JPS609793B2 - 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 them onto an anion exchange resin. There are known methods to do this. It is also known that immobilized glucose isomerase contains porous substances such as silica clay, silica alumina, and activated carbon 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 into a column and the glucose isomerization reaction is performed continuously, the isomerized bran liquid 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 to become acidic was observed.

Such a tendency significantly deviates from the lowest pH or stable pH of glucose isomerase, and it cannot be said that the reactivity or stability of glucose isomerase is fully satisfactory. Furthermore, even when immobilized glucose isomerase is added to a glucose solution and the isomerization reaction is carried out in a batch manner, the isomerized rice bran 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 present inventors have discovered that when producing glucose isomerase, the desired objective can be achieved by adding a refractory phosphate to the immobilized glucose isomerase in a neutral or alkaline aqueous solution, and have thus arrived at the present invention. .

That is, the present invention provides a method for producing immobilized glucose isomerase by agglutinating glucose isomerase-producing microbial cells with chitosan, and the immobilized glucose isomerase is insoluble in a neutral or alkaline aqueous solution at any stage. This is a method for producing immobilized glucose isomerase, which is characterized in that it contains a phosphate. The immobilized glucose isomerase produced by the method of the present invention has significantly enhanced enzyme reactivity, greatly 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. is obtained by culturing it in a medium containing cornstarch 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. The microbial cells used may be either cultured microbial cells as they are or heat-treated microbial cells.Furthermore, the microbial cells may be dispersed or suspended in a buffer solution with a pH of 5 to 9, particularly 5 to 7.0. In this case, the concentration of bacterial cells is usually 0.50% by weight, preferably 0.5-4% by weight.
It is.

Chitosan in the present invention is a solution obtained by dissolving chitosan itself or its solution in an acid such as acetic acid, formic acid, hydrochloric acid, or an acidic solution of these acids.The concentration of this solution is 0.005 to 2.0%. Preferably it is 0.1-1% by weight.

The method of producing immobilized glucose isomerase by agglutinating glucose isomerase cells with chitosan in the present invention is also a method in which chitosan or a solution thereof is mixed with a liquid containing cells to perform an agglutination reaction. There is a method in which bacterial cells are added to a tochitosan solution and mixed 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 mix it with a monkey to disperse the microbial cells in the chitosan solution before carrying out 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.

The phosphates that are sparingly soluble in neutral or alkaline aqueous solutions used in the present invention are those with a solubility of 9 nod' or less of 100 at pH 6 or higher; Acid "Metaphosphoric acid" is a metal salt of a broad concept of acids, including pyrophosphoric acid, hypophosphoric acid, phosphorous acid, and pyrophosphorous acid and hypophosphorous acid.

Specifically, IJ calcium phosphate, magnesium phosphate,
Examples include phosphates such as magnesium hydrogen phosphate, calcium metaphosphate, metaphosphates such as magnesium metaphosphate, pyrophosphates such as calcium pyrophosphate, and magnesium pyrophosphate. Particularly preferred are magnesium hydrogen phosphate, calcium metaphosphate, Magnesium pyrophosphate, calcium topyrophosphate, etc.The amount of phosphate added is preferably an amount that suppresses the pH drop of the isomerized sugar solution, but it varies depending on the type of phosphate, but it is usually Preferably it is between 1 and 2% by weight.

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

For example, even after adding phosphate to the bacterial cell suspension, it is possible to perform the agglutination reaction by mixing it with a chitosan solution, or when adding the chitosan solution to the bacterial cell suspension and performing the agglutination reaction, the phosphate may be added independently. Furthermore, after the microbial cells aggregated with chitosan are molded and before drying, they may be mixed with a phosphate or a dispersion thereof. Some of the phosphates used in the present invention are slightly soluble even in neutral or alkaline aqueous solutions.

The pH of the dispersion of such phosphates may deviate from around neutrality. Therefore, by adjusting the amount of acid or alkali added to the bacterial cell suspension or chitosan solution, the pH during the aggregation reaction can be adjusted to 6. It is desirable to maintain this.
The immobilized glucose isomerase containing phosphate according to the present invention prevents the pH of the reaction system from decreasing even if the phosphate is dissolved during the isomerization reaction.

Therefore, the pH of the isomerized rice bran solution is the optimum pH for the enzyme, which is approximately stable pW.
This maintains the stability and reactivity of the enzyme.Also, the phosphate contained in the immobilized glucose isomerase immediately reacts with the acidic substances produced during the isomerization reaction. As a result of dissolution, the immobilized glucose isomerase becomes porous, but the diffusion resistance of glucose decreases and the reactivity increases.Furthermore, in the present invention, since glucose isomerase cells are aggregated with chitosan, other The cationic coagulant is also extremely strong compared to coagulation with polyvinyl imidazoline or enteric acrylamide, and has very little pressure loss even if it contains phosphate.

Therefore, during the isomerization reaction, not only is the flow rate of the glucose solution high, but also there is almost no change in the flow rate between the start of the isomerization reaction and the end of the operation. The present invention will be explained below using examples.

In the examples, % simply indicates weight %. For enzyme preparation, the international glucose isomerase unit was used, and glucose solution (glucose concentration 2M, 0.02MMgS04, 0.00%
001MCoc12, PH6.85) and reaction temperature 60q
The enzyme activity that isomerizes 1 French mole of glucose per minute at 0 is defined as 1 unit (abbreviated as IGm).

The following method was followed for each measurement item.

m Swelling degree It is expressed as the volume (volume) occupied in water when fixed glucose isomerase is suspended in water and allowed to settle.

[2} Hardness After the granular immobilized glucose isomerase was left in a 40 W/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 degree of disintegration of immobilized glucose isomerase during a 1-hour Takapestle reaction when measuring disintegrating activity.

'4} Sedimentation property It was expressed as the sedimentation rate in a 40W/W% glucose solution.

Example 1 Glucose Isomerase Production Frozen bacterial cells 1.5k9 (solid content: 38% IGIU') were suspended in 60 liters of water and divided into 6 equal parts.

To this suspension, magnesium hydrogen phosphate, magnesium pyrophosphate, magnesium phosphate, calcium phosphate, and calcium pyrophosphate were added at 10% per bacterial cell (dry matter),
The pH was then adjusted to 7.0. 0.2% prepared in advance
An acetic acid solution of chitosan (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 at 70℃, it was separated for 30 minutes.
~40 mesh of immobilized glucose isomerase was obtained. Activity, firmness and swelling of the obtained immobilized glucose isomerase? The dust level is shown in Table 1.

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.
009MMgS04, 0.001MCoc12, pH7
．． 0) Glucose isomerase immobilized on 20 [12
0101U) was added and the reaction was carried out at 60qC 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+fructose×100 After the reaction, the isomerization rate and the pH of the high-fructose sugar solution are shown in Table 2.

Table 2 Example 2 The 30-40 mesh magnesium hydrogen phosphate-containing immobilized glucose isomerase obtained in Example 1 was packed into a column, and a 50% glucose solution was allowed to flow down under gravity. The flow rate was checked every hour.

The results are shown in Table 3. For comparison, Table 3 also shows the case of immobilized glucose insulin which was similarly flocculated with other flocculants, Primafloc C-7 and A-10, and contained magnesium hydrogen phosphate.

As is clear from Table 3, according to the immobilized glucose isomerase aggregated with chitosan and containing magnesium hydrogen phosphate, the flow rate of the glucose solution remained almost unchanged even after 4 hours of total bare cropping.

Example 3 Ten volumes of the immobilized glucose isomerase obtained in Example 1 were packed into a double cylindrical column (2 excitations, 0 x 18 mm), and a continuous isomerization reaction was carried out.

The results are shown in Table 4. Table 4 (Husband 1) Maximum flow rate of glucose solution that can be flowed while maintaining an isomerization rate of 45%

Claims (1)

[Claims] 1. In the method of producing immobilized glucose isomerase by agglutinating glucose isomerase-producing bacterial cells with chitosan, it is possible to add phosphate, which is sparingly soluble in a neutral or alkaline aqueous solution, to the immobilized glucose isomerase at any stage. Characteristic method for producing immobilized glucose isomerase.