JPS6013674B2 - insoluble bioactive - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an immovable biologically active substance containing biologically active substances such as enzymes and microorganisms in a porous material having pores.

Recently, bioactive substances such as enzymes and microorganisms are immobilized on carriers,
Research has been conducted to effectively utilize expensive enzymes and microorganisms by using immobilized enzymes in reactions, and in some cases, immobilized enzymes are used industrially. This immobilized enzyme is one in which the enzyme is enclosed in a polymer, or one in which the enzyme is physically or chemically bonded to the surface of porous alumina or the like.

However, enzymes encased in polymers have poor mechanical strength, and when packed in a reaction tower, etc., they may collapse or open, or the polymer may be exposed to microorganisms. Furthermore, when enzymes are adsorbed onto the surface of porous alumina or the like, the diffusion resistance within the pores is large, making it difficult to obtain sufficient enzyme activity, and the enzymes tend to separate. Furthermore, methods in which enzymes are chemically bonded to the surface of porous materials have drawbacks such as large deactivation of enzymes during bonding. The present invention has been made in view of the above-mentioned drawbacks, and is an insoluble biologically active substance in which a biologically active substance is enclosed within a hollow body, the hollow body having rigidity, and the size of the biologically active substance being larger than that of the biologically active substance. It is composed of a porous material that has through-holes with a small effective diameter, and its purpose is to prevent biologically active substances from flowing into the product and to ensure effective use of biologically active substances.

In the present invention, the biologically active substances contained within the hollow body are enzymes, microorganisms, antigens, antibodies, and hormones, and may be a mixture of two or more of these. In addition, the hollow body that encloses the biologically active substance has rigidity so that it will not be significantly deformed or damaged when packed into a reaction tower, etc. It is composed of a porous material having pores with an effective diameter smaller than the size of the material.

For the hollow body, a core of a desired shape is formed in advance with organic matter such as cellulose, and on the surface of this core, lotus algae, alumina, silica alumina, zeolite, titania, zirconia, acid clay, silica gel, silicon carbide, diatom There are inorganic porous hollow bodies having communication holes formed by gases generated by combustion of organic substances when coating ±-pennite or the like and then firing. The diameter of the hole in these hollow bodies is determined by the selection of firing conditions in the case of inorganic bodies.

However, this diameter does not necessarily need to be smaller than the size of the biologically active substance, and in cases where the biologically active substance is adsorbed within the passage hole surrounding the biologically active substance, the diameter is substantially smaller than the size of the biologically active substance after adsorption. It is sufficient that this effective diameter is smaller than the biologically active substance. For example, if the hollow body is composed of alumina and the bioactive substance encapsulated is glucose isomerase, the glucose isomerase is about 1
It has a size of 00A and has adsorption properties with alumina, so it adsorbs into the passage hole, so the diameter of the alumina lotus passage hole is 300A.
Even if it is A, the effective diameter will be 100 A when glucose isomerase is injected. Therefore, if the pore size is smaller than 300A, glucose isomerase will not flow out to the outside and will be "subsolubilized."On the other hand, if the adsorption between the hollow body and the biologically active substance is weak, the biologically active substance will be absorbed into the hollow body. It is necessary to make a hollow body with a pore diameter smaller than the size.

On the other hand, the external substrate passes through this lotus hole and reacts with the action of the internal biologically active substance, becoming a product and exiting through the lotus hole again to the outside, but the effective meridian of the lotus hole is too small. The effective diameter is preferably large within a range that does not exceed the size of the biologically active substance, since this prevents the passage of substrates and products.

Furthermore, when two or more types of biologically active substances are mixed, it is necessary to use a hollow body with an effective diameter smaller than that of the smaller biologically active substance.

The insolubilized biologically active substance enclosed in such a hollow body is packed into a reaction tower, etc., and comes into contact with a solution containing a substrate, and is used in the process of producing a reaction product. It is often contaminated with various microorganisms, and since the hollow body is inorganic, there is no need to worry about it being contaminated by microorganisms.

In addition, inorganic materials have greater rigidity than organic materials, so they have sufficient mechanical strength even if the wall thickness is made thinner, and the resistance of the substrate and products passing through the lotus hole can be reduced, and the reaction rate can be increased. improves.

Point 9: In an inorganic hollow body, even if harmful bacteria adhere to the biologically active substance, the biologically active substance can be injected after the hollow body is heated and sterilized, so the activity will not be inhibited by the bacteria. I am not a prisoner.

Therefore, in the present invention, inorganic hollow bodies are preferred.

The most preferable hollow body in the present invention is a spherical porous hollow body mainly composed of alumina formed by firing, and has a pore diameter smaller than that of the biologically active material,
It also has at least one injection port for injecting a biologically active substance into the hollow body. The insoluble bioactive substance obtained by injecting the biologically active substance through the injection port and sealing the injection port with an adhesive or the like has a strong mechanical strength because it is spherical. Among them, those having a size of 0.5 to 1 shipboard are preferred because they can be easily filled and discharged into reaction towers with complex shapes and improve the reaction rate of the substrate. The reason for this is not clear, but it is thought that when the hollow body is less than 0.5 cells, clogging occurs in the packed bed and the solution containing the substrate does not reach the surface of the hollow body sufficiently, resulting in a slow reaction rate. It will be done.

In addition, when the hollow body has one or more ribs, the total surface area of the hollow body in contact with the solution in the packed bed becomes smaller, the number of through holes decreases, and the apparent resistance of the substrate or product decreases. It is thought that because the size increases, the reaction rate decreases. As explained above, the insoluble biologically active substance according to the present invention is not adsorbed to the surface unlike the conventional ones, so there is no dust that is released due to the biologically active substance falling off, and the substrate has a thin wall thickness of the hollow body. If it passes through, contact with the active substance is extremely good due to liquid diffusion, so the reaction rate is improved compared to the reaction inside the pores.

Examples of the present invention will be shown below.

Example 1 A spherical porous non-hollow body mainly composed of willow alumina with a pore diameter of approximately 250A and an outer diameter of 2 was washed with demineralized water and further 0.05A in diameter.
It was washed with an aqueous solution of Mg(CH3COO)2 and 0.01 mole/So CoC12, and then soaked in a sufficient amount of glucose isomerase solution for 24 hours to fully adsorb glucose isomerase to the non-hollow body. .

Separately, a porous hollow body mainly made of alumina with a pore diameter of about 90 A, an outer diameter of 2 yam, and a wall thickness of about 0.2 mm (having one injection port of 0.5 mm) was similarly cleaned, and then this hollow body was cleaned in the same manner. After injecting the glucose isomerase solution into the body, the injection port was sealed with epoxy resin.

The non-hollow body and the hollow body each have a diameter of 2.5 c.
Glass tubes with a length of 2.5 cm were filled, and a 50% glucose solution was adjusted to pH 7.0 and temperature to 6yC, and passed through these glass tubes at a rate of 50 cc/min, and the amount of fructose in each passing liquid was determined. It was measured.

These results are shown in Table 1.

The numbers in the table are the rate of glucose conversion to fructose, and are relative to the hollow body on the first day as 100. Table 1 As can be seen from Table 1, the enzyme injected into the hollow body is
Compared to the case where the enzyme is adsorbed to a solid body, the amount of change to fructose is large and the enzyme is active, and this activity only slightly decreases after 30 days.

Example 2 5.1 units/9 of 100 units of urease were mixed 10 units in a 7.0 unit buffer containing dithiothreitol 0.0001 mol/unit and ethylenediaminetetraacetic acid 0.1 mol/unit.
diluted to

This urease was injected into a porous (pore diameter 100A) spherical hollow body (wall thickness 0.2 side, diameter 4 ribs) mainly composed of alumina by the same method as in Example 1.

This 30N hollow body was filled into a glass urea nitrogen analyzer with a diameter of 2 m wide as shown in Fig. 1.

A known solution containing urea nitrogen is poured into the upper part of this hollow body.
The reaction of urea nitrogen with ammonia by urease in the hollow body 1 was detected with an ammonia electrode 2 and measured with a meter 3 to create a calibration curve. Next, serum 3 was diluted with 0.1 mol/l of ethylenediaminetetraacetic acid and buffer solution 15' and injected from the upper part of the hollow body, and the amount of urea nitrogen was measured.

As a result of measuring the same serum 10 times, the average value was 15.1
'/d', the standard deviation was 0.25 9/, so there was little variation. Similarly, measurements were made on a solution of 9/d' of urea nitrogen 30, and the variation was small with an average value of 9/d' of 29.8 and a standard deviation of 9/d' of 0.3. Next, in order to investigate the deactivation of this urease, measurements were carried out at intervals of several days using a solution containing urea nitrogen 30 di/d', and the results were as shown in Table 2, indicating that sufficient activity was maintained even after several days.

Table 2 Next, in order to examine the storage stability of this urease, it was stored at 4°C, and the activity was compared with that immediately after injection into the hollow body.As shown in Table 3, the activity decreased by about 2% even after 6 months. Ta.

Table 3 Example 3 Three microorganisms of Brevibacterium ammoniagenes
The cell suspension obtained by shaking culture for 2 hours and centrifugal D separation was encapsulated in a spherical porous hollow body (pore diameter 800 A, diameter 3 skin) containing alumina as the main component in the same manner as in Example 1. did.

This hollow body was packed in a glass tube, and 1 mol/sodium fumarate solution (pH 8.5 temperature 37°C) was injected from above.
Table 4 shows the results of determining the conversion rate to L-malic acid and examining the activity of microorganisms.

The numbers in the table represent the rate of change to L-malic acid on the first day.
It is a relative thing. As a result, it was revealed that the microorganisms encapsulated in the hollow body were sufficiently durable for practical use.

Table 4

[Brief explanation of the drawing]

FIG. 1 is an example of analysis using the active substance according to the present invention. 1... Active substance, 2... Ammonium electrode, 3... Meter. Ta-no-zu

Claims (1)

[Scope of Claims] 1. A biologically active substance is an insoluble biologically active substance enclosed within a hollow body, the hollow body having rigidity and having communicating holes with an effective diameter smaller than the size of the biologically active substance. An insoluble biologically active substance characterized by being composed of an inorganic porous substance. 2. The active substance according to claim 1, wherein the hollow body is spherical. 3. The active body according to claim 2, wherein the hollow body is alumina. 4. The active substance according to claim 3, wherein the biologically active substance is one of glucose isomerase, urease, and glucose oxidase.