JPH08322585A - Production of palatinose and trehalose - Google Patents

Abstract

PURPOSE: To efficiently produce palatinose and trehalose useful for low- cariogenic sweeteners, etc., by reacting a granular porous chitosan with a specific epoxy compound, immobilizing an α-glucosyltransferase on the resultant carrier and then bringing the prepared carrier into contact with a sucrose solution. CONSTITUTION: A chitosan having 79% deacetylation degree and 46000 average molecular weight is dissolved in a 3.5% aqueous solution of acetic acid and the resultant solution is then dropped into a 7% solution of sodium hydroxide- water-ethanol to coagulate and regenerate the chitosan into a granular and porous substance, which is sufficiently washed with water to afford a granular and porous chitosan having 1mm average grain diameter. The resultant granular and porous chitosan is then reacted with diglycidyl ether of an aliphatic alcohol (e.g. ethylene glycol diglycidyl ether) and phenyl glycidyl ether in water to provide a carrier for immobilizing an enzyme. An α-glucosyltransferase is immobilized on the prepared carrier and the resultant immobilized α- glucosyltransferase is brought into contact with a sucrose solution to efficiently afford palatinose and trehalose useful as low-cariogenic sweeteners, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for efficiently producing palatinose and trehalulose from sucrose using an immobilized α-glucosyltransferase.

[0002]

BACKGROUND OF THE INVENTION Palatinose (also known as isomaltulose) and trehalulose are non-cariogenic disaccharides having a sweet taste in which glucose and fructose are bound by α-1,6 and α-1,1, respectively. Recently, it has been widely used as a low cariogenic sweetener.

[0003] Conventionally, in palatinose and trehalulose, sucrose in which glucose and fructose are bound by α-1,2 is protaminobacte (Protaminobacte).
r), Serratia, Erwinia
genus nia, genus Klebsiella, genus Pseudomonas, Agroba
JP-A-3-180172, which is produced by contact with a glycosyltransferase called α-glucosyltransferase contained in a microorganism such as cterium).
It is disclosed in JP-A-4-169190 and JP-A-5-130886. Further, in order to industrially produce palatinose and trehalulose, a microorganism containing α-glucosyltransferase is added to an aqueous solution of 20 to 60% sucrose and stirred, and the temperature is 15 to 30 ° C.
It is also known that the reaction can be carried out for about 20 hours under the control of around H6, then the microorganisms can be removed from the reaction solution by a centrifugation method or the like and purified by an ion exchange resin or the like. However, in such a manufacturing process, removing the microorganisms from the reaction solution is inefficient because the difference in specific gravity between the reaction solution and the microorganisms is small and the size of the microorganisms is small, and is industrially inappropriate. In order to solve such a problem, for example, JP-B-60-9797 discloses that a microorganism containing an α-glucosyltransferase is included in a gel such as calcium alginate, polyacrylamide, agar, or the like,
It is disclosed that a component of whole cells or crushed cells of a microorganism containing α-glucosyltransferase is adsorbed on a water-insoluble carrier such as DEAE-cellulose or bone charcoal, and optionally crosslinked with glutaraldehyde. In Japanese Patent Publication No. 58-36959,
A microorganism containing a glucosyltransferase,
It is disclosed that calcium alginate gel is entrapped and granulated, and polyethyleneimine is permeated into the granulated product and then crosslinked with glutaraldehyde.

[0004]

In the above-mentioned prior art, among the immobilized α-glucosyltransferases described in JP-B-60-9797, those in which microorganisms are entrapped in calcium alginate gel or the like, and JP-B-58- Three
The immobilized α-glucosyltransferase described in Japanese Patent No. 6959 has a sucrose solution having a pH of 5.8 or higher,
There is a drawback that calcium alginate is converted into water-soluble sodium alginate and eluted by contact with sodium ions and the like contained in roughly purified sucrose, and the physical strength of the gel is lowered. This drawback can be improved by adding a calcium salt to the sucrose solution, but a new problem arises that the reaction solution must be desalted. In addition, the method for entrapping and immobilizing a microorganism containing α-glucosyltransferase involves a low immobilization amount, and a change in the higher-order structure of α-glucosyltransferase having a glycosyl transfer activity and contact with a sucrose solution are comprehensive. There is a drawback that the activity becomes low with a steric hindrance that is hindered by the action. Further, when the immobilized α-glucosyltransferase is inactivated, the gel carrier cannot be regenerated and used repeatedly, which is a big drawback.

As a promising means for solving such a drawback, Japanese Patent Publication No. Sho 60-9797 discloses a method of immobilizing α-glucosyltransferase on a water-insoluble carrier such as DEAE-cellulose. With this method, the amount of immobilization can be increased, the higher-order structure of α-glucosyltransferase is unlikely to change, and
Since the glucosyltransferase is immobilized on the surface of the carrier, it does not inhibit contact with the sucrose solution. Moreover, the carrier can be easily regenerated by treating the inactivated immobilized α-glucosyltransferase with hot alkali to immobilize a new unused α-glucosyltransferase. However, the immobilized α-glucosyltransferase obtained by this method has extremely poor activity persistence,
Initial activity and regeneration are not satisfactory.

From this point of view, the physical strength is excellent,
Immobilized α-glucosyltransferase on a reproducible water-insoluble carrier, excellent initial activity, good activity persistence, α-glucosyltransferase immobilized on a carrier that can be repeatedly used by regeneration. Therefore, there has been a strong demand for development of a method for producing palatinose and trehalulose.

[0007]

Means for Solving the Problems As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that an enzyme obtained by reacting granular porous chitosan with diglycidyl ether of aliphatic polyalcohol and phenylglycidyl ether By immobilizing α-glucosyltransferase on an immobilizing carrier, excellent initial activity and good activity persistence are obtained, and when deactivated, the α-glucosyltransferase is immobilized again after the treatment with hot alkali. As a result, they have found that they can be regenerated and can be used repeatedly, and have reached the present invention.

That is, according to the present invention, α-glucosyltransferase is immobilized on a carrier for enzyme immobilization obtained by reacting granular porous chitosan with diglycidyl ether of aliphatic polyalcohol and phenylglycidyl ether. The present invention provides a method for producing palatinose and trehalulose, which comprises contacting the obtained immobilized α-glucosyltransferase with a sucrose solution.

The enzyme immobilization carrier used in the present invention, which is obtained by reacting granular porous chitosan with diglycidyl ether of aliphatic polyalcohol and phenylglycidyl ether,
It can be produced by the method for producing the enzyme-immobilized carrier previously filed by the applicant in Japanese Patent Application No. 7-39172.

That is, (A) the average molecular weight is 10,000 to
Granular pores obtained by dissolving 230,000 low molecular weight chitosan in an acidic aqueous solution of an organic acid such as formic acid, acetic acid, lactic acid or an inorganic acid such as hydrochloric acid, nitric acid and dropping the chitosan acidic solution into a basic solution. Chitosan is reacted with diglycidyl ether of aliphatic polyalcohol to form crosslinked granular porous chitosan, and then phenylglycidyl ether is suspended in water using a surfactant, and amino groups remaining in the crosslinked granular porous chitosan And then thoroughly washing with water, or (B) the granular porous chitosan obtained as described above is reacted with phenylglycidyl ether in a polar solvent, and then diglycidyl of an aliphatic polyalcohol. After reacting ether mainly with the amino groups remaining in the granular porous chitosan, it is sufficiently washed with water to obtain a carrier for immobilizing an enzyme.

Examples of the diglycidyl ether of the aliphatic polyalcohol used here include polyethylene glycol diglycidyl ether such as ethylene glycol diglycidyl ether and diethylene glycol diglycidyl ether, polypropylene such as propylene glycol diglycidyl ether and dipropylene glycol diglycidyl ether. Examples thereof include glycol diglycidyl ether, glycerol polyglycidyl ether and the like.

The amount of phenylglycidyl ether used here is 20 to 200 per 1 L of the crosslinked granular porous chitosan in the case of (A) in order to exert the excellent performance of α-glucosyltransferase to be immobilized later.
g, preferably 35 to 200 g, and in the case of (B), 50 to 200 g, preferably 7 to 1 L of granular porous chitosan.
It is 5 to 200 g.

Examples of the surfactant used in (A) include a cationic surfactant, an anionic surfactant and a nonionic surfactant. However, generally used nonionic surfactants which do not cause ionic repulsion or adsorption with the amino groups of crosslinked granular chitosan are preferred.

Examples of the polar solvent used in (B) include polar solvents such as dioxane, ethanol and isopropyl alcohol, which are selected and used.

Next, α-glucosyltransferase is immobilized on the enzyme-immobilized carrier thus obtained to obtain immobilized α-glucosyltransferase.
As a method for immobilizing, a usual method can be adopted. For example, a method for treating an enzyme immobilization carrier in a solution containing α-glucosyltransferase for several hours, adsorbing α-glucosyltransferase on the enzyme immobilization carrier, removing the remaining solution, and washing with water, Alternatively, a method of treating the enzyme-immobilized carrier with α-glucosyltransferase by crosslinking for several hours after washing with water to remove the remaining solution and washing with water, or crosslinking the enzyme-immobilized carrier Treatment in the agent solution for several hours,
By binding a part of the functional group of the cross-linking agent and the enzyme immobilization carrier,
After the remaining solution is removed and washed with water, it is treated in a solution containing α-glucosyltransferase for several hours to form an unreacted functional group portion of the cross-linking agent bound to the enzyme immobilization carrier. A method of binding α-glucosyltransferase, removing the remaining solution and washing with water, or the like is adopted, but a method using a crosslinking agent is particularly preferable.

As the cross-linking agent used in the above-mentioned immobilization method, a residual amino group in which the two glycidyl ethers introduced into the enzyme immobilization carrier have not reacted and an amino group possessed by α-glucosyltransferase are included. A compound that crosslinks, that is, a compound having two or more functional groups such as an isocyanate group, an epoxy group, an aldehyde group, and a carbonyl group may be used. Specifically, hexamethylene diisocyanate, ethylene glycol diglycidyl ether,
Examples thereof include glutaraldehyde.

In the reaction using glutaraldehyde, a glutaraldehyde solution of usually 0.1 to 10%, preferably 0.5 to 5% is added to 1 volume of the enzyme-immobilizing carrier.
1 to 10 times volume, preferably 0.5 to 2 times volume is added, and treatment is carried out at room temperature for 10 minutes to 24 hours, preferably 30 minutes to 4 hours to crosslink.

The α-glucosyltransferase used in the above-mentioned immobilization method is not particularly limited as long as it has a transglycosylation activity for producing palatinose and trehalulose from sucrose, and for example, Protaminobacter (Protaminobacter) Genus or Serratia
Genus, Erwinia, Klebsiella
siella) genus, Pseudomonas genus, Agrobacterium (Agrobacterium) genus culture solution containing microorganisms obtained by crushing with a homogenizer obtained unpurified α-glucosyl transferase, such as liquid chromatography Depending on the method, purified α-glucosyltransferase from which impurities have been removed can be used, but it is preferable to use an unpurified α-glucosyltransferase that is industrially easy to operate.

Next, the immobilized α-glucosyltransferase obtained above is brought into contact with a sucrose solution, but the method is not particularly limited, and for example, it is immobilized in a container equipped with a stirring facility containing the sucrose solution. Examples thereof include a method of adding α-glucosyltransferase and stirring for a certain period of time, a method of packing immobilized α-glucosyltransferase in a column and passing a sucrose solution through this column. The latter method is industrially preferable because continuous processing is possible. The concentration of the sucrose solution used in this case is 1 to
60%, preferably 20-60%, pH 4-8, preferably 5-7, temperature 5-60 ° C, preferably 15-4.
0 ° C., liquid passing speed (space velocity) is 0.1 to 5.0 hr ⁻¹ ,
The treatment is preferably carried out for 0.2 to 2.0 hr ⁻¹ . By contacting the sucrose solution with the immobilized α-glucosyltransferase by such a method, a syrup containing the desired palatinose and trehalulose can be obtained. The obtained syrup is subjected to neutralization treatment, desalting treatment, crystallization treatment, ordinary liquid chromatography treatment using an ion exchange resin, etc., if necessary, to give a mixed product of palatinose and trehalulose or a highly pure palatinose product, and Products such as high-purity trehalulose products can be obtained.

The immobilized α-glucosyltransferase used here gradually decreases in transglycosylation activity upon contact with a sucrose solution. When the usage limit is lowered, the carrier for enzyme immobilization can be repeatedly used by a method for regenerating an immobilized enzyme by a usual carrier binding method. That is, the immobilized α-
0.1-5 for 1 volume of glucosyltransferase
N, preferably 0.5 to 1 N sodium hydroxide solution is added in a volume of 0.5 to 10 times, and 40 to 90 ° C., preferably 6
Heat to 0-80 ° C, 10 minutes to 24 hours, preferably 3
After treatment for 0 minutes to 4 hours, the immobilized α-glucosyltransferase is decomposed, washed with water and removed, and then a new α-glucosyltransferase is immobilized again as described above.

The present invention uses a granular porous chitosan as a carrier for immobilizing an enzyme, which is obtained by reacting a diglycidyl ether of an aliphatic polyalcohol and a phenylglycidyl ether to obtain physical strength and initial activity. It is possible to obtain an immobilized α-glucosyl transferase using a carrier which is excellent in the activity, has a good activity persistence, and can be repeatedly used by regeneration. Moreover, by using the immobilized α-glucosyltransferase obtained here, desalting of the reaction solution is unnecessary, and palatinose and trehalulose can be continuously produced from sucrose with high productivity.

[0022]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to this range. The quantification of palatinose and trehalulose was performed by the following method.

Method for Quantifying Palatinose and Trehalulose The reaction solution was injected under the following operating conditions for high performance liquid chromatography to obtain a high performance liquid chromatogram, which was calculated from the area ratio between the peaks of palatinose and trehalulose and the peak of the internal standard substance. . <Operating conditions for high-performance liquid chromatography> Column: ERC-NH1171 6 x 200 mm (manufactured by Elma Optical Co.) Moving layer: 80: Water 20 Flow rate: 1 ml / min Detector: Differential refractometer Sensitivity: 8 to 16 x 10 ^-5 (RIUFS) Injection volume: 20μl

Example 1 210 g of chitosan having a deacetylation degree of 79% and an average molecular weight of 46,000 was dissolved in 2,790 g of a 3.5% acetic acid aqueous solution. The chitosan acidic solution is dropped into a coagulation solution consisting of 7% sodium hydroxide, 20% ethanol and 73% water, and after chitosan is coagulated and regenerated into a granular porous body, it is sufficiently washed with water to have an average particle size of 1 mm.
3,000 ml (wet) of granular porous chitosan were obtained. 1,500 ml of the obtained granular porous chitosan was added with 1,50 ml of water.
0 ml and ethylene glycol diglycidyl ether 7.5
g was added and reacted at 80 ° C. for 1 hour. After completion of the reaction, the product was thoroughly washed with water to obtain 1,470 ml of crosslinked granular porous chitosan.

Next, a prescribed amount of phenyl glycidyl ether and a nonionic surfactant having the composition shown in Table 1 and a brand name of New Pole PE64 (manufactured by Sanyo Kasei Co., Ltd.) were added to water 10.
In addition to 0 ml, each was thoroughly stirred with a stirrer to obtain a suspension.
Then 100 ml of each crosslinked granular porous chitosan was added to the suspension 1
It was added to 00 ml and stirred at 70 ° C. for 4 hours for reaction. After the completion of the reaction, the reaction residual liquid was removed and the product was thoroughly washed with water to obtain 97 ml of each of the enzyme immobilization carriers (carriers A to G).

[0026]

[Table 1]

30 g sucrose, 10 g peptone, 3 meat extracts
g, yeast extract 5 g, disodium hydrogen phosphate 12 water 2 g and sodium chloride 3 g to dissolve in water to 1 L, and a medium 15 L prepared by adjusting the pH to 6.5 to 7.0 with sodium hydroxide solution Put in a mentor and sterilize, then use Protaminob
acter rubrum) inoculated with CBS574.77, temperature 2
8 ° C, aeration rate 13.0 L / min, stirring speed 350 r.
p. After culturing for 16 hours at m, a culture solution having high α-glucosyltransferase activity was obtained. After centrifugation to obtain 3.6 L of a slurry of microbial cells containing α-glucosyltransferase, the whole amount of the slurry was pulverized with a homogenizer to contain α-glucosyltransferase derived from Protaminobacterium rubrum. 3.3 L of enzyme solution was obtained.

25 ml of each of the carriers A to G is weighed and 3.0
% Glutaraldehyde aqueous solution (20 ml) and treated at 25 ° C. for 2 hours and then washed with water to sufficiently remove unreacted glutaraldehyde. Then, each glutaraldehyde-treated carrier for immobilizing each enzyme was stirred in 88 ml of each enzyme solution containing α-glucosyltransferase derived from Protaminobacterium rubrum at a temperature of 25 ° C. for 3 hours, and then washed with water to immobilize α. -Glucosyltransferase A ^* ~
G ^* got 25 ml each.

20 ml of each of the immobilized α-glucosyltransferases A ^{* to} G ^* thus obtained was packed in a column having a diameter of 10 mm and a height of 30 cm, and a 42% sucrose (granulated sugar) aqueous solution having a pH of 5.6 was filled in each column. Was prepared at 25 ° C. and was fed from the upper part of the column at a flow rate of 12.6 ml / hr by a pump, and the reaction solution coming out from the lower part of the column was sampled at regular intervals from the initial stage, and palatinose and trehalulose The content was measured, and this reaction was performed until the content of palatinose and trehalulose became half of the initial reaction. The number of days required up to this time was taken as the half-life, and the total amount of palatinose and trehalulose produced by the half-life was calculated from the measured contents.

Further, 0.5 N of each of the immobilized α-glucosyltransferases A ^{* to} G ^* whose half-life has reached here is added.
100 ml of sodium hydroxide solution was added, and the mixture was heated to 70 ° C. and treated for 4 hours, and then unused α-glucosyltransferases were immobilized again by the same immobilization method. These were packed again in the above-mentioned column, the above-mentioned sucrose solution was supplied, the initial reaction solution coming out from the lower part of the column was sampled, and the contents of palatinose and trehalulose in the reaction solution were measured. The ratio of the content of palatinose and trehalulose measured here compared with the initial content before regeneration immobilization was calculated as the regeneration rate. The results are shown in Table 2.

[0031]

[Table 2]

As is clear from Table 2, the method of the present invention produces a large amount of palatinose and trehalulose, the activity of the immobilized α-glucosyltransferase is very long-lasting, and the regeneration treatment is very good. And trehalulose could be produced efficiently.

(Example 2) 1,500 ml of the granular porous chitosan obtained in Example 1 was weighed and the contained water was sufficiently replaced with isopropyl alcohol to remove it. As shown in Table 3, a predetermined amount of phenylglycidyl ether was added to and dissolved in 100 ml of isopropyl alcohol, and 100 ml of granular porous chitosan was added to the solution, and the mixture was reacted at 70 ° C. for 4 hours.

After the completion of the reaction, the reaction residual liquid was removed, and the product was washed with isopropyl alcohol and then with water. Next is water 100 each
0.5 g ethylene glycol diglycidyl ether
Is added and dissolved, and a granular porous chitosan obtained by reacting a predetermined amount of phenylglycidyl ether is added to the solution, and 8
The reaction was carried out at 0 ° C for 2 hours. After the reaction was completed, the product was thoroughly washed with water to obtain 97 ml of enzyme immobilization carriers (carriers H to M).

[0035]

[Table 3]

25 ml of each of the carriers H to M was weighed, added to 20 ml of 3.0% glutaraldehyde aqueous solution, treated at 25 ° C. for 2 hours, and washed with water to sufficiently remove unreacted glutaraldehyde. Subsequently, each enzyme-immobilized carrier treated with glutaraldehyde was treated with Protaminobacter rubrum CBS574.
Each of 88 ml of the enzyme solution containing 77-derived α-glucosyltransferase was stirred at a temperature of 25 ° C. for 3 hours and washed with water to obtain 25 ml of each immobilized α-glucosyltransferase H ^{* to} M ^* .

20 ml of each of the immobilized α-glucosyltransferases H ^{* to} M ^* obtained here was packed in a column having a diameter of 10 mm and a height of 30 cm, and pH 5.6 was applied to each of the columns in the same manner as in Example 1. Of 42% sucrose solution,
The half-life and the total amount of palatinose and trehalulose produced by the half-life were measured or calculated in the same manner as in Example 1. Further, the immobilized α-glucosyltransferases H ^{* to} M ^* whose half-life reached here were treated with a sodium hydroxide solution in the same manner as in Example 1, and then unused α was reused.
-Glucosyl transferase was immobilized on each column, the column was packed again and the above sucrose solution was supplied, and the reaction solution was sampled to calculate the regeneration rate. The results are shown in Table 4.

[0038]

[Table 4]

As is clear from Table 4, the method of the present invention produces a large amount of palatinose and trehalulose, and the durability of the activity of the immobilized α-glucosyltransferase and the regeneration treatment are extremely good. And trehalulose could be produced efficiently.

(Example 3) (1) Sucrose 375 g, peptone 150 g, meat extract 45
g, yeast extract 75g, disodium hydrogen phosphate-12
15 L of pH 7 medium consisting of 30 g of water and 45 g of sodium chloride was placed in a 30 L jar fermenter for sterilization, and Serratia plymuthica
ATCC15928 was inoculated, the temperature was 28 ° C., the aeration rate was 7.5 L / min, and the stirring speed was 400 r.p.m. p. After culturing for 16 hours at m, a culture solution having a high α-glucosyltransferase activity was obtained, followed by centrifugation to obtain 2.5 L of a slurry of microbial cells containing the α-glucosyltransferase. Next, the whole amount of the slurry was treated with a homogenizer to obtain 2.3 L of an enzyme solution containing α-glucosyltransferase derived from Serratia brimtica (enzyme solution 1).

(2) Sucrose 60 g, peptone 10 g, meat extract 1 g, yeast extract 1 g, disodium hydrogen phosphate-1
2L 2g of water and 3g of sodium chloride are dissolved in water and 1L
And sterilize it by adding a medium adjusted to pH 6.5-7.0 with sodium hydroxide solution to a jar fermenter to sterilize it.
diobacter) MX-232 was inoculated, and the temperature was 28 ° C., the aeration rate was 0.25 L / min, and the stirring rate was 460 r.p.m. p. 4 in m
A culture solution having a high α-glucosyltransferase activity is obtained by culturing for 8 hours, and centrifuged to obtain a slurry of microbial cells containing the α-glucosyltransferase 0.20.
L was obtained. Next, the whole amount of the slurry was treated with a homogenizer to obtain an enzyme solution containing α-glucosyltransferase derived from Agrobacterium radiobacter 0.1.
8 L was obtained (enzyme solution 2).

(3) Sucrose 100 g, peptone 10 g, meat extract 3 g, yeast extract 5 g, disodium hydrogen phosphate.
12 Dissolve 2 g of water and 3 g of sodium chloride in water to prepare 1
L and sterilize by adding a medium adjusted to pH 6.5-7.0 with sodium hydroxide solution to a jar fermenter,
Pseudomonas meso
acidophila) MX-45 was inoculated, the temperature was 28 ° C., the aeration rate was 1.2 L / min, and the stirring rate was 430 r. p. After culturing for 60 hours at m, a culture solution having a high α-glucosyltransferase activity was obtained and centrifuged to obtain 0.15 L of a slurry of microbial cells containing α-glucosyltransferase. Next, the entire amount of the slurry was treated with a homogenizer to obtain 0.14 L of an enzyme solution containing α-glucosyltransferase derived from Pseudomonas mesoacidophila (enzyme solution 3).

(4) Sucrose 50 g, corn steep liquor 3
0 g, yeast extract 5 g, disodium hydrogen phosphate-12
Klebsiella terrigena ATCC was prepared by dissolving 2 g of water and 3 g of sodium chloride in water to make 1 L, and sterilizing the medium prepared with sodium hydroxide solution at pH 6.5 to 7.0 in a jar fermenter.
33257 inoculated, temperature 28 ℃, aeration 1.0L /
min, stirring speed 140 r. p. culture for 24 hours at α
-A culture solution having a high glucosyltransferase activity was obtained and centrifuged to obtain 0.25 L of a slurry of microbial cells containing α-glucosyltransferase. Next, the whole amount of the slurry was treated with a homogenizer to obtain 0.20 L of an enzyme solution containing Klebsiella terrigena-derived α-glucosyltransferase (enzyme solution 4).

25 ml each of the enzyme immobilization carrier D obtained in Example 1 and the enzyme immobilization carrier J obtained in Example 2 were weighed, and the enzyme solutions 1 to 4 obtained by the above method were respectively weighed. 10
After addition to 0 ml and stirring at room temperature for 2 hours, the residual enzyme solution after adsorption was removed. Then, the mixture was placed in 250 ml each of 2.5% glutaraldehyde aqueous solution and treated for 1 hour, then thoroughly washed with water and immobilized α-glucosyltransferase D1, D2, D
25 ml each of 3, D4 and J1, J2, J3, J4 were obtained.

20 ml of each of the immobilized α-glucosyltransferases D1 to D4 and J1 to J4 thus obtained was packed in a column having a diameter of 10 mm and a height of 30 cm, and pH 5 was applied to each of them by the same method as in Example 1. A 42% sucrose aqueous solution of No. 6 was passed through, and the half-life and the total amount of palatinose and trehalulose produced by the half-life were measured or calculated in the same manner as in Example 1. Furthermore, the immobilized α-glucosyltransferases D1 to D4, J having reached the half-life here,
1 to J4 were treated with a sodium hydroxide solution in the same manner as in Example 1, again unused α-glucosyltransferases were immobilized, respectively, and packed again in the column described above to supply the sucrose solution described above, followed by reaction. The solution was sampled and the regeneration rate was calculated. The results are shown in Table 5.

[0046]

[Table 5]

As is clear from Table 5, the immobilized α-glucosyltransferase obtained by the method of the present invention produces a large amount of palatinose and trehalulose against α-glucosyltransferases derived from any microorganisms and is active. The sustainability and regeneration treatment of sucrose were very good, and palatinose and trehalulose could be efficiently produced from the sucrose solution.

(Comparative Example 1) A method for producing a carrier for immobilizing an enzyme similar to that in Example 1 was carried out by using a carrier which was not reacted with phenylglycidyl ether (carrier O) and DEAE-cellulose (carrier P). The enzyme solution obtained in Example 1 was immobilized in the same manner as in Example 1 to obtain immobilized α-glucosyltransferases O ^* and P ^* .

20 ml of each of the obtained immobilized α-glucosyl transferases O ^* and P ^* was used, having a diameter of 10 mm and a height of 30.
cm columns were packed, and a 42% sucrose aqueous solution having a pH of 5.6 was passed through each of them in the same manner as in Example 1 to produce half-life and half-life in the same manner as in Example 1. The total amount of palatinose and trehalulose was measured or calculated. Further, the immobilized α-glucosyltransferases O ^* and P ^* whose half-life reached here were treated with a sodium hydroxide solution in the same manner as in Example 1, and then unused α-glucose was reused.
Glucosyltransferase was immobilized on each column, the column was packed again and the sucrose solution was supplied, and the reaction solution was sampled to calculate the regeneration rate. The results are shown in Table 6.

[0050]

[Table 6]

As is clear from Table 6, in the methods using immobilized α-glucosyltransferases O ^* and P ^* other than the present invention, the productivity of palatinose and trehalulose was low as compared with Examples 1 and 2, and The activity persistence and regeneration treatment were also inferior.

(Comparative Example 2) Sucrose 30 g, peptone 10
g, meat extract 3 g, yeast extract 5 g, disodium hydrogen phosphate-12 water 2 g and sodium chloride 3 g to dissolve the water to 1 L, and the pH is adjusted to 6.5 with sodium hydroxide solution.
15 L of the medium prepared to 7.0 was put in a 30 L jar fermenter for sterilization, and Protaminobacter rubrum CBS57477 was inoculated, and the temperature was 28 ° C., the aeration rate was 13.0 L / min, and the stirring speed was Three
50r. p. After culturing for 16 hours at m, a culture solution having high α-glucosyltransferase activity was obtained. After centrifugation to obtain 3.6 L of a slurry of microbial cells containing α-glucosyltransferase, the whole amount of the slurry was pulverized with a homogenizer to contain α-glucosyltransferase derived from Protaminobacterium rubrum. 3.3 L of enzyme solution was obtained.

2.5 L of the crushed microbial cell solution containing α-glucosyltransferase obtained above was added to
Mix with 2.5 L of 4% aqueous sodium alginate solution,
The mixture was put into a cylindrical extruder equipped with a die having a large number of pores having a diameter of 0.6 mm, and air pressure was applied to push the mixed solution downward from the pores. Below the extruder, 0.
A 20 L container containing 10 L of a 15 M calcium chloride solution was placed, the solution was stirred by a stirrer, and droplets of the mixed solution falling from above were received. The mixed solution was extruded in 10 minutes, and the calcium chloride solution was stirred as it was for 2 hours to obtain 4 kg of alginic acid gel containing crushed microbial cells.

Then, 270 g of 30% polyethyleneimine was diluted with about 3 L of water, neutralized with dilute hydrochloric acid and adjusted to pH 5.
After preparing 5 and making the total amount 4 kg, 4 kg of the alginic acid gel containing crushed microbial cells prepared above was added, gently stirred for 10 minutes, and then filtered to recover the gel.
The gel was added to 10 L of a 10% glutaraldehyde solution, and the mixture was stirred for 30 minutes and washed with water to obtain 3.3 kg of immobilized α-glucosyltransferase in which crushed microbial cells were included in alginate gel.

PH5.6 is obtained by the same method as in the first embodiment.
Of 42% sucrose aqueous solution, and the total half-life and palatinose and trehalulose produced by the half-life were measured or calculated in the same manner as in Example 1. The half-life was 210 days, and the half-life was produced by the half-life. The total amount of palatinose and trehalulose added was 12.5 kg.

As described above, the prior art method was low in productivity of palatinose and trehalulose and inferior in activity sustainability, as compared with the method of the present invention. In addition, since this method of immobilization does not allow regeneration, deactivated immobilization α
-Glucosyltransferase had to be discarded.

[0057]

The method of the present invention uses a carrier for enzyme immobilization obtained by reacting granular porous chitosan having excellent physical strength with diglycidyl ether of an aliphatic polyalcohol and phenylglycidyl ether. By immobilizing α-glucosyltransferase on, the initial activity and the durability of the activity are excellent, and an immobilized α-glucosyltransferase that can be repeatedly used by regeneration is obtained, and a sucrose solution is added to the immobilized-α-glucosyltransferase. By bringing them into contact with each other, the excellent effect that palatinose and trehalulose can be efficiently produced is exhibited.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yoshihide Kawamura 89-5 Oyama, Oyama-machi, Sunto-gun, Shizuoka Prefecture 89-5 (72) Inventor Masaaki Shinonaga 142-3 Toyama, Toyama-cho, Sunto-gun, Shizuoka Prefecture Fumio Miyazawa Kanagawa Prefecture 2065 Mutsuura-cho, Kanazawa-ku, Yokohama-shi

Claims (1)

[Claims] 1. An α-glucosyltransferase is immobilized on a carrier for enzyme immobilization obtained by reacting granular porous chitosan with diglycidyl ether of aliphatic polyalcohol and phenylglycidyl ether, and the immobilization obtained here is immobilized. A method for producing palatinose and trehalulose, which comprises contacting a sucrose solution with a modified α-glucosyltransferase.