JPH0771489B2 - Method for producing transfer sugar - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing a transfer sugar using immobilized cyclodextrin glucanotransferase (abbreviated as CGTase).

According to the method of the present invention, CGTase can selectively undergo a transglycosylation reaction and can efficiently produce neotrehalose, nigerose, kojibiose and the like.

The term "glycosylation reaction" as used herein refers to α-1,4 glucans such as maltose and maltotriose, and α-1,2 (corgibiose), α-1,3 (nigerose), α-1,4 (maltose). , Α-1,6 (isomaltose), α-1,1-β (neotrehalose) and other disaccharides, and the action of producing sugars more than trisaccharides, especially maltose neotrehalose, nigerose, kojibiose, etc. Means a reaction to produce a transfer sugar.

CGTase is essentially an intramolecular transfer of a cyclic sugar (cyclization), an intramolecular transfer of a ring-opening sugar transfer (couping), and a linear sugar intermolecular sugar transfer (disproportion).
ation) and hydrolysis. Since these reactions occur at the same time, various sugars were produced and transfer sugar could not be produced selectively.

[Conventional technology]

To date, various methods have been developed for the immobilization of enzymes, and there are many research cases. For CGTase, see T. Kato a.
nd K. Horikoshi: Immobilized Cyclodextrin Glucanotra
nsferase of an Alkalophilic Bacillus sp.No.38-2, C
abohydrate Research.26,595 (1984), JP-A-63-4269
There is No. 7 bulletin, etc.

However, these methods simply use CGTase
Immobilize on polyacrylonitrile, silica gel, etc.,
The purpose is to express the original CGTase activity as a whole and produce cyclodextrin.
The various actions of CGTase could not be expressed individually.

In addition, the inventors of the present invention have so far developed a Bacillus macerans CGT.
It was reported that the action of ase on starch sugar produced oligosaccharides having a bond other than α-1,4 bond.
A method for producing 1-β bond, a non-reducing disaccharide that is rarely found in the natural world, has been established (JP-A-63-216492).

[Problems to be Solved by the Invention]

Therefore, the present inventors first established a method for individually measuring various actions of CGTase. That is, for cyclodextrin synthetic activity which is an intramolecular transfer, soluble starch as a substrate is subjected to glucoamylase treatment after the reaction is carried out, and all dextrins other than cyclodextrin are converted to glucose, and then silica-NH ₂ type high speed liquid using a column is used. The method for quantifying cyclodextrin by chromatography was used, and for the intermolecular transglycosylation activity, the amount of glucose released using maltose as a substrate was quantified by the glucose oxidase method or high performance liquid chromatography.

Using this method, two types of reaction rates were measured for all immobilized CGTases, an immobilized enzyme agent that selectively expresses either action was found, and earnestly studied for the purpose of preparing it. Have been piled up.

[Means for Solving the Problems]

Therefore, the present inventors collected various immobilized substrates, immobilized CGTase by various methods, and examined using two activity measurement methods. As a result, the activity of CGTase was determined by selecting an immobilization carrier. It was found that they mainly express cyclodextrin synthesizing activity and intermolecular transglycosylating activity, which are internal transfer.

Up to now, the activity of CGTase immobilized on a resin having a hydrophobic binding mode was usually detected by the starch-iodination reaction, so that only glycosyl transfer activity and cyclodextrin synthetic activity were expressed. Even though I was doing it, it was judged as negative.

Since the activity ratios of the CGTase from the crude enzyme to the purified preparation were the same in each purification step, it is suggested that these active sites exist on the same enzyme protein.

The present invention is shown below.

1) A cyclodextrin glucanotransferase immobilized with an anion exchange resin and / or chitosan beads is allowed to act on maltose or a maltose mixture to cause a transglycosylation reaction,
A method for producing transglycosylation, which comprises expressing the transglycosylation reaction at a rate of 30% or more to produce neotrehalose, nigerose or kojibiose.

Although anion exchange resins and chitosan beads were mainly used as the solidified substrate, other immobilized substrates can be applied to the method of the present invention.

Examples of anion exchange resins include commercially available Amberlite from Organo, Diaion from Mitsubishi Kasei, and Dowex from Dow Chemical.

Chitosan is a polysaccharide with a cellulose-like molecular structure in which glucosamine is linked to β-1,4.Since it has an amino group in its molecule, it is easy to use a cross-linking agent such as glutaraldehyde. It is possible to immobilize proteins such as enzymes. Recently, a product in which chitosan is molded into a bead shape, and a beaded product in which chitosan is subjected to various crosslinking treatments to improve physical strength and acid resistance are commercially available.

The immobilization method will be described below, but the immobilization substrate and immobilization method are not limited to those described below, and any carrier that can immobilize CGTase can be used in the method of the present invention.

When an anion exchange resin is used as the immobilization carrier, CGTase, such as Bacillus maceran, is added to 1 g of the simple substance in the adsorption method.
s CGTase (400 international units (cyclodextrin synthetic activity)) was added, and the mixture was shaken at 30 ° C for 2 hours, and then 0.1 M acetate buffer (pH 6.
It was washed with 0) to obtain the immobilized enzyme.

In order to strengthen the immobilization, in the method using glutaraldehyde as a cross-linking agent, 1 ml of 5% glutaraldehyde is added to 1 g of the carrier, shaken at 30 ° C for 2 hours, and washed with a buffer solution as in the adsorption method. , 400 enzymes of Bacillus macerans
A unit was added, and the mixture was shaken for 2 hours and washed with a buffer solution to obtain an immobilized enzyme.

An immobilized enzyme was obtained in the same manner when chitosan beads were used.

When using a cross-linking agent, glutaraldehyde is widely used as a cross-linking agent, but in addition to this, bisdiazobenzidine, hexamethylene diisocyanate, toluene diisocyanate, hexamethylene diisothiocyanate, N,
N-ethylene bismaleimide and the like can be selected or used in combination. When genipin is used as a crosslinking agent, genipin is 1% (w /
v), and the crosslinking reaction between the carrier and the enzyme was carried out at 40 ° C overnight.

The method of the present invention can also be applied to various other immobilization methods such as a covalent bond method, an ionic bond method, a physical adsorption method, a crosslinking method, and an encapsulation method. In addition, according to the present invention, CGTase can be immobilized by immobilization in combination with other immobilization substrates and / or various crosslinking agents.
The possibility of transforming the action is fully predictable.

The enzyme agent for immobilization is not limited to Bacillus macerans, but also B. licheniformis, B. megaterium, B. ohbensis, B.stea.
CG Tas such as rothermophilus and alkalophilic Bacillus sp.
Any product derived from e-producing bacteria can be used, and various purification stages from purified enzyme to crude enzyme preparation are used, but in order to increase the activity per carrier, partial purification by starch adsorption / desorption was performed. It is desirable to use an enzyme preparation and further an enzyme preparation purified by salting out with ammonium sulfate.

Table 1 shows the results of investigating the protein immobilization rate and the activity expression rate of the immobilized enzymes obtained by these methods. In the table, CER is a cation exchange resin, AER is an anion exchange resin, and CB is chitosan beads. The product treated with glutaraldehyde is shown in (G). The protein immobilization rate was determined by measuring the amount of protein in the washing solution and subtracting it from the amount of added protein.The enzyme activity was measured using the above two methods and expressed as the expression rate when the initial activity was 100%. It was Indicates cyclodextrin synthetic activity, and indicates transposition activity. The activity indications here are 1 μmol of cyclodextrin synthesizing ability and glucose producing ability per minute.

Regarding the cation exchange resin, the protein adsorption rate was high in all cases. Regarding the activity expression rate, No. 1 showed a high value for cyclodextrin synthetic activity, but the others showed only trace levels and no metastatic activity was expressed.

For anion exchange resins, the protein adsorption rate differs depending on the carrier, but the activity expression rate after immobilization shows almost no cyclodextrin synthetic activity regardless of the presence or absence of glutaraldehyde treatment, and the transfer activity is 30-40%. The expression rate was%.

The chitosan beads all showed a protein adsorption rate of about 90%, showed almost no cyclodextrin synthetic activity like the anion exchange resin, and had a high activity expression rate of about 50% on the transfer activity.

Thus, roughly, CGTase can be immobilized by anion exchange resin and / or chitosan beads and converted into an enzyme that selectively expresses transglycosylation reaction, and immobilized by cation exchange resin to selectively perform cyclization reaction. It can be converted into an enzyme expressed in.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

〔Example〕

Production Example 1 400 g of a purified enzyme of Bacillus macerans (cyclodextrin synthetic activity) was added to 1 g of a carrier (manufactured by Mitsubishi Kasei, anion exchange resin, WA-20), and the mixture was shaken for 2 hours and washed with an acetate buffer. , Protein immobilization rate 45%, metastasis activity expression rate
41% immobilized enzyme was obtained.

The expression rate of cyclodextrin synthesis activity of this immobilized enzyme is
Remarkably low at 0.2%.

Production Example 2 1 ml of 5% glutaraldehyde (crosslinking agent) was added to 1 g of a carrier (Mitsubishi Kasei, anion exchange resin, WA-20),
After shaking for 2 hours and washing with an acetate buffer, an immobilized enzyme having a protein immobilization rate of 64% and a translocation activity expression rate of 47% was obtained in the same manner as in Production Example 1.

The expression rate of cyclodextrin synthesis activity of this immobilized enzyme is
Remarkably low at 0.4%.

Production Example 3 A protein immobilization rate of 91% was obtained in the same manner as in Production Example 2 except that a carrier (Fuji Spinning Co., Ltd., Chitosan beads, BCW-2510) was used.
An immobilized enzyme having a transfection activity expression rate of 48% was obtained.

The expression rate of cyclodextrin synthesis activity of this immobilized enzyme is
Remarkably low at 0.1%.

Example 1 When 20% maltose was used as a substrate and the immobilized enzyme prepared in Production Example 1 was allowed to act at pH 6.0 and 50 ° C., a transfer sugar containing neotrehalose, nigerose and kojibiose was efficiently produced.

〔The invention's effect〕

By immobilizing CGTase on a selected carrier, the reaction mode of the enzyme is changed and α-
The synthesis reaction of transfer sugars including neotrehalose, nigerose, and cordiose from 1,4 glucan is mainly caused, and these can be selectively produced. Further, the enzyme can be repeatedly used, and the economical efficiency is remarkably improved.

Therefore, according to the present invention, food materials such as neotrehalose, nigerose and kojibiose can be continuously and efficiently manufactured. In addition, in order to increase the purity of these transfer sugars, a combination of an immobilized enzyme reaction step and a commercially available gel filtration agent, ion exchange resin, column chromatography such as activated carbon, or membrane separation concentration using various membranes is used. Can also be considered.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Toshiya Takano 5-25-2 Shirayama, Toride-shi, Ibaraki Prefecture (72) Masao Shioda 2-2-20 Ninomiya, Tsukuba-shi, Ibaraki Prefecture Maison Science No. 202 (72) ) Inventor Nobuya Yamu 2821, Isusu, Kamisu-cho, Kashima-gun, Ibaraki Prefecture No. 2-209, Showa Sangyo Kamisu Shrine, 2-209 (72) Inventor, Hajime Takaku 1-40-12 Minamiono, Ichikawa-shi, Chiba 108, Showa-san, Ichikawa 56) References JP-A-61-185188 (JP, A) JP-A-61-185196 (JP, A) JP-A-63-44886 (JP, A) JP-A-63-196290 (JP, A)

Claims (3)

[Claims] 1. A cyclodextrin glucanotransferase immobilized using an anion exchange resin and / or chitosan beads is allowed to act on maltose or a maltose mixture to cause mainly a transglycosylation reaction to bring about 30% of the transglycosylation reaction. A method for producing a transfer sugar, which comprises expressing neotrehalose, nigerose or kojibiose by expressing at the above ratios. 2. The method according to claim 1, wherein glutaraldehyde is used for immobilizing the enzyme. 3. Maltose or maltose mixture 20 w /
The method according to claim 1, which is used at a concentration of w% or more.