JPS639829B2 - - 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 belonging to the genus Aspergillus and capable of producing 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

Sorbitol is currently produced industrially 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 a screening for the purpose of obtaining microorganisms capable of producing sorbitol from soil etc. As a result, the culture, bacterial cells, or bacterial cells of the genus Aspergillus grown in pentose were obtained. The present invention was completed by discovering that freeze-dried bacterial cells reduce the substrate glucose to sorbitol in high yield.

As the preferable Aspergillus bacteria used in the present invention, G.
-18 strain is exemplified as a representative strain, and the mycological properties of this strain are shown in Table 1.

Table 1 Mycological properties of Aspergillus terreus G-18 Culture conditions: Cultured on Tuapetsk agar at 25℃ for 14 days Colony properties: 35 mm Cream to golden yellow Slightly fluffy and thick Underside colorless Microscopic morphology: Conidial head, columnar, apical capsule, hemisphere Shape: Phialide 2-tiered 12-15μm Conidiophore Long 500μm or more Conidia Spherical 2.5μm smooth surface Based on the above mycological properties, the ``Manual of
Manual of the Aspergilli, Thom & Raper [1945]
As a result of searching according to ``Raper [1945]'', this strain was found to belong to Aspergillus terreus, and was named Aspergillus terreus G-18.
It has been deposited with the Microbial Technology Research Institute as Microbiological Research Institute No. 7264.

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.
The growth rate can also be increased by adding various vitamins, minerals, and appropriate organic substances to the medium in addition to the above-mentioned components.

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, reduced nicotinamide and adenine dinucleotide phosphate (hereinafter referred to as "NADPH") are used as coenzymes.
By allowing sorbitol to coexist, the productivity of sorbitol can be dramatically improved.
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 by giving 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 mycelium of Aspergillus terreus G-18 (Keiken Bokuyori No. 7264) was inoculated.
The cells were cultured at 30°C for 3 days. The cultured bacterial cells were washed twice with phosphate buffer and suspended again 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 obtained in the above operation, the pH was adjusted to 7.5, and the reaction solution was reacted at 30° C. for 6 hours. 30 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 Cells of Aspergillus terreus G-18 (Feikoken Bibori No. 7264) obtained by culturing in the same manner as in Example 1 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. 122 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 Reaction solution obtained by the method of Example 1 using Aspergillus terreus G-18 (Feikoken Bibori No. 7264)
Sorbitol was purified by passing 20 ml through a column of ion exchange resin (Aminex A4: trade name).
The sorbitol obtained was 54 μmoles.

Claims (1)

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