JPS5853915B2 - 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 suppressing calorie intake as much as possible, its use in low-calorie IJ-foods to prevent obesity or beauty foods has recently attracted attention.

In addition, a sugar solution obtained by isomerizing glucose has a sweetness about 12 to 14 times that of sucrose, and has recently been rapidly used in fields such as soft drinks, canned goods, and frozen desserts due to its economic efficiency.

Traditionally, D-fructose has been produced by hydrolyzing sucrose to separate D-fructose, but in recent years the method of isomerizing glucose by allowing glucose isomerase to act on glucose has been expanded on an industrial scale. waiting.

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 are added to a 50-kg glucose solution, and the isomerization reaction is 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. You are 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 cellulose triacetate or the like as a building material.

These methods do not provide an industrially used immobilized enzyme that is satisfactory in all respects such as production cost, safety, stability, 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 compared to untreated bacterial cells, the residual activity value remained at an initial level of 40-50%.
%, which was not sufficient for industrial purposes.

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

(1) Enzyme activity is sufficiently maintained for long-term use.

(il) Do not elute impurities.

(iii) The column should not be clogged when packed.

(IV) Physically robust in glucose solution.

(V) It should be easy to handle.

(VD Enzymes are inexpensive.

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 is characterized by treating glucose isomerase-producing bacteria with tartaric acid, lactic acid, or maleic acid.

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

roseochromogenes), Streptomyces 0 olivaceus (Jot 1vace11s)
, Streptomyces californicus (S, cal
Streptomyces venuceus (S, venuceus) stress Streptomyces such as butomyces virginiae (S, virginiae), Pseudomonas hydrohira (PseBdomonas)
Bacillus megaterium (Bacillus hydrophila) and other Pseudomonas bacteria,
A wide range of microbial cells can be mentioned, including bacteria of the genus Bacillus such as Bacillus (a terium ), bacteria of the genus Brevibacterium, bacteria of the genus Lactobacillus, and bacteria of the genus Aerobacterium.

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 0.1 to 50% by weight, preferably 0.1 to 50% by weight.
The weight is 5 to 40 φ.

The acid used in the present invention is tartaric acid, lactic acid, or maleic acid, and these acids are food additives, safe, and used in the processing of various foods.

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

In addition, 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 film or partially deacetylated chitin film on the bacterial surface is A double-sided effect of reinforcement 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 0.1 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 one reason is that substances that promote the oxidative inactivation of glucose isomerase are removed from the bacterial cells, considering that the solution soaked with bacterial cells is markedly colored. It is presumed that it is.

The conditions for acid treatment include an acid concentration of 0.5 for treating bacterial cells.
% or higher, but a concentration of 2 to 8 φ is generally preferred.

The effect can be seen by adjusting the pH of the acid aqueous solution to a range of pH 1, particularly pH 5 to 7, which 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 O1IM, yagnesium sulfate 0. OIM
, phosphate buffer (0.05M, pH 7.2) at a reaction temperature of 70'C for 11 minutes to isomerize 1 inch of glucose, and the enzyme activity to produce fructose is defined as 1 unit.

Example 1 Streptoyaces pheochromogenes (Strep
tomyces phaechromogenes)
(Feikoken Bibori No. 221) with corn staple liquor,
After aerated culture in a medium containing xylose, potassium phosphate, magnesium sulfate, and cobalt chloride, cells were obtained by centrifugation.

50 g of the thus obtained bacterial cells (solid content: 30 articles) (activity value: 752 units/g) were suspended in a small amount of water to obtain a suspension of 80.529.

Various acid solutions (concentration 5, pH
5,75) 100 ml was added and left at room temperature for 2 to 3 hours.

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

The acid-treated bacterial 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 2
40 units) into a glucose solution (glucose 50 (W/V
) Excellent, magnesium sulfate 0.005M, cobalt chloride 0
．． 001M, phosphate buffer solution (0.05M) and 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) was used as an isomerization 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.

The isomerization rate and the isomerization rate maintenance rate are shown in Table 1.

For comparison, bacterial cells not subjected to acid treatment are also shown in Table 1.

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

Example 2 50 g of bacterial cells (solid content 80%) (activity value 752 units/, 9) 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. Processed.

Collect the bacterial cells by centrifugation, and add 50 g of the bacterial cells to 3.31 g of water.
suspended in.

Of this suspension, 0.31 cells were used as control cells.

To the remaining bacterial cell suspension 81, 0.1M acetate buffer (pH 5) 11 in which 0.2% chitosan was dissolved was added,
It was stirred with a mixer.

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

Divide this bacterial body into 9 g each and use various acid solutions (5 concentrations,
(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 the same glucose solution as in Example 1.
m1 and maintain the pH at 6.8-7.2 at 60°
The reaction was carried out at C 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.

The isomerization rate and the isomerization rate maintenance rate are shown in Table 2.

Example 3 Bacteria capable of producing glucose isomerase, 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.

The bacterial cells were collected by centrifugation, and the bacterial cells 5 (Di') were dissolved in water 3.
31.

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

After dissolving this in a to% acetic acid solution, the pH was adjusted to 7.0 with 40 g of 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 acetic acid solution 11, the pH was adjusted to 5.0 with caustic soda.

To this 0.2% partially deacetylated chitin solution, 31 ft of the above prepared bacterial cell suspension was added and stirred with a mixer.

This suspension was centrifuged at 4.00 Orpm 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 allowed to stand at room temperature for 2 hours, and 10.00 Orp.
The bacterial cells were separated by centrifugation for 10 minutes at m, and the cells were washed twice with water.

After vacuum drying the bacterial cells at 80°C for 16 hours, they were crushed,
It was made into an immobilized enzyme product.

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

Table 3 shows the isomerization rate and the isomerization rate maintenance rate.

Claims (1)

[Claims] 1. A method for producing immobilized glucose isomerase, which comprises treating gelcole isomerase-producing bacteria with tartaric acid, lactic acid, or maleic acid.