JP6073585B2 - Process for producing 1,5-D-anhydroglucitol - Google Patents

Description

  The present invention relates to a method for producing 1,5-D-anhydroglucitol by a microorganism.

1,5-D-anhydroglucitol (hereinafter 1,5-AG) is a polyol having a structure in which the hydroxyl group at the 1-position of glucose is reduced, and since many animals and plants biosynthesize it, It is a widely distributed substance. 1,5-AG is metabolically stable in the animal body, and in rats and mice, of the radioactivity given as 1,5-AG, the amount excreted in exhalation during the following 48 hours is less than 1% of the total. There is a report that there is (see Non-Patent Document 1), and it can be used as a low calorie or non-calorie sweetener. Furthermore, it is used industrially as a research reagent and a clinical test reagent.
As a method for preparing 1,5-AG, a chemical synthesis method from β-D-glucopyranose pentaacetate (see Non-Patent Document 2) has been reported. The synthesis method is carried out by dissolving β-D-glucopyranose pentaacetate in ether, followed by Br conversion with hydrogen bromide and deacetylation with lithium aluminum hydride.

Also reported is a method for preparing 1,5-AG by extracting, isolating, and crystallizing 1.5-AG from protea leaves with organic solvents such as ethanol and hexane. (See Non-Patent Document 3).
These chemical synthesis and extraction from plants are multi-step and complicated processes, and use organic solvents such as ether and hexane. The need arises. Furthermore, questions remain from the viewpoint of safety.
As a means for solving these problems, a production method is required which has a simple production process and does not use an organic solvent such as ether.

It has been reported that 1,5-AG is biosynthesized by Escherichia coli (see Non-Patent Document 1). However, biosynthesis by Escherichia coli is not intended for industrial production, and the amount of 1,5-AG produced is about several micrograms per liter, which is not satisfactory for industrial production.
Furthermore, a method for preparing 1,5-AG by hydrogenation to 1,5-D-anhydrofructose (hereinafter 1,5-AF) in the presence of a palladium catalyst has been reported (see Non-Patent Document 4). ) 1,5-AM is generated in addition to 1,5-AG, which is about 1/5 of the reaction product and is difficult to obtain efficiently.

  1,5-AF, which can be a raw material for 1,5-AG, is a saccharide that can be prepared by degrading α-1,4-glucan such as starch with α-1,4-glucan lyase, and its production technology is It has been proposed (see Patent Document 1). Development of a method for efficiently converting 1,5-AF to 1,5-AG is desired, but in recent years, 1,5-AF has been brought into contact with microorganisms as an industrial production method for 1,5-AG. Has been proposed (see Patent Document 2). In this production method, exogenous 1,5-AF is converted to 1,5-AG by microorganisms. However, the above method is not always satisfactory industrially because the production amount of 1,5-AG is low. In addition, since the culture broth after culturing contains products other than 1,5-AG or unreacted raw materials, a separation step is required to produce high-purity 1,5-AG (patented) In the process, the recovery rate of 1,5-AG is lowered. In addition, a method (see Patent Document 3) that can separate and recover 1,5-AG with high purity and high recovery rate has been proposed. However, since the 1,5-AG liquid is diluted in the separation step, before and after the separation step. In order to produce at an industrial level, the 1,5-AG manufacturing process must be simplified.

JP 2005-168454 A JP 2008-54531 A JP 2012-7907 A JP 2009-215231 A

Biochemistry Vol. 69, No. 12, pp. 1361-1372, (1997) J. et al. Am. Chem. Soc. 72, 4547-4553 (1954) Phytochemistry Vol. 22, No. 9, 1959-1960 (1983) Carbohydrate Research 337 (2002) 873-890

  The present invention solves such conventional problems and provides a method for producing 1,5-AG using a microorganism having a high conversion ability to 1,5-AG using 1,5-AF as a starting material. It is to provide.

As a result of intensive studies, the present inventors have found a strain of Geotricum candy dam species having a high ability to convert 1,5-AF to 1,5-AG, and use carbohydrates other than glucose in this strain. Thus, depending on the strain, it was found that 1,5-AF can be converted to almost all 1,5-AG with high productivity, and the present invention has been achieved.
That is, in the present invention, a strain of Geotricum candy dam species having a high ability to convert 1,5-AF to 1,5-AG is contacted in a medium containing carbohydrates other than glucose, and 1,5-AF is 1 , 5-AG, and the resulting 1,5-AG is collected.

  According to the present invention, the productivity of 1,5-AG is improved by using a strain of Geotricum candidum, and further, by using a carbohydrate other than glucose, 1,5-AF is substantially converted to 1,5-AG. Since it can be converted, it is possible to make almost no substances other than 1,5-AG exist in the culture broth after completion of fermentation. This not only improves the productivity of 1,5-AG, but also eliminates the need for a separation step in the manufacturing process, which makes it possible to manufacture 1,5-AG by a simple method, leading to cost reduction. Therefore, 1,5-AG can be produced industrially advantageously. Furthermore, since the yield of 1,5-AG is high, the amount of drainage discharged per 1,5-AG production can be reduced, and an ecological effect can be expected.

  Specific examples of strains of Geotricum candy dam species having high ability to convert 1,5-AF to 1,5-AG include NBRC5767, NBRC4601, NBRC5959, and NBRC9542. These strains can be obtained from the Independent Administrative Agency of Biotechnology National Institute of Technology and Evaluation of the Biotechnology Headquarters of the National Institute of Technology and Evaluation. However, as for the strain of Geotricum candy dam species used in the present invention, all strains of Geotricum candy dam species having the ability to convert 1,5-AF to 1,5-AG can be used. It is not limited to the above strains.

The strain of Geotricum candy dam species of the present invention is a mutant strain obtained by ultraviolet irradiation, N-methyl-N-nitrosoguanidine (NTG) treatment, ethylmethanesulfonate (EMS) treatment, nitrite treatment, acridine treatment, etc. Alternatively, any strain such as a genetically modified strain induced by a genetic technique such as cell fusion or a genetic recombination method may be used.
As a culture solution containing 1,5-AF for culturing microorganisms, a medium containing a carbohydrate other than glucose, a nitrogen source, inorganic ions, and, if necessary, an organic nutrient source can be used. Examples of carbohydrates other than glucose include fructose, maltose, sucrose, glycerol, mannose and the like. Fructose is particularly preferred. As organic nutrient sources, for example, yeast extract, malt extract, peptone, meat extract, corn steep liquor, casein degradation product containing vitamins, amino acids and the like are appropriately used. As the inorganic ions, for example, magnesium ions, phosphate ions, calcium ions and the like are appropriately used. A 1,5-AF aqueous solution that was separately sterilized by filter was added to the medium to prepare a medium for collecting 1,5-AG.

The culture conditions are not particularly limited. For example, the culture can be performed at a pH of 3 to 7 and a temperature of 20 to 40 ° C. under aerobic conditions, and the culture can be performed for about 2 to 7 days while maintaining an appropriate pH and temperature.
The 1,5-AG thus produced in the culture broth is purified from the culture by a well-known means that is usually performed. Specifically, cells and solids were removed by centrifugation and diatomaceous earth filtration, followed by decolorization with activated carbon, desalting with an ion exchange resin, concentration, and crystallization. Examples of the crystallization method include a method of precipitating crystalline 1,5-AG from an aqueous solution of 1,5-AG (Patent Document 4 (Japanese Patent Laid-Open No. 2009-2152315)), but is particularly limited. It is not something.
The 1,5-AG thus produced in the culture solution is purified and collected by well-known means that are usually carried out without going through a separation step. Specifically, after removing cells and solids by centrifugation and diatomaceous earth filtration, decolorization with activated carbon, desalting with ion exchange resin, and concentration. After concentration, it was crystallized.

The amount of 1,5-AG produced in the culture medium can be measured quickly by HPLC, and the culture was terminated when the amount of 1,5-AG produced reached the maximum. Detailed conditions for the HPLC measurement are shown below.
Separation column: Shodex SP810-MCIGELCK08S connection (manufactured by Showa Denko KK, Mitsubishi Chemical), mobile phase: distilled water, flow rate: 1.0 mL / min, column temperature: 40 ° C., detection: differential refractive index detection Measurement was performed under the conditions of a vessel and a sample supply amount: 20 μL.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these Examples. Unless otherwise specified,% used in the following description is capacity (w / v)%. The production amount of 1,5-AG was determined from the calibration curve of a standard sample prepared in advance for the peak area of 1,5-AG detected by the above HPLC measurement. Moreover, the conversion rate to 1,5-AG shows the ratio by which 1,5-AF added in the culture medium was converted into 1,5-AG. Examples 1 to 3 are reference examples.

Example 1 (Comparison of 1,5-AG productivity of each yeast)
A medium (pH 6.0) consisting of 2.0% glucose, 2.0% peptone and 1.0% yeast extract, 50 ml was dispensed into a 300 ml shake flask, and a slant medium (glucose 2.0%, Peptone 2.0%, yeast extract 1.0%, agar powder 1.5%, pH 6.0), the most yeast among the yeasts described in Patent Document 1 (Japanese Patent Laid-Open No. 2005-168454) Schizo Saccharomyces pombe (NBRC 1608) and Geotricum candy dam (NBRC5767), which are highly productive of 1,5-AG, were brought into contact with each platinum ear and cultured with shaking at 30 ° C. and 130 rpm overnight. This was used as a seed culture solution. 50 ml of the same composition medium as described above was dispensed into a shake flask, and 1,5-AF solution sterilized with a 0.45 μm pore size filter was added to 1%. 1 ml each of the seed culture solution was added to this medium, followed by shaking culture at 30 ° C. for 3 days at 130 rpm. After removing the cells by centrifugation, 1,5-AG produced in the culture solution was quantified by HPLC. The results are shown in Table 1. The production of 1,5-AG was about 1.6 times higher for NBRC 5767 than for NBRC 1608.

Example 2 (Comparison of 1,5-AG productivity of each Geotrichum genus)
50 ml of a medium (pH 6.0) composed of 2.0% glucose, 2.0% peptone and 1.0% yeast extract was dispensed into a 300 ml shake flask, and slant medium (glucose 2.0%, peptone 2 0.02%, yeast extract 1.0%, agar powder 1.5%, pH 6.0), each strain shown in Table 2 was inoculated with 1 platinum ear, and cultured overnight at 30 ° C. and 130 rpm with shaking. This was used as a seed culture solution. 50 ml of the same composition medium as described above was dispensed into a shake flask, and 1,5-AF solution sterilized with a 0.45 μm pore size filter was added to 1%. 1 ml of the seed culture solution was added to this medium, and cultured with shaking at 130 rpm for 2 days at 30 ° C. After removing the cells by centrifugation, 1,5-AG produced in the culture solution was quantified by HPLC. The results are shown in Table 2. From this, among species of the genus Geotricum, a species having a high 1,5-AG productivity was a Candida species.

Example 3 (Comparison of 1,5-AG productivity of each Geotricum candy dam)
50 ml of a medium (pH 6.0) composed of 2.0% glucose, 2.0% peptone and 1.0% yeast extract was dispensed into a 300 ml shake flask, and slant medium (glucose 2.0%, peptone 2 0.03%, yeast extract 1.0%, agar powder 1.5%, pH 6.0), each strain shown in Table 3 was inoculated with 1 platinum ear, and cultured overnight at 30 ° C. and 130 rpm with shaking. This was used as a seed culture solution. 50 ml of the same composition medium as described above was dispensed into a shake flask, and 1,5-AF solution sterilized with a 0.45 μm pore size filter was added to a concentration of 6%. 1 ml of the seed culture solution was added to this medium, followed by shaking culture at 130 rpm for 3 days at 30 ° C. After removing the cells by centrifugation, 1,5-AG produced in the culture solution was quantified by HPLC. The results are shown in Table 3. Among the Geotricum candy dams used, the strain with the highest 1,5-AG productivity was NBRC 5767.

Example 4 (Comparison of 1,5-AG productivity by type of carbohydrate)
50 ml of a medium (pH 6.0) composed of 2.0% glucose, 2.0% peptone and 1.0% yeast extract was dispensed into a 300 ml shake flask, and slant medium (glucose 2.0%, peptone 2 1 platinum ear inoculated with Geotricum candy dam (NBRC 5767) cultured in 0.0%, yeast extract 1.0%, agar powder 1.5%, pH 6.0) and shaken overnight at 30 ° C. and 130 rpm. did. This was used as a seed culture solution. 5 ml of the same composition medium as described above was dispensed into a test tube, and the carbohydrates shown in Table 4 were similarly sterilized so that the 1,5-AF solution sterilized with a 0.45 μm filter was 4%. It added so that it might become 1%. 0.1 ml of the seed culture solution was added to this medium, and cultured with shaking at 130 rpm for 3 days at 30 ° C. After removing the cells by centrifugation, 1,5-AG produced in the culture solution was quantified by HPLC. The results are shown in Table 4. With the carbohydrates other than glucose used, the productivity of 1,5-AG was improved, and the most productive carbohydrate was fructose.

Example 5 (Relationship between carbohydrate concentration and 1,5-AG productivity)
50 ml of a medium (pH 6.0) composed of 2.0% glucose, 2.0% peptone and 1.0% yeast extract was dispensed into a 300 ml shake flask, and slant medium (glucose 2.0%, peptone 2 1 platinum ear inoculated with Geotricum candy dam (NBRC 5767) cultured in 0.0%, yeast extract 1.0%, agar powder 1.5%, pH 6.0) and shaken overnight at 30 ° C. and 130 rpm. did. This was used as a seed culture solution. 50 ml of the same composition medium as described above was dispensed into a shake flask, and the carbohydrates shown in Table 5 were similarly sterilized so that the 1,5-AF solution sterilized with a 0.45 μm pore size filter was 6%. It added so that it might become each concentration. 1 ml of the seed culture solution was added to this medium, followed by shaking culture at 130 rpm for 3 days at 30 ° C. After removing the cells by centrifugation, 1,5-AG produced in the culture solution was quantified by HPLC. The results are shown in Table 5. When 2% of fructose is added, the 1,5-AG productivity is the highest, the production amount of 1,5-AG is 49 g / L, and the conversion rate to 1,5-AG is 81.2%. At this time, the culture broth contained almost no substance other than 1,5-AG.

Claims (2)

A strain of Geotricum candidum sp. Is contacted in a medium containing 1,5-D-anhydrofructose and carbohydrates other than glucose to produce 1,5-D-anhydroglucitol and the produced 1,1 A method for producing 1,5-D-anhydroglucitol, which comprises collecting 5-D-anhydroglucitol. The strains of Geotricum candidum are NBRC 5767, NBRC 4601, NBRC 5959 or NBRC 9542.
The method for producing 1,5-D-anhydroglucitol according to claim 1.