JPS5930400B2 - Method for producing sorbitol using microorganisms - 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, using live cells, treated cells, crushed cells, cell extracts, etc. of microorganisms of the genus Penicillium. The present invention relates to a method for efficiently producing sorbitol from glucose in the presence of reduced nicotinamide adenine dinucleotide phosphate (hereinafter referred to as NADPH).

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 C1 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 such a method inevitably requires high temperature (
160℃), Takako (170fu/crA) and Stingray.
In addition to consuming a large amount of hydrogen, 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.

In animal cell systems, the mechanism for reducing glucose to sorbitol is found in the spermatozoa of sheep (H, G, Hers Bioche).
m.

Biophys, Acta 37 (1960)
127-138) or in the lens (M, Lou Bi
ochem, Biophys.

Acta 141 (1967) 547-559), but microorganisms are known to aerobically ferment xylitol and reduce it to It merely discloses the method.

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 tests on many types of microorganisms and found that Penicillium cells, cell extracts, and freeze-dried cells grown in pentose were found to be MADPH.
The present inventors have completed the present invention by discovering that the substrate glucose can be reduced to sorbitol in high yield in the presence of sorbitol.

The Penicillium genus microorganism used in the present invention is Penicillium chrysogenum (Penicillium chrysogenum).
sogenum), Penicillium citrinum (Pe
nicillium citrinum), among others, Penicillium chrysogenum MR-
FEB-25 strain is preferred.

In growing the bacteria according to the present invention, D-xylose, D-arabinose, D-
In a liquid medium containing pentose such as ribose and supplemented with yeast extract or cornstap liquor, etc.
Culture at 5°C for 2-10 days.

Any culture method can be adopted as long as it is under aerobic conditions, such as aerated culture, shaking culture, rotary drum method, and ram method.

Furthermore, the growth rate can be further increased by adding various vitamins, minerals, peptone, and organic substances such as yeast extract to the medium in addition to the above-mentioned components.

Even if the live bacteria obtained by collecting and washing the bacterial cells grown in this way are used as they are, some effects can be observed.
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 an organic solvent such as acetone or ether.

Furthermore, crushed bacterial cells and bacterial cell extracts prepared by ultrasonication or using a crusher such as Vibrogen Cell Mill can be used.

To produce sorbitol, the treated bacterial cells, crushed bacterial cells, bacterial cell extracts isolated therefrom, or mixtures thereof obtained by the above method are added to a glucose solution with a concentration of 1 to 60%, and the mixture is heated at 10 to 60°C. The reaction is preferably carried out at 25 to 35°C.

At this time, the productivity of sorbitol is dramatically improved by coexisting NADPH as a coenzyme.

The amount of NADPH added varies greatly depending on the preparation method of the bacterial cells used or the extraction procedure of the bacterial cells, and is 0 per mole of glucose.
．． 1 mmol to 20 mol, preferably 0.1 mol to 10
It is a mole.

The time required for the reaction also varies 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 complete, sorbitol is separated from the mother liquor.

For separation and purification of sorbitol, a combination of known methods such as centrifugation, ultrafiltration, and ion exchange methods is utilized.

Hereinafter, the present invention will be explained in detail by giving Examples.

'Example 1 50ml of a sterilized medium with the composition shown in Table 1 was placed in a 5007721 volume digestion flask, and P en, chrysog
enumMR-PE B-25 strain and Pen, ci
One platinum loop of trinumIF06026 was inoculated at 30°C.
, and cultured for 6 days.

The cultured bacterial cells were washed twice with phosphate buffer and suspended in 50 ml of phosphate buffer.

This bacterial cell suspension was sonicated for 20 minutes at 14,000 f.
.

After centrifugation for 20 minutes, both supernatants were used for sorbitol production.

A reaction solution having the composition shown in Table 2 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.

Pen, chrysogenum has 58 μmole
45 μmol for S, Pen, and citrinum
es sorbitol was obtained, respectively.

Table 1 D-Xylose 8? KH2P
O40,1f MgSO4・7H200,05g CaCA2・2H200,01f NaC70,01iii' Casamino acids 0.4 Feast extract 0.1 2 pH5,0100ml Table 2 Centrifugal supernatant 5ml Glucose 3.6m molesNAD
PH0.2m moles phosphoric acid buffer solution 0.7m moles distilled water
Confirmation of the 5mβ Total vol 10m12 product was performed by thin layer chromatography, high performance liquid chromatography and gas chromatography.

Quantification was performed using high performance liquid chromatography.

Example 2 Pen, citrinum I AM 7316 strain was cultured in the same manner as in Example 1, and the resulting bacterial cells were washed twice with phosphate buffer and then freeze-dried.

A reaction composition solution shown in Table 3 was prepared using the obtained freeze-dried bacterial cells, and reacted at 30°C for 16 hours.

From this reaction solution, 630 μm oles of sorbitol was obtained.

Table 3 Freeze-dried bacterial cells 0.51 Glucose 10 m molesNADP
H2m moles phosphoric acid buffer solution 3.5m moles distilled water
Example 3 20 ml of the reaction solution obtained by the method of Example 1 using Pen and chrysogenum was passed through a column of ion exchange resin (Aminex A5: trade name) to purify sorbitol.

The sorbitol obtained was 108 μm01eS.

Claims (1)

[Scope of Claims] 1. Live bacterial cells, treated bacterial cells, crushed bacterial cells, and bacterial cell extracts obtained by aerobically cultivating Penicillium microorganisms using a medium containing pentose as the main nutrient source. A method for producing sorbitol using a microorganism, which comprises producing sorbitol by allowing at least one of the substances to act on glucose in the coexistence of reduced nicotinamide adenine dinucleotide phosphate. 2. The method for producing sorbitol using a microorganism according to claim 1, wherein the Penicillium genus microorganism is Penicillium chrysogenum. 3 Penicillium chrysogenum is MR-FEB-25
A method for producing sorbitol using a microorganism according to claim 2, which is a strain.