JPS60110291A - Porous gel containing immobilized biocatalyst and its production - Google Patents

Abstract

PURPOSE:To increase the effective area for reaction, and to enable the smooth release of bubbles from the gelled material in the gas-forming reaction, by including minute continuous pores in a gelled materials imobilized with a biocatalyst. CONSTITUTION:A solution of a gel substrate is mixed with a biocatalyst, gelatinized, and cooled to freeze the solvent in the gelled product. The solidified solvent in the frozen gel is sublimed under reduced pressure to obtain a porous gel. Continuous pores having pore diameter of 1-100mum can be produced by the freezing process, and the catalytic activity per unit area of the untreated gel can be increased to 1.2- several times.

Description

[Detailed Description of the Invention] [Field of Application of the Invention] The present invention provides a biocatalyst-immobilized porous gel with a large reaction area, in which biocatalysts, so-called enzymes, organelles, and cells are immobilized, and a method for producing the same. Regarding.

[Background of the invention]

Conventionally, enzymes have been used in the form of aqueous solutions during reactions, making repeated use difficult. In recent years, it has become possible to immobilize enzymes and microbial cells and handle them in a form similar to ordinary solid catalysts.

Among various immobilization methods, the gel entrapment method, in which enzymes are encapsulated in a hydrophilic polymer matrix, has the advantage of being relatively easy to operate and can be performed under mild conditions, and therefore has a wide range of applications. Specifically, IC is prepared by dissolving or dispersing enzymes and bacterial cells in a gel-based solution such as carrageenan, agar, acrylamide, or urethane, and then gelling the solution. When the form of use is granular, the particle size of these gel particles is adjusted in the order of diameters 05 to 5 from the viewpoint of practicality.

During the reaction, substrate molecules diffuse from the surface of the gel particles into the interior of the matrix. However, under practical reaction conditions, only the surface layer of the gel participates in the reaction VC, and the inside does not contribute to the reaction. That is, the gel entrapment method targets axial points where a large reaction area cannot be obtained. Of course, as the particle size becomes smaller, the reaction area increases, but it becomes difficult to retain the gel particles in a fixed bed or a fluidized bed. In addition, gel particles have low mechanical strength at 1"1. When used in a fixed bed, they are consolidated, resulting in large pressure loss, and when used in a fluidized bed,
Stirring tends to destroy the particles. Furthermore, during the reaction to generate gas, the Shigel particles are likely to be destroyed by air bubbles generated inside the gel. These are not limited to gel particles,
This is a common drawback of all forms of gelatinized products, such as gel membranes.

[Purpose of the invention]

The purpose of the present invention is to improve the above-mentioned drawbacks of the gel entrapment method, and to develop a new biological material that has a large reaction area and high mechanical strength, and that can smoothly release bubbles from inside the gel during the gas production reaction. An object of the present invention is to provide a catalyst-immobilized gelled product and a method for producing the same.

[Summary of the invention]

To summarize the present invention, the first invention of the present invention relates to a porous gelled material with a biocatalyst immobilized thereon, in which microscopic continuous cells are formed in the gelled material with a biocatalyst immobilized thereon. It is characterized by being inclusive.

The second invention of the present invention is an invention relating to a method for producing the porous gelled product of the first invention, which includes a first step of adding and mixing a biocatalyst to the gel base bath liquid, and a first step of adding and mixing a biocatalyst to the gel base bath liquid. The second step is to process the mixture obtained in the second step to prepare a gelled product, the third step is to cool the gelled product obtained in the second step and freeze the solvent in the gelled product, and the frozen gelled product obtained in the third step is It is characterized by including each step of a fourth step of sublimating and removing the solidified solvent under reduced pressure.

Furthermore, the third invention of the present invention is an invention regarding another method for producing the porous gelled product of the first invention,
The first step is to add and mix the biocatalyst and fibrous material to the gel base liquid.
A second step of treating the mixture obtained in the first step to prepare a gelled product; a second step in which the gelled product obtained in the second step is brought into contact with an enzyme that decomposes only the fibrous material, and the fibers in the gelled material are and a sixth step of decomposing and removing the solid matter.

The present inventors have conducted intensive studies on a method for producing gel particles in order to increase the effective area of the gel particles by providing pores that penetrate the gel particles. As a result, the present invention relating to providing a large number of pores in gel particles without impairing the catalytic activity of the gel particles was completed.

The manufacturing method according to the present invention can be roughly divided into two types: freezing method and decomposition removal method.

As the target biocatalyst that can be applied to the present invention, a wide range of known ones that can be immobilized by conventional entrapment methods can be used. That is, enzymes, organelles, cells, etc. are targeted.

As the gel base, known materials used in the entrapment method can be used. For example, carrageenan, alginic acid, agar, and the like are representative examples of materials that can be sizolized by heating and sigelized by cooling. Mannan can be used in the case of an alkali-resistant enzyme, and gelatin can also be used in the case of use at a relatively low temperature of 30° C. or lower. In addition, there are acrylamides and methyl methacrylates that are cured with visible light, ultraviolet rays, radiation, and radical initiators, and those that are crosslinked with a crosslinking agent to form a three-dimensional matrix to form a sigella, such as dextran and Ze'Latin. It can be fully used. In addition to gels that swell with water, polyurethane, polystyrene, and the like that swell with organic solvents can also be used.

The concentration of these materials is adjusted so that a gel breaking strength of 1f/crn2 or more can be obtained. In the first step,
Biocatalyst stabilizers, for example enzyme stabilizers such as serum albumin, may be added.

Each manufacturing method of the present invention will be explained in more detail below.

First, in the freezing method of the second invention, freezing is preferably carried out for 60 minutes or less at a temperature of -3°C or less, which is higher than the melting point of the solvent used.

If the gel is frozen for one hour or more in a temperature range of -2° C. below the melting point of the solvent, the gel will shrink and a solidified solvent will be formed outside the gelled product, which will easily destroy the gelled product. The frozen gelled product can be dried under reduced pressure of 10wIHg or less.

When producing a porous gelled product by the freezing method of the present invention, a fifth step may be included in which the dried gelled product obtained in the fourth step described above absorbs a solvent such as water or a pH buffer. . Further, a fifth step of mechanically compacting the dried gelled product obtained in the fourth step described above, and a solvent such as water or a pH buffer solution is added to the compacted dried gelled product obtained in the fifth step. The sixth step of swelling may also be included.

Furthermore, in each of the above-mentioned methods, the treatment in the second step may include particle formation, linearization, or film formation treatment. However, when performing the granulation, linearization, or film formation treatment, there are two methods: 0) The process in the second step involves forming the mixture obtained in the first step into particulates, linearizing or into a film, and then forming each particulate body or linear body. Alternatively, there is a method of gelling each membranous body.

The compaction described above is optimally carried out at a pressure of 1 kg/am" or higher. If the compaction is carried out outside the pressure range mentioned above, after compaction, when the solvent is absorbed, the dry gel will lose 50 to 50% of its original volume.
Restore to 95chi. Preferably 1.5 kg7cm2
If it is compacted in the above manner, even if it swells, it can be kept at 3D% or less of its original volume.

Furthermore, during compaction, it is preferable that the solvent content of the gelled product be 20 or less. This is because if the solvent content exceeds 40%, the material will swell and swell with the solvent after compaction and will return to more than 90% of its original volume.

According to the freezing method of the present invention described above, continuous cells with a pore size of 1 to 100 μm could be formed.

As a result, the enzyme activity per unit volume of untreated gelled material could be improved by 1.2 to several times.

Furthermore, it has been found that even if the dried gelled material is compressed into pieces and swelled in water, it will return to less than its original volume. Moreover, it was also found that the activity per unit dry weight of gelled product remained unchanged or only slightly decreased. That is, we succeeded in significantly improving the enzyme activity per unit volume compared to the original untreated gelled product. It has also been found that the mechanical strength of the gelled product is significantly improved, and that bubbles generated inside the gelled product can be smoothly released to the outside in response to a gas generation reaction.

Next, the decomposition and removal method, which is the third aspect of the present invention, will be specifically explained.

Fibrous substances include cellulose, starch, DNA,
RNA etc. are used.

As cellulose, natural fibers such as cotton and filter paper, or processed natural fibers such as cellophane and soap can be used. When these fibers are newly prepared, spinning is extremely suitable since the diameter of the fibers can be adjusted. The diameter of the fiber is CtS ~ 10 of the gel diameter, and the length is 5 of the diameter.
It can be used in a range of 0 to 150%. Further, the amount to be added is in the range of 10 to 500 cm per ml of gel.

If the fiber diameter, length, and amount added are less than the lower limit of the above range, it will be difficult to smoothly conduct the liquid into the gelled product.
Catalytic activity does not improve. On the other hand, if it exceeds the upper limit of the above range, it becomes impossible to maintain practical mechanical strength as a porous gel.

The resulting mixture is then processed to obtain a gelled product, and then
The gelled product is brought into contact with a hydrolyzing enzyme of the polymeric fibers, and the fibers are decomposed into the interior of the gelled product from the portion where the fibers are applied on the surface. For contact with the hydrolytic enzyme, methods such as immersion in a hydrolytic enzyme solution or coating the gelled product with the enzyme are used. It is necessary to use a hydrolytic enzyme that not only has high substrate specificity for fibers but also has a purity that does not contain grotease as an impurity above a practical concentration. Cellulase is used for cellulose, α-amylase is used for starch, DNAsθ is used for DNAIC, and RNase is used for RNA.

Conditions for hydrolase treatment vary depending on the stability of the target enzyme and the reaction characteristics of the hydrolase. That is, it is carried out under conditions that do not impair the activity of the target compound and are as suitable as possible for the hydrolytic action of each hydrolytic enzyme used. After the hydrolysis treatment is completed, wash with water or a buffer solution stable to enzyme activity, if necessary.

In the above-mentioned decomposition and removal method, the second step may also include a particleization treatment, as in the case of the freezing method of the present invention, and in that case, the above-mentioned (4) or ( Either method B) may be adopted.

Even in the decomposition and removal method described above, a large number of through holes were formed in the gelled product, and the enzyme activity per unit volume of the gelled product increased.

[Embodiments of the invention]

Hereinafter, the present invention will be explained in more detail with reference to Examples and Comparative Examples, but the present invention is not limited to these Examples.

Incidentally, the attached FIGS. 1 to 6 are graphs showing the relationship between reaction time and enzyme activity.

Comparative Example 1 0.15 f of carrageenan and 4.85 r of water were heated in an autoclave at 120°C for 15 minutes to make 5 t of carrageenan solution.
I got k. The above carrageenan solution was cooled to 40°C.

To this carrageenan solution, an aqueous solution of 10 Iv of urease (originated from sea cucumber) and 20 Iv of bovine serum albumin (used as an enzyme stabilizer): 5 I: 3 d of water was added and mixed.

The mixed solution was immediately poured onto a flat plate to form a plate-shaped gel with a thickness of 1.7 to 2.4 square centimeters, which was cut into 2×2 square pieces.

The interior of the resulting gel particles was observed by microscopic photography and was found to consist of a homogeneous transparent gel. Then, the gel particles were separated at 0.5 f, and 10 thiurea 5+++/!
, 20vt of 1M potassium citrate buffer (pH & 7) and 5d of water were added, and the mixture was shaken back and forth at 40°C for 30 minutes (
amplitude 3 crn, 30 strokes/min). The time course of the amount of annia-N produced by the decomposition of urea was measured.

The results are shown in FIG. In other words, Fig. 1 is a graph showing the relationship between reaction time (minutes) (horizontal axis) and enzyme activity (2 gN-NH3/f untreated gel) (vertical axis), and the graph of the examples described later is also shown. show.

On the other hand, the gel particles α1t were collected, water SmTh was added, and 40
After shaking at °C for 1 hour, centrifuge (45002, 10 minutes)
Then, the packed volume (packθ+l vo1u
mθ) was measured. The results are shown in Table 1.

Example 1 Gel particles 2f of the same batch prepared in Comparative Example 1? It was placed in an eggplant flask and frozen at -50°C within 2 minutes. The frozen gel particles were dried under reduced pressure of 1 mmHg or less to obtain a dry gel of 46 cm. When this dried gel was allowed to absorb water and viewed under a microscope, the formation of numerous continuous cells was observed.

Next, 0.5 f in terms of gel particles before drying was collected from the dried gel particles, and the time course of decomposition of shiurea was measured using the same procedure as in Comparative Example 1. The results are shown in FIG. On the other hand, Q, 11 was fractionated from the dried gel particles in terms of gel before drying, and the υ packed volume was measured using the same procedure as in Comparative Example 1. The results are shown in Table 1.

Example 2 Dry gel particles 3 from the same batch as prepared in Example 1
0.0 mm (gel equivalent before drying: 13 f) was compacted at a pressure of 10 kg/cm'' to form a flake.

Ten squares of this flaky dry gel were placed in water, and the packed volume was measured in the same manner as in Example 1. The results are shown in Table 1. In addition, the gel after being swollen with water was observed under a microscope. The continuous cells of Example 1 were compacted.

For Comparative Example 1, Examples 1 and 2, the urease activity of gel particle dry matter 1 and the urease activity of packed volume 1 d are summarized and shown in Table 1. The urease activity based on dry weight is that of untreated gel particles (Comparative Example 1)
Compared to this, the freeze-drying gel particles (Example 1) improved by 1.5 times, and the freeze-drying-consolidation gel particles (Example 2) improved by 3 times. On the other hand, the packed volume of 12 kg of dry matter in Example 2 can be reduced to one quarter of that in Comparative Example 1. Therefore, the activity of the packed volume 1 of Example 2 was improved by a factor of 1105.2 compared to that of Example 2, which greatly contributed to making the fixed bed more compact.

1st Table Arrow Initial velocity dust calculated from 3 amounts of N-NU generated in 10 minutes Example 3 Purified agar Q, 15 f, water 4.85 S' at 90°C.
The agar solution St' (obtained) was heated for 15 minutes. The above agar solution was cooled to 40°C. Urease 3 ov was added to this agar solution.
g (origin from sea cucumber, specific activity 4 U/■, 1 U: 1 μmol N
- NH3/min) and bovine serum albumin (used as a stabilizer for enzymes) at 30% in water and 1.6% in O2.
31F, and fibers (fiber diameter 50 to 60 μm) obtained by chopping absorbent cotton to lengths of 2 to 3+n+n were added and mixed. Immediately pour the mixture onto a flat plate to a thickness of 1.7 to 2.3
It was solidified and molded into a mosquito plate shape. Then, 100q cellulase (Trichoderma origin, 100FPA-U/q) k7m
7! To a solution dissolved in 005M sodium acetate buffer (pH 4, 5), 1.52 of the above fiber-containing urease-fixed agar gel particles were added and treated at 40°C for 2 hours. When the fiber-containing shiurease-fixed agar gel particles were observed under a microscope after cellulase treatment, it was observed that the cellulose fibers that were introduced before the cellulase treatment disappeared and that through-holes were created in the disappeared areas. It was. The gel particles after cellulase treatment were separated and collected, and added to 1M citrate buffer (pH 6, 7).
)20m7! and adding 10 d of 10% urea solution to 40
The mixture was shaken reciprocally at ℃ for 2 hours (amplitude 3crn1 filter 0 strokes/min).

Figure 2 shows the time course of the amount of ammonia N produced by the decomposition of urea.

That is, FIG. 2 is a graph showing the relationship between reaction time (minutes) (horizontal axis) and enzyme activity (μ9N −NU3/y) (vertical axis), and also shows the results of the comparative example described later.

Comparative Example 2 The same batch of fiber-containing shiurease-fixed gel particles prepared in Example 3, 1.5 t'x, Q without adding cellulase,
05 M sodium acetate buffer (pH 4,7),
The temperature was kept at 40°C for 2 hours in the same manner. After the above treatment, a urea decomposition test was conducted in the same manner as in Example 3. Figure 2 shows the time course of ammonia N production.

Comparative Example 3 After storing the same batch of fiber-containing Vase-immobilized gel particles (1.5 fI) prepared at pH 5Tf for 2 hours at 5°C, a urea decomposition test was conducted in the same manner as in Example 3. The time course of ammonia N production is also shown in Figure 2.

Comparative Example 4 In preparing Vase-immobilized gel particles, urease-immobilized gel particles were prepared in the same manner as in Example 3 by adding CL6f water to a cotton fiber substitute. This gel particle 1.52
A urea decomposition test was conducted in the same manner as in Example 3. The time course of ammonia N production is also shown in Figure 2.

The results of Example 3 and Comparative Examples 2 to 4 are summarized in terms of urease activity per 12 gel particles and are shown in Table 2. Example 3 showed about 1.4 times the activity as compared to the comparative example, and the activity of the present invention was increased by 40 times compared to the conventional simple comprehensive method.

2nd Table Using the initial velocity calculated from the amount of N-NHg generated in 10 minutes Comparative Example 5 Alginic acid 0.4? , water 4.6 f'ft105℃, 1
After heating for 0 minutes, alginic acid solution 5f was obtained. Add 1 lactase (originated from Satucharomyces yeast) to the above alginate solution.
An aqueous solution dissolved in water 2- was added to 01Iv1 and mixed. The mixed solution was dropped into a dicalcium chloride solution and solidified to obtain spherical particles with a diameter of 1.5 to 4 mm. Next, the gel particles were washed with water, 0.5f was collected, 1d of 0.1M potassium phosphate buffer pH 6.5 was added, and ortho-nitrophenol-β-D-galactopyrano7d ( 05 aqueous solution 3- of 0mGF) was added. The mixture was reacted at 30° C. for 10 minutes, cooled on ice, and all the enzyme-immobilized particles were separated in a furnace. Next, the amount of produced orthophenol in the p solution was determined colorimetrically at a 20 nm. 10
Lactase activity was calculated from the initial rate calculated from the amount of orthophenol produced per minute. On the other hand, gel particles IIL
1 t was collected, 5 mft of water was added, and the mixture was shaken at 30°C for 10 minutes, centrifuged (45009, 10 minutes), and the packed volume was measured. Table 6 shows the lactase activity of packed volume and packed volume, and the lactase activity of dry comb.

Example 4 Two grams of gel particles of the same patch prepared in Comparative Example 5 were placed in an eggplant flask and frozen within 1 minute in liquid nitrogen. The frozen gel particles were dried under reduced pressure of 1 mHg or less to obtain dry gel 40q. When this dried gel was soaked with water and viewed under a microscope, the formation of numerous continuous cells was observed. Next, 0.5f in terms of pre-drying gel particles was collected from the dried gel particles, and diortho.
The initial rate of decomposition of nitrophenol-β-D-i59 topyranondo was determined. On the other hand, 0.1 g in terms of gel before drying was collected from the dried gel particles, and the packed volume was measured using the same procedure as in Comparative Example 1. The results, lactase activity as per packed volume and lactase activity as per dry weight are shown in Table 3.

Example 5 Dry gel particles 2 from the same batch as prepared in Example 4
0TIl! (Gel equivalent before drying: 1.3 t) t" 8
, compressed by pressing at a pressure of kg/1yn2, 2X2
It was made into a flaky shape of m+. 10 q of this flaky dry gel was placed in water, and the packed volume was measured in the same manner as in Example 4. The results are shown in Table 3.

Table 3 Example 6 0.2 f of carrageenan and 4.82 g of water were added to an autoclave V-
Carrageenan solution 51 was obtained by heating at 110° C. for 5 minutes in a vacuum chamber. The above carrageenan solution was cooled to 40°C. To this carrageenan solution, 25 rug of Vase (originated from sea cucumber, specific activity 4 U/η, 1 U: 1 μmol N - N
1.06 S of an aqueous solution of H37 min) and bovine serum albumin xoI+9 (used as an enzyme stabilizer) dissolved in 1 F of water.
', and 0.62 of fibers (fiber diameter 60-60 μm) obtained by cutting absorbent cotton into pieces of length 2-6W11 were added and mixed.

Immediately pour the mixed solution onto a flat plate, solidify and mold it into a floating plate of 1.7-2.3 cm, 1.5-2.5 x 1.5-2.
5 verses x 1. D ~ 1.5 mm thick pieces were shredded to prepare urease-immobilized carrageenan gel particles containing cellulose fibers. Next, the above fiber-containing Vase-immobilized carrageenan gel particles 1. 59 was added and treated at 40°C for 2 hours. As a result of the cellulase treatment, the cellulose fibers that had been observed before the treatment disappeared, and through-holes were observed in the disappeared portions, as observed at 4fL. The gel particles after cellulase treatment were separated and collected, and mixed with 1M citrate buffer (pH 6,7) 20mi and 10 thiurea solution 10
The mixture was shaken for 2 hours at 40°C (amplitude 3).
crn, 30 strokes/min). FIG. 5 shows the time course of the amount of ammonia N produced by decomposition of urea.

That is, FIG. 6 is a graph showing the relationship between reaction time (minutes) (horizontal axis) and enzyme activity (py N -NH3/ml (vertical axis)), and also shows the results of a comparative example described later.

Comparative Example 6 The same batch of fiber-containing urease-fixed carrageenan gel particles prepared in Example 6 (1.59 i), Q without cellulase added, was added to 05M sodium acetate buffer (pH 4,7) and incubated at 40°C in the same manner. , and kept warm for 2 hours. After the above treatment, a urea decomposition experiment was conducted in the same manner as in Example 6. The time course of ammonia N production is also shown in Figure 3.

Comparative Example 7 Fiber-containing ciurease-fixed carrageenan gel particles of the same length prepared in Example 3 1.5 f Th 2 at 5°C
After storage for a period of time, a urea decomposition test was conducted in the same manner as in Example 6. The time course of ammonia nitrogen production is also shown in Figure 3.

Comparative Example 8 In preparing urease-immobilized gel particles, 0.6 t of water was added to the cotton fiber substitute, and urease-immobilized gel particles were prepared in the same manner as in Example 3. This gel particle 1.5
A urea decomposition test was conducted on the sample g in the same manner as in Example 6. The time course of ammonia N production is also shown in Figure 3.

The results of Example 6 and Comparative Examples 6 to 8 are summarized by Vase activity of 12 gel particles and are shown in Table 4. Example 6 showed about 1.4 times more activity than the comparative example, and the present invention showed a 40% increase in activity compared to the conventional simple inclusion method.

Table 4 Example 7 A liquid mixture of the following five components was poured between the glass walls with gaps i and 5 an, and allowed to stand at 15°C for 40 minutes to gel.
A gel plate having a thickness of 1.5 layers was prepared.

A21NTIC14,8sd Trishydroxymethylaminomethane 57- Distilled water & 3- B Niacrylamide 61 Methylenebisacrylamide α161F Potassium ferricyanide 1,4■ Distilled water 141d O: [1,14qb Ammonium persulfate 40@gD=
Urease rJ, 3f (originated from sea cucumber, specific activity 4 U/
q, 1ty: 1 fmol n -NH3/min) bovine serum albumin α5fc used as enzyme stabilizer) Distilled water 9.4 m B: Starch fiber (fiber length 2-Sm, fiber diameter 50-15
0 μm 1 fiber is prepared by cutting a thin film of potato starch paste dried into a film) 10f The above gel plate is 1.5~L 5 x 1.5~2.
Urease-immobilized polyacrylamide gel particles containing starch fibers were prepared by chopping into pieces the size of 5 snowflakes.Next, 50η of α-amylase (hay resistant origin, 7oOU/!) was prepared.
, IU: maltose from 1f molar starch/3 min) Kjaer's 0.05 M potassium phosphate buffer (pH 6,8
) was added with 1.5 f of the above fiber-containing α-Vase-fixed polyacrylamide gel particles and treated at 37°C for 15 minutes. 5. Observation using an optical microscope. It was observed that the introduced starch fibers disappeared and through holes were formed in the disappeared parts. Separate and collect the gel particles after α-amylase treatment,
1M citrate buffer (pH 6,7) 20m7! and io
n-urea solution 107 was added and shaken reciprocally at 40° C. for 2 hours (amplitude Scm, 50 strokes/min). Measure the time course of the amount of ammonia N produced during the decomposition of urea, calculate the apparent specific activity per 2 α-amylase-untreated gels from the initial rate, and 4. The value of OU/f was obtained.

Comparative Example 9 Urease-fixed fiber-filled polyacrylamide gel particles of the same batch prepared in Example 7 11.5? , 0.05M without adding α-amylase! Potassium J phosphate buffer (
pH 6,8) and kept at 37°C for 15 minutes in the same manner. After the above treatment, a urea decomposition experiment was conducted in the same manner as in Example 7.

The initial velocity was measured from the time course of the amount of ammonia N produced by the decomposition of urea, and the apparent specific urease activity of the α-amylase-untreated gel was calculated, and a value of 2.7 U/f was obtained. .

Comparative Example 10 After storing 1.52 of the same batch of fiber-containing ciurease-fixed polyacrylamide gel particles prepared in Example 7 at 5° C. for 2 hours, a urea decomposition test was conducted in the same manner as in Example 7. The initial rate was measured from the time course of the amount of ammonia N produced, and the apparent specific urease activity of 2 gels without α-amylase treatment was calculated to obtain a value of 2.6 U/f.

Comparative Example 11 During the preparation of urease-immobilized gel particles, urease-immobilized gel particles were prepared in the same manner as in Example 7 by adding 1Of water to the starch fibers. This gel particle 1.
52 was subjected to a urea decomposition test in the same manner as in Example 7.

The initial rate is measured from the time course of the amount of ammonia N produced,
2. Calculate the apparent Vase specific activity 5c of α-amylase-free 2. What is the value of yU/f? Uta.

Example B Carrageenan α22, water 4.89 k Heated at 110° C. for 5 minutes in an autoclave to obtain carrageenan solution 5f.

The above carrageenan solution was cooled to 40°C. This carrageenan solution t1rc, invertase 30 [Candida utilis]
Origin, specific activity 20 units U/■, 1U varnish cloth 1f moles WfZ minutes] water 1fK aqueous solution 1.03 f
and fibers made by shredding absorbent cotton into 2 to 3 pieces (fiber diameter 30
~60 μm) was added and mixed. The mixed solution was poured onto a flat plate overnight and solidified into a film having a thickness of 0.8 to 1.2 points. Then 150μ of cellulase (originated from Trichoderma, 1o OFPA-ty/1Iv) was added to 7-0.0
The above fiber-containing ciurease-immobilized carrageenan gel membrane 1.
5r (4o, x4o) was soaked and treated at 40°C for 1 hour. Due to the cellulase treatment, the cellulose fibers that had been observed before the treatment disappeared, and it was observed that through holes were formed in the disappeared areas. The gel particles after cellulase treatment were separated and collected, 20 tnl of α1M acetate buffer (pH 4,7) and 10 dj of 5% sucrose solution were added, and incubated at 45°C for 2 hours.
It was shaken back and forth for an hour (amplitude 3 crn 1 TJO strokes/min). The initial rate was measured from the time course of the amount of glucose produced by the decomposition of sucrose, and the apparent specific urease activity of the cellulase-free gel 2 was calculated.
The value of U/y was obtained.

Comparative Example 12 The same batch of fiber-containing cyinvertase-immobilized carrageenan gel membrane tst prepared in Example 8 was added to 0.05 M sodium acetate buffer (pH 6,8
) and kept at 40°C for 1 hour in the same manner. After the above treatment, the same t-domain-c' sucrose decomposition experiment as in Example 8 was conducted. The initial rate was measured from the time course of the amount of glucose produced by the decomposition of sucrose, and the apparent specific invertase activity per cellulase-untreated gel was calculated to obtain a value of 28 U/f.

Comparative Example 13 The same batch of fiber-containing cyinvertase-fixed carrageenan gel membrane prepared in Example 8 was stored at 5° C. for 2 hours, and then a sucrose decomposition experiment was conducted in the same manner as in Example 8. The initial rate of glucose production (due to the decomposition of sucrose) was measured over time, and the apparent specific invertase activity of the cellulase-free gel was calculated.
The value of U/y was obtained.

Comparative Example 14 In preparing an invertase-immobilized carrageenan gel membrane, a gel membrane was prepared in the same manner as in Example 8 by adding 109 water to the cellulose fibers. This gel film 1.
A sucrose decomposition experiment was conducted in the same manner as in Example 8 using 5f.

The initial rate was measured from the time course of the amount of glucose produced by the decomposition of sucrose, and the apparent specific activity of invertase was calculated from the cellulase-free gel.
The value of f was obtained.

Example 9 A carrageenan solution 61 containing urease and absorbent cotton fibers was prepared in the same manner as in Example 6, and this was dropped into a 5-tripotassium chloride solution (6°C) and formed into 2- to 3- beads. The above gel particles 1.51 were separated and treated with cellulase in the same manner as in Example 6. As a result, it was confirmed that the cellulose fibers that had been observed before the treatment had disappeared, and that through-holes had been created in the disappeared areas. The gel particles after cellulase treatment were separated, and the apparent specific activity of the cellulase-untreated gel was calculated in the same manner as in Example 6 to obtain a value of 59 U/t.

Comparative Example 15 Same batch of gel particles prepared in Example 9, s t
'Z without adding cellulase [added to LO5M sodium acetate slowing solution (pH 4,7) and calculated the apparent specific activity per 2 cellulase-free gels in the same manner as 2.4 U/
We obtained a value of 2 for y.

Comparing the specific activities of Examples 6 to 9 and Comparative Examples corresponding to each family, the apparent specific activity of the Examples was 1.4 to 1.6 times that of the Comparative Examples. It's improving.

Comparative Example 16 A liquid containing the following four components was mixed in a width of 1.5 x 1.5 x
The mixture was poured into a groove on a 60 m acrylic plate and allowed to stand at 15° C. for 40 minutes to gel, thereby preparing a linear gelled product.

A) I N Hot 4.8m7! Trishydroxymethylaminomethane 3,7 tng Distilled water 6.3- B) Acrylamide 62 MethyVn Bis 7 Acrylamide Q, 16r Potassium ferricyanide 1.4 tng Distilled water 14- 0) 0.14% Ammonium persulfate A4.0 m7! D) Maltase (Saccharomyces yeast origin 0.22 (21
0ro U) Distilled water 9.4d The above linear gelled product 2. Of'r, separate and add 5℃ water 5
was soaked in the water for 3 hours and washed. Next, 10 d of 0.1 M potassium phosphate buffer pH 6.8 was added, and to this was added 13 yd of a 0.5 t aqueous solution of ortho-nitrophenol-β-D-glucopyranoside as a substrate. After reacting the above liquid mixture at 37° C. for 10 minutes, the mixture was cooled on ice to obtain an enzyme-immobilized linear gel. This gelled product was colorimetrically determined based on the amount of orthophenol produced f 420 nm in the filtered filtrate. Maltase activity was calculated from the initial velocity calculated from the amount of orthophenol produced in 10 minutes. On the other hand, separate the linear gelled product o, s t and add 5M water! , and centrifuged (4500t, 10 minutes) to measure the packed volume. Table 5 shows maltase activity based on packed volume and packed volume, and maltase activity based on dry weight.

Example 10 Linear gelled product 22 of the same batch tested in Comparative Example 16
The sample was washed by immersing it in 5°C water for a medium hour. The gelled product was then placed in an eggplant flask and frozen at -50°C within 2 minutes. The frozen linear gel particles were dried under reduced pressure of 1°C mHg or less to obtain a dry gel of 0.27S'. Put this into a concave mold with a diameter of 1 crn and a depth of 1 crn and mold for 3 k.
It was pressed at a pressure of P/6n2 to obtain a disc-shaped Matsutra.

This mat was cut into 2×2 m squares. Then Il
IMIJ potassium buffer pH 6,8'f! :10f
Add nl, remove V this group ￥lL, C 0.5% aqueous solution of ortho-nitrophenol-β-ri-glucobyranoside r-
was added. The mixture was allowed to react at 37° C. for 10 minutes, cooled on ice, and the immobilized enzyme particles were separated in a furnace. 10
The maltase activity was calculated from the initial velocity calculated from the amount of orthophenol produced per minute. On the other hand, a corresponding amount of gelled product 052 immediately after gelling was prepared by cutting a mat prepared in the same manner, and water 5- was added thereto, followed by centrifugation (
45008', 10 minutes), and the packed volume was measured. The maltase activity per packed volume and the maltase activity per dry weight are shown in Table 5.

Table 5 Comparative Example 17 Carrageenan αsy, water 4.5t 1 was heated in an autoclave at 120°C for 15 minutes to form a carrageenan y solution H5t.
I got 2. The above carrageenan solution 2, Ωt, was cooled to 4D°C. In addition, sucrase (originated from yeast of the genus Satucharomyces) α1 f (1500 U), calcium chloride 0.2
Add an aqueous solution of 2 dissolved in water 3- and pour it into a container with an internal dimension of 50 x 50 mm and a depth of 10 ffill+, which is lined with a square analytical filter paper (model 174) with 50 strips on the side, and let it stand still at room temperature. It was left to solidify. A membrane-like gelled product with a thickness of 2 mm containing filter paper was immersed in 5° C. water for 6 hours to be washed. The gel was then divided into two equal parts of 25×50111+1. Put one of the gelled products above in a '1100 go beaker and add 10ml of 10ml sucrose solution! , α5M acetate buffer p
Add H5, 9, 5ml 1k, shake gently, 40
The reaction field was incubated at ℃ for 10 minutes. Sucrase activity of the gelled product was calculated from the amount of glucose produced in the reaction solution. On the other hand, the volume of the gelled product was measured, and the sucrase activity per gel volume and the activity per dry weight were calculated and shown in Table 6.

Example 11 Comparative Example 17 The unused 25×50fi gelled product prepared in Mo was placed in a wide-mouthed 1-ton eggplant flask, and the flask was frozen at -45° C. with the flask attached to the wall.

This frozen gelled product was dried under a reduced pressure of f 1 tm Hg or less to obtain a dry film 92■. This dry film is dried at a humidity of 80%.
% in air for 1 hour, then press a hydraulic press machine (50 kg
/crn2) to obtain a paper-like membrane.

The above membrane was placed in a 100-beaker, and sucrase activity and wet membrane volume were measured in the same manner as in Comparative Example 17. The sucrase activity per wet membrane volume and the activity per dry weight were calculated and shown in Table 6.

Table 6 Comparative Example 18 Purified agar [115S', water 4.85? was heated in an autoclave at 120°C for 90 minutes to obtain agar solution 51.

The above agar solution was cooled to 40°C.

Add this agar solution to 200 ■ baker's yeast cake (42 ■ dry matter)
A solution suspended in 1 d of water was added and immediately poured onto a flat plate to solidify. Plate-shaped gel (thickness 1.8~Z6-
) was cut into a 2×2 square. The above gel particles Q, 52 were separated into a 100-Erlenmeyer flask, 10% glucose solution 5-11M acetate buffer pH 5,6,5rnt, water 10d.
'i was added and shaken reciprocally at 30°C for 10 minutes (amplitude 2
．． 5 cm.

After the reaction, the cells were cooled to 5° C., the cells were centrifuged, and the ethanol concentration in the p solution was measured by gas chromatography. On the other hand, the gel particles were added to 0.5 fk water, the volume of the gel particles was measured, and the alcohol production rate per wet gel particle volume and the alcohol production rate per dry weight were calculated and shown in Table 7.

Example 12 Same batch of gel particles o, sr6 prepared in Example 18
It was collected, placed in a 50 (] d eggplant flask, and frozen at -45°C within 1 minute. This frozen product was dried under reduced pressure of lmmHg or less to obtain dry gel particles. Each individual particle was pressed with a press machine at (5 kg/crn2) to obtain a flake-like material. Using this flake-like material, the ethanol production rate and immersion in water were determined in the same manner as in Comparative Example 18. The volume of the sample was measured and the results are shown in Table 7.

Table 7 [Effects of the Invention] As explained in detail above, according to the present invention, the specific activity per unit volume of the immobilized bovine body catalyst particles by the gel entrapment method can be significantly improved, and the reaction tank can be reduced accordingly. It is remarkable that by reducing the effective volume, it becomes possible to react efficiently.

[Brief explanation of the drawing]

FIGS. 1 to 3 are graphs showing the time course of ammonia nitrogen production by urea decomposition on each side in terms of the relationship between reaction time and enzyme activity. Patent Applicant: Hitachi, Ltd. Agent: Hiroshi Nakamoto Figure 1 and Figure 2: Flaws (Time: 4 minutes) Continued from page 1 @ Inventor: Yoji Odawara Hitachi Wheels 3-chome Office

Claims (1)

[Claims] 1. A biocatalyst-immobilized porous gelled material, characterized in that the biocatalyst-immobilized gelled material contains minute continuous cells in the gelled material. λ The first step of adding and mixing the biocatalyst to the gel base solution,
A second step in which the mixture obtained in the first step is processed to prepare a gelled product, a third step in which the gelled product obtained in the second step is cooled and the solvent in the gelled product is frozen, and a gelled product obtained in the third step is A method for producing a biocatalyst-immobilized porous gelled material, comprising the following steps: a fourth step of sublimating and removing the solidified solvent in the frozen gelled material under reduced pressure. 5 Add the solvent fj-Nishi 1! to the dried gelled product obtained in the fourth step. +7 (The method for producing a biocatalyst-immobilized porous gelled material according to claim 2, which also includes the method of producing a porous gelled material with immobilized biocatalysts. 4. The dried gelled material obtained in the fourth step. Claim 2, which also includes the steps of a fifth step of mechanically compacting and a sixth step of adding a solvent to the compacted dry gel obtained in the fifth step to swell it. The method for producing a biocatalyst-immobilized porous gelled material according to claim 1. & The method according to any one of claims 2 to 4, wherein the treatment in the second step includes a granulation treatment. A method for producing a biocatalyst-immobilized porous gelled product. & The biocatalyst according to claim 5, wherein the treatment in the second step is to gel the mixture obtained in the first step and then to form particles. A method for producing an immobilized porous gelled material.l The treatment in the second step is to granulate the mixture obtained in the first step and then gel each particle separately. A method for producing a biocatalyst-immobilized porous gelled material.a The treatment in the second step comprises gelling the mixture obtained in the first step and then linearizing it. The method for producing a biocatalyst-immobilized porous gelled material according to any one of Item 4. 9. The treatment in the second step includes linearizing the mixture obtained in the first step and then gelling each linear body. A method for producing a biocatalyst-immobilized porous gelled material according to any one of claims 2 to 4. 10. Weaving the dried gel linear body obtained in the fourth step. 11. The method for producing a biocatalyst-immobilized porous gelled material according to claim 8 or 9, which also includes a fifth step of accumulating and compacting. 11. The second step. The production of a biocatalyst-immobilized porous gelled product according to any one of claims 2 to 4, wherein the treatment in step 1 gels the mixture obtained in the first step and then forms a membrane. Method. 12. Any one of claims 2 to 4, wherein the treatment in the second step is to form the mixture obtained in the first step into a film and then gel each film. The method for producing a biocatalyst-immobilized porous gelled product as described in 1.5. The first step of adding and mixing a biocatalyst and a fibrous material to a gel base liquid, and processing the mixture obtained in the first step to produce a gelled product. a second step of preparing a gel, and a third step of contacting the gelled product obtained in the second step with an enzyme that decomposes only the fibrous material to decompose and remove the fibrous material in the gelled material. 1. A method for producing a biocatalyst-immobilized porous gel, comprising: 14. The method for producing a biocatalyst-immobilized porous gelled material according to claim 13, wherein the treatment in the second step includes a granulation treatment. 15. The method for producing a biocatalyst-immobilized porous gelled material according to claim 14, wherein the second step comprises gelling the mixture obtained in the first step and then forming particles. 16. The biocatalyst-immobilized porous gelled material according to claim 14, wherein the treatment in the second step is to granulate the mixture obtained in the first step and then gel each particle. manufacturing method.