JPH0583235B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to the immobilization of enzymes using gelatin as a matrix. [Prior Art] In order to improve the usability of enzymes, various matrices have been used to immobilize enzymes. For example, there is a method in which enzymes are immobilized in cellulose, polyacrylamide, collagen, or the like. However, enzymes immobilized by these methods have problems such as insufficient enzymatic activity and high manufacturing costs, and various techniques using matrices other than these have been developed. Among these, we focused on the excellent properties of gelatin as a carrier for enzyme immobilization.
JP-A-52-57390 discloses a technique for immobilizing enzymes in gelatin. Specifically, after adding an enzyme to a gelatin solution, the solution is cast onto a suitable plate-like material such as a titanium plate, air-dried to form a thin film, and then this thin film is treated with formaldehyde, glutaraldehyde, etc. The immobilized enzyme is obtained by immersing it in aldehyde and gelling it. Also, in Special Publication No. 52-18794,
A technique in which a non-proteolytic enzyme is added to an aqueous solution of a gelatinized protein such as gelatin, gelled, and then reacted with a bi- or polyfunctional protein reagent to crosslink the molecules of the gelled protein to immobilize the enzyme. is disclosed. It is true that gelatin is an excellent matrix for immobilizing enzymes, but the above methods have problems such as the use of various reagents, which increases the deactivation of the enzyme, and the contamination of bacteria after the reagents are used. It was hot. Furthermore, S. Bachman et.al.'s paper Starch/Sta¨rke 33 (1981)
Nr.2.S.63-66 describes a method for obtaining an immobilized enzyme by irradiating a gelatin solution with radiation to make gelatin insoluble, adding an enzyme, and then crosslinking using glutaraldehyde. has been done. However, the immobilization of the enzyme was insufficient in this method, and there were problems similar to those of the technique described in the above-mentioned publication. [Problems to be Solved by the Invention] Therefore, an object of the present invention is to provide a method for immobilizing enzymes by simple operations without using reagents such as glutaraldehyde or formaldehyde.
Another object of the present invention is to provide a method for immobilizing enzymes under sterile conditions. [Means for Solving the Problems] The present invention provides that, after gelatinizing an enzyme-containing gelatin aqueous solution, the gelatin is insolubilized by irradiating the gelatin with radiation to effectively immobilize the enzyme in the gelatin matrix, and at the same time, the gelatin This was done based on the knowledge that germs inside were sterilized. That is, the present invention provides a method for producing an immobilized enzyme, which comprises gelatinizing an enzyme-containing aqueous gelatin solution and then irradiating the gelatin with radiation to insolubilize the gelatin to immobilize the enzyme within the gelatin gel. . In the present invention, an enzyme-containing gelatin aqueous solution is first prepared. At this time, add 2 to 10% gelatin by weight.
(hereinafter abbreviated as %), preferably from 5 to 10%, and the enzyme from 0.01 to 10%, preferably from 0.1 to 2%, with the balance being water. In other words, if the gelatin concentration is less than 2%, the contraction of the gelatin gel due to radiation irradiation will increase, making it easier for the enzyme to separate from the gelatin matrix during immobilization. On the other hand, if the gelatin concentration exceeds 10%, no improvement in gelatin strength can be expected, and the gelatin gel will be tightly bonded, resulting in a decrease in enzyme activity yield. As the enzyme used in the present invention, any non-proteolytic enzyme can be used. Specifically, invertase, sucrase,
Satucarase, β-glycosidase, catalase, lysozyme, amyloglucosidase, lactase, maltase, amylase, urease, lipase, esterase, glucose isomerase, glucose oxidase, dehydrogenase, penicillinase, fructosyltransferase, α-glucosyltransferase ,
Examples include one enzyme selected from the group of melibiase, naringinase, and pectinase, or a mixture of two or more enzymes. Among these, hydrolases such as invertase and isomerases such as glucose isomerase are particularly preferred. Furthermore, as the enzyme, the enzyme itself can of course be used, but
The particles may be enlarged with a stabilizer such as a phosphate, or microbial cells containing enzymes, such as yeast and actinomycetes, or mixtures thereof may be used. In addition, when preparing the above-mentioned enzyme-containing gelatin aqueous solution, the content ratio of enzyme and gelatin is adjusted to 1/1.
(Weight ratio) below, preferably 1/50 to 1/5. In other words, if the content of gelatin is less than the amount of enzyme, the gel-forming ability of gelatin will decrease, and the enzyme will fall off even after radiation irradiation. The above enzyme-containing gelatin aqueous solution can be prepared by any method, but first, after dissolving a predetermined amount of gelatin in water heated to about 40 to 60°C,
It is desirable to cool the solution to a temperature of 15-20°C and then add the enzyme. In other words, the lower the liquid temperature, the more inhibited the deactivation of the enzyme. In the present invention, the enzyme-containing gelatin aqueous solution is then gelled. Although this gelation can be carried out by various methods, it is preferable to gelatin by cooling the aqueous solution to 0 to 10°C. In other words, gelling by cooling at this temperature has the effect of reducing the decrease in enzyme activity. In the present invention, desired shape,
For example, it can be formed into various shapes such as granules, membranes, plates, and fibers, but it is desirable to have the final shape at the time of gelation, especially considering the sterilizing effect of radiation that will be performed afterwards. Next, in the present invention, the gelatin gel containing the enzyme is irradiated with radiation. As the radiation used here, any of α rays, β rays, γ rays, X rays, and electron beams can be used, but
Preferably, a line is used. In addition, when electron beams or β-rays are used, it is preferable to make the gel into a thin film, or when it is in the form of particles or fibers, reduce the thickness as much as possible and irradiate the gel with electron beams or β-rays. . In the present invention, the above-mentioned radiation irradiation is used to insolubilize gelatin and at the same time firmly retain the enzyme in the gelatin matrix, or to sterilize the gelatin gel on which the enzyme is immobilized. be. Therefore, the radiation dose should be 2 to 30 kg Gy, preferably 5 to 20 kg.
It is better to use Gy. In other words, the radiation dose is 2 kg Gy.
If it is less than that, the insolubilization of gelatin is insufficient and the bactericidal effect is also small. On the other hand, if irradiation exceeds 30 kgy, enzyme deactivation will increase. still,
The temperature of the gel during irradiation with radiation is preferably 10 to 20°C to prevent the gel from dissolving. In addition, in order to scavenge excess radicals generated by radiation irradiation and prevent their effects on enzymes, antioxidants such as tocopherol, BHT, BHA, and ascorbic acid can be added as radical scavengers. On the other hand, sensitizers such as alkali halides such as potassium bromide and potassium iodide, vitamin K ₅ , N-ethylmaleide, and sodium acetate may be added to improve the sensitivity of contaminated microorganisms to radiation and reduce the irradiation dose. can. The immobilized enzyme finally obtained by the above method uses gelatin as a matrix,
It contains about 85 to 95% water. Therefore, the immobilization method of the present invention is particularly useful for immobilizing hydrolytic enzymes such as invertase and isomerizing enzymes such as glucose isomerase, which are difficult to deactivate in the presence of water, among the enzymes mentioned above. The present invention is based on the above-mentioned structure, but in addition to the above-mentioned components, various additives such as various fungicides, preservatives, enzyme stabilizers such as phosphates, and agents that improve enzyme action are used. can be added. [Effects of the Invention] When enzymes are immobilized by the method of the present invention, gelatin has a large ability to retain enzymes, so there is almost no elution of enzymes, and there is an advantage that continuous use is possible. Furthermore, since the gel strength is improved by radiation irradiation, the gel is less likely to break even after long-term use, and the adhesion to the device is improved, which is advantageous in terms of handling. Furthermore, since the enzyme-containing gel is sterilized by radiation irradiation, a sterile immobilized enzyme can be obtained, and there is also the advantage that a sterile product can be obtained when a sterile substrate or the like is treated. Therefore, the immobilized enzyme obtained by the method of the present invention, such as invertase (β-fructofuranosidase), can be immobilized and used to produce invert sugar, such as decomposing sucrose into glucose and fructose, or It is widely used to immobilize isomerase and produce isomerized sugar (mixed sugar of glucose and fructose) from glucose. Next, the present invention will be explained with reference to examples. [Example] Example 1 Add 0.05M acetate buffer (PH
5.0) Add 100ml to prepare a 5% gelatin solution, cool this solution to 20℃, and then add
ml of invertase (manufactured by Toyobo Co., Ltd.)
100 mg was added and mixed. Next, mix the above mixture at 5°C.
After cooling to gelatin and shaping into particles, the gelatin was irradiated with 10 kiloGy of gamma rays in a cobalt-60 irradiation pool at 15°C to obtain an enzyme immobilized in gelatin. The performance of the immobilized enzyme thus obtained was measured as follows. Γ Enzyme reaction Using 1% sucrose solution adjusted to pH 5.0 with 0.05M acetate buffer as a substrate, add 0.5mg of enzyme (enzyme alone or immobilized enzyme produced by the above method) to 50ml of this solution. The enzyme activity was determined from the amount of reducing sugar produced by carrying out the enzymatic reaction at 30°C. Here, 1 unit (unit of activity) is the amount of enzyme that decomposes 1 μmol of substrate in 1 minute at 30°C. As a result, the activity of invertase immobilized by the above method was 43.0 units/mg.
The activity of unirradiated invertase was 152.6 units/mg. Therefore, the activity of the enzyme immobilized by the above method, that is, the activity yield, was 100×43.0/152.6=28.2%. Γ Enzyme activity due to repeated use Substrate solution (sucrose concentration 1%, pH 5.0) in 50ml
The activity was determined from the amount of reducing sugar produced 10 minutes after adding immobilized enzyme containing 0.5 mg of invertase.
Next, the immobilized enzyme was separated and washed with distilled water, and the washed immobilized invertase was added to the substrate solution in the same manner as before to determine the activity. This operation was repeated 10 times, and the activity for each time was determined as a relative value when the first time activity was taken as 100%. The results are shown in Table-1.

[Table] Table 1 shows that the enzyme activity is sufficiently maintained even after repeated use. Γ Enzyme activity due to continuous use A column was filled with immobilized enzyme containing 20 mg of invertase (5% gelatin, initial enzyme concentration 2 mg/ml, irradiation dose 10 kg Gy), and a 5% sucrose solution (PH5.0) was added to the column at SV= 5.0, and the retained enzyme activity (% sucrose decomposition) was determined. In addition, SV is space
Velocity (space velocity) is the value obtained by dividing the volume of liquid that passes through an enzyme column in one hour by the volume of immobilized enzyme. The results are shown in Table-2.

[Table] Table 2 shows that even after 168 hours, the sucrose decomposition ability was as high as 96.5%, indicating that the method of the present invention resulted in less elution of the enzyme and firm immobilization. Comparative Example A gelatin aqueous solution with a concentration of 4% was irradiated with cobalt 60 gamma rays (15 kiloGy) to insolubilize the gelatin, and then dried at 50°C. Next, grind the dried gelatin,
Select gelatin particles with a diameter of 0.4 mm or less, and apply invertase (Toyobo Co., Ltd.) to 500 mg of gelatin.
10 mg (manufactured by M.D.) were mixed and added to 4 ml of water to moisten the mixture. Thereafter, the enzyme was washed with acetone, immersed in glutaraldehyde (10%) for 1 minute for immobilization, and then washed with water to obtain an immobilized enzyme. The activity yield of this immobilized enzyme was determined in the same manner as in Example 1, and was found to be 13.9%. Example 2 An immobilized enzyme was prepared in the same manner as in Example 1 except that the gelatin concentration and radiation dose were changed, and the activity yield was determined. The results are shown in Table-3.

[Table] From Table 3, when the gelatin concentration is low (1%), the activity yield is low, but when it exceeds 2%, the activity yield becomes almost constant, and as the irradiation dose increases, the activity yield decreases. I understand that. Example 4 Using an enzyme other than invertase as the enzyme, an immobilized enzyme was prepared in the same manner as in Example 1 except for the conditions shown in Table 4, and the activity yield was determined. The results are shown below.

【table】

Claims (1)

[Claims] 1. After gelatinizing an enzyme-containing gelatin aqueous solution,
A method for producing an immobilized enzyme, which comprises immobilizing the enzyme within a gelatin gel by insolubilizing gelatin by irradiating the gelatin with radiation. 2 The gelatin aqueous solution contains 2 to 10% gelatin by weight.
A manufacturing method according to claim 1 containing: 3. The manufacturing method according to claim 1, wherein gelation is performed by cooling. 4. The manufacturing method according to claim 1, wherein the radiation dose is 2 to 30 kiloGy. 5. The manufacturing method according to claim 1, wherein the enzyme is a non-proteolytic enzyme. 6. At least one enzyme selected from the group of invertase, β-glucosidase, catalase, lysozyme, amyloglucosidase, lactase, maltase, amylase, urease, lipase, esterase, glucose isomerase, glucose oxidase, dehydrogenase, and penicillinase. The manufacturing method according to claim 5.