998, and use NCBI-BLAST of the National Center of Biotechnology Information to determine the final strain species from the base sequence homology. ing.

  On September 6, 2004, the strain belonging to Methicobia chorensis was applied to the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (2-5-8, Kazusa-Kamazu, Kisarazu City, Chiba Prefecture). Deposited (NITE P-19).

  As the culture method, a nutrient medium required by yeast belonging to the genus Methynicobia, for example, a nutrient medium containing a carbon source, a nitrogen source, an inorganic salt, etc., preferably a slightly acidic to neutral liquid medium, L-psicose The yeast having the ability to produce L-talitol can be inoculated and cultured at a temperature of about 30 ° C. for 48 to 60 hours under aerobic conditions. In particular, L-arabitol is most suitable as a carbon source, and L-arabinose, D-xylose, erythritol and the like are also suitable as a carbon source. It is desirable to culture aerobically in a liquid medium containing a carbon source and various other nutrient sources.

  Viable cells obtained by the culture method as described above are brought into contact with an aqueous solution containing L-psicose, preferably under aerobic conditions such as shaking tow, stirring with aeration, and injecting oxygen, and the sugar The quality can be converted to L-talitol. In this conversion, coexistence of D-sorbitol, glycerol, etc. in the reaction solution is advantageous because the conversion rate from L-psicose to L-talitol increases.

  The yeast used for this conversion is optional to use it in the culture solution or to use viable cells separated from the culture solution.

  The aqueous solution containing L-talitol produced and accumulated by the various methods described above is separated and collected from insoluble matter such as bacterial cells by an appropriate separation method such as centrifugation or filtration.

The obtained aqueous L-talitol solution can be produced by methods such as ammonium sulfate salting out, deproteinization by zinc hydroxide adsorption, decolorization by activated charcoal adsorption, desalting by H-type or CO ₃ type ion exchange resin, if necessary. The syrupy L-talitol product can be collected by concentration. Furthermore, it can be crystallized by fractionation, purification, and concentration by column chromatography using an ion exchange resin, and a high purity crystal product of 99% or more can be easily obtained.

  The L-talitol produced in this way is usually obtained in a high yield of 70 w / w% or more with respect to the raw material L-psicose and is suitable as an industrial production method for supplying a large amount at a low cost.

  Therefore, L-talitol can be effectively used as a raw material, an intermediate and the like not only for reagent use but also for industrial use such as food industry, pharmaceutical industry and chemical industry.

Casamino acid 0.4 w / v%, yeast extract 0.1 w / v%, monopotassium phosphate 0.1 w / v%, magnesium sulfate heptahydrate 0.05 w / v%, calcium carbonate dihydrate 0. 100 ml of a culture solution consisting of 01 w / v%, L-arabinose 1.0 w / v% and deionized water was placed in 6 500 ml shake tow flasks, autoclaved at 121 ° C. for 15 minutes, and allowed to cool. To this, 0.1 v / v% of the culture solution of Methicobia pullulans LA1 previously cultured for 48 hours in the same medium was inoculated and cultured at 30 ° C. for 48 hours with shaking.

After cultivation, the cells are collected by centrifugation, and the obtained living cells are added to 100 ml of 0.05 M PIPES buffer (pH 7.0) containing 0.5 w / v% L-psicose and 1 w / v% D-sorbitol. The mixture was added and mixed in 50 ml aliquots in two 500 m shake tow flasks and shaken at 30 ° C. for 24 hours to convert L-psicose into L-talitol. After 24 hours, the cells were removed by centrifugation.

1/10 volume of 25 w / v% zinc sulfate was added to the obtained supernatant, the pH was adjusted to 7.6, and the supernatant was collected by centrifugation. This supernatant was decolorized using activated carbon according to a conventional method, and then “Diaion SK1B” (H type, trade name manufactured by Mitsubishi Kasei Kogyo Co., Ltd.) and “Amberlite IRA611” (CO ₃ type, Organo Corporation). The product was desalted using the product name) and concentrated under reduced pressure to obtain a transparent syrup having a concentration of about 60%.

A fraction containing a high L-talitol content was collected by column chromatography using Dowex 50WX2 (Ca-type cation exchange resin, trade name manufactured by Dow Chemical Co., Ltd.), produced, concentrated, and crystallized from L-talitol. And crystallized L-talitol. The yield of crystalline L-talitol based on L-psicose was about 50% per solid.

In order to identify the product thus obtained, the physicochemical properties were examined by experiment, and the properties were described in “Journal of Fermentation and Bioengineering”, Vol. 85, pp. 84-88. (1998) and compared with the measured value using D-talitol prepared from D-psicose as a standard substance.

(1) Analysis by high performance liquid chromatography This product was analyzed by high performance liquid chromatography (Hitachi LaChrom: column, Hitachi GL-C611; eluent, ^10-4 M sodium hydroxide, 60 ° C; flow rate, 1 ml / min; detection). , Hitachi L-3350), the elution position was 21.6 minutes, the same as D-talitol as the standard substance. The melting point of this product was 88.0 ° C, consistent with D-talitol.

(2) Specific rotation Measured value [α] D = −3.20 ° of this product
Measured value of standard reagent D-talitol [α] D = + 3.20 °

(3) Infrared absorption spectrum Figure 1 shows the infrared absorption spectrum of the product measured by the KBr tablet method. This spectrum was in good agreement with the infrared absorption spectrum of standard D-sorbitol shown in Fig. 2.

(4) ¹³ C-NMR measurement of ¹³ C-NMR measurement of the product of "Biochemistry (Biochemistry)", carried out according to the method described in Vol. 13, 146 to 153 pages (1974) As shown in Table 1, the chemical shift agreed well with that of standard D-talitol.

As is clear from the above results, the measurement results of high performance liquid chromatography, melting point, infrared absorption spectrum, and ¹³ C-NMR of this product are in good agreement with the values of standard D-talitol, and the specific rotation is confirmed. The standard D-talitol and the value of this product matched, but + and-were reversed, and the product obtained by the present invention was judged to be L-talitol. This product can be used not only as a reagent as a rare saccharide, but also as a raw material and intermediate for food industry, pharmaceuticals, chemical industry, etc.

  Using the culture solution having the same composition as in Example 1, the cells were cultured in the same manner, and then centrifuged to collect the cells. The obtained viable cells were mixed with 50 ml of 0.05 M PIPES buffer (pH 7.0) containing 3 w / v% L-psicose and 1 w / v% D-sorbitol, and the resulting mixture was placed in a 500-m shake tow flask. The mixture was shaken at 30 ° C. for 48 hours to convert L-psicose into L-talitol. During the conversion reaction, the coexisting D-sorbitol was added so that the reaction solution concentration became 1 w / v% every 12 hours. After 72 hours, the cells were removed by centrifugation, and were decolorized with activated carbon, desalted with ion exchange resin and concentrated in the same manner as in Example 1. A transparent syrup having an L-talitol content of 75% and a concentration of about 70% was obtained with a yield of about 67% per solid relative to the raw material.

  This product can be advantageously used as a raw material and intermediate for food industry, pharmaceutical industry, chemical industry, etc.

The present invention establishes a method for easily producing L-talitol which has been extremely difficult to obtain by a biochemical method. In particular, it has been found that L-talitol can be produced using L-psicose as a raw material using microorganisms belonging to the genus Methynicobia, which is extremely advantageous for the production method of L-talitol.
That is, L-psicose used as a raw material is an enzyme reaction using ketose 3-epimerase described in JP-A-6-125576, JP-A-8-154696, etc. by the present inventors, and JP-A-11 A ketohexose that can be mass-produced by biochemical methods from D-fructose as a starting material, D-psicose, and allitol by enzymatic reaction using polyol dehydrogenase described in No. 056583 Therefore, mass production of L-talitol became possible. Therefore, the method of the present invention is suitable as an industrial production method of L-talitol, facilitates the supply of a large amount and at a low cost, and is not only used as a reagent as a rare saccharide but also in the food industry and pharmaceuticals that could not be predicted in the past. It opens the way for a wide range of industrial applications such as industry and chemical industry.

IR spectrum of the product IR spectrum of D-talitol

Claims (7)

Metschnikowia koreensis LA1 (NITE P-19) has the ability to produce L-talitol from L-psicose.   In a method for producing L-talitol, which contacts L-psicose as a raw material and converts it to L-talitol, using a yeast belonging to the genus Methynicobia having the ability to convert L-psicose to L-talitol as the microorganism. A process for producing L-talitol.   The method for producing L-talitol according to claim 2, wherein the contact with the microorganism is carried out by adding L-psicose as a raw material to the cell culture system of the microorganism and culturing.   The method for producing L-talitol according to claim 2, wherein the contact with the microorganism is carried out by adding L-psicose as a raw material to the cells obtained by culturing the microorganism and incubating the same.   The method for producing L-talitol according to claim 3 or 4, wherein the microorganism is brought into contact with L-psicose as a raw material in the form of an aqueous solution containing L-psicose. The method for producing L-talitol according to any one of claims 2 to 5, wherein the yeast belonging to the genus Methynicobia is Metschnikowia koreensis . 7. The method for producing L-talitol according to claim 2, wherein the produced L-talitol is recovered.

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