JPS639830B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing sorbitol from glucose by a microbial method, and more specifically to a method for efficiently producing sorbitol using a microorganism that belongs to the genus Fusarium and has the ability to produce sorbitol. be.

Sorbitol has a unique sweet taste, has low reactivity, is harmless, and has a moisturizing effect, so it is used as an additive in foods, toothpaste, cosmetics, etc., and is also used as an intermediate raw material in the production of vitamin C and surfactants. It is widely used as

Currently, sorbitol is industrially produced by catalytic reduction of glucose using a Ni catalyst such as Raney nickel, but this method requires high temperature (160°C) and high pressure (170 kg). / ^cm2 )
In addition to consuming a lot of energy, it also requires a pressure-resistant container, and there is an inherent danger of explosion due to the handling of hydrogen.

The present inventors differed in concept from the above method and attempted a method for producing sorbitol under mild conditions by microbiologically reducing glucose. The mechanism for reducing glucose to sorbitol has been demonstrated in animal cell systems in the spermatozoa of sheep (HGHers Biochem.
Biophys.Acta 37 (1960) 127-138) or in the lens (M.Lou Biochem.Biophys.Acta 141
(1967) 547-559).
There are no examples of microorganisms producing sorbitol, and only Japanese Patent Publications No. 45-24834 and No. 46-23038 disclose a method of aerobically fermenting and reducing xylose to xylitol. Further, even if the strain disclosed in the above invention was cultured in a glucose medium, sorbitol could not be obtained.

Therefore, the present inventors conducted screening for the purpose of obtaining microorganisms capable of producing sorbitol from soil and the like.

As a result, it was discovered that Fusarium cultures, cells, cell extracts, and freeze-dried cells grown in pentose can reduce the substrate glucose to sorbitol in a high yield, and the present invention was developed based on this finding. It was completed.

The preferable Fusarium genus bacteria used in the present invention is G-13, which was newly isolated from soil.
This strain is exemplified as a representative strain, and the mycological properties of this strain are as shown in Table 1.

Table 1 Mycological properties of Fusarium oxysporum G-13 Culture conditions: Potato dextrose agar 25℃, 10
Condition of daily culture colony: 85 mm or more, white fluff, some sectors are blue, colorless on the back Microscopic form: Large conidia, 3-5 cells, 22-28 x 4-5 μm, small conidia (+), oval, 7.5-8.5 x 2.5-3.0 μm, thickness Hymenospore (+) Phialide short Based on the above mycological properties, “Dematiaceous hyphomycetes [1971]
hyphomycetes [1971])”
This strain is recognized to belong to the Fusarium genus Oxysporum, and Fusarium Oxysporum G-
13 and has been deposited with the Microbial Technology Research Institute as Microbiological Research Institute No. 7262.

When growing the bacteria according to the present invention, 2 to 15% of pentose sugars such as D-xylose, D-arabinose, and D-ribose are used as a carbon source, and yeast extract or cornstarch liquor is used. Use the added liquid medium. Culture at 25-35℃ for 2-10 days
Do it for days. Any culture method can be adopted as long as it is under aerobic conditions, such as aeration culture, shaking culture, and rotating drum method. In addition to the above ingredients, the culture medium also contains various vitamins,
The growth rate can also be increased by adding inorganic substances or suitable organic substances.

Next, sorbitol can be obtained by contacting the culture thus obtained with glucose, but some effect can be observed even if the live bacteria obtained by washing the cells is used as is.
However, it is much more effective to use treated bacterial cells such as freeze-dried bacterial cells, freeze-thawed bacterial cells, and treated bacterial cells with organic solvents such as acetone and ether. Also,
It is also possible to use crushed bacterial cells and bacterial cell extracts prepared by ultrasonication or by using a crusher such as Vibrogen Cell Mill.

For glucose reduction, 1 to 60% of the treated bacterial cells, crushed bacterial cells, bacterial cell extracts isolated therefrom, or mixtures thereof obtained by the above method are used.
Add to the glucose solution at a concentration of 10-60℃, preferably
React at 25-35°C. At this time, the productivity of sorbitol can be dramatically improved by coexisting reduced nicotinamide and adenine dinucleotide phosphate (hereinafter referred to as "NADPH") as coenzymes.
The amount of NADPH added varies greatly depending on the method of preparing the bacterial cells used or the extraction procedure for the bacterial cells, and is 0.1 mmol to 20 mol, preferably 0.1 mol to 10 mol, per mol of glucose. The time required for the reaction also varies greatly depending on the conditions; for example, when a bacterial cell extract is used, the reaction is completed in 30 minutes to 14 days.

After the reaction is completed, sorbitol is separated and purified from the mother liquor by using a combination of known methods such as centrifugation, ultrafiltration, and ion exchange.

Hereinafter, the present invention will be explained in detail with reference to Examples. The product was confirmed by thin layer chromatography, high performance liquid chromatography, and gas chromatography. Quantification was performed using high performance liquid chromatography.

Example 1 50 ml of a sterilized medium having the composition shown in Table 2 was placed in a 500 ml digestion flask, and the mycelia of Fusarium oxysporum G-13 (Feikoken Bacteria No. 7262) were inoculated.
The cells were cultured at 30°C for 3 days. The cultured bacterial cells were washed twice with phosphate buffer and resuspended in the same buffer. This bacterial cell suspension was mixed with vibrogen cernil (Edmund
(manufactured by Biiler) for 20 minutes, centrifuged at 14,000 g for 20 minutes, and the supernatant fraction was used for sorbitol production.

A reaction solution having the composition shown in Table 3 was prepared using the supernatant fraction in the above operation, the pH was adjusted to 7.5, and the reaction solution was reacted at 30° C. for 6 hours. 36 μmoles of sorbitol was obtained in the reaction solution.

Table 2 D-xylose 8g K ₂ HPO ₄ 0.1g MgSO ₄・7H ₂ O 0.05g CaCl ₂・2H ₂ O 0.01g NaCl 0.01g Casamino acids 0.4g Yeast extract 0.1g PH5.0 100ml Table 3 Centrifugal supernatant 5ml Glucose 3.6m moles NADPH 0.2m moles Phosphate buffer 0.7m moles Distilled water 5ml Total volume 10ml Example 2 Fusarium oxysporum G-13 obtained by culturing in the same manner as in Example 1 (Feikoken Bacteria No. 7262)
The cells were washed twice with phosphate buffer and then freeze-dried.

A reaction composition solution shown in Table 4 was prepared using the obtained freeze-dried bacterial cells, and reacted at 30°C for 16 hours. 168 μmoles of sorbitol was obtained in the reaction solution.

Table 4 Freeze-dried bacterial cells 0.5g Glucose 10m moles NADPH 2m moles Phosphate buffer 3.5m moles Distilled water 40ml Example 3 20 ml of the reaction solution obtained by the method of Example 1 using Fusarium oxysporum G-13 (Feikokuken Bibori No. 7262) was passed through a column of ion exchange resin (Aminex 4: trade name) to remove sorbitol. was purified.
The sorbitol obtained was 66 μmoles.

Claims (1)

[Claims] 1. A Fusarium bacterium that belongs to the Fusarium genus and is characterized in that it produces sorbitol by culturing a strain having the ability to produce sorbitol in a medium containing pentose as the main carbon source, and then contacting the resulting culture with glucose. Method for producing sorbitol by.