JPH0716411B2 - Immobilized enzyme - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial field of application> The present invention relates to an immobilized enzyme used for producing cyclodextrin.

<Prior Art> Cyclodextrin glucanotransferase produced by Bacillus macerans (hereinafter abbreviated as CGTase)
It has been known for a long time that it acts on potato starch, corn starch and the like to produce cyclodextrin (hereinafter abbreviated as CD). In addition, this CD contains α-CD consisting of 6 glucoses and β-C consisting of 7 glucoses.
D, γ-CD consisting of 8 glucoses, etc. are included.

By the way, conventionally, in the production of CD, α-amylase or CGTase was added to a starch suspension to liquefy it, and then these enzymes were inactivated by heating, and further CGTase was added to the liquefied starch solution to obtain a CD.
It is performed by a batch method of reacting for 24 hours.

<Problems to be Solved by the Invention> However, when a CD is produced by such a method, the reaction time is extremely long, and the CGTase used for producing the CD is a batch type, and therefore it can be reused. Instead of being disposable, there are drawbacks such as high enzyme costs.

Therefore, the inventors conducted various studies on immobilization of CGTase using an ion exchange resin, and proposed an immobilized enzyme in which a weakly basic anion exchange resin adsorbed CGTase in a specific range of amount (Japanese Patent Application No. 60-25118). did.

However, it has been found that the above CGTase-immobilized enzyme cannot withstand long-term use because the production amount of CD is significantly reduced when it is used for a long time. It is one of the important requirements that the immobilized enzyme can be used for a relatively long time, and the economical value of the immobilized enzyme that cannot withstand the long-term use is small.

Therefore, the present inventors have conducted intensive studies in order to obtain an immobilized enzyme having a CD-producing ability that can last for a relatively long time, and as a result, have a carrier for adsorbing CGTase, which has an alcoholic hydroxyl group, has a reduced performance. It was found to be extremely important for prevention.

<Means for Solving Problems> The present invention is based on these findings, and is characterized in that CGTase is adsorbed on a carrier having an anion exchange group and an alcoholic hydroxyl group of 0.5 mmol / g (dry resin) or more. And the immobilized enzyme.

<Operation> The present invention will be described in detail below.

As described above, when the CD is produced using the carrier in which the CGTase is adsorbed on the weakly basic anion exchange resin previously proposed by the present inventors, the CD is produced after the fifth day as shown in the examples described later. The production amount of abruptly decreases, and the production amount becomes 40% or less on the 7th day. As a result of diligent studies on the cause, it is considered that the polymer substance in the source liquid is responsible for blocking the pores of the ion exchange resin matrix. That is, the weakly basic anion exchange resin is a copolymer of styrene and divinylbenzene or a copolymer of acryl and divinylbenzene with an amine group, and since the matrix is hydrophobic, polymer substances are not adsorbed. It is easy, and the polymer substance once adsorbed is not easily desorbed. In particular, like a liquefied starch solution, a stock solution that suppresses the production of monosaccharides due to the hydrolysis of starch as much as possible contains a large amount of a high molecular weight high molecular weight substance, and the high molecular weight substance is easily adsorbed on the ion exchange resin. It will cause performance degradation.

On the other hand, as shown in Examples described later, when a carrier having an alcoholic hydroxyl group is used as the carrier, CD can be stably generated for an extremely long time.

It is considered that when the alcoholic hydroxyl group is added to the carrier, the carrier becomes hydrophilic, so that the polymer substance in the undiluted solution as described above is not adsorbed, or once adsorbed, it is immediately desorbed during the passage.

In any case, when a carrier having an alcoholic hydroxyl group is used, compared to the case of using a normal weakly basic anion exchange resin having no alcoholic hydroxyl group as a carrier, its CD-forming ability can be significantly prolonged. .

As the carrier used in the present invention, for example, unsaturated carboxylic acid glycidyl ester such as meta or acrylic acid glycidyl ester, crotonic acid glycidyl ester, etc. are reacted with a crosslinking agent such as divinylbenzene, ethylene glycol dimethacrylate, polyethylene glycol dimethacrylate, etc. An anion exchange group composed of a tertiary amine such as diethylaminoethyl or diethylaminopropyl or a quaternary amine such as trimethylylamine or tripropylamine is added to the thus obtained matrix. In order to add an alcoholic hydroxyl group, a material having an alcoholic hydroxyl group from the beginning is used as a raw material used for polymerizing the mother body, or when adding an anion exchange group to the mother body or after adding an anion exchange group. Thus, the alcoholic hydroxyl group can be added in various processes for producing the carrier.

For example, one production example of a carrier having an anion exchange group and an alcoholic hydroxyl group used in the present invention is shown below.

That is, when glycidyl methacrylate is reacted with dimethylaminoethanol to introduce an amine group, the following reaction occurs. The epoxy ring is opened to introduce an amine group and an alcoholic hydroxyl group is generated.

The carrier used in the present invention is one in which an alcoholic hydroxyl group is added as described above, but if the addition amount is small, the object of the present invention cannot be achieved. For example, if the added amount of alcoholic hydroxyl groups is less than 0.5 mmol / g (dry resin), the duration of the CD-forming ability is short, so at least 0.5 mmol / g (dry resin) or more alcoholic hydroxyl groups should be applied to the carrier. Need to be added.

Next, as the matrix structure, there are a gel type and a giant network structure (MR type), and the latter is advantageous because the enzyme is easily adsorbed because the pore size of the particle is larger. The carrier has a particle size of 0.05 to 0.6 mm, preferably 0.1 to 0.6 mm.
0.2mm is preferable. That is, if the particle size is too small, when the immobilized enzyme is packed in a column and the stock solution is passed through this column, pressure loss increases. On the other hand, if the particle size is too large, the specific surface area becomes small and the amount of enzyme to be adsorbed becomes small, so that the expected performance cannot be obtained.

Next, explaining the method of adsorbing CGTase on the carrier, first, the carrier is regenerated with an alkaline solution to convert the exchange group into a free base form or a hydroxide compound ion form, and then a buffer solution having a pH of about 6 such as acetic acid. Pre-treatment is performed by washing with sodium acetate solution, phosphoric acid / sodium phosphate solution. CGTase is brought into contact with and adsorbed on a fixed amount of the carrier thus prepared.

As the method of contacting the carrier with CGTase, the carrier used in the present invention and the enzyme solution are put in a container and adsorbed while stirring by a batch method, or the carrier is packed in a column, and the enzyme solution is flown downward or upward. To pass. In this case, the effluent may be recirculated and adsorbed. The contact time is 0.
It is adsorbed in 5 to 4 hours, preferably about 1 hour.

Next, the amount of CGTase adsorbed on the carrier used in the present invention will be described.

Conventionally, when immobilizing an enzyme by adsorbing it on a carrier such as a weakly basic anion exchange resin, the larger the adsorption amount of a normal enzyme such as a hydrolase or an isomerase, the higher the titer of the immobilized enzyme obtained. Is usually large, and the performance of the immobilized enzyme is also excellent.

However, since the CGTase is a transferase, it behaves differently from general enzymes, and an increase in the adsorbed amount does not necessarily lead to an increase in performance, and it is also found that if the adsorbed amount is excessively increased, the performance is decreased. did.

FIG. 2 shows that the carrier used in the present invention was loaded with immobilized enzymes in which various amounts of CGTase were adsorbed on each column, and 4% liquefied starch solution was passed through at a flow rate of SV1 to SV3. It shows the amount of CD produced.

As is clear from FIG. 2, the amount of CGTase adsorbed per unit resin is small or large, but the amount of CD produced in the treatment liquid is small. That is, when the adsorbed amount of CGTase is small, the enzyme activity is weak and CD is not sufficiently produced, and when the adsorbed amount exceeds a certain value, the enzymatic activity is too strong and the CD once produced is decomposed.

Therefore, the adsorption amount of CGTase adsorbed on the carrier is 0.2 to 5 mg (activity 15 to 15 mg as protein per 1 g of wet resin).
It is better to adsorb it within the range of 360 IU), preferably 0.5 ~
It is advisable to adsorb in the range of 2 mg. The wet resin in the present invention refers to the same state as a generally commercially available ion exchange resin that adsorbs water having a water content of about 50 to 60%.

<Effects> As described above, the carrier used in the present invention can adsorb CGTase very easily, and the adsorbed CG
Tase can be continuously produced by a very simple operation without desorbing Tase, and by packing the immobilized enzyme in a column and passing a liquefied starch solution through it. The phosphatase can maintain extremely stable performance over a very long period of time.

Therefore, compared to the batch method or the conventional immobilized enzyme of CGTase, the consumption of the enzyme is extremely small, and CD can be economically produced.

Examples will be described below in order to further clarify the effects of the present invention.

Example-1 By adding tripropylamine to a polymer of glycidyl methacrylate and ethylene glycol dimethacrylate, 1.42 meq and 1.85 mmol of anion exchange group and alcoholic hydroxyl group were added per 1 g of dry resin, respectively. CGTa on a carrier with a particle size of about 0.18 mm by the following procedure
se was adsorbed.

That is, 4 g of the carrier in a column with a diameter of 10 mm and a height of 200 mm.
(5.2 ml), then 25m of 1N sodium hydroxide solution
After passing l, the solution was washed with water to give the carrier in the OH form. Then, pretreat by passing 500 ml of 1/10 M acetic acid / sodium acetate buffer solution (pH 6.0), remove the pretreated carrier from the column, put it in a 100 ml beaker, and add CGTase solution (active 6
1.2 IU / ml) 6 ml and pure water 2 ml were added and reacted for 1 hour with stirring to adsorb the enzyme. The immobilized enzyme thus obtained was 0.68 mg of CGTa as protein per 1 g of wet carrier.
se is adsorbed. This immobilized enzyme was packed in the column again, and 200 ml of the above-mentioned buffer solution was passed through, and then 4% liquefied starch solution was passed through this column at a temperature of 50 ° C. and a flow rate of SV1 to produce CD production over a long period of time. It was measured.

For comparison, weak base anion exchange resin Amberlite
C using IRA-93 in the same manner as the carrier used in the present invention.
GTase was adsorbed and then passed through in the same manner as described above to measure the amount of CD produced in the treatment solution. The results are shown in FIG.

As shown in FIG. 1, the immobilized enzyme of the present invention has extremely excellent performance as compared with the conventional immobilized enzyme having a weakly basic anion exchange resin as a carrier.

Example-2 In the same carrier used in the present invention as used in Example-1, 0.29 as a protein per wet carrier was prepared by the same method as in Example-1.
Four kinds of immobilized enzymes adsorbing mg, 0.68 mg, 1.0 mg, 5.0 mg of CGTase were prepared, these immobilized enzymes were packed in a column, and a 4% liquefied starch solution at a temperature of 50 ° C and a flow rate SV1 ~ The solution was passed through in step 3, and the amount of each CGTase adsorbed and the amount of CD produced by the treated solution in each SV were measured. The results are shown in FIG. The CD production amount means the weight when starch is changed to CD.

As shown in FIG. 2, it is shown that the CD production amount becomes small when the adsorption amount of CGTase is too small or too large at each flow rate.

[Brief description of drawings]

1 and 2 show the results of the examples,
FIG. 1 is a graph showing the amount of CD produced when the immobilized enzyme of the present invention and a conventional immobilized enzyme using a weakly basic anion exchange resin as a carrier are packed in a column and a liquefied starch solution is passed through the column. The vertical axis shows the CD production amount, and the horizontal axis shows the liquid passage time. The second
The figure is a graph showing the relationship between each flow rate, the amount of enzyme adsorbed, and the amount of CD produced in the immobilized enzyme of the present invention. The vertical axis shows the amount of CD produced and the horizontal axis shows the amount of enzyme produced.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kozo Hara 2-6-3-104 Namiki, Kanazawa-ku, Yokohama-shi, Kanagawa

Claims (2)

[Claims] 1. Anion exchange group and 0.5 mmol / g (dry resin)
An immobilized enzyme characterized in that cyclodextrin glucanotransferase is adsorbed on the carrier having the above alcoholic hydroxyl group. 2. Cyclodextrin glucanotransferase as a protein in an amount of 0.2 to 5 mg per 1 g of a wet carrier.
The immobilized enzyme according to claim 1, which is adsorbed in the range of.