JPS5937075B2 - Method for producing sorbitol using Hansenula bacteria - 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 Hansenula. 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°C) and high pressure (170 kV/c 11 yen), which consumes a lot of energy, requires a pressure-resistant container, and has the 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 is found in animal cell systems, such as in sheep spermatids (H, G, Hers Biochem).

Biophys, Acta 37 (1960) 1
27-138) or in the lens (M, Lou Bi
ochem.

Biophys, Acta 141 (1967)
547-559), but
Regarding microorganisms, there are only a few
No. 6-23038 merely discloses a method for 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 tests on many types of microorganisms, and found that the substrate glucose was used in the presence of Hansenula cells grown in pentose, cell extracts, and freeze-dried cells NADPH. The present invention was completed by discovering that sorbitol can be reduced to sorbitol in high yield.

The Hansenula genus used in the present invention is /'% Hansenula.
Hansenula anomala
I AM12210, Hansenula polymorpha I
Among these, Hansenula anomala IAM12210 is preferred.

In growing the bacteria according to the present invention, the carbon source is 2 to 15%, D-xylose, D-arabinose, D-
In a liquid medium containing pentose such as ribose and supplemented with yeast extract or cornstap liquor, etc.
Incubate for 2-10 days at 5°C.

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

The bacteria grown in this way are collected, washed, and the resulting live bacteria can be used as is to some extent, but it is still effective to some extent. It is much more effective to use treated microbial cells such as those that have been treated.

Furthermore, crushed bacterial cells and bacterial cell extracts prepared by ultrasonication or using a crusher such as Vibrogen Cell Mill can also 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. Preferably 25-35°C
React with.

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 mmIJ mol to 20 mol, preferably 0.1 mol to 10
It is a mole.

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 complete, sorbitol is separated from the mother liquor.

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

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

Example 1 A 500 ml digestion flask was filled with 50 r/ll of a sterilized medium having the composition shown in Table 1 and was incubated with Hansenula anomala IAM1.
2210 and Hansenula polymorpha are IF010
One platinum loop of each of the 71 strains was inoculated and cultured at 30°C for 6 days.

The cultured cells were washed twice with phosphate buffer and suspended in phosphate buffer so that the turbidity at 610 nm was 25.

This bacterial cell suspension was subjected to ultrasonic treatment for 20 minutes, and 14,000
g.

The mixture was centrifuged for 20 minutes, and the supernatant fraction was used for the production of vine beets.

A reaction solution having the composition shown in Table 2 was prepared using both supernatants obtained in the above operation, the pH was adjusted to 7.5, and the reaction solution was reacted at 30° C. for 6 hours.

35 μm for Hansenula anomala IAM12210 strain
oles1 Hansenula polyhansenula IF01071
Each strain yielded 21 μmoles of sorbitol.

Product confirmation was performed by thin layer chromatography, high performance liquid chromatography, and gas chromatography.

In addition, quantitative determination was performed using high performance liquid chromatography.

Example 2 Cells of Hansenula anomala IAM12210 obtained by culturing in the same manner as in Example 1 were washed twice with a phosphate buffer solution 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 the reaction solution, 151 μmoles of vine beets were obtained.

Example 3 Reaction solution 2077!71! obtained by the method of Example 1 using Hansenula anomala IAM12210 strain. was passed through a column of ion exchange resin (Amifox A4: trade name) to purify sorbitol.

The amount of sorbitol obtained was 65 μmoles.

Claims (1)

[Scope of Claims] 1. Live bacterial cells, treated bacterial cells obtained by aerobically cultivating Hansenula microorganisms using a medium containing pentose as the main nutrient source,
1. A method for producing sorbitol using a microorganism, which comprises producing sorbitol by allowing at least one of a crushed bacterial cell material and a bacterial cell extract 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 Hansenula microorganism is Hansenula anomala or Hansenula polymorpha. 3. The method for producing sorbitol using a microorganism according to claim 2, wherein Hansenula anomala is IAM12210 and Hansenula polymorpha is IFO1071.