JPH0555109B2 - - Google Patents

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention relates to a method for producing an enzyme immobilization carrier used for immobilizing an enzyme, which is a protein that catalyzes a biochemical reaction, for use in an industrial production process. It is something.

(Prior Art) In order to carry out biological reactions using enzymes industrially, it is common to adsorb enzymes to polysaccharides or synthetic polymers, or to immobilize them by enclosing them in gel. However, since these carriers are organic materials, they tend to have low mechanical strength, making them unsuitable for scale-up. Therefore, it is preferable to immobilize the enzyme on the surface of ceramic, which has high mechanical strength and is resistant to heat and chemicals for sterilization. However, even those belonging to the category of ceramics, such as murai and cordierite, have a specific surface area of around 1 m ² /g, so they have the disadvantage that the amount of enzyme that can be immobilized is small. Siliceous porous glass beads are also known as carriers for enzyme immobilization, but although they have the advantage of having a relatively large specific surface area of 75 m ² /g, they have the disadvantages of complicated manufacturing processes and extremely high costs. It was hot.

Recently, attempts have been made to use sepiolite, which is made of magnesium silicate, as this type of carrier. For this purpose, it is necessary to remove impurities that cause a decrease in the specific area from the sepiolite raw stone and use a high-purity sepiolite raw material.
In general, sepiolite rough contains a large amount of carbonate minerals such as dolomite, so in the past, the parts with many impurities had to be removed from the sepiolite rough and then further refining was required. The ratio of carriers became extremely low, and there was a problem that they could not economically compete with other carriers.

(Problems to be Solved by the Invention) The present invention solves the above-mentioned conventional problems and has a large specific surface area of around 100 m ² /g to immobilize a larger amount of enzyme than conventional methods. This method was completed with the aim of producing a carrier for enzyme immobilization that can be produced from raw sepiolite, which has a lot of impurities, and which has been discarded in the past.

(Means for Solving the Problems) The present invention involves crushing and sieving sepiolite raw stone containing 16% or more of ignition loss and 5% or more of CaO to obtain a constant particle size, and then heat-treating it within a temperature range of 800 to 1000°C. The material is characterized by being metasepiolite or protoenstatite.

The sepiolite rough used in the present invention is a lump of fibrous magnesium silicate,
It has a variety of shapes, including cork-like, leather-like, and lump-like shapes, but its crystal structure is unique, resembling small pieces of talc stacked together as bricks. This kind of sepiolite rough stone has many pores, and when examining the pore size distribution, it is found that it has peaks around 10 Å and 200 Å. It is thought that pores around 10 Å are due to the above-mentioned crystal structure, and pores around 200 Å are due to the fiber diameter. Sepiolite rough is mainly produced in Spain, Turkey, China, Korea, etc. As mentioned above, sepiolite rough contains large amounts of carbonate minerals such as dolomite (CaCO ₃ / MgCO ₃ ). These impurity contents are expressed as loss on ignition and CaO content when analyzing the chemical composition. In the present invention, raw sepiolite with a high content of dolomite, etc., having a loss on ignition of 16% or more and a CaO content of 5% or more, which has been previously discarded, is used.

In the present invention, such sepiolite raw stone with a high ignition loss and CaO is crushed, preferably 30 to 200
After being sieved into mesh and sized to a constant particle size, it is heat-treated within a temperature range of 800 to 1000°C, which is higher than the thermal decomposition temperature of carbonate minerals, without adding water.
Through such heat treatment, sepiolite loses its water of crystallization and turns into a ceramic, transitioning to a metasepiolite or protoenstatite state, but the crystallographic structure of this phase has not been fully elucidated at present. However, the present inventors have confirmed that this heat treatment causes a change in the crystal structure of sepiolite, causing a change in the pore size distribution. In other words, as shown in Figure 1, raw sepiolite with many impurities shows a broad pore size distribution with pores of 300 Å or less in raw form, as shown by the broken line, but when it is heat-treated at a low temperature of about 500°C, The pore size distribution shows a pore size distribution with a larger peak between 200 and 400 Å, and furthermore, when heat treated at around 900°C as in the present invention, the pore size distribution shows a pore size distribution with a sharp and large peak around 400 Å, as shown by the solid line. .

According to the inventor's estimation, this variation in pore size distribution is caused by the thermal decomposition of dolomite, which is present in large amounts in the fiber structure of sepiolite, to generate CO ₂ gas, and this CO ₂ gas is This is caused by the spreading of sepiolite fibers. However, when heat treated at a temperature exceeding 1000°C, the peak value decreases, which is thought to be because so-called sintering begins to occur. Note that a heat treatment time of about 1 hour is sufficient, and a support with much higher strength than raw sepiolite can be obtained.

The metasepiolite or protoenstatite thus obtained is suitable as a carrier for enzyme immobilization because it has sufficient pores with a pore diameter of 200 Å or more suitable for immobilizing enzymes, and is shown in Figure 2. As shown in the graph of the present invention, the horizontal axis shows the heat treatment time of sepiolite, and the vertical axis shows the amount of invertase immobilized per gram of carrier expressed in units of enzyme activity. It can be seen that the largest amount of immobilization can be obtained when heat treatment is performed at 800 to 1000°C. If the heat treatment temperature is less than 800℃, sufficient change in crystal structure will not occur, making it impossible to obtain a pore size distribution suitable for enzyme immobilization, and if the heat treatment temperature exceeds 1000℃, so-called sintering will occur. This is thought to result in a decrease in the number of pores, resulting in a decrease in the amount of enzyme immobilized. Furthermore, sepiolite rough stones with a loss on ignition of less than 16% or a CaO content of less than 5% have a small amount of carbonate minerals such as dolomite mixed in, so the above-mentioned fluctuations in pore size distribution are not sufficiently affected. It is not suitable for the present invention, which aims to effectively utilize waste, because it is expensive and has been effectively used as a material with a low impurity content.

(Example) Next, preferred embodiments of the present invention will be shown.

Raw sepiolite from Spain containing 23% loss on ignition and 9.8% CaO is crushed using a crusher, sieved into granules with an average particle size of 80 to 100 mesh, and then placed in an electric furnace in air. Heat treatment was performed at 900°C for 1 hour. The pore size distribution of the obtained carrier was measured by the method of differentiating the cumulative porosity.
The pore diameter peaks at 400 Å and the specific surface area is 100 m ² /
It was hot at g. This metasepiolitic carrier is
% gamma-aminopropyltriethoxysilane, washed with acetone, air-dried overnight, treated with 1% glutaraldehyde aqueous solution for 3 hours, and treated with 1% invertasenoacetic acid buffer (PH
4.0) and maintained for 1 hour, and further washed with buffer to immobilize invertase on the carrier. Next, the following operation was performed to measure the invertase activity immobilized on the carrier.

First, acetic acid buffer 30 containing 1% sutucarose.
After constant temperature of ml at 30℃, soak the above carrier, and sample 100ml each while stirring with a stirrer.
The reaction was completely stopped by adding a certain amount of 0.1N Na ₂ CO ₃ . Next, the product glucose was quantified by Wako Pure Chemical's glucose B test method, and the amount of glucose produced per unit time was calculated. Expressing the results as 1 unit of enzyme activity that produces 1 μmol of glucose per minute, it is 290 units/g, which is 10 units/g for the conventional cordierite carrier and 3 units/g for the mullite carrier. In contrast to Corning's alumina bead carrier for enzyme immobilization, which was 160 units/g, an extremely large value was obtained.

(Effects of the Invention) As is clear from the above description, the present invention utilizes extremely cheap raw sepiolite, which has been discarded in the past due to its high content of impurities, and has a large specific surface area to immobilize enzymes. It has pores of 200 Å or more suitable for immobilization, and can immobilize a much larger amount of enzyme than conventional cordierite, mullite, etc., and can immobilize enzymes that are equivalent to or greater than expensive glass beads or ceramic beads. We succeeded in obtaining a carrier for enzyme immobilization that can be immobilized. Therefore, the present invention can greatly contribute to the development of industry by solving the problems of the conventional technology.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the pore size distribution of the carrier for enzyme immobilization of the present invention, and FIG. 2 is a graph showing the relationship between the heat treatment temperature of sepiolite and the amount of invertase immobilized.

Claims (1)

[Claims] 1 Sepiolite raw stone containing ignition loss of 16% or more and CaO 5% or more is crushed and sieved to a constant particle size, and then heat-treated within a temperature range of 800 to 1000℃ to produce metasepiolite or protoenstatite. Characteristic method for producing carriers for enzyme immobilization.