JPS5857152B2 - Method for immobilizing glucose isomerase - Google Patents

Description

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

Fructose is an isomer of glucose, and has approximately twice the sweetness of glucose, and exhibits the same sweetness as glucose when ingested at about half the amount of glucose.

Therefore, it has the advantage of providing sweetness while minimizing calorie intake.

In addition, a sugar solution containing fructose isomerized from glucose exhibits a sweetness that is approximately 1.2 to 1.4 times sweeter than sugar.
In recent years, it has been rapidly used in fields such as soft drinks, canned goods, and frozen desserts.

Conventionally, in order to produce liquid sugar by isomerizing glucose using glucose isomerase, frozen glucose isomerase-producing microbial cells are added to a concentrated glucose solution of about 45 to 50% by weight, and the process is carried out at a temperature of about 70°C. for a long time,
After the isomerization reaction occurs, isomerized liquid sugar is obtained through various steps such as filtration, decolorization, and deionization.

However, the above method has a major drawback in that at the end of the isomerization reaction, the oxygen 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 bonding method using a porous glass aromatic amino derivative as a carrier, an ionic carrier bonding method using an ion exchanger such as DEAE-cellulose or Duolly A7 as a carrier, and polyacrylic acid gel. There are also comprehensive methods using materials such as collagen or cellulose triacetate.

These methods lack physical strength even if the enzyme is immobilized, and may not be usable on a large scale, or the immobilized enzyme may swell and become clogged during continuous reactions, or may not be able to withstand physical strength. However, the enzyme activity is low and the productivity is very poor, so that a sufficient immobilized enzyme for industrial use has not yet been obtained.

A method is also known in which glucose isomerase is immobilized on the bacterial cells by heating the bacterial cells to a temperature of 55° C. or higher and 90° C. or lower (Japanese Patent Publication No. 19030/1983).

Although this method significantly reduced the decrease in enzyme activity compared to untreated bacterial cells, the residual activity value remained at an initial level of 40-55%.
It was 0%, which was still not sufficient.

In view of the above circumstances, the present inventors conducted various studies on the immobilization of intracellular enzyme glucose isomerase, and found that
After treating the bacterial cells with an inorganic acid or organic acid or its salt, chitosan mayo is a bacteria treated with an inorganic acid or an organic acid or its salt by adding and mixing partially acylated chitosan to a relatively high concentration. By forming a chitosan or partially acylated chitosan film on the body surface, the coloring of the isomerized liquid during the glucose isomerization reaction is extremely small.
The present invention was achieved by discovering that an immobilized enzyme with significantly high activity per dry matter of the immobilized enzyme can be obtained.

That is, the present invention involves treating a glucose isomerase producing bacterium with an inorganic acid or an organic acid or a salt thereof, and then mixing and reacting chitosan or partially acylated chitosan of 8 weight range or more with the dried product of the bacterium. This is a method for immobilizing glucose isomerase, which is characterized by:

Examples of the glucose isomerase-producing microbial cell in the present invention include Streptonyoes phaeochromogenes.
genes), Streptomyces frasiae (S,
fradiae), Streptomyces roseochromogenes (S, roseochromogenes), Streptomyces olivaceus (S, olivaceus)
us), Streptomyces califol-cas (S,
cal if orni cas), Streptomyces venuceus (S, venuceus), Streptomyces virginia (S, virginias),
actinomycetes such as Pseudomonas hydrohira (Fs
Bacillus megaterim (Bacillus megaterim)
A wide range of microorganisms can be mentioned, including bacteria of the genus Bacillus such as 1lus megateriwn), 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

The bacterial cells used in the present invention, especially after culturing the bacterial cells,
It is preferable to use the bacterial cells isolated from the culture solution as they are or those 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 dispersed or suspended in a buffer solution is usually 0.
．． The weight range is 1 to 50% by weight or 0.5 to 10% by weight.

As the inorganic acid or organic acid or its salt used in the present invention, P is within a range that does not deactivate glucose isomerase.
Those having a pH of 4.5 to 7.0 and having a buffering effect are particularly preferred.

Such inorganic acids include phosphoric acid, hydrochloric acid, sulfuric acid, and polymerized phosphoric acid, while organic acids include succinic acid, tartaric acid, citric acid, acetic acid, lactic acid, fumaric acid, maleic acid, and malic acid. Ammonium salts as acid salts,
There are sodium salts, potassium salts, magnesium salts, etc.

The concentration of the above-mentioned inorganic acid or organic acid or its salt varies depending on the type thereof, but is generally preferably 0.1 to 10% by weight of the solution.

Chitosan is a white amorphous powder obtained by deacetylating chitin, and is a basic polysaccharide consisting of glycosamine.

Partially acylated chitosan is one obtained by partially acylating the above-mentioned chitosan with a suitable acylating agent, such as an organic acid anhydride or an organic acid chloride, or one obtained by deacetylating chitin.

The chitosan or partially acylated chitosan in the present invention is the above-mentioned chitosan or partially acylated chitosan or a solution thereof, such as an acid such as acetic acid, formic acid, phosphoric acid, or hydrochloric acid, or a solution obtained by dissolving these acids or an acidic solution of these acids. It is.

The concentration of this solution ranges from 0.05 to 2.0 weight range, preferably from 0.1 to 1.0 weight range.

In the method of the present invention, glucose isomerase-producing bacteria are first treated with an inorganic acid or an organic acid or a salt thereof.

Regarding the conditions for treatment with an acid or its salt, an effect is recognized when the concentration of the acid or its salt used to treat the bacterial cells is 0.3% or more, but a concentration of 2 to 8 is generally preferred.

Effects are observed when the pH of the aqueous solution of the acid or its salt is within a range that does not deactivate glucose isomerase (especially at pH 5 to 7).

The concentration of bacterial cells in the above treatment is 0.01 to 5, especially 0.
．． It is preferable that it is the 5th to 1st section.

It is necessary to wash the treated bacterial cells with water to remove the acid or its salt.

There is no need to dry the washed bacterial cells.

In the method of the present invention, glucose isomerase producing bacteria treated with an inorganic acid or an organic acid or a salt thereof are then mixed with chitosan or partially acylated chitosan and reacted.

The amount of chitosan or partially acylated chitosan or its solution mixed with the bacterial cells is such that the amount of chitosan or partially acylated chitosan is 8 weight range or more, preferably 10.0 to 50.0 weight range, based on the dry weight of the bacterial cells. The amount is such that

If the amount of chitosan or partially acylated chitosan added is less than 8% by weight based on the dry bacterial cells, the chitosan or partially acylated chitosan film will be too weak, resulting in poor stability of the immobilized enzyme. be.

That is, enzymes are likely to be detached from the bacterial cells, and destruction due to mechanical impact is likely to occur.

Specifically, the method of mixing the above-mentioned bacterial cells and chitosan or partially acylated chitosan includes a method of adding and mixing chitosan or partially acylated chitosan or a solution thereof to a liquid containing bacterial cells, and a method of adding and mixing chitosan or partially acylated chitosan or a solution of chitosan or partially acylated chitosan. There are methods such as adding and mixing bacterial cells.

From the viewpoint of operability in production of immobilized enzymes, the microbial cells are added to the chitosan or partially acylated chitosan solution and stirred well, or the microbial cells are kneaded to disperse the microbial cells in the chitosan or partially acylated chitosan solution. is preferred.

If necessary, bacterial cells are separated from the mixture obtained by the above method by operations such as filtration and centrifugation.

In the method of the present invention, after treatment with an acid or a salt thereof, the bacterial cells are mixed and reacted with chitosan or partially acylated chitosan, molded if necessary, and then dried to obtain an immobilized enzyme product.

When the molded bacterial cells are treated with an alkali having a pH of 8 to 10, the chitosan or partially acylated chitosan film is further hardened.
Note that this is preferable because durability is improved.

The drying conditions are preferably within a temperature range that does not disqualify the enzyme.

Various methods such as vacuum drying and solar drying are employed as the drying method.

The immobilized enzyme obtained by the method of the present invention not only has excellent physical strength, but also has high activity per dry matter of the immobilized enzyme.
In addition, the degree of swelling is low even when immersed in glucose solution, and the activity per immersed enzyme volume is high.This means that there is no clogging when packed in a column, and the isomerization reaction rate is high, allowing glucose solution to be pumped at a high rate (initial 5V = 1 or more), and the productivity of isomerized sugar is extremely high (satisfactory on an industrial scale).

It also maintains the characteristic that the glucose isomerization solution is extremely less colored.

In particular, by using a relatively large amount of chitosan or partially acylated chitosan on the bacterial cells, the chitosan or partially acylated chitosan film formed on the surface of the bacterial cells acts as an adhesive between the bacterial cells. This improves the moldability of.

The present invention will be explained below using Examples.

Note that the glucose isomerase activity was measured using the international glucose isomerase unit, using a glucose solution (glucose concentration: 2 M, 0.02 M magnesium sulfate, 0.02 M magnesium sulfate, 0.02 M magnesium sulfate, 0.02 M magnesium sulfate,
001 cobalt chloride, pH 6,85) and reaction temperature 6
One unit is the enzyme activity that isomerizes 1 μmol of glucose in 1 minute at 0°C.

The swelling degree is expressed by the volume occupied in water when the immobilized enzyme 11 is suspended in water.

In addition, the strength is shown by the weight when a weight is placed on one particle of the immobilized enzyme after swelling it in water with a particle size of 20 to 30 mesh, and the weight begins to soak.

The isomerization reaction is carried out using a glucose solution (glucose 50 (w/
v)%, 0.05M phosphate buffer, 0.005M magnesium sulfate, 0. OOIM cobalt chloride) 50-
Add 80 IGIU of immobilized enzyme to pH 6,8-7.
The temperature was maintained at 60C for 20 hours.

After the reaction was completed, the immobilized enzyme was collected by filtration, and a glucose solution having the same composition was added again to repeat the soaking reaction.

Damaged dust indicates the damaged state of the immobilized enzyme after repeating the isomerization reaction five times.

Example 1 Strephtomyces phaeochromogenes (Strep
tomyces Phaechromogenes)
(Moisture 70, 420.3 I
G I U/f (dry matter) 1.5 to 9] was well suspended in 7.5 t of water.

To this suspension, 7.5 t of a 5-functional citric acid solution adjusted to pH 6.0 with a 5-functional caustic soda solution was added and mixed, and after being left at room temperature for 1 hour, the bacterial cells were filtered.

Next, the bacterial cells were thoroughly washed with water, suspended in water again, and weighed at 60 tons.
A bacterial cell suspension (PH7.0) was obtained.

This suspension was divided into 6 equal parts of 1 (l), and a previously prepared 0.2% chitosan-acetic acid solution (PH6.0) was added to the bacterial cells (
The amount of chitosan is 5 parts, 8 parts, 15 parts, 2 parts relative to dry matter)
The mixture was added with stirring at a ratio of 0% and 30% to react with the bacterial cells.

The microbial cells having a chitosan membrane thus obtained were dehydrated using a centrifugal dehydrator, extruded using an extruder having a nozzle with a pore size of 1 rra, dried overnight at 30°C, and pulverized appropriately. , sieve to remove fine parts, 20-30
An immobilized enzyme of Metsuyu was obtained.

The properties of the obtained immobilized enzyme are shown in Table 1.

Glucose isomerization reaction was repeatedly performed using the immobilized enzyme obtained by the above method, and the isomerization rate was measured.

The results are shown in Table 2. As is clear from Tables 1 and 2, when the amount of chitosan relative to the bacterial cells (dry matter) is 8 weight ratio or more, a robust immobilized enzyme with low swelling degree and high physical strength can be obtained. When using a transactivating enzyme, a long-term isomerization reaction of glucose is possible because the decrease in activity is small and there is no damage.

Example 2 Streptomyces pheochromogenes (StreP
Glucose isomerase-producing bacterial cells obtained by culturing S. tmyces Phaechromgenes (Feikoken Bibori No. 221) [solid content].

0, 592I GIU/PC (dry matter)] 1320f was suspended in 13.21% of a 5.0% succinic acid solution whose pH was adjusted to 6.0 with a caustic soda solution and left at room temperature for 2 hours.

Thereafter, the bacterial cells were filtered and washed thoroughly with water.

Then, the cells were suspended in water again to obtain a cell suspension of 521.

A pre-adjusted 0 and 2 chitosan-acetic acid solution (PH
6.0) The above bacterial cell suspension was mixed in 26 tons with gradual stirring so that the amount of chitosan based on the bacterial cells (dry matter) was 15.8%.

The microbial cells having a chitosan membrane thus obtained were washed and dehydrated, extruded using an extruder having a nozzle with a hole diameter of 1r+m+, and then dried with ventilation at 30° C. overnight.

After pulverizing the mixture, fine particles were removed by sieving to obtain 20 to 30 meshes of immobilized enzyme A3701.

Same glucose isomerase producing bacterial cell [solid content 25.0
592 IGIU/f (dry matter)] 1320S' was treated with co-phosphoric acid solution in the same manner as above, and the bacterial cells (dry matter) were added to 26 t of 0.06% chitosan-acetic acid solution (PH6,0).
Immobilized enzyme B530r was obtained by adding chitosan in an amount of 4.7%.

The properties of the obtained immobilized enzymes A and B are shown in Table 3.

The immobilized enzyme A or B51 was packed into a double cylinder with an inner diameter of 2.21 m and a length of 100 cm.

In the case of immobilized enzyme A, the column volume was 180m, whereas in the case of immobilized enzyme B, it was 275-.

65°C hot water is circulated through the outer tube to bring the internal temperature to 65°C.
It was maintained at C.

A 40% glucose solution (PH8.5, magnesium sulfate 4X10-4M) was poured into this column, and the flow rate was adjusted so that the isomerization rate was 42%. In contrast, immobilized enzyme B was able to withstand continuous operation for hours, whereas immobilized enzyme B began to swell significantly after 100 hours of continuous operation, resulting in clogging and a significant drop in flow rate.

FIG. 1 shows the course of the flow rate when the isomerization rate of immobilized enzymes A and B was maintained at 42%.

From this result, when immobilized enzyme A is used at a flow rate of up to 5v=0.25, 15 g of glucose per IKg of immobilized enzyme is used.
It was possible to process 00Kg.

Example 3 Chitosan (trade name Fronac N, manufactured by Kyowa Yushi) 801
was added to a 10% acetic acid solution (1,600 ml), and methanol S and Ot were further added thereto.

Next, after thoroughly stirring this solution at °C, 0.5 mol of acetic anhydride (2 mol as acetic acid) was added to the hexosane residue of chitosan.
1.7ff) was added, stirred well, and left at room temperature overnight.

After the reaction was completed, 4 tons of methanol was added and left to stand overnight, the resulting gel was collected and distilled water was added to make the solution 40%.

After further standing overnight, the resulting gel was separated and freeze-dried to obtain a 64.5S' powder.

After dissolving the partially acetylated chitosan thus obtained in acetic acid, 50% caustic soda solution was added to the solution to pH 6.0.
A 0.2% partially acetylated chitonsan solution was prepared.

On the other hand, the same glucose isomerase producing bacterial cells as in Example 1 were heat-treated at 70C for 30 minutes [solid content 34.
Section 5, 631.4 IGIU/f (dry goods)] 1.2 to 7
water 6. Suspend with Ot and add P with 5% caustic soda solution.
5-functional sodium metaphosphate solution adjusted to H6.0 6.0
4 was added and treated at room temperature for 1 hour.

Next, this was suction-filtered, the sodium metaphosphate solution was separated, and the bacterial cells were washed with water.

Furthermore, this bacterial cell was suspended in water to give a concentration of 13.44/,
2.24 I! , divided into 6 equal parts.

The above 0.2 ql) partially acetylated chitosan was stirred into 2.2 At of this bacterial cell suspension so that the amount of partially acetylated chitosan (dry matter) relative to the bacterial cells (dry matter) was as shown in Table 4. I added while doing so.

The bacterial cells having the partially acetylated chitosan film thus obtained were dehydrated, extruded using an extruder having a nozzle with a hole diameter of 1 rrrrn, and then dried.

Next, this was appropriately pulverized and sieved to obtain 20 to 30 meshes of immobilized enzyme.

The properties of these immobilized enzymes are shown in Table 4.

Using the above immobilized enzyme, Glucose isomerization The reaction was repeated.

The results are shown in Table 5. As is clear from Tables 4 and 5, when the amount of partially acetylated chitosan is 8 weight percent or more relative to the bacterial cells (dry matter), the degree of swelling is low, excellent physical strength is maintained, and glucose An immobilized enzyme with high isomerase activity can be obtained.

Example 4 Glucose isomerase-producing bacterial cells were heat-treated in the same manner as in Example 3 [solid content 26.3%, 450 IGIU/y (
(Dry matter): 1486 g was suspended in 2.4 t of water, divided into 2 equal parts (1) and 2 t, and immobilized enzymes were produced using the following two methods.

The first method is to add a 10% citric acid solution whose pH has been adjusted to 6.0 with a 5% caustic soda solution to a 1.2/- bacterial cell suspension, stir and mix, and then leave it for 2 hours to separate the bacterial cells. Filtered.

This was suspended in water to obtain OL. Pre-prepared 0.2% chitosan-acetic acid solution (PH6.0) 6.3
t to bacterial cell suspension 9. It was added** to OA with vigorous stirring and allowed to react.

The bacterial cells having a chitosan membrane thus obtained were filtered and dehydrated, then extruded using an extruder having a nozzle with a pore diameter of 1 rran, and dried.

This dried product was crushed appropriately to obtain 20 to 40 meshes of immobilized enzyme C.

The second method is to add water to 1.2 t of bacterial cell suspension and perform step 9. I became an office lady.

This bacterial suspension 9. 0.2% with OL adjusted in advance
The mixture was added to a chitosan-acetic acid solution (PH 6.37) with vigorous stirring in the same manner as in the first method, and reacted.

The bacterial cells having a chitosan membrane thus obtained were filtered and dehydrated, and then immersed in 2.4 t of a 5-chain citric acid solution (PH6,0) at room temperature for 2 hours, followed by dehydration, washing, and extrusion molding.

After drying the molded product, it was appropriately crushed to obtain 20 to 40 meshes of immobilized enzyme.

The properties of the obtained immobilized enzymes C and D are shown in Table 6.

Using the immobilized enzyme C or D, the isomerization reaction of glucose was repeated and the isomerization rate was measured.

The results are shown in Table 7.

[Brief explanation of the drawing]

FIG. 1 shows the course of flow rate when the isomerization rate of immobilized enzymes A and B was maintained at 42%.

Claims (1)

[Claims] 1. A cell characterized by treating glucose isomerase producing bacteria with an inorganic acid or an organic acid or a salt thereof, and then mixing and reacting the dry product of the bacteria with chitosan or partially acylated chitosan of 8 weight range or more. Method for fixing course isomerase.