JP2611951B2 - Method for producing maltooligosaccharides - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing maltooligosaccharides, and more particularly, to a method for producing maltooligosaccharides efficiently by allowing a starch substrate to act on an immobilized enzyme.

[Prior art and problems to be solved by the invention]

Focusing on the usefulness of maltooligosaccharides, research on maltooligosaccharides has been actively conducted, but at present only maltose is industrially mass-produced. Maltotriose, maltopentaose and the like are only produced in small quantities for reagents and the like.

A method for producing maltooligosaccharides using starch as a raw material has been proposed.For example, maltopentaose production is performed by adding α-amylase to a starch suspension, liquefying the mixture, inactivating the enzyme, and further liquefying the starch. There is a batch method in which a maltopentaose-forming enzyme is added to and reacted. In addition, a method is also employed in which an enzyme is adsorbed on a polymer carrier such as an adsorption resin, packed into a column, and a substrate is passed through the column to obtain a reaction product.

However, the batch method requires a long time for the reaction, and the enzyme cannot be reused. In addition, in the immobilization method using a column, since starch is used as a substrate, aging occurs in the column, and the enzyme is easily detached from the carrier due to a flow rate or the like,
There are problems such as difficulty in prolonged operation. Moreover,
When the adhesion time between the adsorption resin and the substrate is prolonged, the reaction proceeds while the substrate is clogged on the surface of the resin or the maltooligosaccharide generated at the upper part of the column moves to the lower part, and the purity of the target product is reduced.

Therefore, the present inventors have repeatedly studied a carrier for a maltooligosaccharide-forming enzyme, and found that immobilization of the enzyme on a porous hollow membrane enables repeated use and long-term operation of the enzyme. Based on the above, the present invention has been completed.

[Means for solving the problem]

The present invention provides an immobilized enzyme obtained by adsorbing a maltooligosaccharide-forming enzyme to a porous hollow fiber membrane, wherein at least one of starch, a starch composition fraction and a starch degradation reaction product is used.
An object of the present invention is to provide a method for producing maltooligosaccharides, which comprises contacting a seed substance.

Regarding the maltooligosaccharide-forming enzyme used in the present invention, those derived from animals, plants, microorganisms and the like can be arbitrarily used regardless of their origin. Hereinafter, maltooligosaccharide-forming enzymes that can be used in the present invention will be exemplified.

Maltose (G ₂ ) producing enzyme (1) β-amylase of plant origin: barley origin (J. In
st. Brewing., 63 , 24 (1957)), soybean origin (Biochem.
J., 95 , 621 (1965)), wheat origin (Cereal chem., 43 , 6).
2 (1966)), sweet potato origin (Biochemistry, 4 714 (196)
5)) (2) β-amylase of microbial origin: Bacillus cereus origin (JP-B-56-20835), Bacillus polymixer origin (Arch. Biochem. Biophys., 104 , 388-345 (1964)),
Bacillus megaterium origin (Agric. Biol. Chem., 38 , 10
23-1029 (1974)), Bacillus genus BQ10 origin (fermentation engineering,
57 , 102-113 (1979)), origin of Pseudomonas genus BQ6 (fermentation engineering, 57 , 102-113 (1979)) (3) α-maltose-forming amylase: origin of Streptomyces praecox NA-237 (starch science, 25 , 155
-161 (1978)), Streptomyces tosaensis origin (Japanese Patent Laid-Open No. 50-125046), Streptomyces hygroscopicus origin (Showa 47, Abstracts of the 1971 Agricultural Chemistry Congress, p. 86 (1972)) Maltotriose (G ₃ ) Synthase (1) Origin of Streptomyces griseus
-373237, the same 57-6915, starch Science, 26, 175 (1979)) (2) Bacillus subtilis origin (1983 Japan agrochemical tournament Abstracts 169 pp. (1983)) maltotetraose (G ₄₎ generating enzyme (1) Origin of Pseudomonas stuccieri (Arch.Bio
chem. Biophys., 145 , 105 (1971)) (2) Origin of Pseudomonas saccharophila
61-202687, the 61-202700) maltopentaose (G ₅₎ forming enzyme (1) Bacillus licheniformis origin (Arch.Bioch
em. Biophys., 155 , 290 (1973), Japanese Patent Publication No. 50-2552) (2) Bacillus cereus origin (Agric. Biol. Chem., 4)
9 2379 (1985), ibid, 49, 3369 (1985)) (3) Pseudomonas sp. KO-8940 origin (JP 60-188065) maltohexaose (G ₆₎ forming enzyme (1) Aero Arthrobacter Earogenesu origin (Amylase Symposium, 6 , 31 (1971), Biochem. Biophys. Act)
a., 410 333 (1975)) (2) Bacillus circulans origin (Agric. Biol. C
hem., 46 , 1539 (1982), Starch Science, 29 , 145 (1982)) (3) Bacillus circulans origin (Starch Science, 2
9, 107 (1982)) maltoheptaose (G ₇₎ forming enzyme (1) cereal origin α- amylase (Biochem. J.., 56, 86
(1954), Arch. Biochem. Biophys. 94 , 121 (1961), Ibid., 99 , 105 (1962), Starch Science, 24 , 42 (1977). It is important to use these enzymes efficiently in producing maltooligosaccharides.
Therefore, in the present invention, these enzymes are immobilized and used.

In the present invention, the maltooligosaccharide-forming enzyme is adsorbed and immobilized on the porous hollow membrane. Since the membrane module used in the present invention has a structure in which the substrate flows from the outside to the inside of the membrane, clogging of the polymer substrate inside the membrane is small, and a smooth reaction can be performed. Both hollow and capillary types can be used for the membrane. The material of the membrane is MF membrane (microfiltration membrane) made of cellulose, such as cellulose acetate and cellulose nitrate, fluorine, polypropylene, etc., and polyamide, Made of polyimide, polysulfone, polyacrylonitrile, etc.
A UF membrane (ultrafiltration membrane) or the like can be used.

In the present invention, permeation of a polymer substrate can be performed smoothly by changing the exclusion limit molecular weight of the membrane according to the molecular weight of the enzyme, and the exclusion limit molecular weight is 8,000 or more, usually 8,000 to
Use 100,000 membranes.

The enzyme can be immobilized on the membrane by any method. For example, there is a method in which the enzyme is activated by introducing a functional group onto the porous surface of the membrane. In the present invention, the immobilization of oxygen by the covalent bonding method is performed. Is preferred. Among the covalent bonding methods, a membrane activation method using a crosslinking reagent is most effective. Known crosslinking agents can be used, for example, glutaraldehyde, hexamethylene diisocyanate, toluene diisocyanate, hexamethylene diisothiocyanate, bisdiazobenzidine, benzidine-2,2'-disulfonic acid, N, N '-Ethylenebismaleimide, N, N'-polymethylenebisiodoacetamide and the like, and glutaraldehyde is particularly preferable.

As another covalent bonding method, a diazo coupling method in which an enzyme is immobilized on an aromatic amino group of a membrane; a membrane having a carboxyl group is converted into a derivative such as azide, chloride, carbodiimide, isocyanate, and the like, and liberated in an enzyme protein. A peptide method for bonding to an amino group; a method using a peptide condensation reagent such as Woodwork reagent K and a carbodiimide reagent; further, a free amino group in an enzyme protein,
A covalent bonding method such as an alkylation method in which a phenolic hydroxyl group or the like is immobilized on a film having a functional group such as halogen can be selected and applied in consideration of the material of the film.

The immobilization of the enzyme on the membrane and the enzyme reaction test are carried out using an enzyme-immobilized membrane reactor connected to a membrane module.
The main functions of the device are as follows. That is, an enzyme reaction is carried out by circulating a substrate solution through a substrate tank and an enzyme-immobilized membrane module by a pump, a product permeates a membrane, an unreacted liquid is returned to a substrate tank through a bypass, and is subjected to a reaction again. The enzyme can be immobilized by putting an enzyme solution in place of the substrate solution, connecting a permeate line to the substrate tank, and circulating by a pump. In addition, the same amount of the substrate solution as the membrane permeate can be automatically replenished from the reservoir tank, and continuous operation can be performed. Further, the circulating flow rate, the operating pressure, the temperature and the like can be monitored on the apparatus main body panel, and can be kept constant.

When the immobilization of the enzyme is described using maltopentaose-forming enzyme as an example, using a capillary membrane,
Set in a polysulfone housing. This is 1 ~
A 5% glutaraldehyde solution is circulated at room temperature for 7-8 hours to activate the membrane surface. If this treatment is performed at 40 ° C., the activation is completed in 4 to 5 hours. Next, the glutaraldehyde remaining in the membrane module is washed with distilled water.

After washing, a 0.2 to 10 mg protein / ml maltopentaose-forming enzyme solution is circulated and immobilized at a low temperature of 10 ° C or lower. After the immobilization, the unreacted enzyme in the substrate tank, piping, and module is recovered at a low temperature, and then the unreacted enzyme in the porous membrane is recovered by back washing, and then washed with a buffer.

 Next, the substrate used in the present invention will be described.

First, starch obtained from any raw material such as potato, sweet potato, corn, waxy corn, barley, wheat, rice, tapioca, and sago can be used. Also, as the composition fraction of starch,
For example, there are amylose and amylopectin, and as the decomposition reaction products of starch, for example, roasted dextrins such as white dextrin, yellow dextrin, and british gum; oxidized starch, low-viscosity denaturation (enzymes,
Modified starch such as starch; starch derivatives such as starch ethers and starch esters represented by starch phosphate, acetic acid starch, etc .; irradiation with radiation or neutron beam, high-frequency treatment or wet heat treatment Physically treated starch such as dried starch; α-starch and the like. These starches can be used alone or in combination of two or more.

The reaction to be carried out by bringing the substrate into contact with the immobilized enzyme may be performed by setting conditions in consideration of the properties of the enzyme to be used, the properties of the substrate, and the like. For example, when producing maltopentaose,
The reaction temperature is 40 ~ 45 ℃ considering the stability of the enzyme and the optimum temperature.
The substrate concentration may be 0.1 to 30%, preferably 0.5 to 10%. The pH of the substrate is preferably 4 to 10, but is preferably 8 to 8.5 in terms of stability and the most efficient generation of maltopentaose. It should be noted that the circulation flow rate during operation does not significantly affect the production rate of maltopentaose. Further, it is preferable that the operation pressure is slightly higher than the low pressure because the permeation speed is higher and the purity of maltopentaose in the reaction product is higher. In terms of sanitation, the introduction of an ultraviolet sterilizer into the line can prevent the incorporation of sterilization.

In the case of maltopentaose, a production rate of about 35% can be obtained, but it is desirable to terminate the reaction when maltopentaose is produced at about 30% in order to suppress by-products of other oligosaccharides.

After completion of the enzymatic reaction, the target maltooligosaccharide is separated and purified from the reaction solution by a conventional method.

〔Example〕

Porous hollow membrane (Nitto Denko Corporation, membrane module NT)
E-370, effective membrane area 0.1 m ² ) was connected to an enzyme-immobilized membrane reactor Membrane Master BM-1 (manufactured by Nitto Denko Corporation),
Activation of the membrane, immobilization of maltopentaose-forming enzyme on the membrane, and production of maltopentaose were performed. That is,
5% glutaraldehyde solution circulating flow rate 1 / min, pressure
The membrane was circulated at room temperature for 7 hours under the condition of 0.6 kg / cm ² to activate the membrane. Then, the activated film was heated at room temperature for 1 hour.
After washing with distilled water, maltopentaose-forming enzyme (Pseudomonas sp. KO-8940 origin;
188065) was immobilized. Immobilization was performed using a mild pentaose-forming enzyme (activity: 16.71 U / ml) with a protein amount of 1 mg / ml.
ml was circulated at a flow rate of 0.8 / min, a pressure of 0.45 kg / cm ² , and a temperature of 5 ° C. for 14 hours. The enzyme immobilization rate (protein adsorption rate) was 51.2%. For washing after the adsorption, 20 mM Tris-HCl buffer (pH 8) was used.

Using this maltopentaose-forming enzyme-immobilized membrane, a substrate was passed through to conduct a reaction test. As a substrate, 2% soluble starch (pH 8, manufactured by Junsei Chemical) was used, and the solution was passed through a filter paper after filtration. Liquid passing temperature 45 ° C, circulation flow 0.6 / min, pressure 0.4kg / cm ²
, The permeate was sampled by a fixed amount, and the reaction product was analyzed and confirmed by high performance liquid chromatography using a Bio-Rad HPX42A column. The result is shown in FIG.

Next, FIG. 2 shows the results when a 6% dextrin aqueous solution (pH 8, Paindex # 100, manufactured by Matsutani Chemical) was used as a substrate under the same operating conditions.

As is clear from the figure, maltopentaose is stably generated. In addition, as shown in FIG. 3, in a repeated test using the same membrane when soluble starch was used as the substrate, the enzyme was used after 20 operation cycles with one reaction test and one backwash. Almost no decrease in the activity, and maltopentaose can be produced by stabilization.

[Effects of the Invention] According to the present invention, maltooligosaccharides can be efficiently produced from starches. In particular, since the immobilized enzyme reaction is performed using a membrane, the reaction can be controlled by pressure, and the reaction can be stably performed for a long time without clogging of the membrane with the substrate. Another feature of the present invention is that the enzyme can be used repeatedly.

[Brief description of the drawings]

1 and 2 are graphs showing the relationship between the amount of permeate and the production rate of a reaction product in the embodiment of the present invention, and FIG. 3 is a graph showing the relationship between the number of operations and the sugar production rate in the embodiment of the present invention. It is a graph shown.

Claims (2)

(57) [Claims] 1. An immobilized enzyme having maltooligosaccharide-forming enzyme adsorbed on a porous hollow fiber membrane, wherein at least one of starch, a starch-containing fraction and a starch degradation reaction product is added.
A method for producing maltooligosaccharides, which comprises contacting seed substances. 2. The method according to claim 1, wherein the porous hollow membrane is a membrane having an exclusion limit molecular weight of 8,000 or more.