JPH0320232B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for entrapping enzymatically active substances into a protein gel lattice. Attempts were made in the late 1960s to immobilize physiologically active substances, especially enzymes, using polymeric carriers and use them continuously and economically, including the production of amino acids, high fructose sugar, 6-aminopenicillamine, and low-lactose milk. The technology has already entered the practical stage, and the technology is currently being actively developed for bioreactors and biosensors. Such immobilization methods include (1) adsorption and immobilization on an insoluble carrier, (2) covalent bonding on an insoluble carrier, (3) cross-linking polymerization between enzymes, and (4) ) A method of entrapping it in the lattice of a polymer carrier is known. Since (1) has a weak binding strength, the enzyme is easily detached from the carrier depending on the external environment such as pH and buffer solution. Since (2) and (3) are immobilized under relatively harsh conditions, their disadvantages include deactivation of the enzyme, changes in tertiary structure, and complicated immobilization operations. Therefore, the inclusive method (4) can be considered as a method that can be prepared under mild conditions. As the enveloping material, natural materials such as agar, alginate, collagen, and gelatin are available, and chemically synthesized materials include polyacrylamide, photocrosslinkable resin prepolymers, urethane prepolymers, and the like. Reaction conditions are often limited for natural materials. For example, agar gel and gelatin gel begin to dissolve at reaction temperatures of 40°C or higher, so the reaction must be carried out at low temperatures. Furthermore, chemically synthesized materials are also known to have drawbacks, such as enzymes being deactivated by polymerization accelerators and initiators during immobilization. The present inventors have compensated for the above-mentioned drawbacks by immobilizing enzyme active substances under mild conditions using natural materials, making them resistant to changes in the external environment such as temperature and pH, and ensuring safety. As a result of conducting various studies in an attempt to develop a problem-free immobilization product, it was found that enzymes can be immobilized by preparing a protein gel using transglutaminase and entrapping the enzymatically active substance in the lattice of the protein gel. They discovered this and completed the present invention. That is, in the present invention, transglutaminase is added to 1 g of protein in a protein-containing solution with a protein concentration of 2% by weight or more.
This is an enzyme immobilization method characterized by adding one or more units to a protein gel to form a gel, and entrapping an enzyme active substance in the lattice of the protein gel. The gelling carrier protein used in the present invention is not limited by its origin, and any protein such as vegetable protein or animal protein can be used. Examples of vegetable proteins include defatted oilseed products (defatted soybeans, etc.) and proteins separated from them. Furthermore, examples of animal proteins include milk protein, gelatin, and collagen.
A solution containing 2% by weight or more of these proteins is prepared. It is desirable that the protein-containing solution has a relatively high concentration, usually 2% by weight or more, preferably 5% to 15% by weight. The enzyme active substance used in the present invention is not particularly limited as long as it does not inhibit the protein gelation reaction of transglutaminase, and examples include glycosidase, mannosidase, glucose oxidase, catalase, diafluorase, glutamate dehydrogenase, and ribonuclease A. etc. can be mentioned. It is also possible to mix two or more of these. These enzymatically active substances to be immobilized may be blended into a protein-containing solution. The amount may be appropriately selected and blended within a range that does not inhibit subsequent gelation by transglutaminase or reduce the solubility of the protein serving as a carrier. The specific amount to be added is preferably about 0.5% by weight for a 10% protein solution, and about 1% by weight for a 5% protein solution. To this mixed solution of protein and enzyme active substance, one unit or more of transglutaminase prepared by the method described in Japanese Patent Application No. 57-31978 (Japanese Unexamined Patent Publication No. 58-149646) is added per 1 g of protein. By gelation, the enzymatically active substance can be encapsulated in the lattice of the protein gel. If the amount of transglutaminase added is less than 1 unit, the solution becomes highly viscous, and even if more than 2000 units are added, the effect does not change much. ,
Gel strength and gel lattice size can be adjusted. When the thus obtained protein gel containing the enzymatically active substance was frozen and dried, its structure was observed under a scanning microscope, and the protein gel formed a network structure. It is proven that it is formed. Furthermore, if an enzymatically active substance coexists, it is considered that the enzymatically active substance is enclosed and immobilized within the lattice of this protein gel. In this way, a protein gel having enzymatic activity entrapped in the protein lattice is obtained. Since this protein gel is insoluble in water, the reaction can be carried out continuously. In addition, this protein gel is stable even when heated to 100°C, is stable in all pH ranges, and can withstand any reaction conditions suitable for immobilized enzyme active substances, and can be used for long periods of time. It can be stably used for reaction for a period of time. Furthermore, the immobilized enzyme obtained by the present invention is
Since the gelling carrier is a natural protein and the general agent is also an enzyme, it has little effect on living organisms and is effective as a method for immobilizing enzymes used in the production of food and pharmaceutical products. The present invention will be described below with reference to Examples, but the present invention is not limited to these Examples in any way. Example 1 A 5% by weight solution of _αS1 casein was added to 0.1M Tris-
It was prepared with a hydrochloric acid buffer (containing 5mM _CaCl2 , 20mM dithiothreitol, PH7.6). Add glucose oxidase (Boehringer,
After adding 1 mg (13 units) (manufactured by Mannheim Yamanouchi Pharmaceutical Co., Ltd.) and thoroughly dissolving it, 0.01 unit of transglutaminase prepared by the method described in Japanese Patent Application No. 57-31978 was added per 1 mg of α _S1 casein. The mixture was shaken and incubated at 37° for minutes to obtain a protein gel (wet weight: 870 mg). The thus obtained immobilized glucose oxidase α _S1 casein gel was mixed with MBTH-DEA solution (EDTA・2Na 100 mg, 3-Methyl-2-
benzothiazolinone-hydrazone 17mg,
Dissolve 0.4ml of diethylaniline in 0.1M acetic acid to make a final volume of 1) 2.0ml, POD solution (peroxidase diluted to 8 purpurogalin units/ml with 0.1M sodium acetate solution) 0.5ml, substrate solution (0.01M Make 15% glucose solution with acetic acid) 0.5ml
and reacted for 15 min at 37°, then 0.5NHCl
1 ml was added to stop the reaction. The O of this liquid.
D. Method of measuring absorbance at 590nm (R.
Bentley, Methods in Enzymology, Vol.
86 (1966)). The immobilized glucose oxidase measured in this way showed 0.145 units (0.166 units/
g). Similarly, the stability of this immobilized glucose oxidase α _S1 casein gel was measured for two months, and the results were as shown in Table 1.

[Table] Example 2 A 5% by weight solution of α _S1 casein was prepared in 0.1M Tris-HCl buffer (containing 5mM CaCl ₂ , 20mM dithiothreitol, pH 7.6), and 1ml of this solution was prepared.
After adding 1 mg (2350 units) of catalase (manufactured by Wako Pure Chemical Industries, Ltd.) to the solution and thoroughly dissolving it, 0.01 unit of transglutaminase was added per 1 mg of α _S1 casein, and the protein gel was incubated at 37° with shaking for 30 minutes. (wet weight 910 mg). Immobilized catalase α _S1 thus obtained
Casein gel in 3ml of 0.1M phosphate buffer (PH7.0)
added to. Add 25μl of 30% hydrogen peroxide to this,
The decrease in absorbance at OD240nm was measured at 25°, and the activity was determined based on the amount of decomposing 1 μmol of hydrogen peroxide per minute as 1 unit. Immobilized catalase α _S1 measured in this way
The casein gel showed an activity of 567 units.
(623 units/g) Example 3 A 5% by weight solution of _αS1 casein was added to 0.1M Tris-
Prepared with hydrochloric acid buffer (containing 5mM CaCl ₂ , 20mM dithiothreitol, PH7.6), and this solution 1
1 mg of glucose oxidase and 1 mg of catalase per ml
After sufficiently dissolving, add 0.01 unit of transglutaminase per 1 mg of α _S1 casein.
The mixture was shaken and incubated at 37° for 30 minutes to obtain a protein gel (wet weight: 905 mg). When the activity was measured using this immobilized glucose oxidase and catalase α _S1 casein gel in Examples 1 and 2, the results were as follows.
The same results were obtained. Example 4 A 5% by weight solution of _αS1 casein was added to 0.1M Tris-
Prepared with hydrochloric acid buffer (containing 5mM CaCl ₂ , 20mM dithiothreitol, PH7.6), and this solution
Add 1 mg (15 units) of diafluorase (manufactured by Boehringer Mannheim Yamanouchi) to 0.25 ml, dissolve thoroughly, add 0.01 unit of transglutaminase per 1 mg of α _S1 casein, shake and incubate at 37° for 30 minutes, and dissolve the protein gel. obtained (wet weight 210
mg). The thus obtained immobilized diafluorase α _S1 casein gel was mixed with 2.4 ml of distilled water, 200 mM Tris-HCl buffer (PH7.5), and 0.1 μM NADH (reduced form of nicotinamide adenine dinucleotide).
ml, 1.2mM DCPIP (2-6-
dichlorophenolindophenol) added to 0.1ml, 25
Then, the change in absorbance at 600 nm per minute was determined (1 unit is the amount by which the absorbance at 600 nm is decreased by 1 per minute). (F. Kaplan et al., Aroh.
Biochim. Biophys. 132 , 91 (1966). The immobilized diafluorase α _S1 casein gel thus determined showed 7.2 units (34 units/g). Similarly, the stability of this immobilized diafluorase α _S1 casein gel was measured over a period of 2 months, and the results were as shown in Table 2.

[Table] Example 5 A 5% by weight solution of α _S1 casein was added to 0.1M Tris-
Prepared with a buffer solution (containing 5mM CaCl ₂ , 20mM dithiothreitol, pH 7.6) and 0.25ml of this solution.
Add 1 mg (50 units) of ribonuclease A (manufactured by Boehringer Mannheim Yamanouchi Co., Ltd.) to the solution, dissolve thoroughly, and dissolve transglutaminase into α _S1 casein 1.
0.01 unit per mg was added and incubated at 37° for 30 minutes with shaking to obtain immobilized ribonuclease A α _S1 casein gel (wet weight 198 mg). The resulting gel was subjected to the Kunitz method (M.Kunitz, J.Gen.
Physiol., 24 , 15 (1940)) showed 16 units of activity. (84 units/g) Example 6 A 5% by weight solution of _αS1 casein was added to 0.1M Tris-
Prepared with hydrochloric acid buffer (containing 5mM CaCl ₂ , 20mM dithiothreitol, PH7.6), and this solution
1 mg of glycosidase (manufactured by Seikagaku Corporation) in 0.25 ml
(5 units) was added and dissolved, and 0.01 unit of transglutaminase was added per 1 mg of α _S1 casein, and the mixture was shaken and incubated at 37° for 30 minutes to obtain an immobilized glycosidase α _S1 casein gel (wet weight: 205
mg). The activity of this gel was measured using p-nitrophenyl-α-D-glucopyranoside as a substrate (N.
A.Khan anh NREaton, Biodhim.Biophys.
Acta., 146 , 173 (1967)) showed 1 unit (4.8 units/g). Example 7 A 5% by weight solution of α _S1 casein was added to 0.1M Tris-
Prepared with hydrochloric acid buffer (containing 5mM CaCl ₂ , 20mM dithiothreitol, PH7.6), and this solution
1 mg of mannosidase (manufactured by Seikagaku Corporation) in 0.25 ml
(7.1 units) was added and dissolved, and 0.01 unit of transglutaminase was added per 1 mg of α _S1 casein, and the mixture was incubated with shaking at 37° for 30 minutes to obtain an immobilized mannosidase α _S1 casein gel (wet weight: 220
mg). When the activity of this product was measured using p-nitrophenyl-α-D-mannopyranoside as a substrate, it showed 3.4 units (15 units/g). Example 8 A 5% by weight solution of α _S1 casein was added to 0.1M Tris-
Prepared with hydrochloric acid buffer (containing 5mM CaCl ₂ , 20mM dithiothreitol, PH7.6), and this solution
50 μl (5 units) of glutamate dehydrogenase 50% glycerol solution (manufactured by Wako Pure Chemical Industries) in 0.25 ml
0.01 unit of transglutaminase was added per 1 mg of α _S1 casein, and the mixture was shaken and incubated at 37° for 30 minutes to obtain immobilized glutamate dehydrogenase α _S1 casein (wet weight: 264 mg). The immobilized glutamate dehydrogenase thus obtained was incubated at pH 8.0 and 30° for 1 minute using α-ketoglutarate and sodium acetate as substrates.
The activity was measured using the amount that reduced the absorbance at 340 nm by 0.1 as one unit. (Enzymes, 11 , 293
(1975)) The activity of immobilized glutamate dehydrogenase thus determined was 1.26 units.
(4.77 units/g) Application example (Production of glucose-free dried egg white) The immobilized glucose oxidase, catalase α _S1 casein gel (wet weight 3 g) prepared according to Example 3 was subdivided and dispersed in 0.01 M acetate buffer. After standing at 5° for a day and night, it was filtered to obtain an amorphous spherical gel. PH of 50 ml of egg white with 2NHCl prepared with 0.9% NaCl
Adjusted to 5.0-6.0. To this, add 0.9% NaCl solution 2
ml of the above gel was added, and the mixture was stirred at 37° for 2 hours under an oxygen stream. This was filtered through gauze, and the resulting egg white solution was spray-dried to obtain enzyme-treated dried egg white. Separately, dried egg white obtained by the same treatment without the addition of gel was left at 5° for 3 weeks. Suspend 500 mg of both in 50 ml of water and homogenize in a graduated cylinder (2000 rpm, 3 minutes)
When the foam volume was measured, the enzyme-treated dried egg white had a volume 1.25 times larger.

Claims (1)

[Claims] 1. Enzyme immobilization characterized by adding 1 unit or more of transglutaminase per 1 g of protein to a protein-containing solution with a protein concentration of 2% by weight or more to form a gel, and enclosing an enzyme active substance in the lattice of the protein gel. method.