JPH0795959B2 - Method for producing fructose by glucose-isomerase - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing fructose from glucose.

[Conventional technology]

Glucose isomerase is an enzyme that reversibly converts glucose into fructose, and it has been known that this enzyme is produced by some actinomycetes, lactic acid bacteria, and Bacillus subtilis. It is industrially used as an enzyme.

[Problems to be solved by the invention]

However, the glucose isomerases discovered so far require expensive xylose or xylan when culturing the bacteria that produce them, and require metal ions such as magnesium, manganese, and cobalt when expressing the enzyme activity. There was a drawback to It is also known that the Michaelis constant (Km), which shows the dependency of the enzyme on the substrate concentration, is as high as several hundred millimoles, and in the case of a low-concentration glucose solution,
It had the drawback that it was not converted to fructose.

Further, the conventional glucose isomerase has a higher affinity for xylose than glucose, and when xylose is mixed, the ability to convert glucose into fructose is also reduced.

Therefore, as a result of intensive studies, the present inventors have found that the anaerobic bacterium Bifidobacterium has glucose isomerase activity, and as a result of further studies, it is obtained from Bifidobacterium. Glucose isomerase activity does not require metal ions such as magnesium, manganese, and cobalt when the activity is expressed, and the Km value is about
It was found that glucose at a concentration as low as 3 mM showed a converting effect to fructose. Furthermore, it was also found that xylose and xylan are not required at all when the cells are cultured to obtain this activity. Thus, the glucose isomerase activity obtained from Bifidobacterium is
The inventors have found that the activity is completely different from that of the conventional one, and have completed the present invention.

[Means for solving problems]

TECHNICAL FIELD The present invention relates to a bacterium or enzyme obtained by culturing a Bifidobacterium genus in the production of fructose, and a method for converting glucose into fructose using a treated product thereof. Hereinafter, the present invention will be described in detail.

The conversion to fructose according to the present invention refers to a reaction in which glucose is converted to fructose as a raw material, and therefore, cells of Bifidobacterium are crushed and used, or only the enzyme is incomplete or , Completely purified and isolated, and then used. Furthermore, crushed bacterial cells, incomplete and completely purified products of the enzyme are treated with alginic acid, an ion exchange resin,
The method of immobilizing with an immobilizing agent such as ion exchange cellulose, polyacrylamide and a photo-curing resin is also included.

In order to specifically explain the present invention, as an example of the genus Bifidobacterium used in the present invention, Bifidobacterium longum, Bifidobacterium address sense, Bifidobacterium infantes, Bifidobacterium bifidum , Bifidobacterium breve, Bifidobacterium liberrum, etc., but the present invention is not limited to these strains, and includes all strains classified into the genus Bifidobacterium.

The culture of these strains is usually performed at 37 ° C. under static or anaerobic conditions.

As a medium for culturing these strains, glucose, peptone, a medium for culturing ordinary Bifidobacterium containing yeast extract is sufficient, and when magnesium ion, marble or the like is added to the medium, the production amount of glucose isomerase Is significantly increased.

The glucose isomerase activity was measured as follows. An appropriate amount of enzyme solution was added to 0.4 ml of 20 mM Tris buffer (pH 7.5) and 0.1 ml of 0.1 M glucose solution, and the total volume of the reaction solution was adjusted to 1 ml with 20 mM Tris buffer (pH 7.5) and reacted at 37 ° C for 10 minutes. Let After completion of the reaction, the enzyme is deactivated and the fructose produced by the cystinecarbazole method is quantified. Under this condition, the amount of the enzyme solution required to produce 1 μmol of fructose is 1 unit per 1 ml of the reaction solution.

〔Example〕

 Examples will be described below.

Example 1 Polypeptone 30 g, glucose 30 g, yeast extract 12 g, salt 1
0 g, the MgSO ₄ · 7H ₂ O1g, after dissolved by adding water 2, the pH value
Adjust to 7.2, add 30 g of marble, and sterilize by autoclaving at 121 ° C for 20 minutes. After cooling, Bifidobacterium addresscentes is inoculated, and static culture is performed at 37 ° C for 20 hours. After the culture, 20g of glucose was added to the enzyme solution (200 units of glucose isomerase) obtained by crushing the collected bacterial cells by centrifugation, and the total amount was 100ml with water.
And reacted at pH 7 for 16 hours at 37 ° C. As a result, 6 g of fructose was produced.

Example 2 A medium having the same composition as in Example 1 was inoculated with Bifidobacterium liberolum, and static culture was carried out at 37 ° C. for 20 hours.
The obtained bacterial cells are crushed to obtain an enzyme solution. Glucose (40 g) was added to the enzyme solution (glucose isomerase activity: 180 units), the total amount was adjusted to 100 ml with water, and the reaction was carried out at pH 7.0 at 37 ° C for 14 hours. As a result, 10 g of fructose was obtained.

Claims (1)

[Claims] 1. A method for producing fructose from glucose using glucose isomerase obtained from cultured cells of the genus Bifidobacterium.