JP3194160B2 - Method for producing L-tagatose - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing L-tagatose, and more particularly to a method for producing L-tagatose by using a bacterium belonging to the genus Klebsiella and capable of producing L-tagatose from galactitol. The present invention relates to a method for producing L-tagatose.

[0002]

2. Description of the Related Art L-tagatose is a monosaccharide classified as ketohexose and is a rare saccharide which hardly exists in nature. In principle, L-tagatose can be produced by isomerizing L-galactose or L-talose in the presence of an alkaline agent such as caustic soda or pyridine by an organic chemical method. . However, in practice, it is difficult to obtain L-galactose or L-talose as a raw material, and no suitable method for producing a sufficient amount of L-tagatose has been reported to date.

[0003]

In recent years, the biochemical industry has been rapidly developed, and the development of new carbohydrates is desired in the field of carbohydrate chemistry. L-tagatose has not been established as a mass production method and is not commercially available as a reagent.
Therefore, it has not yet been used as an industrial raw material for the food industry, the pharmaceutical industry, the chemical industry, and the like.

[0004]

Means for Solving the Problems The present inventors have made intensive studies for the purpose of producing L-tagatose in large quantities and at low cost by biochemical means.

As a result, it has been found that bacteria belonging to the genus Klebsiella and capable of producing L-tagatose from galactitol readily convert galactitol in an aqueous solution into L-tagatose. L
-It was confirmed that tagatose was produced in high yield, and the present invention was completed.

That is, in the present invention, the bacterium used to produce L-tagatose from galactitol is a bacterium belonging to the genus Klebsiella and having the ability to produce L-tagatose from galactitol. One example is Klebsiella Pneumoniae.
a pneumoniae) IFO 3317 or
Mutants thereof can be advantageously used.

The bacterium belonging to the genus Klebsiella and capable of producing L-tagatose from galactitol as referred to in the present invention is a microorganism capable of producing L-tagatose from galactitol upon contact with an aqueous solution containing galactitol. For example, Klebsiella pneumoniae I
FO 3317, or a suitable bacteria such as the mutant strain is usually, these bacteria were cultured xylitol, D- talitol, L- Arabinoru, in a nutrient medium containing a carbon source such as glycerol, preferably, shaking, Cultivation is performed under aerobic conditions such as aeration and agitation, and galactitol in the aqueous solution is converted into L-tagatose during the culture or by using the obtained bacteria (live cells), and the L-tagatose produced is converted. You just need to collect it.

[0008] As a culture method, nutrient sources required by bacteria belonging to the genus Klebsiella, for example, a carbon source, a nitrogen source,
A medium having an ability to produce L-tagatose from galactitol is inoculated on a medium containing an inorganic salt or the like, preferably a slightly acidic or slightly alkaline liquid medium, at a temperature of about 20 to 3.
The cells may be cultured under aerobic conditions at 5 ° C. for 1 to 10 days. Among others, as a carbon source, for example, xylitol,
It is desirable to cultivate aerobically in a liquid medium containing one or more carbon sources such as D-talitol, L-arabitol, and glycerol together with various other nutrient sources.

The bacteria (live cells) obtained by the above-described culturing method are brought into contact with an aqueous solution containing galactitol, preferably under aerobic conditions such as shaking, aeration and oxygen injection. To convert the saccharide to L-tagatose. At this time, it is convenient to add ethyl alcohol, glycerol, D-mannitol, or the like to the reaction solution because the conversion speed is increased.

The bacteria used for this conversion need not be limited to the viable cells isolated from the culture solution as they are, for example, bacteria in which the viable cells are encapsulated in hollow fibers made of a semipermeable membrane, agar, gelatin, etc. It is also convenient to repeatedly use galactitol to convert it into L-tagatose as bacteria immobilized in various forms such as beads, sheets, and the like, which are wrapped with alginate and the like.

The aqueous solution containing L-tagatose produced and accumulated by the above-described various methods is separated from bacteria by an appropriate separation method, for example, a method such as centrifugation or filtration, and collected.

The obtained aqueous L-tagatose solution may be used, if necessary, for example, by a method such as salting out ammonium sulfate, deproteinization by adsorption of zinc hydroxide, decolorization by adsorption of activated carbon, and desalting by an H-type or OH-type ion exchange resin. It can be purified and concentrated to obtain a syrupy L-tagatose product. Further, column chromatography using an ion exchange resin, for example, Dowex 50WX4 and Dowex WGR manufactured by Dow Chemical Co., Ltd., Amberlite XT-1008, Amberlite IRA47 manufactured by Tokyo Organic Chemical Industry Co., Ltd., Mitsubishi Product name: Diaion SK106, Diaion WA11 manufactured by Kasei Kogyo Co., Ltd.
Can be crystallized by fractionation, purification and concentration by column chromatography using
A high-purity crystal sample as described above can be easily obtained.

[0013] The L-tagatose thus produced is usually about 40% w / w% based on the starting material galactitol.
It is suitable as an industrial production method that can be obtained at a high yield as described above and that is supplied in large quantities and at low cost.

Therefore, L-tagatose can be freely used as a raw material, an intermediate or the like in the food industry, pharmaceutical industry, chemical industry and the like.

Hereinafter, embodiments will be described.

[0016]

Example 1 Ammonium sulfate 0.2 w / v%, monopotassium phosphate 0.24 w / v%, dipotassium phosphate 0.1 w / v%
56w / v%, magnesium sulfate heptahydrate 0.01w /
v%, yeast extract 0.5 w / v%, xylitol 2 w /
v% and deionized water in 100 ml each
Transfer to 20 00 ml shake flasks,
Minutes after autoclaving, Klebsiella pneumoniae
One loopful of IFO 3317 was inoculated, and cultured at 30 ° C. for 2 days with shaking.

After the culture, the cells are collected by centrifugation, and about 20 g of the obtained viable cells are added to 1 L of 0.05 M phosphate buffer (pH 7.0) containing 2% w / v galactitol, and mixed. About 100 ml of this mixture was dispensed into 500 ml shake flasks and shaken at 30 ° C. for 2 days to convert galactitol to L-tagatose. The bacteria were then removed by centrifugation.

The supernatant obtained was mixed with 1% of 25% w / v zinc sulfate.
The mixture was adjusted to pH 7.6 by adding / 10 volume and centrifuged to collect the supernatant. The supernatant is 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 Diaion WA
Desalting was performed using No. 30 (OH type, trade name manufactured by Mitsubishi Kasei Kogyo Co., Ltd.), and concentrated under reduced pressure to obtain a transparent syrup having a concentration of about 60% and containing galactitol as an impurity as an intermediate.

Purification and concentration by column chromatography using Dowex 50WX4 (calcium type cation exchange resin, trade name manufactured by Dow Chemical Co.)
-L-tagatose crystals were collected by crystallizing and separating L-tagatose.

The yield of L-tagatose based on galactitol was about 50% per solid.

In order to identify the product thus obtained, the physicochemical properties were examined by experiment and the properties were compared with those in the literature, or the measurement of the reagent D-tagatose commercially available from Sigma as a standard substance. Value.

(1) Melting point Measured value of this product 134-135 ° C D-tagatose literature value 134-135 ° C L-tagatose literature value 133-135 ° C Literature: Chapman & Hall (Chapman)
and Hall), "Dictionary of Organic Compounds (Dictionary of Organic Compounds)
f Organic Compounds) ", No. 509
7 pages (1982)

(2) Measurement of specific rotation Measurement value of this product Reagent D-measured value of tagatose Literature value of D-tagatose Literature: (1) Same as for melting point

(3) Infrared absorption spectrum The infrared absorption spectrum of this product measured by the KBr tablet method is shown in FIG. This spectrum was in good agreement with the infrared absorption spectrum of reagent D-tagatose measured separately.

(4) Confirmation of reducing substance with sodium borohydride This product and the reagent D-tagatose were reduced with sodium borohydride, and the product was identified. The reduction products from the product and reagent D-tagatose were both galactitol and talitol.

(5) Analysis by high performance liquid chromatography This product was analyzed by high performance liquid chromatography (880-PU, manufactured by JASCO Corporation; column, GL-C611, Hitachi, Ltd.).
Eluent, ^10-4 M sodium hydroxide, 60 ^<0> C;
1 ml / min; detection, analyzed by Shimadzu RID-6A), the elution position was different from ketohexose such as reagents D-psicose, D-fructose and D-sorbose as standard substances. It was the same 21.5 minutes.

As is apparent from the above results, the melting point, infrared absorption spectrum, analysis of the reduced product, and high performance liquid chromatography were performed for D-tagatose and L-
The values corresponded well to the literature values of tagatose or the values of the reagents as their standard substances, and the specific rotation was + or-reversed, although it coincided well with the measured value of D-tagatose. The obtained product is determined to be L-tagatose.

The product can be advantageously used not only as a reagent as a rare saccharide, but also as a raw material and an intermediate for the food industry, pharmaceuticals, chemical industry and the like.

[0029]

Example 2 Xylitol 2 in the culture solution of Example 1
Except for adding galactitol in an amount of 2 w / v% in addition to w / v%, 30 L of a culture solution having the same composition as in Example 1 was taken in each of two 30 L jar fermenters, and 12 L was taken.
After sterilizing at 0 ° C. for 20 minutes, cool to 30 ° C.
Klebsiella pneumoniae (Klebsie shaking for 1 day incubation at 30 ° C. using a culture medium of the same composition as in Example 1
lla pneumoniae) A seed culture of IFO 3317 was inoculated at 1 v / v%, aerated and agitated at 30 ° C. for 5 days to convert galactitol into L-tagatose, and then centrifuged to separate cells and supernatant. The resulting supernatant was decolorized by activated carbon adsorption, then desalted using an ion exchange resin, concentrated, purified by column chromatography and concentrated according to the method of Example 1. Tagatose crystals were collected. The yield of L-tagatose based on galactitol was about 50% per solid.

This product, as in the case of Example 1,
The physical and chemical properties of tagatose were shown.

The product is used not only as a reagent for rare saccharides, but also as a raw material for food industry, pharmaceutical industry, chemical industry, etc.
It can also be advantageously used as an intermediate.

[0032]

As is apparent from the above, the present invention establishes a method for easily producing L-tagatose by a biochemical method, which has been extremely difficult to obtain conventionally. In particular, the fact that L-tagatose can be produced from a cheap sugar alcohol called galactitol as a raw material using a microorganism belonging to the genus Klebsiella is extremely advantageous for a method for producing L-tagatose.

Therefore, the method of the present invention is suitable as an industrial method for producing L-tagatose, facilitates the supply of large amounts at low cost, and is not only expected to be used as a reagent as a rare saccharide but also could not be expected in the past. It opens the way for use in the food, pharmaceutical, and chemical industries.

[0034]

[Brief description of the drawings]

FIG. 1 is a diagram showing an infrared absorption spectrum of L-tagatose obtained by the method of the present invention.

──────────────────────────────────────────────────続 き Continued on the front page (58) Fields surveyed (Int. Cl. ⁷ , DB name) C12P 19/02 BIOSIS (DIALOG) WPI (DIALOG)

Claims (2)

(57) [Claims] An L-tagatose is produced by contacting a bacterium belonging to the genus Klebsiella and capable of producing L-tagatose from galactitol with an aqueous solution containing galactitol to produce L-tagatose, and collecting the L-tagatose. -A method for producing tagatose. 2. A genus Klebsiella bacterium belonging to the, Klebsiella pneumoniae (Klebsiella PNE
umoniae) The method for producing L-tagatose according to claim 1, which is IFO 3317 or a mutant thereof .