JP2795281B2 - Immobilized enzyme - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention <relates> relate immobilized enzyme used in producing maltopentaose (hereinafter shortly referred G _5).

<Prior Art> Bacillus licheniformis origin, Bacillus cereus origin, G ₅ generates enzyme potato starch bacteria such as Pseudomonas · spKO-8940 origin produced, to generate the G ₅ acts on the corn starch and the like known Have been. Also the conventional G ₅
Manufacturing is liquefied by adding α- amylase to the starch suspension, followed After inactivating the enzyme, G ₅ in the liquefied starch solution
It is performed by a batch method in which a production enzyme is added and a reaction is performed for a long time. Further still the G ₅ forming enzyme was adsorbed on a polymer carrier such as adsorption resin, by filling the column are also employed a method for obtaining the reaction product was passed through a liquefied starch solution to this.

However, the batch method requires a long time for the reaction, and the enzyme cannot be reused.

In the method of adsorbing and immobilizing an enzyme on a carrier, the enzyme is easily desorbed due to the weak adsorbing power to the carrier, which makes it difficult to operate for a long time. The polymer substance is adsorbed to the carrier by a hydrophobic bond, and closes the pores of the carrier, which causes a so-called organic contamination, which makes it difficult to operate for a long time.
One of the important requirements is that the immobilized enzyme can be used for a relatively long time, and an immobilized enzyme that cannot withstand long use has a small economic value.

<INVENTION AND SUMMARY Problems> The present invention solves the conventional problems described above, G ₅ forming enzyme was adsorbed on the carrier is not disengaged during the enzyme reaction solution, maintaining high activity for a long time and to provide a G ₅ generates immobilized enzyme capable.

<Means for Solving the Problems> In order to achieve the above-mentioned object, the present inventors have conducted intensive studies, and as a result, have found that a carrier having a large network structure composed of a polymer of unsaturated carboxylic acid glycidyl ester is used as a carrier. with a basic anion-exchange resins having a primary amine as an ion exchange group, the immobilized enzyme with intervening glutaraldehyde to the carrier as a crosslinking agent was obtained by combining the G ₅ generates enzyme in the enzyme reaction solution It has been found that high activity can be maintained for a long time without separation.

The present invention is based on this finding, and uses a basic anion exchange resin having a primary amine as an ion exchange group as a carrier in a matrix having a large network composed of a polymer of unsaturated glycidyl carboxylate as a carrier, Glutaraldehyde as a cross-linking agent in primary amines
It relates immobilized enzyme, characterized in that to bind the G ₅ forming enzyme.

<Operation> Hereinafter, the present invention will be described in detail.

As described above, using an immobilized enzyme having adsorbed bind G ₅ forming enzyme in the synthetic resin, as shown in Examples to be described later to produce a G _5, generation rate G ₅ is 2 or 3 days suddenly drops Will come. This is considered to be due to the fact that the bond between the carrier and the enzyme is an ionic bond, so that the bond is weak and the adsorbed enzyme is released.

Carrier used in the present invention is not to adsorbed by ionic binding to G ₅ forming enzyme, via glutaraldehyde to a primary amine, G ₅ generates enzyme firmly so immobilizing the G ₅ forming enzyme by the so-called covalent bond Can be held.

First, the carrier used in the present invention will be described. For example, the carrier was polymerized with glycidyl methacrylate or glycidyl acrylate, glycidyl crotonate using ethylene glycol dimethacrylate or polyethylene glycol dimethacrylate as a crosslinking agent. A, a matrix having a huge network consisting of a polymer of unsaturated glycidyl carboxylate,
For example, a primary amine is added as an ion exchange group by reacting ethanolamine, propanolamine and ammonium.

The reaction formula when ethanolamine is reacted with a base obtained by polymerizing glycidyl methacrylate and the above-mentioned crosslinking agent is as shown in the following formula (1).

As shown in the formula (1), the epoxy ring is cleaved during the exchange group introduction reaction, and an exchange group consisting of a primary amine is added to one side, and an alcoholic hydroxyl group is generated on the other side. Is obtained, which is excellent as a carrier used for the enzyme reaction.

The physical structure of the matrix having a huge network structure is an average particle diameter of 0.2 to 1 mm, a pore diameter of 100 to 2,000 mm, and a pore volume of about 0.5 to 1.5 ml / g.

Referring now to an example of the immobilization method of the G ₅ forming enzyme, it first contacted the buffer pH8.0 to the carrier, the carrier is buffered and then dissolved in the same buffer 0.5-10
% Glutaraldehyde solution and 0.5 ~ at 4 ~ 40 ℃
Contact for 2 hours, then wash off excess glutaraldehyde with buffer. In this way, the adjusted carrier and G _{5-synthesizing} enzyme solution and a 1 hour or more, preferably washed away after immobilizing the G ₅ forming enzyme by contacting 2-5 hours, the excess G ₅ forming enzyme in buffer Can be obtained by G ₅ The immobilization amount of generated enzymatic 150 IU / g- wet carrier or as good as the enzyme activity, and preferably from a range of 300~500IU / g- wet carrier.

The reaction of bonding an enzyme with glutaraldehyde as a cross-linking agent on a primary amine of a carrier as described above is exemplified as in the formulas (2) and (3).

Carrier _{-NH 2 + OHC- (CH 2)} 3 -CHO → carrier _{-N = CH- (CH 2) 3} -CHO ...... (2) carrier _{-N = CH- (CH 2) 3} -CHO + enzyme → carrier -N = CH- (CH ₂ ) ₃ -CH = N-enzyme (3) As a method for contacting the carrier with glutaraldehyde or a solution, the column is filled even in a batch method and the solution is passed through an ascending or descending flow. May be implemented.

Then using an immobilized enzyme of the present invention obtained as described above, when describing a process operation for generating the G ₅ from starch, and filling to form a packed bed of immobilized enzyme immobilized enzyme reactor , it is possible to obtain a processing solution containing G ₅ in the effluent and the substrate to the filler layer passing liquid downflow or upflow. The flow rate depends on the amount of the immobilized G5 producing enzyme, but is preferably about SV 0.1 to ₅ .

Immobilized enzyme of the present invention is as shown in (2) and (3), since G ₅ forming enzyme is covalently conjugated via glutaraldehyde to a primary amine of a carrier, different from the ionic bond its binding force is strong, hence G ₅ forming enzyme from the carrier during the enzymatic reaction is never disengaged.

When such an enzymatic reaction is continued for a long time,
G ₅ forming enzyme is inactivated, but when brought into contact with warm alkaline solution in this case immobilized enzyme, it is possible to desorb the G ₅ forming enzyme was deactivated from the carrier.

That is, first, the immobilized enzyme used in the reaction is washed with water, and then an alkali solution having a concentration of 0.5 to 10%, particularly preferably 2 to 5%, such as sodium hydroxide, potassium hydroxide, or sodium carbonate solution is heated to 40 to 70 ° C. at 0.5 hours or more, preferably in contact for 1 to 3 hours to desorb G ₅ forming enzyme together with glutaraldehyde, washing the carrier.

Thereafter buffered carrier in the manner described above, to implement the glutaraldehyde treatment, and re-fixed in contact with G ₅ forming enzyme solution, subjected to generation of G _5. Incidentally method of contacting alkali solution upon inactivation G ₅ of forming enzyme desorption may be either be a method of passing liquid alkaline solution or packed bed also batchwise.

Immobilized enzyme of the present invention as has been <Effects> description, carrier and G ₅
Since glutaraldehyde is interposed between the produced enzymes as a cross-linking agent and immobilized by so-called covalent bonds,
Its binding force is strong, there is no separation of G ₅ forming enzyme in the enzyme reaction and no organic contamination since the carrier is a hydrophilic, it is possible to maintain a high activity over a long period of time.

The use of immobilized enzymes of the present invention therefore, since G ₅ forming enzyme as a batch method is not a disposable one-time, desorbs less its consumption, and the G ₅ forming enzyme was deactivated from the carrier As a result, the carrier can be reused, and when these are combined, the economic merit is very large.

Hereinafter, examples of the present invention will be described in order to clarify the effects of the present invention.

Reference Example (Carrier Manufacturing Method) A mixed solution of 200 g of glycidyl methacrylate, 50 g of ethylene glycol dimethacrylate, 2 g of benzoyl peroxide and 250 g of toluene was mixed with 2 g of polyvinyl alcohol.
Was added to 1,000 ml of dissolved water. The mixture was reacted at 60 ° C. for 4 hours with stirring to carry out polymerization. After cooling, the product was filtered, washed, and vacuum dried at 60 ° C. for 16 hours to obtain 205 g of a white opaque spherical resin.

100 g of the obtained spherical resin was added to 500 ml of ethanolamine, and reacted by stirring at 110 to 120 ° C. for 6 hours. After cooling, the product was filtered, washed and vacuum dried at 60 ° C. for 16 hours to obtain 117 g of the product.

The particle size of this resin is 100 to 500 μm, the pore volume measured by the mercury porosimeter method is 112 ml / g dry resin, and the specific surface area based on pores having a pore diameter of 100 mm or more is 53.3 m.
² / g dry resin.

EXAMPLES Pseudomonas spKO- was prepared by the following procedure for a carrier having a primary amine as an ion-exchange group in a matrix having a large network consisting of a polymer of unsaturated glycidyl carboxylate obtained by the production method shown in Reference Example. the G ₅ forming enzyme 8940 origin was bound.

That is, first, 50 ml of the carrier is packed in a column, and 4%
After passing 250 ml of a sodium hydroxide solution at 50 ° C., the mixture was washed with pure water. Next, a 0.1 M Tris-HCl buffer (pH 8.
0) After buffering by passing about 1,000 ml, the carrier was taken out of the column into a beaker, 100 ml of a 5% glutaraldehyde solution dissolved in the same buffer was added, and the mixture was allowed to react for 1 hour with stirring. The mixture was separated into solid and liquid, and excess glutaraldehyde was further washed with a buffer.

Take 5 g of wet resin adjusted by the above method in a beaker,
The buffer solution 8ml and G ₅ forming enzyme (401IU / ml) and 8ml added,
The reaction was carried out for 2 hours with stirring to immobilize the enzyme, followed by solid-liquid separation with a glass filter, and further washing of the excess enzyme with a buffer to obtain immobilized enzyme A of the present invention. The thus obtained immobilized enzyme A has an activity of 41 g / g of the wet carrier.
0 IU of the enzyme was immobilized.

5 g of the immobilized enzyme was packed into a jacketed column having an inner diameter of 10 mm and a height of 200 mm, and a 4% liquefied starch solution (Paindex, a product of Matsutani Chemical Industry Co., Ltd. dissolved in water) was passed through at a temperature of 40 ° C. The solution was passed at a flow rate of SV 0.1 and pH 8.0. Thereafter continuously passing fluid at the same flow rate, it was measured after day changes in the production rate of G _5.

For comparison, a conventional adsorption resin (porous synthetic resin obtained by copolymerizing styrene and divinylbenzene) was wetted.
5g is taken in a beaker, the buffer solution 8ml and G ₅ forming enzyme (401
(IU / ml) and immobilized while reacting for 2 hours with stirring to obtain immobilized enzyme B.

Thus immobilized enzyme obtained B is G ₅ forming enzyme activity 420IU per wet resin 1g had been immobilized. The same 4% liquefied starch solution with an immobilized enzyme and the aforementioned liquid passage under the same conditions to measure the yield of G _5.

FIG. 1 shows the results obtained using the immobilized enzyme A of the present invention and the immobilized enzyme B of the comparative example.

Immobilized enzyme B from Figure 1 whereas generation rate G ₅ from around day 2 is suddenly lowered, the immobilized enzyme A of the present invention for more than one month, G ₅ production rate of about 30% And showed extremely excellent performance.

[Brief description of the drawings]

Figure 1 is a graph showing the yield of G ₅ with liquefied starch solution for the immobilized enzyme B of comparative example immobilized enzyme A of the present invention in Example, G ₅ production rate on the vertical axis, horizontal axis Shows the number of days passed.

Claims (1)

(57) [Claims] 1. A primary anion-exchange resin comprising a matrix having a macro net structure comprising a polymer of unsaturated glycidyl carboxylate and a primary anion exchange resin having a primary amine as an ion-exchange group as a carrier. An immobilized enzyme characterized in that maltopentaose-forming enzyme is bound to glutaraldehyde as a crosslinking agent.