JPS6137912B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing an immobilized enzyme active substance. BACKGROUND OF THE INVENTION Enzyme reactions have traditionally been used to produce useful substances or to decompose substances harmful to humans or animals or metabolites harmful to humans and animals. The present inventors first added 10 sulfate groups to the molecular structure.
An excellent immobilized enzyme active substance can be obtained by adding and mixing an enzyme active substance (e.g., enzyme, microbial cells) to an aqueous solution of polysaccharide containing W/W% or more, and then gelling the mixture. He discovered that this could be done and filed a patent application (Japanese Patent Application Laid-Open No. 53-6483). As a result of further various studies, the present inventors found that prior to gelling a mixture of an aqueous solution of a polysaccharide containing 10 W/W% or more of sulfate groups in its molecular structure and an enzyme active substance, The present inventors have discovered that if a multicationic polymer compound is present in the liquid, an immobilized enzyme active substance with extremely stable enzyme activity can be obtained, and the present invention has been completed. That is, the present invention has a sulfate group in the molecular structure of 10W/W.
An enzyme active substance and a polycationic polymer compound are added to and mixed with an aqueous solution of a polysaccharide containing % or more, and then the polysaccharide in the mixture is gelled, thereby injecting the enzyme into the gel lattice of the polysaccharide. This is a method for producing an immobilized enzyme active substance, which consists of entrapping an active substance. Polysaccharides containing 10W/W% or more of sulfate groups in the molecular structure used in the method of the present invention include:
Examples include carrageenan, furcerelan, cellulose sulfate, and the like. Carrageenan is a member of the red algae (Rodophyceae) Gigartinaceae.
It is a polysaccharide containing 20 to 30 W/W% of sulfate groups extracted and purified from seaweed belonging to Solievianceae.
For example, Genyugel wG (trade name manufactured by Copenhagen Pectin Factory), Genyugel
Examples include GwG (ibid.) and Genius Bisco J (ibid.). Furthermore, Furcelleran is a polysaccharide containing 12 to 16 W/W% of sulfate groups extracted from Furcellaria fastigiota, a type of red algae (Rodophyceae); for example, Furcelleran manufactured by Litex in Denmark is mentioned. Further, as the cellulose sulfate ester, for example, Kelco SCS (trade name manufactured by Kelco Corporation) can be mentioned. Further, examples of the enzymatically active substance used in the method of the present invention include enzymes, microbial cells, and the like. Enzymes are not particularly limited, and include, for example, oxidoreductases (e.g., amino acid oxidase,
uricase, catalase, etc.), transposases (e.g., aspartate acetyltransferase, glycine aminotransferase, amino acid aminotransferase, etc.), hydrolytic enzymes (e.g., asparaginase, acetylcholinesterase, aminoacylase, etc.), lyases (e.g., asparaginase, acetylcholinesterase, aminoacylase, etc.), (e.g., fumarase, aspartate decarboxylase, aspartase, citrate lyase, etc.), isomerase (e.g., alanine racemase, glucose isomerase, glutamate racemase, etc.), ligase (e.g., asparagine synthetase, glutathione synthase, glutamine synthetase, etc.) Examples include.
The microbial cells are not particularly limited as long as they can be used as enzyme sources, and include, for example, microbial cells containing the enzymes mentioned above. These microbial cells may be live cells, or may be freeze-dried, freeze-thawed, acetone-treated, heat-treated, or the like. The enzymatically active substance used in the method of the present invention may be of a single enzyme type or may be of a multiple enzyme type. Furthermore, two or more enzyme active substances may be used simultaneously. Further, the multi-cationic polymer compound used in the method of the present invention includes a polymer compound having a cationic functional group and having a molecular weight of 1,000 to several million. Such polycationic polymer compounds include, for example, aminoalkylated water-insoluble polysaccharides (e.g., aminohexylated agarose, aminohexylated cellulose), dialkylaminoalkylated water-insoluble polysaccharides (e.g., diethylaminoethylated water-insoluble polycationic polymer compounds such as crosslinked dextran), or polyalkyleneimines (e.g. polymethyleneimine,
Polyethyleneimine, etc.), copolymers of dimethylaminoethyl methacrylate and alkylamide, polythiourea hydrochloride, polyvinylpyridine hydrochloride, and water-soluble multicationic polymer compounds such as chitosan. Among these polycationic polymer compounds, polyalkyleneimine or chitosan is particularly preferred. In carrying out the method of the present invention, first, an aqueous solution of a polysaccharide (hereinafter simply referred to as polysaccharide) containing 10 W/W% or more of sulfate groups in its molecular structure is prepared. The aqueous solution can be easily prepared by adding and mixing the polysaccharide to warm water at 30°C to 90°C. The concentration of polysaccharides added is 0.05-20W/W%,
Particularly preferred is about 0.4 to 10 W/W%. Next, an enzyme active substance and a polycationic polymer compound are added and mixed to the thus obtained polysaccharide aqueous solution to prepare a mixed solution. In preparing the mixed solution,
An enzyme active substance dissolved or suspended in water, physiological saline, or an appropriate PH buffer (PH 1 to 13, preferably PH 4 to 10) is mixed with an aqueous polysaccharide solution kept at a temperature of 15 to 70 degrees Celsius. This is preferably carried out by adding and mixing water or an appropriate buffer solution or suspension of the polycationic polymer compound maintained at 15°C to 70°C to the mixed solution. In this case, the concentration of the polycationic polymer compound added is 0.05% relative to the polysaccharide.
~200W/W%, preferably 0.1~50W/W% is appropriate. Next, the polysaccharide in the mixture of the polysaccharide, enzyme active substance, and polycationic polymer compound obtained above is gelled to enclose the enzyme active substance in the gel lattice of the polysaccharide. As a gelling means,
This can be easily carried out by cooling the liquid mixture. For example, the mixture should be heated to about -60°C.
By standing at ~25°C for about 10 minutes to 10 hours, the polysaccharide gels, and at the same time, the enzymatically active substance is entrapped within the gel lattice of the polysaccharide. The gel thus obtained can be molded into a suitable shape. In addition to cooling the mixed liquid as described above, the gelling method may include contacting the mixed liquid with ammonium ions or metal ions having an atomic weight of 24 or more (for example, potassium ions, magnesium ions, calcium ions, aluminum ions, etc.). can also be adopted. When the immobilized enzyme active substance obtained by the method of the present invention as described above is used in an enzyme reaction, the enzyme active substance does not leak out from within the gel lattice, so it not only exhibits high enzyme activity for a long period of time, but also retains its shape. It also has excellent properties such as strength, strength, and elasticity, so it can be used stably for long periods of time in enzyme reactions. In particular, the sol transition temperature of the immobilized enzyme active substance obtained by the method of the present invention shifts to a significantly higher temperature side, so that the gel does not disintegrate even in the absence of a gel preservative such as potassium ions. , can perform normal functions. Furthermore, since the immobilized enzyme active substance obtained by the method of the present invention has high stability against organic solvents such as ethanol and acetone, it is necessary to carry out the enzyme reaction in the presence of organic solvents that may denature the enzyme. can be used effectively. Furthermore, the immobilized enzyme active substance obtained by the method of the present invention has the advantage of being highly stable against protein denaturants such as urea and guanidine hydrochloride. Furthermore, since the enzyme activity of the immobilized enzyme active substance obtained by the method of the present invention is stable even at high temperatures, the enzyme reaction can be carried out at high temperatures and the productivity of products per unit time can be increased. There are also advantages. Example 1 Corn steep liquor 2.0%, malonic acid 2.0%,
Ammonium citrate 0.5%, potassium phosphate 0.2% and magnesium sulfate heptahydrate 0.05
Brevibacterium flavum ATCC14067 was inoculated into 500 ml of a medium (PH7.0) containing
Cultured with shaking for hours. 8.0 g (wet weight) of bacterial cells collected from the culture solution by centrifugation was suspended in 8 ml of physiological saline, and added to the suspension was a 5.0% Genyugel wG (carrageenan from Copenhagen Pectin Factory) aqueous solution kept at 50°C in advance. Add 30ml and mix well. To this mixture was added 4 ml of a 1.5% Epomine p-1000 (polyethyleneimine manufactured by Nippon Shokubai Chemical Co., Ltd.) aqueous solution (adjusted to pH 6.2 with sodium phosphate) kept at 50°C and mixed well. This mixture was left to cool at 4° C. for 30 minutes. The thus obtained gel was shaped into a cube with sides of 3 mm, immersed in 120 ml of a 1M sodium fumarate aqueous solution containing 0.6% bile powder, and left at 37°C for 24 hours. By washing the gel with a 2% potassium chloride aqueous solution, 50.2 g (wet weight) of an immobilized Brevibacterium flavum agent having fumarase binding properties was obtained. This immobilized Brevibacterium flavum agent
6.35g of 1M sodium fumarate aqueous solution (PH7.0)
40 ml was added and reacted at 37°C with shaking.
1 ml of each reaction solution was collected 15 and 30 minutes after the start of the reaction, and hydrochloric acid was added to precipitate and remove unreacted fumaric acid, and the amount of L-malic acid in the supernatant was reduced to 2.7 ml.
- Quantitated by a method of reacting with naphthalene diol to develop color, L- in the reaction solution over 15 minutes
Fixed Brevibacterium spp.
When the fumarase activity of flavum drugs was calculated,
The gel showed 910 μmoles/hr/ml, which corresponded to 50% of the enzyme activity of the bacterial cells used. Example 2 Glucose 2.0%, fumaric acid 0.5%, urea 0.2%,
Phosphate-potassium 0.2%, magnesium sulfate 7
Brevibacterium was added to 500 ml of medium (PH7.0) containing 0.05% hydrate and 1.0% corn steep liquor.
Inoculated with Ammoniagenes IAM1645 and kept at 30℃
Cultured with shaking for 24 hours. 8.0% of the bacterial cells collected by centrifugation were suspended in 8 ml of physiological saline, and 5.0% of the bacterial cells, which had been pre-warmed at 45°C, were added to the suspension.
% Genyugel wG (carrageenan from Copenhagen Pectin Factory) aqueous solution was added and mixed well. This mixture was heated to 45°C.
% Epomin p-1000 (polyethyleneimine manufactured by Nippon Shokubai Chemical Co., Ltd.) aqueous solution (adjusted to pH 6.2 with sodium phosphate) and mixed well. This mixture was left to cool at 4° C. for 30 minutes. After molding the gel thus obtained into a cube of 3 mm on a side, 2%
By washing with an aqueous potassium chloride solution, the immobilized Brevibacterium with fumarase activity was removed.
48.8 g of ammoniagenes agent was obtained. The L-malic acid production ability of the immobilized Brevibacterium ammoniagenes agent was 526 μmoles/hr/ml gel;
This corresponded to 60% of the enzyme activity of the bacterial cells used. Example 3 Ammonium fumarate 0.5%, fumaric acid 1.14
%, corn steep liquor 2%, meat 2%,
Magnesium sulfate 0.05%, dipotassium phosphate 0.2
Escherichia coli ATCC11303 was inoculated into 200 ml of a medium (PH 7.0) containing 20% of the total concentration of E. coli, and the bacteria were collected after culturing at 30°C for 16 hours. 2.5 g (wet weight) of the obtained bacterial cells was weighed and suspended in 2.5 ml of the cultured solution, and the temperature of the solution was adjusted to 40°C. Add 45% to the suspension in advance.
11.9 ml of a 4.7% Genyugel wG (carrageenan from Copenhagen Pectin Factory) aqueous solution kept at ℃ was added and mixed well. to this mixture
1.9 ml of a 1.0% Epomin p-1000 (polyethyleneimine manufactured by Nippon Shokubai Chemical Co., Ltd.) aqueous solution (adjusted to pH 6.2 with sodium phosphate) kept at 45° C. was added and mixed well. This mixed solution was allowed to stand at room temperature for 30 minutes, and then allowed to cool at 4° C. for 30 minutes. The thus obtained gel was shaped into a cube with sides of 3 mm, and then washed with a 2% aqueous potassium chloride solution. Double the volume of the gel to 1M
19 g (wet weight) of an immobilized Escherichia coli agent having aspartase activity was obtained by activation in an aqueous ammonium fumarate solution (containing 1 mM magnesium chloride) at 37° C. for 48 hours. The aspartase activity of the immobilized E. coli agent was determined using Leuconostoc metsucenteroides P-60.
Bioassay method using J.Biol.Chem.
172 , 15 (1948)), 13100
μmoles/hr/ml gel, which corresponded to 39% of the enzyme activity of the bacterial cells used. Example 4 Monosodium glutamate 3.2%, Measto 0.5
Pseudomonas dacne IAM1152 was inoculated into 500 ml of a medium (PH7.3) containing 0.05% of potassium phosphate, 0.05% of potassium phosphate, and 0.01% of magnesium sulfate, and the bacteria were collected after culturing with shaking at 30°C for 24 hours. 5 of the obtained bacterial cells
g (wet weight) was suspended in 5 ml of physiological saline, and the temperature of the solution was adjusted to 45°C. The suspension is heated to 50℃ in advance.
18 ml of a warmed 50% Genyugel wG (carrageenan from Copenhagen Pectin Factory) aqueous solution was added and mixed well. Add this mixture to 50℃.
2 ml of a 1.5% Epomine P-100 (polyethyleneimine manufactured by Nippon Shokubai Chemical Co., Ltd.) aqueous solution kept warm was added thereto and mixed well. This mixture was left to cool at 4° C. for 30 minutes. The thus obtained gel was shaped into a cube with sides of 3 mm and washed with a 2% aqueous potassium chloride solution to obtain 31 g (wet weight) of an immobilized Pseudomonas ducne agent having L-aspartate β-decarboxylase activity. Ta. The L-aspartate β-decarboxylase activity of the immobilized Pseudomonas dacne agent was 3240 μmoles/hr/ml gel;
This corresponded to 50% of the enzyme activity of the bacterial cells used. In addition, the measurement of L-aspartate β-decarboxylase activity was performed using 4 g of immobilized Pseudomonas dacne agent.
1 ML ammonium aspartate aqueous solution containing 10 ^-4 M pyridoxal phosphate (pH with ammonia)
5.5) was added, the reaction was carried out with shaking at 37°C for 1 hour, and the produced L-alanine was quantified by a bioassay method using Leuconostoc titroborum ATCC8081. Example 5 In Example 1, 1.5% Epomin p-1000
(Polyethyleneimine manufactured by Nippon Shokubai Chemical Co., Ltd.)
Process in the same manner as in Example 1, except that instead of 4 ml of the aqueous solution, a solution of 0.5 g of Fronatsuk N (chitosan manufactured by Kyowa Yushi Kogyo Co., Ltd.) dissolved in 4 ml of 0.1 M acetic acid solution (temperature 50°C, pH 6.5) was used. As a result, 49.8 g (wet weight) of an immobilized Brevibacterium flavum agent having fumarase activity was obtained. The fumarase activity of the immobilized Brevibacterium flavum agent is
The gel showed 970 μmoles/hr/ml, which corresponded to 53% of the enzyme activity of the bacterial cells used. Example 6 In Example 1, 1.5% of Epomin p-1000 (polyethyleneimine manufactured by Nippon Shokubai Chemical Co., Ltd.)
Instead of 4 ml of aqueous solution, use Sumifloc FC-240 (trade name of a copolymer of dimethylaminoethyl methacrylate and acrylamide manufactured by Sumitomo Chemical Co., Ltd.)
A solution of 0.5g dissolved in 4ml of 0.1M acetic acid (temperature
50° C., pH 6.5) was used in the same manner as in Example 1 to obtain 49.7 g (wet weight) of an immobilized Brevibacterium flavum agent having fumarase activity. The fumarase activity of the immobilized Brevibacterium flavum agent is 950 μmoles/hr/
ml gel, which shows the enzyme activity of the bacterial cells used.
%. Example 7 In Example 1, 1.5% Epomine p-1000
(Polyethyleneimine manufactured by Nippon Shokubai Chemical Co., Ltd.)
By treating in the same manner as in Example 1, except that instead of 4 ml of the aqueous solution, a suspension of 0.5 g of AH-Sepharose 4B (trade name manufactured by Pharmacia) in 4 ml of water (temperature 50°C) was used. 48.8 g (wet weight) of an immobilized Brevibacterium flavum agent having fumarase activity was obtained. The fumarase activity of the immobilized Brevibacterium flavum agent is 957μ
moles/hr/ml gel, which corresponded to 53% of the enzyme activity of the bacterial cells used. Example 8 In Example 1, 1.5% Epomine p-1000
(Polyethyleneimine manufactured by Nippon Shokubai Chemical Co., Ltd.)
Instead of 4 ml of aqueous solution, use aminohexylated cellulose (J.Solid-Phase Biochemistry., 3 , 161).
(1978)) by suspending 0.5 g (wet weight) in 4 ml of water (temperature: 50°C) in the same manner as in Example 1, immobilized Brevibacterium having fumarase activity was obtained.・Flavum agent
51.2g (wet weight) was obtained. The fumarase activity of the immobilized Brevibacterium flavum agent is 843μ
moles/hr/ml gel, which corresponded to 46% of the enzyme activity of the bacterial cells used. Example 9 In Example 1, 1.5% Epomine p-1000
(Polyethyleneimine manufactured by Nippon Shokubai Chemical Co., Ltd.)
Instead of 4 ml of aqueous solution, DEAE-Sephadex A
-25 (product name manufactured by Pharmacia) 0.5g (wet weight) suspended in 4ml of water (temperature 50℃)
50.0 g (wet weight) of an immobilized Brevibacterium flavum agent having fumarase activity was obtained by treating in the same manner as in Example 1 except for using .
The fumarase activity of the immobilized Brevibacterium flavum agent was 930 μmoles/hr/ml gel;
This corresponded to 51% of the enzyme activity of the bacterial cells used. Experimental Example 1 Immobilized Brevibacterium flavum agent prepared in Example 1 (hereinafter abbreviated as A), immobilized Brevibacterium flavum agent prepared in Example 5 (hereinafter abbreviated as E), Example 6 The immobilized Brevibacterium flavum agent prepared in Example 7 (hereinafter abbreviated as F), the immobilized Brevibacterium flavum agent prepared in Example 7 (hereinafter abbreviated as G), Example 8
Using the immobilized Brevibacterium flavum agent prepared in Example 9 (hereinafter abbreviated as H), the immobilized Brevibacterium flavum agent prepared in Example 9 (hereinafter abbreviated as I), and a multicationic polymer compound. The following experiments (A) to (E) were conducted using a control immobilized Brevibacterium flavum agent (hereinafter abbreviated as J) prepared without the use of a control agent. A control immobilized Brevibacterium flavum agent was prepared as follows. (Preparation of control immobilized Brevibacterium flavum agent) 8.0 bacterial cells obtained under the same culture conditions as in Example 1
g (wet weight) was suspended in 8 ml of physiological saline, and 34 ml of a 5.0% Genyugel wG (carrageenan manufactured by Copenhagen Pectin Factory) aqueous solution previously kept at 50°C was added to the suspension and mixed well. This mixed solution was left to cool at 4° C. for 30 minutes. The thus obtained gel was shaped into a cube with sides of 3 mm, immersed in a 1M aqueous sodium fumarate solution containing 0.6% bile powder, and left at 37°C for 24 hours. By washing the gel with a 2% potassium chloride aqueous solution,
50.0 g (wet weight) of a control immobilized Brevibacterium flavum agent having fumarase activity was obtained.
The fumarase activity of the immobilized Brevibacterium flavum agent was measured in the same manner as in Example 1.
The gel showed 900 μmoles/hr/ml, which corresponded to 49% of the enzyme activity of the bacterial cells used. Experiment A (Stability during continuous enzyme reactions) 6.3 g each of immobilized Brevibacterium flavum agents A and E to J were packed into a column with a jacket tube (1.6 cm x 12 cm), and the column was filled with a 1M aqueous sodium fumarate solution ( PH7.0) was continuously passed day and night at 37°C at a flow rate of 6 ml/hr.The effluent was sampled at appropriate times to measure the fumarase activity of the immobilized Brevibacterium flavum agent. 45℃ and
It was also carried out at a temperature of 50°C. Furthermore, the initial activity of each immobilized Brevibacterium flavum agent was 50
The number of days required for the percentage decrease (half-life) was calculated from the following formula. The results are shown in Tables 1 to 3 below. Kd=2.303/tlogn ₀ /n t1/2=0.693/Kd However, Kd=degradation rate constant (1/day) t=reaction time (day) _n0 =initial enzyme activity n=enzyme after t days Activity t1/2 = half-life

【table】

【table】

[Table] Experiment B (thermal stability) 2.0 g each of immobilized Brevibacterium flavum agents A and J were added to 0.1 M potassium phosphate buffer (PH7.0)
It was immersed in 2.0 ml of water and heat-treated at 60°C for 1 hour. Next, the fumarase activity after the heat treatment was measured, and the residual rate of fumarase activity was calculated from the following formula, and the results shown in Table 4 below were obtained. Residual rate of fumarase activity (%) = fumarase activity after heat treatment / fumarase activity before heat treatment × 100

[Table] Experiment C (Stability in PH4.5 solution) Immobilized Brevibacterium flavum agents A, E
2.0 g of each of J and J were immersed in 5 ml of 0.5M acetate buffer of PH4.5 and treated at 37°C for 1 hour. Next, the fumarase activity after treatment with the PH4.5 solution was measured, and the residual rate of fumarase activity was calculated from the following formula, and the results shown in Table 5 below were obtained. Residual rate of fumarase activity (%) = fumarase activity after treatment with PH4.5 solution / fumarase activity before treatment with PH4.5 solution x 100

[Table] Experiment D (Stability in ethanol-containing solution) Immobilized Brevibacterium flavum agents A and E
2.0 g of each of J and J were immersed in 2.0 ml of a 2% aqueous potassium chloride solution containing 0.46 g of ethanol, and treated at 37° C. for 30 minutes. Next, the fumarase activity after the treatment with the ethanol-containing solution was measured, and the residual rate of fumarase activity was calculated from the following formula, and the results shown in Table 6 below were obtained. Residual rate of fumarase activity (%) = fumarase activity after treatment with ethanol-containing solution / fumarase activity before treatment with ethanol-containing solution × 100

[Table] Experiment E (Stability in urea-containing solutions) 2 g each of immobilized Brevibacterium flavum agents A and J were immersed in 2.0 ml of a 2% potassium chloride aqueous solution containing 0.36 g of urea, and treated at 37°C for 30 minutes. . Next, fumarase activity was measured after treatment with a urea-containing solution,
When the residual rate of fumarase activity was calculated from the formula below, the results shown in Table 7 were obtained. Residual rate of fumarase activity (%) = fumarase activity after treatment with urea-containing solution / fumarase activity before treatment with urea-containing solution x 100

[Table] Experimental Example 2 Immobilized Brevibacterium ammoniagenes agent (hereinafter abbreviated as B) prepared in Example 2 and control immobilized Brevibacterium ammonia prepared without using a polycationic polymer compound The following experiment was conducted using Genes agent (hereinafter abbreviated as K).
(F) to (I) were performed. A control immobilized Brevibacterium ammoniagenes agent was prepared as follows. (Preparation of control immobilized Brevibacterium ammoniagenes agent) 8.0 bacterial cells obtained under the same culture conditions as in Example 2
After suspending g (wet weight) in 8 ml of physiological saline,
By treating in the same manner as in the preparation of the control immobilized Brevibacterium flavum agent in Experimental Example 1, 49.7 g (wet weight) of a control immobilized Brevibacterium ammoniagenes agent having fumarase activity was obtained. The L-malic acid producing ability of the immobilized Brevibacterium ammoniagenes agent is 509μ
moles/hr/ml gel, which corresponded to 58% of the enzyme activity of the bacterial cells used. Experiment F (thermal stability) 2.0 g each of immobilized Brevibacterium ammoniagenes agents B and K were immersed in 2.0 ml of 0.1 M potassium phosphate buffer (PH7.0) and heat-treated at 55° C. for 1 hour. Next, the fumarase activity after the heat treatment was measured, and the residual rate of fumarase activity was determined according to Experiment B of Experimental Example 1.
When calculated using the same formula, the results shown in Table 8 below were obtained.

[Table] Experiment G (Stability in PH4.5 solution) 2.0 g each of immobilized Brevibacterium ammoniagenes agents B and K were immersed in 5 ml of 0.5 M acetate buffer and treated at 37° C. for 1 hour. Next, the fumarase activity after treatment with the PH4.5 solution was measured, and the residual rate of fumarase activity was calculated using the same formula as in Experiment C of Experimental Example 1, and the results shown in Table 9 below were obtained.

[Table] Experiment H (Stability in ethanol-containing solution) 2.0 g each of immobilized Brevibacterium ammoniagenes agents B and K in 2% ethanol containing 0.46 g
It was immersed in 2.0 ml of potassium chloride aqueous solution and treated at 37°C for 30 minutes. Next, the fumarase activity after the treatment with the ethanol-containing solution was measured, and the residual rate of fumarase activity was determined using the same formula as in Experiment D of Experimental Example 1, and the results shown in Table 10 below were obtained.

[Table] Experiment I (Stability in urea-containing solutions) 2.0 g each of immobilized Brevibacterium ammoniagenes agents B and K were immersed in 2.0 ml of a 2% potassium chloride aqueous solution containing 0.36 g of urea, and the mixture was heated at 37°C for 30 minutes. Processed. Next, the fumarase activity after treatment with the urea-containing solution was measured, and the residual rate of fumarase activity was determined using the same formula as in Experiment E of Experimental Example 1, and the results shown in Table 11 below were obtained.

[Table] Experimental Example 3 The immobilized E. coli agent prepared in Example 3 (hereinafter abbreviated as C) and the control immobilized E. coli agent (hereinafter referred to as L) prepared without using a polycationic polymer compound. The following experiment (J) was conducted using A control immobilized E. coli agent was prepared as follows. (Preparation of control immobilized E. coli agent) Bacterial cells 2.0 obtained under the same culture conditions as in Example 3
g (wet weight) was suspended in 2.0 ml of the culture completion solution, and the temperature of the solution was adjusted to 40°C. Add 45% to the suspension in advance.
4.1% Genyugel wG (carrageenan manufactured by Copenhagen Pectin Factory) kept at ℃
11.0 ml of aqueous solution was added and mixed well. This mixture was allowed to stand at room temperature for 30 minutes, and then left to cool at 4° C. for 30 minutes. The thus obtained gel was shaped into a cube with sides of 3 mm, and then washed with a 2% aqueous potassium chloride solution. By activating the gel by keeping it at 37°C for 48 hours in a 1M ammonium fumarate aqueous solution (containing 1mM magnesium chloride), 15g of an immobilized Escherichia coli agent having aspartase activity was obtained.
(wet weight) was obtained. The aspartase activity of the immobilized E. coli agent is 12400 μmoles/hr/
ml gel, which shows the enzyme activity of the bacterial cells used.
%. Experiment J (thermal stability) Immobilized Escherichia coli agents C and L each 1.0 g
Soaked in 10.0ml of 2% potassium chloride aqueous solution and heated at 55℃.
Heat treatment was performed for 30 minutes. Next, the aspartase activity after the heat treatment was measured, and the residual rate of aspartase activity was calculated from the following formula, and the results shown in Table 12 below were obtained. Remaining rate of aspartase activity (%) = Aspartase activity after heat treatment / Aspartase activity before heat treatment × 100

[Table] Experimental Example 4 The immobilized Pseudomonas dacne agent prepared in Example 4 (hereinafter abbreviated as D) and the control immobilized Pseudomonas dacne agent prepared without using a polycationic polymer compound (hereinafter referred to as M The following experiment (K) was conducted using A control immobilized Pseudomonas dacne agent was prepared as follows. (Preparation of control immobilized Pseudomonas dacne agent) 5.0 bacterial cells obtained under the same culture conditions as in Example 4
g (wet weight) was suspended in 5 ml of physiological saline, and the temperature of the solution was adjusted to 45°C. The suspension is heated to 50℃ in advance.
20 ml of a 5.0% Genyugel wG (carrageenan manufactured by Copenhagen Pectin Factory) aqueous solution kept warm was added and mixed well. Add this mixture to 4
The mixture was left to cool at ℃ for 30 minutes. The thus obtained gel was shaped into a cube with sides of 3 mm and washed with a 2% potassium chloride aqueous solution to obtain 30 g (wet weight) of a control immobilized Pseudomonas dacne agent having L-aspartate β-decarboxylase activity. I got it.
The L-aspartate β-decarboxylase activity of the immobilized Pseudomonas dacne agent was 3240 μmoles/
hr/ml gel, which corresponded to 50% of the enzyme activity of the bacterial cells used. Experiment K Immobilized Pseudomonas dacne agents D and M each
Soak 2.0g in 2.0ml of 0.1M phosphate buffer with pH 7.0.
Heat treatment was performed at 60°C for 30 minutes. Next, the L-aspartate β-decarboxylase activity after the heat treatment was measured, and the residual rate of L-aspartate β-decarboxylase activity was calculated from the following formula, and the results shown in Table 13 below were obtained. Ta. Remaining rate of L-aspartate β-decarboxylase activity (%) = L-aspartate β-decarboxylase activity after heat treatment/L-aspartate β-decarboxylase activity before heat treatment × 100

【table】

Claims (1)

[Scope of Claims] 1. Add and mix an enzyme active substance and a polycationic polymer compound to an aqueous solution of a polysaccharide containing 10 W/W% or more of sulfate groups in the molecular structure, and then add and mix the polysaccharide in the mixed solution. 1. A method for producing an immobilized enzyme active substance, which comprises encapsulating the enzyme active substance in a gel lattice of the polysaccharide by gelling the polysaccharide. 2. The production method according to claim 1, wherein the polysaccharide is carrageenan. 3. The production method according to claim 1 or 2, wherein the enzymatically active substance is a microbial cell. 4. Claim 3, wherein the polycationic polymer compound is polyalkyleneimine, chitosan, a copolymer of dimethylaminoethyl methacrylate and acrylamide, aminohexylated agarose, or dimethylaminoethylated crosslinked dextran.
Manufacturing method described in section.