JPS5853916B2 - Method for producing immobilized glucose isomerase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing immobilized glucose isomerase useful for isomerizing glucose to fructose.

Fructose is an isomer of glucose, and is about twice as sweet as glucose, making it the sweetest substance among sugars.

Therefore, since it can provide sweetness while minimizing calorie intake, its use in low-calorie foods to prevent obesity or beauty foods has recently attracted attention.

In addition, sugar solution produced by isomerizing glucose has a sweetness that is about 1.2 to 1.4 times sweeter than sucrose, so it has been rapidly used in fields such as soft drinks, canned goods, and frozen desserts due to its economic efficiency. There are two S.

Traditionally, D-fructose has been produced by hydrolyzing sucrose to separate D-fructose, but in recent years, a method in which glucose isomerase acts on glucose to isomerize it has been developed on an industrial scale. It's expanding.

By the way, in order to produce isomerized liquid sugar from glucose using glucose isomerase, it generally takes about 40 to
Glucose isomerase-producing bacteria were added to a glucose solution weighing 50% by weight, and the isomerization reaction was allowed to occur for a long time at a temperature of about 70°C, followed by isomerization through various steps such as filtration, decolorization, and deionization. I am getting liquid sugar.

However, the above method has a major drawback in that after the isomerization reaction is completed, the enzyme activity decreases to about half of that before the reaction, and when a new isomerization reaction is performed, the lost amount of enzyme must be compensated for.

Therefore, it is strongly desired to immobilize enzymes to enable long-term use of enzymes and continuous enzymatic reactions.

Various methods have been known for immobilizing glucose isomerase, regardless of whether it is a free enzyme or an intracellular enzyme.

For free enzymes, for example, a covalent carrier bonding method using a porous glass aminosilane derivative as a carrier, an ionic carrier bonding method using an ion exchanger such as DEAE-cellulose or Duolite as a carrier, polyacrylamide gel, collagen, etc. Alternatively, there is a comprehensive method using a composite material such as cellulose triacetate.

These methods do not provide an industrially used immobilized enzyme that is satisfactory in all respects such as production cost, stability, safety, and durability.

Also known is a method in which glucose isomerase is immobilized within the bacterial cells by heating the bacterial cells to a temperature of 55° C. or higher and 90° C. or lower.

Although this method significantly improved the decrease in enzyme activity by one point compared to untreated bacterial cells, the residual activity value was still lower than the initial level of 40 to 5.
0%, which was not industrially sufficient.

It is particularly strongly desired that an immobilized enzyme agent for glucose isomerase that satisfies industrial applications has the following properties.

m Enzyme activity must be sufficiently maintained for long-term use.

(11) Do not elute impurities.

(111) No clogging when filling the column.

+1V) Physically robust in glucose solutions.

M. Easy to handle.

(VD Enzymes are cheap.

For these purposes, the present inventors have conducted extensive studies on the immobilization of glucose isomerase as an enzyme within bacteria, and have discovered that the desired purpose can be achieved by treating bacterial cells with a specific acid. invention has been achieved.

That is, the present invention is a method for producing immobilized glucose isomerase, which comprises treating glucose isomerase-producing bacteria with succinic acid, malic acid, or fumaric acid.

Examples of glucose isomerase-producing bacterial cells include Streptomyces pheochromocennes (Strept.
omyces phaeochromgenes),
Streptomyces fradiae (S, fradiae)
), Streptomyces roseochromogenus (S.

roseochromogenes), Streptomyces.

olivaceus (S, olivaceus), Streptomyces californicus (S, cal 1forn
icas), Streptomyces venuseus (S, v
enuceus), Streptomyces baaginia (
actinomycetes such as S. virginiac, Pseudomonas hyd.
Pseudomonas bacteria such as Bacillus megaterium (rophi la),
A wide range of microbial cells can be mentioned, including bacteria of the genus Bacillus such as B.

These bacteria were cultured in a medium containing corn staple liquor alone or defatted soybeans as a nitrogen source, carbohydrates such as starch and pheasant rose as a carbon source, and potassium phosphate, cobalt chloride, and magnesium hydrochloride as inorganic salts. It is obtained by

In particular, the bacterial cells used in the present invention are obtained by culturing the above-mentioned bacterial cells,
It is preferable to use the bacterial cells isolated from the culture solution as they are or the bacterial cells that have been heat-treated.

Further, the bacterial cells may be dispersed or suspended in a buffer solution having a pH of 5 to 9, particularly 5 to 7.0.

The concentration of bacterial cells is usually 1 to 50% by weight, preferably O11 to
The weight is φ40.

In the method of the present invention, by acid-treating bacterial cells containing glucose isomerase, the stability of intracellular enzymes can be significantly improved.

Furthermore, when a mixture of bacterial cells and chitosan or a mixture of bacterial cells and partially deacetylated chitin is treated with acid, the enzyme is stabilized by the acid and the chitosan membrane or partially deacetylated chitin membrane on the bacterial surface is strengthened by the acid. A double-sided effect is achieved.

The mixing amount of chitosan or partially deacetylated chitin is
It is preferable that the amount of chitosan or partially deacetylated chitin is 8 weight φ or more based on the dried bacterial cells.

The reason why the enzyme is significantly stabilized by acid treatment is not clear, but considering that the bacterial cell soaking solution is significantly decolored, it is possible that substances that promote the oxidative inactivation of glucose isomerase are removed from the bacterial cells. It is speculated that this is because

Regarding the conditions for acid treatment, the effect is seen at a concentration of 0.5 weight φ or more for treating bacterial cells, but a concentration of 2 to 8 weight φ is generally preferred.

The effect can be seen by controlling the pH of the acid aqueous solution within a range that does not deactivate glucose isomerase.

An immobilized enzyme product can be obtained by drying the acid-treated bacterial cells, if necessary, or by molding them into a bottom mold and drying them.

The drying conditions are preferably within a temperature range that does not deactivate glucose isomerase, particularly at 50° C. or lower.

As the drying method, various methods such as vacuum drying, solar drying, and freeze drying are employed.

The immobilized glucose isomerase obtained by the method of the present invention has significantly less deactivation and durability than conventional bacterial cells that have been simply heat-treated, and can be used for a long period of time, and the enzyme reaction can be carried out continuously. can.

The present invention will be explained below using Examples.

Note that glucose isomerase activity is determined by glucose solution (
Glucose concentration 0.1M, magnesium sulfate 0. OIM
, phosphate buffer o, 05 M, pH 7,:2)
Using a reaction temperature of 70 DEG C., 1.5 g of glucose is isomerized in 1 minute, and the enzyme activity to produce fructose is defined as 1 unit.

Example 1 Streptomyces pheochromogenes (Strep
tomyces phaechromogenes)
(Feikoken Bibori No. 221) was aerated and cultured in a medium containing corn stapler xylose, potassium phosphate, magnesium sulfate, and cobalt chloride, and then centrifuged to obtain bacterial cells.

50 g of the bacterial cells (solid content 30%) (activity value 752 units/g) thus obtained were suspended in a small amount of water to obtain 80.52 g of suspension.

Add various acid solutions (concentration 5, pH 5) to 7.32 g of this suspension.
, 75) LooTLl was added and left at room temperature for 2 to 8 hours.

The cells were then centrifuged at 10.00 Orpm for 10 minutes to obtain bacterial cells.

The acid-treated cells were washed twice with water, vacuum dried at 30° C. for 14 hours, and ground to obtain an immobilized enzyme product.

This immobilized enzyme product (glucose isomerase activity 24
0 units) to a glucose solution (glucose 50W/V column,
Magnesium sulfate 0.005M, cobal chloride) O, O
OIM, IJ phosphate buffer solution 0.05M) 20rrLl
In addition, the mixture was reacted by shaking at 60° C. for 1 day while maintaining the pH at 6.8 to 7.2.

A monomer shaker J type (manufactured by Taiyo Kagaku Kogyo KK) was used as an isomerism reaction apparatus, and the L-shaped test tube containing the glucose solution was shaken at 38 times/min.

After the reaction was completed, the bacterial cells were collected by centrifugation, and a glucose solution with the same composition was added again to perform the isomerization reaction.

This isomerization reaction was repeated three times.

Table 1 shows the isomerization rate and the isomerization rate maintenance rate.

Note that the isomerization rate and the isomerization rate maintenance rate were calculated according to the following formula.

Example 2 50 g of bacterial cells (solid content 30φ) (activity value 752 units/g) having the ability to produce glucose isomerase, which were cultured in the same manner as in Example 1, were suspended in a small amount of water and heated at 80°C for 2 minutes. provided.

Collect the bacterial cells by centrifugation, and add 50 g of the bacterial cells to 3.5 g of water. :l
suspended in.

This suspension is 0.34! ' was used as a control cell. 0.2 pieces of Gitosan was dissolved in the remaining bacterial suspension 31.
, 1M acetate buffer (pH 5) IA was added and stirred in a mixer.

This suspension was centrifuged at 400 rpm for 10 minutes to obtain 100.7 g of bacterial cells.

Divide the bacteria into 9g portions and use various acid solutions (concentration 5φ).
, pH 5,75) and stirred well.

This solution was allowed to stand at room temperature for 2-4 hours, and was heated to 10.00 O
The cells were separated by centrifugation at rpm for 10 minutes, and washed with water twice.

The cells were vacuum-dried at 30° C. for 16 hours and then ground to obtain an immobilized enzyme product.

The obtained immobilized enzyme product (glucose isomerase activity 240 units) was added to 20T of the same glucose solution as in Example 1.
60°C while maintaining pH 6.8-7.2.
The reaction was carried out for 20 hours.

After the reaction was completed, the bacterial cells were collected by centrifugation, and a glucose solution with the same composition was added again to repeat the isomerization reaction three times.

Table 2 shows the isomerization rate and the isomerization rate maintenance rate.

Example 3 Bacteria capable of producing glucose isomerase, which were cultured in the same manner as in Example 1, were suspended in a small amount of water and heated at 80° C. for 2 minutes.

Collect the bacterial cells by centrifugation, and convert the bacterial cells 50.9 to 3.31
suspended in water.

Of this suspension, 0.31 cells were used as control cells. On the other hand, 5 g of chitin powder was deacetylated in 200 ml of 40 weight φ caustic soda at 60° C. in the presence of nitrogen gas for 12 hours, and after cooling, the mixture was centrifuged and the precipitate was washed until it became neutral.

After dissolving this in a lo% acetic acid solution, the pH was adjusted to 7.0 with 40φ caustic soda, and the solution was left in a cold room overnight.

Then, it was centrifuged, washed with water, twice with ethanol, twice with ether, and dried.

After dissolving 2 g of the partially deacetylated chitin thus obtained in 1 liter of acetic acid solution, the pH was adjusted to 5.0 with caustic soda.

The above prepared bacterial cell suspension 31 was added to this 0.2% partially deacetylated chitin solution, and the mixture was stirred with a mixer.

This suspension was centrifuged at 400 rpm for 10 minutes to obtain 100 g of bacterial cells.

Divide this bacterial body into 9 g each and use various acid solutions (concentration 5% p).
H5,75) and stirred well.

This solution was left at room temperature for 2 hours, and 1 O,000 rpm
The bacterial cells were separated by centrifugation at m for io minutes, and washing with water was repeated twice.

After vacuum-drying the bacterial cells at 30°C for 16 hours, they were crushed,
It was used as an immobilized enzyme preparation.

The obtained immobilized enzyme product (glucose isomerase activity 240 units) was subjected to an isomerization reaction in the same manner as in Example 1.

The isomerization rate and the isomerization rate maintenance rate are shown in Table 3.

Claims (1)

[Claims] 1. A method for producing immobilized glucose isomerase, which comprises treating glucose isomerase-producing bacteria with succinic acid, malic acid, or fumaric acid.