JPH0458312B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to an immobilized enzyme used in producing cyclodextrin.

<Conventional technology> Cyclodextrin glucanotransferase (hereinafter referred to as
It has long been known that CGTase (abbreviated as CGTase) acts on potato starch, corn starch, etc. to produce cyclodextrin (hereinafter abbreviated as CD). Further, this CD includes α-CD consisting of 6 glycoses, β-CD consisting of 7 glycoses, γ-CD consisting of 8 glycoses, etc.

By the way, conventionally, CD production has been carried out by adding α-amylase or CGTase to a starch suspension and liquefying it, then inactivating these enzymes by heating, and then adding CGTase to the liquefied starch solution and allowing it to react for about 24 hours. It is done by law.

<Problems to be solved by the invention> However, when producing CDs using this method, the reaction time is very long, and the CGTase used for CD production is batch-type, so it cannot be reused. Since it is disposable, it has drawbacks such as high enzyme costs.

Therefore, the present inventors conducted extensive research on the immobilization of CGTase for the purpose of continuous production of CD and effective use of enzymes, and as a result, CGTase was extremely effectively adsorbed and immobilized on weakly basic anion exchange resin. We also found that the amount of CGTase immobilized per unit resin greatly affects the amount of CD produced, and that the intended purpose could not be achieved unless adsorption was within a certain range.

<Means for Solving the Problems> The present invention is based on these findings, and contains CGTase produced from Bacillus macerans as a protein in the range of 0.5 to 30 mg per gram of wet resin of the weakly basic anion exchange resin. This invention relates to an immobilized enzyme characterized by being adsorbed.

<Effect> The present invention will be explained in detail below.

The weakly basic anion exchange resin used in the present invention has polyamine, primary/secondary amine, tertiary amine, etc. as the main exchange group, and the base of the resin is a copolymer of styrene and divinylbenzene, a copolymer of acrylic and divinylbenzene, etc. It is a copolymer or phenolic type, and Amberlite (registered trademark) IRA
−93, IRA−94, IRA−68, IRA−47, IRA−
35, IR-45, Diaion (registered trademark) WA10,
WA20, WA30, Revachit (registered trademark) MP64,
You can use MP62 or something similar.

There are various types of weakly basic anion exchange resins, ranging from those with strong basicity to those with weak basicity. Although sometimes referred to as a resin, such weakly basic anion exchange resins with relatively strong basicity can also be used in the present invention. Furthermore, although there are two types of parent structures, a so-called gel type and a giant network structure (MR type), the latter is more advantageous because the pore size of the particles is larger, so that enzymes can be easily adsorbed. The particle diameter of the ion exchange resin used is 0.05 to 0.6 mm, preferably 0.1 to 0.2 mm. That is, if the particle size is too small, pressure loss will increase when the immobilized enzyme is packed into a column and the liquefied starch is passed through the column. On the other hand, if the particle size is too large, the surface area becomes small, the amount of enzyme to be adsorbed becomes small, and the volume of the immobilized enzyme becomes large, which is economically disadvantageous.

Next, to explain the method of adsorbing CGTase onto a weakly basic anion exchange resin, first, the ion exchange resin is regenerated with an alkaline solution to make the exchange group into a free base type, and then a buffer solution with a pH of around 6, such as acetic acid Perform pretreatment by washing with sodium solution and phosphoric acid/sodium phosphate solution. To a certain amount of the ion exchange resin prepared in this way,
Contact and adsorb CGTase. The amount of CGTase added is 0.05 to 1.5 mg/ml as protein [activity 5 to
Use an enzyme solution with a concentration of 250 THU (TildenHudson units/ml) in an amount approximately 10 times the volume of resin.
Preferably 0.15 to 0.4 mg/ml as protein (active
It is recommended to use an enzyme solution with a concentration of 20 to 60 THU in an amount 10 times the amount of resin. Further, as a contact method, a resin and an enzyme solution are placed in a container and adsorbed while stirring in a batch method, or the resin is packed in a column and the enzyme solution is passed in a downward or upward flow. In this case, the effluent may be recirculated for adsorption, and the contact time is 0.5 to 4 hours, preferably about 1 hour.

Next, adsorb it on a weakly basic anion exchange resin.
Explain the amount of CGTase.

Conventionally, when enzymes are adsorbed and immobilized on carriers such as ion-exchange resins, the larger the amount of normal oxygen adsorbed to the ion-exchange resin, such as hydrolytic enzymes and isomerases, the better the immobilization obtained. Usually, the titer of the enzyme is high, and the performance of the fixed titer enzyme is also excellent.

However, since the CGTase is a transferase, its behavior differs from that of general enzymes, and it has been found that increasing the amount of adsorption does not necessarily lead to an increase in performance, and that increasing the amount of adsorption excessively actually reduces its performance. did.

Figure 1 shows the results when a column was filled with immobilized enzymes with various amounts of CGTase adsorbed on weakly basic anion exchange resin Amberlite IRA-93, and a 4% liquefied starch solution was passed through the column at a flow rate of SV1 to SV3. in the processing solution.
This shows the amount of CD produced.

As is clear from Figure 1, per unit resin
Whether the amount of CGTase adsorbed is small or large, the amount of CD generated in the treatment solution will be reduced. That is,
When the adsorption amount of CGTase is small, the enzyme activity is weak and CD
If not enough is produced and the amount of adsorption exceeds a certain value, the enzymatic activity will be too strong and the once produced CD will be decomposed.

Therefore, the amount of CGTase adsorbed on the weakly basic anion exchange resin is 1
0.5 to 30 mg of protein per g (activity 70 to
5000 THU), preferably in the range of 1 to 20 mg. The term "wet resin" in the present invention refers to a commercially available resin that has adsorbed water with a moisture content of about 50 to 60%. CGTase is also adsorbed to strongly basic anion exchange resins or synthetic adsorbents, and exhibits the same behavior as the weakly basic anion exchange resins, but its CD production is inferior to the latter, making it undesirable as a carrier.

<Effect> As explained above, by passing the liquefied starch solution through the alum filled with the immobilized enzyme of the present invention,
Since CDs can be produced continuously, the operation for producing CDs is simple, and when the immobilized enzyme of the present invention is packed in a column and used, it is possible to adsorb CGTase so that the amount of CDs produced is increased as much as possible. Since the amount is adjusted, it is possible to produce CD most economically without wasting the enzyme.

Examples will be described below to make the effects of the present invention more clear.

Example 1 Weakly basic anion exchange resin Amberlite IRA
Fill a column with a diameter of 10 mm and a height of 200 mm with 5 ml of -93 particles with a particle size of approximately 100 mesh. Next, pass 100 ml of IN-sodium hydroxide solution through the solution, and wash with water to obtain a free base form. Next, administer one dose of 1/10M acetic acid/sodium acetate buffer (PH6.0) solution.
After washing with water, remove this resin from the column and place it in a 100ml beaker, and add the CGTase enzyme solution (protein
3.05mg/ml, activity 475THU/ml) 5ml and pure water 44ml
Add and react for 1 hour while stirring with a stirrer (approximately 200rpm) to adsorb the enzyme. This immobilized enzyme is packed into the column again, and 20 ml of the aforementioned buffer solution is passed through it. After washing with water, a 4% liquefied starch solution was passed through this column at a temperature of 50°C and a flow rate of SV1.
When the amount of CD produced in the treatment solution was measured, it was 4.32%. Note that the amount of CD produced refers to the percentage by weight when starch is converted to CD.

For comparison, instead of weakly basic anion exchange resin, strong basic anion exchange resin Amberlite was used.
Exactly the same method except using IRA-900
When CGTase was adsorbed and then a liquefied starch solution was passed under the same conditions, the CD concentration of the treated solution was 2.3%, and the strongly basic anion exchange resin produced much less CD than the weakly basic anion exchange resin. Ta.

Example 2 Using the same method as in Example 1, 0.15 mg, 0.7 mg of protein was added to Amberlite IRA-93, the same weakly basic anion exchange resin used in Example 1, per 1 g of wet resin of the weakly basic anion exchange resin. 2.9mg,
Six types of immobilized enzymes with 5.8 mg, 15 mg, and 25 mg of CGTase adsorbed were prepared, these immobilized enzymes were packed into a column, and 4% liquefied starch was added at a flow rate of 50°C.
Pass the liquid through SV1 to 3, and measure the adsorption amount of each CGTase and each SV.
The amount of CD produced in the treatment solution was measured. The results are shown in FIG. Note that the CD production amount refers to the amount of CD produced by starch.
It shows the weight % when changing to.

As seen in Figure 1, each flow velocity
Even if the amount of CGTase adsorbed is too small or too large,
It has been shown that the amount of CD produced is reduced.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the relationship between the amount of enzyme adsorption and the amount of CD produced at each flow rate, with the vertical axis showing the amount of CD produced and the horizontal axis showing the amount of enzyme adsorption.

Claims (1)

[Claims] 1. An immobilized enzyme characterized in that cyclodextrin glucanotransferase produced from Bacillus macerans is adsorbed as a protein in the range of 0.5 to 30 mg per gram of wet resin of a weakly basic anion exchange resin.