KR810001825B1 - Process for the renewal of an insolubilized glucose isomerase - Google Patents

Abstract

A method is described for the immobilization of glucose isomerase on an anion exchange resin and, following the isomerization of glucose to fructose, regeneration of the support. After isomerization, the column was washed with 500 ml of IN H2SO4, desalted H2O, 0.1N NaOH, desalted H2O, IN H2SO4, respectively. Fresh glucose isomerase was readsorbed and the isomerization repeated.

Description

Regeneration method of insoluble glucose isomerase

The present invention relates to a method for regenerating insoluble glucose isomerase, and more specifically, insoluble glucose isomerase obtained by adsorbing and supporting glucose isomerase on synthetic anion exchange resin to fructose glucose. The method relates to a method of desorbing reduced glucose isomerase from this insoluble glucose isomerase and then adsorbing and supporting the colored glucose asomase on the carrier. .

Glucose isomerase is an enzyme that reversibly converts glucose and fructose into bacteria, such as Pseudomonas genus, Visilus genus, Brevibacterium genus, Lactobacillus genus, and Streptomyces genus. It is found in a wide range of microorganisms such as actinomycetes and other yeasts. Industrially, glucose isomerase produced in actinomycetes, particularly Streptomyces spp.

Glucose isomerase is also known to be used for isomerization of glucose. In particular, glucose isomerase extracted from cells is adsorbed and supported on a carrier to insolubilize it, and it is industrially advantageous to use it as a solid catalyst. Do. As carriers for insolubilizing glucose isomerase, DEAE Sephadex, DEAE cellulose, ion exchange resins, and the like are known (Japanese Food Industry Chemical Publication No. 14, No. 12, December 1967, Tsumura Shinzo, Ishikawa Continuous Isomerization Reaction of Glucose with Masako Enzyme, U.S. Patent No. 3,788,945, Japanese Patent Application Publication Nos. 49-80160 and 51-53582), among which ion exchange resins contain a large amount of glucose isomera per unit volume. It is an excellent carrier because it can absorb an aze and also has high and stable activity with adsorbed enzymes. To perform isomerization of glucose using an insoluble glucose isomerase supported by an ion exchange resin, insoluble glucose isomerase is added to the aqueous glucose solution and stirred, or the column is filled with a column to prepare an aqueous glucose solution from the top. It is a nucleus.

Insoluble glucose isomerase gradually decreases its activity during the isomerization reaction, but its initial activity is halved 15 to 60 days after the start of the reaction.

Insoluble glucose isomerase, whose activity is reduced by half, is a method of removing glucose isomerase from the reaction system and desorbing glucose isomerase, as described in Japanese Patent Publication No. 48-21514. A method of passing an aqueous solution of an inorganic salt such as potassium chloride or sodium chloride of about 0.2 to 0.5 M from the top of a column filled with an anion exchange resin is known.

The present inventors treated the insoluble glucose isomerase with reduced activity after using the isomerization reaction by the above method to desorb the adsorbent from the carrier, and then adsorbed the highly active glucose isomerase and then used in the isomerization reaction. After that, the adsorption of isomerase, isomerization reaction and desorption of adsorption components were repeated.

However, as the above process is repeated, the amount of detachment of the adsorption component from the carrier is decreased, and the glucose isomerase adsorption capacity of the carrier after regeneration is also deteriorated, thereby lowering the glucose isomerase activity value per unit volume of the carrier. When the process was repeated several times, it was recognized that it could not be industrially used as an insoluble glucose isomerase carrier.

Therefore, the present inventors earnestly studied a method of regenerating insoluble glucose isomerase, and as a regenerant, when the aqueous solution of the mine is used in combination with the aqueous solution of alkali, the electrolyte salt, the aqueous solution of the mine, or a mixture thereof, Adsorption components such as glucose isomerase, coarse protein, and metal salts with reduced activity from insoluble glucose isomerase can be detached with good efficiency, and the carrier after desorption of the adsorption component is glucose isomera. The present invention has been achieved by finding that the aze adsorption capacity is almost made up, and that the activity of the insoluble glucose isomerase thus reproduced is sufficiently expressed to a sufficient degree.

That is, an object of the present invention is to regenerate an insoluble glucose isomerase agent after use in an isomerization reaction and to prepare an insoluble glucose isomerase agent having high activity per unit volume. Insolubilized glucose isomerase obtained by adsorption-supporting glucose isomerase on a synthetic anion exchange resin according to the method of the present invention is used in a reaction for isomerizing glucose to fructose, and then the insoluble glucose isomerase agent is used in the mineral aqueous solution. And an aqueous alkaline solution to remove the components adsorbed on the carrier and finally to make the ionic form of the carrier a salt form, adsorbing glucose asomase to it.

The present invention will now be described in detail.

In the present invention, various known synthetic anion exchange resins are used as carriers of glucose isomerase, but quaternary ammonium type porous basic anions based on styrene-divinylbenzene copolymers are used. Exchange resins such as Ambaride 938, Ambarite 904 (Rohm Haas Co.) or Diamond Ion HPA 11 (manufactured by Mitsubishi Chemical Co., Ltd.) are suitable.

Glucose egg white risjo is easily extracted from the cells by treating glucose isomerase producing bacteria such as the bacterium of Streptomyces genus with ultrasonic or egg white risjo.

Insoluble glucose isomerase is prepared by adsorbing glucose isomerase to a synthetic anion exchange resin.

To adsorb glucose isomerase to the synthetic anion exchange resin, the resin of the aqueous solution of glucose isomerase extracted from the cells may be taken into consideration, stirred as necessary, and the resin may be taken out and washed with water after an appropriate adsorption time. Glucose isomerase can also be adsorbed onto the resin while fluidizing the resin by filling the column with a column and supplying an aqueous solution of glucose isomerase from the bottom of the column.

Since the ion type of the resin is higher in the salt type (SO ₄ type, CI type, etc.) than the free type (OH type), the adsorption of glucose isomerase to the resin of the glucose isomerase. The resin is treated with an acid or salt aqueous solution to leave the ionic form of the resin in salt form beforehand.

The amount of glucose isomerase is expressed as active unit U (1U is the amount of enzyme that isomerizes glucose to produce 1 mg of fructose under specific conditions). Adsorption of excess glucose isomerase on the resin lowers the value of the activity expression rate (see later description). Thus, the amount of glucose isomerase adsorbed on the resin is generally 500 to 4000 U / ml-Resin [U / ml-Resin; Amount of glucose isomerase adsorbed on 1 ml of swelled resin], preferably within the range of 1000 to 200 U / ml-Resin.

The insoluble glucose isomerase obtained in this manner was charged to a column, and the aqueous solution of glucose at 40 to 60% concentration was controlled at pH6.5 to 8.5 and 55 to 70 ° C while the liquid space velocity was adjusted so that the isomerization rate was 35 to 50%. Pass through the column at the temperature of. In addition, in the aqueous glucose solution, metal ions such as magnesium, cobalt, and iron are contained in order to prevent a decrease in glucose isomerase activity.

When the reaction is carried out under the above conditions, the glucose isomerase is halved in 30 to 50 days after the start of the reaction, and when 50 to 85% of its initial activity is lost, regeneration of insoluble glucose isomerase is performed. do.

In regeneration of an insoluble glucose isomerase agent in the present invention, first, an insoluble glucose isomerase having reduced activity is treated with an aqueous solution of a mineral acid and an aqueous alkali solution and the components adsorbed on the carrier are removed.

Among the carriers of insoluble glucose isomerase after use in the isomerization reaction, not only glucose isomerase with reduced activity, but also various metal salts, sugars, and the like added to the protein, pigment, and other glucose solutions in the glucose isomerase extract Although adsorbed, these adsorption components are efficiently desorbed by treatment by a combination of an aqueous mine solution, an aqueous alkali solution and an electrolyte salt.

It should be noted that in the present invention, it is not necessary to remove all of the adsorbed components of the carrier. That is, since the purpose of the regeneration treatment in the present invention is to restore the glucose isomerase adsorption capacity and the activity capacity almost to the original to the carrier, it is necessary to consume a large amount of regenerant and to remove the adsorption component more than necessary. It is not advantageous as a rule.

Therefore, in the present invention, the insoluble glucose isomerase after adsorption adsorbs the amount of isomerase of 500 to 4000 U / ml-Resin, and the adsorbed component at the time when the active expression rate becomes 70%, preferably 80% or more. It is preferable to stop the desorption of.

In the present invention, the aqueous mineral solution has a good desorption effect on the whole of the components adsorbed on the carrier, that is, on minerals such as magnesium salts, iron salts and cobalt salts, and proteins, pigments and sugars containing glucose isomerase.

On the other hand, the aqueous alkali solution does not have an excellent elution effect directly with respect to the adsorbent component, but has an action of swelling the resin, and in particular, the treatment of the alkaline aqueous solution between the mine aqueous solution treatments, such as the mine aqueous solution treatment-alkali aqueous solution treatment-mine aqueous solution treatment Insertion of the resin repeats swelling shrinkage of the resin, and as a result, desorption of the adsorption component is promoted.

Moreover, since the mixed aqueous solution of electrolyte salt and alkali is effective also in desorption of the pigment | dye adsorb | sucked to a support | carrier, it is preferable to use the mixed aqueous solution of electrolyte salt and alkali as said alkali aqueous solution process.

In the present invention, aqueous solutions of hydrochloric acid and sulfuric acid are used as the aqueous solution of mineral acid, and aqueous solutions of alkali metal hydroxides such as sodium hydroxide and potassium hydroxide are used as the aqueous alkali solution, and their aqueous solution concentration is 0.2 to 2, preferably 0.5 to 2 rules.

As the electrolyte salt, chlorides and sulfates of alkali metals such as sodium chloride, potassium chloride, sodium sulfate and potassium sulfate are used.

In the method of the present invention, to treat insoluble glucose isomerase with a regenerant, the regenerant may be passed through the top of a column packed with an insoluble glucose isomerase.

After passing through the regenerant, it is preferable to pass 2-15 times the amount of demineralized water of the insoluble glucose isomerase agent through the column and wash it.

Although the treatment temperature of insoluble glucose isomerase by a regenerant may be normal temperature, since the desorption efficiency of an adsorption component improves with higher temperature, it is preferable to set it as the range of 50-75 degreeC in consideration of heat resistance of resin.

After treatment with the regenerant, the ionic form of the carrier is salted to increase the glucose isomerase adsorption capacity of the carrier. In order to make the ionic form of the carrier into a salt form, the carrier may be treated with an aqueous alkali solution, followed by treatment with an aqueous solution of a mineral acid or an all salt.

Of course, in the case of using the aqueous photoacid solution as the final regenerant treatment, it is not particularly necessary to perform the treatment for making the ionic form of the carrier into the salt form.

Therefore, the most suitable regeneration treatment method in the method of the present invention also takes into account the desorption effect of the adsorption component, so that (1) mine solution aqueous solution treatment-alkali solution treatment-mine solution treatment or (2) mine solution aqueous solution-alkali + electrolyte salt Mixed aqueous solution treatment-mine aqueous solution treatment.

After treatment with the regenerant, the salt-formed carrier is once again adsorbed with new glucose isomerase and used repeatedly as an insoluble glucose isomerase.

In addition, according to the regeneration treatment method of the present invention, it is possible to provide an excellent insoluble glucose isomerase agent by the same carrier over a long period of time by exhibiting a good regeneration effect on an insoluble glucose isomerase agent that is repeated many cycles. There is a number.

As described above, according to the method of the present invention, glucose isomerase and other adsorption components having reduced activity from the insoluble glucose isomerase after use of the isomerization reaction can be dehydrated with good efficiency, By adsorbing new glucose isomerase with a high adsorption rate on the carrier, it is possible to prepare insoluble glucose isolasease having high activity expression rate again.

Next, although an Example demonstrates this invention further in detail, this invention is not limited to a following example, unless the summary is exceeded.

The phrases “glucose isomerase activity”, adsorption rate, “insoluble glucose isomerase activity” and “insoluble glucose isoamase activity expression rate” in the present specification are defined as follows. Extraction of glucose isomerase, adsorption of glucose isomerase to a carrier, and isomerization reaction in Examples and Comparative Examples were carried out by the methods described below.

(1) glucose isomerase activity (U / ml)

An aqueous solution in the glucose substrate solution [1 equal volume mixture of molar concentrations of D- glucose, 0.05 molar concentration of MgSO ₄ .7H ₂ O and 0.5 molar phosphate buffer (pH 7.2) of the] 0.6ml containing glucose isopropyl camera Kinase It is added and water is added to make 2 ml whole quantity.

The mixture is maintained at 70 ° C. for 60 minutes to conduct isomerization reaction. 2 ml of 0.5 mole HClO _{4 is} added to the reaction mixture to stop the reaction, and the amount of furacrose produced is determined by the cysteine carbazole-sulfuric acid method.

From this value, the activity of the glucose isomerase-containing liquid per unit amount is calculated. 1 U means the amount of enzyme that produces 1 mg of fructose under the above conditions.

(2) adsorption rate (%)

Total activity (A) is measured with respect to the enzyme extract before treatment with a carrier. After the enzyme is adsorbed onto the carrier, the carrier is filtered off and washed. Subsequently, the total ringtone (B) of the filtrate and washings is measured. Adsorption rate is represented by the following equation.

(3) Insolubilized Glucose Isomemerase Circulation (U / ml-Resin)

The insoluble glucose isomerase of about 1-10 ml (swelled state) was added to 300 ml of the above-mentioned glucose substrate solutions, and water was added thereto to make 1 L of the total amount, and then subjected to an isomerization reaction at 70 ° C. for 60 minutes with gentle stirring. I was. After completion of the reaction, the insoluble glucose isomerase was separated from the reaction solution, and the amount of fructose produced in the reaction solution was quantified by the cysteine carbazole-sulfuric acid method. Calculate.

Under the above conditions, the amount of enzyme that produces 1 mg of fructose is 1 U.

(4) cyclic expression rate (%) of insoluble glucose isomerase

으로 표시한다.

Indicated by.

Extraction of Glucose Isomerase

Glucose isomerase ＂Nagase＂ (Streptomyces, Paclomogenes, Nagase Sangyo Co., Ltd.) was suspended in water, and a small amount of risso steam was added thereto, followed by stirring to obtain glucose isomerase from the cells. Extracted. The glucose isomerase activity in this extract (hereinafter referred to as glucose extract A) was 240 U / 1 ml.

In addition, the glucose isomerase extract was prepared by the same method as the glucose isomerase extract separately, and the glucose isomerase was 200 U / ml (hereinafter referred to as glucose isomerase extract B).

Adsorption of Glucose Isomerase to Carrier

Porosity Strong basic anion exchange resin Dion ion HPA 11 (trade name, manufactured by Mitsubishi Chemical Co., Ltd., with styrene-divinylbenzene crosslinked copolymer structure having quaternary ammonium ion exchanger) by one to a salt type (SO ₄ type) in advance, and then, a suspension of the resin from glucose isopropyl camera azepin extraction solution of 10-fold amount relative to the swollen state such resin volume, and stirred at 50 ℃ 6 sigan adsorbed glucose isopropyl camera kinase .

Isomerization reaction

A 100 ml of an insoluble glucose isomerase prepared by adsorbing glucose isomerase to a carrier by the above-mentioned method was filled into an outer tube subcolumn having an inner diameter of 20 ml, and glucose (3 mol concentration) and magnesium sulfate (5 An aqueous solution of pH 7.5 containing millimolar concentration) and iron sulfate (0.1 mmol concentration) at 60 ° C. Glucose isomerized by passing 30 days from the top of the column while adjusting the value of the liquid space velocity so that the average isomerization rate is 45%.

[Examples 1 and 2]

Insoluble glucose isomerase (Sorption rate 100%, Adsorption amount 2400U / ml-Resin) prepared by adsorbing glucose isomerase extract A to a carrier with a strong basic anion exchange resin HPA 11 of SO ₄ type Isomerization of glucose was performed by the method of A using. After completion of the reaction, about 1 L of demineralized water was passed through the top of the column to wash insoluble glucose isomerase, and then 600 ml of 1 N sulfuric acid solution followed by 2 L of demineralized water was passed through the top of the column at room temperature.

By this operation, 50% of the amount of protein adsorbed on the support was further desorbed 97% of iron content adsorbed on the support.

Next, the glucose isomerase extract A was again adsorbed to the resin after regeneration. The adsorption rate was 100% (adsorption amount 2400U / ml Resin).

Also, using this insoluble glucose isomerase, the isomerization reaction was continued according to the method of A, and finally, the adsorption-isomerization reaction of the glucose isomerase was repeated four times, and then the glucose When the isomerase extract A was adsorbed, the adsorption rate was lowered to 51% (adsorbed amount 1224 Uml Resin).

After using this insolubilized glucose isomerase agent for isomerization, 20 ml of each was taken and filled in another column, and the aqueous solution shown in Table 1 below was passed through from the top of the column to regenerate the resin.

In addition, the adsorption rate and the active expression rate of the insoluble glucose isomerase prepared by adsorbing the glucose isomerase extract to the resin after regeneration are also shown in this table. As apparent from this table, the adsorption capacity can be restored to the carrier on which glucose isomerase adsorption and the like by the regeneration method of the present invention are reduced.

TABLE 1

Examples 3 to 5 and Comparative Examples 1 to 5

An insoluble glucose isomerase agent [adsorption rate 100%, adsorption amount 2000U / ml Resin] prepared by adsorbing a strong basic anion exchange resin HPA 11 of SO ₄ type and adsorbing glucose isomerase extract B The column was packed and isomerized by the method of A. After the reaction was added, 500 ml of 1 N sulfuric acid, demineralized water, 1 N sodium hydroxide aqueous solution, 1 N sulfuric acid and 1 N sulfuric acid were sequentially passed from the top of the column at room temperature to regenerate the carrier. Subsequently, glucose isomerase extract B was adsorbed onto the carrier after regeneration to carry out isomerization reaction again.

Thus, the regeneration cycle of the adsorption-isomerization reaction-carrier of the glucose isomerase extract was repeated. As a regeneration method of the carrier in five cycles, 500 ml of the various regenerants and demineralized water shown in Table 2 were passed through the column to desorb the protein powder and iron powder adsorbed on the carrier.

Thereafter, glucose isomerase extract B was adsorbed on the carrier to prepare insoluble glucose isomerase. Iron desorption rates, protein desorption rates, adsorption rates and activity expression rates for adsorbed enzymes are also shown in Table 2.

TABLE 2

Claims (1)

Glucose isomerase is adsorbed and supported on the synthetic anion exchange resin, and an insoluble glucose isomerase agent used in the reaction for isomerizing glucose to fructose is used for hydrochloric acid, a mineral acid solution such as sulfuric acid, sodium hydroxide and potassium hydroxide. Decomposed components adsorbed on the resin by treating with an aqueous alkaline salt solution, an aqueous solution of an electrolyte salt such as sodium chloride, sodium sulfate, potassium sulfate or a mixture thereof, and then the ionic form of the resin is salted, and glucose isomerase is added thereto. A method for regenerating insoluble glucose isomerase characterized by adsorbing.