JPS609794B2 - 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.

In recent years, it has been carried out industrially to immobilize bacterial cells having glucose isomerase activity on a carrier and to efficiently isomerize glucose using the immobilized glucose isomerase.

Immobilized glucose isomerase can be produced by, for example, aggregating and immobilizing bacterial cells with polyvinyl imidazoline or a soluble polymer flocculant such as positive acrylamide or chitosan, or by adsorbing onto an anion exchange resin. etc. are known. It is also known to incorporate porous substances such as diatoms, silica alumina, and activated carbon into immobilized glucose isomerase in order to increase the expression rate of glucose isomerase activity in bacterial cells. However, when the immobilized glucose isomerase obtained in this way is packed in a column and the glucose isomerization reaction is performed continuously, the isomerized sugar solution becomes acidic in the early stage of the isomerization reaction. Alternatively, as the reaction rate decreased in the latter stage of the isomerization reaction, a tendency toward acidity was observed.

Such a head orientation significantly deviates from the optimum pH or stability axis of glucose isomerase, and it cannot be said that the reactivity or stability of glucose is fully satisfactory. Furthermore, even when immobilized glucose isomerase is added to a glucose solution and the isomerization reaction is performed in a batch manner, the isomerized sugar solution tends to become acidic as the reaction progresses.

The present inventors conducted various studies to resolve these tendencies without reducing the activity and physical strength of immobilized glucose isomerase, and found that glucose isomerase-producing bacterial cells were aggregated and immobilized with chitosan. The inventors have discovered that when producing glucose isomerase, the desired objective can be achieved by incorporating carbonate into the immobilized glucose isomerase, and the present invention has been achieved.

That is, the present invention is characterized in that in a method for producing immobilized glucose isomerase by aggregating glucose isomerase producing bacterial cells with chitosan, carbonate is added to the immobilized glucose isomerase at an arbitrary step. This is a method for producing immobilized glucose isomerase. The immobilized glucose isomerase obtained by the method of the present invention has significantly enhanced enzyme reactivity, significantly improved glucose isomerization ability, stable glucose isomerization ability, and excellent operability.

Examples of the glucose-isomerase-producing microbial cells used in the present invention include microbial cells of the genus Streptomyces, Bacillus, Arthrobacter, and Pseudomonas that produce glucose-isomerase. These bacteria are grown in a medium containing corn stave liquor alone or other nitrogen sources as a nitrogen source, carbohydrates such as starch and xylose as a carbon source, and potassium phosphate, cobalt chloride, and magnesium chloride as inorganic salts. Obtain by culturing. The cells used may be cultured cells as they are or heat-treated cells. 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. In this case, the concentration of bacterial cells is usually 0.1 to 5% by weight, preferably 0.5 to 4% by weight. Chitosan in the present invention is chitosan itself or a solution thereof, such as an acid such as acetic acid, formic acid, or hydrochloric acid, or a solution obtained by dissolving it in an acidic solution of these acids.
The concentration of this solution is 0.05-2. % by weight, preferably from 0.1 to 1% by weight. The method of producing immobilized glucose isomerase by agglutinating glucose isomerase cells with chitosan in the present invention is specifically a method of mixing chitosan or a solution thereof into a liquid containing cells and performing an agglutination reaction. There are methods such as adding bacterial cells to a chitosan solution and mixing them to perform an agglutination reaction.

From the viewpoint of operability in producing the immobilized enzyme, it is preferable to add the microbial cells to the chitosan solution and mix well, or to disperse the microbial cells in the chitosan solution by kneading, and then perform the agglutination reaction. After the agglutination reaction, the bacterial cells are filtered, centrifuged, or compressed to have a moisture content of 20 to 50%, and then granulated using a granulator and dried as is, or after being shaped using a sizing machine and then dried. do.

Examples of carbonates used in the present invention include magnesium carbonate, manganese carbonate, barium carbonate, calcium carbonate, and cobalt carbonate. , soluble in acidic solutions.

These substances are contained in the immobilized glucose isomerase, neutralize acidic substances in the isomerized sugar solution, and suppress a decrease in the pH of the sugar solution. Furthermore, these substances do not exhibit an inhibitory effect on enzyme reactions and do not pose any problem in immobilization. The amount of carbonate added is preferably an amount that suppresses decomposition of the isomerized sugar solution, and although it varies depending on the type of carbonate, it is usually added to the raw material bacterial cells (
It is desirable that the amount is 1 to 2% by weight based on the dry matter.

The use of carbonate in the present invention is carried out at any stage.

For example, carbonate may be added to a bacterial cell suspension and then mixed with a chitosan solution to perform an agglutination reaction, or when a chitosan solution is added to a bacterial cell suspension and an agglutination reaction is performed, the carbonate is dispersed. The liquid may be added independently. Alternatively, after molding the microbial cells aggregated with Chitosaso, it may be mixed with carbonate before drying. Some carbonates used in the present invention are slightly soluble in neutral or alkaline solutions. The dispersion of such carbonates may deviate from the neutral range. Therefore, it is desirable to maintain the pH at 6 to 7 during the aggregation reaction by adjusting the amount of acid or alkali added to the bacterial cell suspension or chitosan solution. In the carbonate-containing immobilized glucose isomerase according to the present invention, carbonic acid is dissolved during the isomerization reaction, thereby preventing a decrease in the pH of the reaction system.

Therefore, the pH of the isomerized sugar solution is the optimum pH for the enzyme, and the stable pH
Since the temperature is maintained at H, the stability and reactivity of the enzyme are extremely good. In addition, the carbonate contained in the immobilized glucose isomerase immediately reacts and dissolves the acidic substances generated during the isomerization reaction, and as a result, the immobilized glucose isomerase becomes porous and has resistance to glucose diffusion. decreases and reactivity increases. The present invention will be explained below using Examples. In the examples, % simply means weight %.
shows.

Enzyme activity was measured using the international glucose isomerase unit, and glucose solution (glucose concentration 2M, 0.02MM
4, 0.001M CoC12, pH 6.85) at a reaction temperature of 60q0, the enzyme activity to isomerize 11 moles of glucose per minute was defined as 1 unit (abbreviated as IGIU). In addition, each measurement item followed the method below.

[1' Swelling] It is expressed as the volume (volume) occupied in water when one volume of immobilized glucose isomerase is suspended in water and allowed to settle.

(2) Hardness After the granular immobilized glucose isomerase was left in a 40W'W% glucose solution (pH 8.5) at room temperature overnight, the hardness was examined according to the following method.

In other words, the material was pinched between the index finger and thumb, and a panel of three people visually judged the firmness. Hardness to withstand industrial use is 3.5
I think that's all. '3' represents the extent to which immobilized glucose isomerase is degraded during the 1-hour Takamiki reaction when measuring the disintegrating activity.

(2) Sedimentation property It was expressed as the sedimentation rate in a 40W/W% glucose solution.

Example 1 Glucose Isomerase Production 1.5 kg of frozen bacterial cells (solid content: 38% IGIU) was suspended in water and divided into 6 equal parts.

Add magnesium carbonate to this suspension per bacterial cell (dry matter) at 1
%, 2.5%, 5%, 10%, 20% and the pH was adjusted to 7.0 by adding acetic acid. A pre-prepared 0.2% chitosan acetic acid solution (pH 6.0) was added to the above bacterial cell suspension while stirring carefully to cause an agglutination reaction. The aggregated bacterial cells were dehydrated to obtain a chitosan bacterial cell agglutination reaction product. After molding this and drying it under hot air of 60 qo, it was separated into stripes and
~40 mesh of immobilized glucose isomerase was obtained. Table 1 shows the activity, hardness, and degree of swelling of the immobilized glucose isomerase obtained. Table 1 In order to investigate the differences in the isomerization reactions of these immobilized glucose isomerases, a glucose isomerization reaction was carried out.
The isomerization rate was measured.

The isomerization reaction was performed using a glucose solution (glucose 50%, 0.
008MMgS04, 0.001MCoC12, pH 7
．． 0) Immobilized glucose isomerase (120
1 GIU) was added thereto, and the reaction was carried out at 6 oq○ for 1 hour. After the isomerization reaction was completed, the enzyme was collected, and a glucose solution with the same composition was added again to repeat the isomerization reaction. Fructose isomerization rate = glucose 10 fructose x 100 After the reaction, the isomerization rate and the pH of the high-fructose sugar solution are shown in Table 2.

Table 2 As is clear from Tables 1 and 2, the immobilized glucose isomerase containing magnesium carbonate has almost no change in physical properties, has a high isomerization rate, and has a fairly good retention rate.

Example 2 Magnesium carbonate-containing immobilized glucose isomerase (MgC03 content 10%) obtained by the same method as Example 18
(4301 CIU/event) and as a control, immobilized glucose isomerase without magnesium carbonate (8.9 CIU/event).
391101U/Y) each with a double cylindrical column (2 enemies J
A continuous isomerization reaction was carried out by packing the reactor into a 18-ft.

The continuous isomerization reaction was carried out using a 50% crystalline glucose solution (original
4, pH 8.25) was passed through a thermometer kept at 65°C to maintain the isomerization rate at 45%.

FIG. 1 shows the flow progress of the glucose solution. As is clear from FIG. 1, the pH of the control immobilized glucose isomerase decreases over time, whereas the pH of the immobilized glucose isomerase containing magnesium carbonate does not decrease. Therefore, the isomerization reaction rate is fast. Further, the immobilized glucose isomerase containing magnesium carbonate has smooth isomerization ability with little sudden fluctuation, and maintains the isomerization rate at 45%, so the amount of glucose solution to be passed can be predicted very easily. In addition, immobilized glucose lysomerase l
The glucose isomerization capacity per k9 was 2400 k9 in the case containing magnesium carbonate, while it was 2000 kg in the case without magnesium carbonate. Example 3 Magnesium carbonate in Example 1 was replaced with basic cobalt carbonate [father o (O
H) 2.

The activity, hardness and swelling degree of the obtained immobilized glucose isomerase are shown in Table 3. Table 3 Using these immobilized glucose isomerases, an isomerization reaction was carried out in the same manner as in Example 1.

The results are attached to Table 4. Table 4 Example 4 Glucose Isomerase Production Frozen bacterial cells 1.2k9 (solid content 29.0%) were suspended in 52.8ml of water, and 34.89ml of magnesium silicate was added thereto and mixed well.

In the same manner as in Example 1, a 0.2% chitosan acetic acid solution was added to the above cell suspension while stirring thoroughly to cause aggregation. After aggregation, the mixture was dehydrated to obtain a chitosan cell aggregate. This was divided into 6 equal parts, and basic cobalt carbonate [XoC031 Father o (OH) 21 days 20] was added to 0% of the original bacterial cells (dry matter).
%, 2%, 4%'' 6%, 8% or 10% and mix well.

After molding this, it was thoroughly dried under hot air at 60°C.
Table 5 shows the activity, hardness, and degree of swelling of the immobilized glucose isomerase obtained by the above method.

Table 5 Next, as in Example 1, glucose solution (glucose 5
0%, magnesium sulfate 0.08M "cobalt chloride 0.08M"
4901 U/night of each immobilized glucose isomerase was added to 001M) 20' and the reaction was carried out at ~60 qo for 20 hours, and the isomerization rate and pH of the reaction solution were examined.

In addition, each of the above-mentioned immobilized glucose isomerase was packed into a double cylindrical column (2 x 18 x 18 x 2 x 2 x 10 x 10 x 10 x 15 x 10 x 10 x 10 x 10 x 10 x 10 x 10 x 10 x 18 x 25 x 18 x 10 x 20 x 100 x 100 x 100 cm. When the isomerization ability of glucose (isomerization rate 45%) was investigated, the results were as shown in Table 6.Table 6

[Brief explanation of the drawing]

FIG. 1 shows changes over time in the supply rate and pH of the glucose solution when the isomerization reaction was carried out while maintaining the isomerization rate at 45%. Figure 1

Claims (1)

[Claims] 1. A method for producing immobilized glucose isomerase by agglutinating glucose isomerase-producing microbial cells with chitosan, the method comprising making the immobilized glucose isomerase contain a carbonate.