JPS59113889A - Preparation of immobilized enzyme or immobilized microbial cell - Google Patents

Abstract

PURPOSE:To prepare immobilized enzyme or immobilized microbial cell having high mechanical strength and capable of keeping the activity of the enzyme or microbial cell stably for a long period, by using an alginic acid salt and a carboxyl-containing polysaccharide as the gel components. CONSTITUTION:(A) Enzyme or microbial cells and (B) a water-soluble polycationic polymer compound such as polyethyleneimine, chitosan, etc. are added to (C) an aqueous solution containing a polysaccharide containing carboxymethyl group in the molecule (e.g. sodium aliginate, carboxymethylcellulose, carboxymethylpullullan, etc.) and/or a water-soluble protein such as albumin, gelatin, etc., and the obtained mixture is made to contact with an aqueous solution containing calcium ion or aluminum ion to effect the gelatination of the solution and immobilize the enzyme or the microbial cells. If necessary, the surface of the obtained gel is treated with a polyfunctional crosslinking agent such as glutaraldehyde.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing an immobilized enzyme or an immobilized microorganism. More specifically, in an aqueous solution consisting of (sodium lyalginate), (il) a polysaccharide having a carboxymethyl group in the molecule and/or a water-soluble protein,
After adding and mixing enzymes or microbial cells with a water-soluble multicationic polymer compound, the enzymes or microbial cells are immobilized by contacting with an aqueous solution containing calcium ions or aluminum ions to form a gel, or The immobilized enzyme or immobilized microbial cells thus obtained is further reacted with a polyfunctional crosslinking agent.
The present invention relates to a method for producing an immobilized enzyme or an immobilized microbial cell, which is characterized by surface treatment.

In recent years, the use of immobilized enzymes, which are made by insoluble enzymes, has become increasingly popular for the purpose of using the catalytic elements of biological reactions as catalysts in the production processes of foods, medicines, and chemical products. It is attracting attention as an important technology. A method for similarly utilizing microorganisms having 19 element activity as immobilized microorganisms and a technique for immobilizing living microorganisms and utilizing them as immobilized proliferating microorganisms are being developed.

Many methods have already been reported for producing identified enzymes or immobilized microbial cells, but methods using alginic acid, carakeenan, etc., which are easy to produce and can be applied to immobilization of various enzymes or microbial cells, have been reported. A method of immobilizing polysaccharide using gel is known. However, immobilization using these polysaccharides requires gelling agents such as alkali metals and alkaline earth metals, and since gelation by these gelling agents is reversible, after immobilization, the immobilized enzyme and When immobilized microbial cells are brought into contact with a solution that does not contain a gelling agent or a solution that contains a degelling agent such as phosphate ions, the gel dissolves and the immobilized enzyme or immobilized microbial cells collapse. There was a serious problem. In the case of immobilization using alginate gel, it is important to coexist gelling agents such as calcium ions or aluminum ions at a certain concentration in the reaction solution during the reaction with the immobilized enzyme or the immobilized microbial cells. Not only can it have an unfavorable effect on the body's activity, but it also poses problems in the separation and purification of reaction products. In addition, in order to use immobilized enzymes or immobilized microbial cells as catalysts for practical use, it is necessary to develop an immobilization method that can maintain constant catalytic activity and gel mechanical strength for a long period of time. .

The present inventors have conducted intensive studies on methods to overcome the drawbacks of immobilized enzymes or immobilized microbial cells using alginate gel, and have found that (+) sodium alginate, (11) carboxymethyl group in the molecule. to an aqueous solution consisting of a polysaccharide and/or a water-soluble protein,
Immobilized enzymes or immobilized microbial cells produced by adding and mixing enzymes or microbial cells with a water-soluble polycationic polymer compound and then contacting with an aqueous solution containing calcium ions or aluminum ions to form a gel can be used during the reaction. It has been found that even in the absence of a gelling agent, not only is it possible to maintain stable catalytic activity and gel shape for a long period of time, but also the mechanical strength of the gel is high enough for practical use. Furthermore, by carving the immobilized enzyme or immobilized microbial cells thus obtained into 8-fold shapes and surface-treating them by reacting them with a polyfunctional cross-linking agent, it is possible to make the enzyme stable even without a gelling agent during the reaction. It was discovered that the detachment of enzymes or microbial cells from the inside of Zukel is extremely small, and that it is possible to obtain immobilized enzymes or immobilized microbial cells that are stable for a long period of time (7) The present invention has been completed.

The present invention will now be explained. Enzymes or microbial cells used in the present invention are not particularly limited, and examples of enzymes include oxidoreductases such as alcohol dehydrogenase, D-amino acid oxidase, and catalase, transketolase, adenylate kinase, and hexokinase. Enzymes, hydrolase enzymes such as β-galactositase, penicillinase, lipase, esterase, lyase enzymes such as fumarase, aspartase, threonine aldolase, β-tyrosinase, isomerase enzymes such as glucose isomerase, alanine isomerase, glutathione synthase and glue case enzymes such as curtamine synthase. Also, as microbial cells, bacteria, yeast,
There are no particular limitations on microorganisms that have enzymatic activity, such as Kahi, actinomycetes, etc., and intracellular organelles that have enzymatic activity, cell fractions, enzymes or processed products of microbial cells can also be immobilized. It can also be used for Furthermore, in the specific immobilization method of the present invention, it is also possible to immobilize microbial cells while they are still alive.

As the sodium alginate used in the present invention, sodium alginate extracted from commercially available algae (viscosity 50 to 1000 Cps at 1% concentration) is sufficient.

The polysaccharide having a carboxymethyl group in the molecule used in the present invention is carboxydimethyl cellulose (CMC).
), carboxymethyl pullulan dimethyl starch, etc. Carboxymethyl pullulan, carboxymethyl dextran, and carboxymethyl starch can be easily synthesized by known methods with reference to the synthesis method of carboxydimethyl cellulose. By using a polysaccharide with a carboxymethyl group in the molecule as a gel component, the ionicity of the immobilized element or microbial cells in the gel is increased, and the interaction between enzymes, microbial cells, and polyethyleneimine is increased. It has the effect of strengthening ionic bonds (thus forming a gel with high mechanical strength) and stably maintaining the activity of enzymes or microorganisms for a long period of time.

Albumin, gelatin, soluble collagen, etc. are suitable as l-type proteins, and egg albumin is preferred because it is inexpensive and easily available in large quantities. Using water-soluble protein as a gel component for immobilized enzymes or immobilized microbial cells has the effect of increasing the ionicity within the gel and stably maintaining the activity of enzymes or microbial cells for a long period of time. .

In addition, when the surface is treated with a polyfunctional crosslinking agent, multiple crosslinks including protein crosslinks are formed, which increases the mechanical strength of the gel and facilitates the release of enzymes or microorganisms from within the gel. It is effective in preventing the gel from collapsing.

By simultaneously using a polysaccharide having a carboxymethyl group in its molecule and a water-soluble protein as gel components, a synergistic effect can be expected.

Examples of the water-soluble polycationic polymer compounds used in the present invention include polyalkyleneimines (for example, polymethyleneimine, polyethyleneimine, polypropyleneimine, etc.), chitosan, and neutralized salts of these compounds. It will be done.

Among these water-soluble polycationic polymer compounds, polyethyleneimine accounts for 10 to 30 (W/V)%.
Like chitosan, it is commercially available as an aqueous solution and is conveniently available.

The aqueous solution containing calcium ions or aluminum ions used as a kelizing agent in the present invention is not particularly limited, and may be any solution containing a water-soluble compound containing calcium ions or aluminum ions, calcium chloride or aluminum chloride. Aluminum is inexpensive and preferred for use.

Polyaldehydes are suitable as the polyfunctional crosslinking agent used in the present invention, and examples include dialdehyde starch, glyoxal, malonaltehyde, succinic aldehyde, and glutaraldehyde, but glutaraldehyde is particularly suitable. preferable. These polyfunctional crosslinking agents cause a crosslinking reaction with each component contained in the gel on the surface of the immobilized enzyme or immobilized microbial cells to form a crosslinked product 7, and then release the enzyme or microbial cells from within the gel. It is effective in preventing the desorption of

To explain in more detail the specific manufacturing method of the present invention,
For example, if a polysaccharide having a carboxymethyl group in the molecule is used as a gel component, water can be added to physiological saline together with sodium alginate, and the mixture can be autoclaved if necessary. When water-soluble protein is used as a gel component, it is added after being subjected to a separate sterilization process other than heat sterilization, if necessary. Subsequently, a water-soluble multi-cationic polymer compound was added and mixed well.
An aqueous solution or culture solution of enzymes or microorganisms is added to form a mixed solution.

The temperature of sodium alginate in the final mixed solution to which enzymes or microorganisms are added is Q, and the concentration is preferably 5 (w/v)% to 10 (w/v)%, preferably 1
(w/v,)% to 6 (w/v,)% concentration.

The concentration of polysaccharides with carboxymethyl groups in the molecule is 0
．． 2 (w/v) * to 6 (fv) qt,
The concentration of water-soluble protein is 0.1 (VV,)%
from 10-(VV)% concentration, preferably 0.5 (
(w/v)% to 5 (w/v)% concentration. When both are used, the total concentration is l (rv)96
A concentration of about 10 to 10 (VV)% is preferable. The concentration of the water-soluble polycationic polymer compound is 0.05 (W/V
-) %L/10 (vv) % dark weight <, preferably 0.1 (rv) % to 5 (W/V)
% concentration. It is preferable to mix the water-soluble polycationic polymer compound to sodium alginate at a ratio of about 0.02 parts to 1.5 parts to 1 part of sodium alginate. When immobilizing an enzyme, the concentration of the enzyme is 0. Ol(vy'V)% to q(V
V, )% concentration is preferable, and when immobilizing microbial cells, the concentration of microorganisms is 0.01 (wet Xi/V).
A concentration range of 50% (wet weight/V)% is preferred. When immobilizing live microorganisms, the number of viable bacteria in the gel can be increased by culturing the immobilized microorganisms in a medium containing immobilized microorganisms, so the concentration of microorganisms at the time of immobilization can be varied over a wide range. It is possible to take

The thus prepared mixed solution is brought into contact with an aqueous solution containing calcium ions or aluminum ions to form a gel, thereby producing immobilized enzymes or immobilized microbial cells. The concentration of calcium ions or aluminum ions is preferably 0.01M to 1.0M, preferably 0.02M to 0.5M.

As a method for preparing the gel, a bead-shaped immobilized product can be obtained by dropping the mixed solution into an aqueous solution containing calcium ions or aluminum ions using a pipette, syringe, or high-pressure automatic injection pump. Alternatively, a film-like immobilized product can be obtained by casting the mixed solution on a glass plate and then contacting it with an aqueous solution containing calcium ions or aluminum ions. Sodium alginate forms a reversible gel when it comes into contact with calcium ions or aluminum ions, but it also forms strong ionic bonds with water-soluble polycationic polymer compounds, forming an irreversible and stable gel. Ru.

The contact time between the mixed solution and the aqueous solution that combines calcium ions or aluminum ions is 0.5 to 2 hours.
A time range of 0 hours is sufficient, a pH range of PHa, o to pH 11.0, and a temperature range of 4°C to 50°C. It is best to allow contact within a range that does not cause any negative effects.

The immobilized enzyme or immobilized microbial cells produced in this way are then extremely stable even when immersed in a reaction solution without calcium ions or aluminum ions or in an aqueous solution containing phosphate ions for a long time. The mechanical strength of the gel is also high, and no dissolution of the gel is observed. Furthermore, when surface treatment is performed by reacting these immobilized enzymes or immobilized microbial cells with a polyfunctional crosslinking agent as necessary,
The final concentration of polyfunctional crosslinker was 0.005 (w/l
)% to 0.5 (w/v)% concentration tL, <
Ha0.01 (w/v) *suI,'[,0,2(y
? )% concentration is suitable. The temperature of the crosslinking reaction is preferably in the range of 4°C to 50°C, and the reaction time is preferably 5 minutes to 1 hour, but it is preferably determined depending on the concentration of the polyfunctional crosslinking agent.

This surface treatment not only makes it extremely stable even when immersed in a reaction solution without calcium ions or aluminum ions or in an aqueous solution with phosphate ions, but also provides high mechanical strength with a strengthened gel surface. The enzyme or the immobilized microbial cells can be obtained, the enzyme or the microbial cells can be prevented from being detached from the inside of the gel, and stable activity can be obtained for a long period of time. The present invention will be described below with reference to examples, but the present invention is not limited to the following examples unless it exceeds the spirit and spirit thereof.

Example 1 Sodium alginate (80 (lP8) IF and carboxymethyl cellulose (OMC) 06t were dissolved in 25ml of distilled water, and then autoclaved at 120°C and IKV/2 for 20 minutes. Allowed to cool. After that, add 0.7 tnl of polyethyleneimine aqueous solution (aO('gg/4)%) and mix well, then add a medium containing 1% crucose, 0.5% peptone, 08% yeast extract, and 0.3% malt extract. , pH
A mixed solution was prepared by adding culture solution 5- (wet weight of bacterial cells: 0.8 P) of Satucharomyces cerevisiae (Sake Yeast Association No. 7) cultured at 80° C. for 24 hours at 5-0. This mixed solution was added dropwise with a pipette to a previously sterilized aqueous solution of calcium chloride with a concentration of 0.1M, and the mixture was stirred at 80° C. for 1 hour to complete gelation. Thereafter, it was filtered to obtain immobilized yeast 32-.

10 m of the immobilized yeast obtained in this manner was packed in a column with a jacket tube and used as a medium containing 0% glucose, 0.2% yeast extract, 0.25 g6 ammonium chloride, 0.1% sodium chloride, and diphosphoric acid. Potassium 0.55%, Magnesium chloride 7H200,025%, Citric acid 0.8
8% liquid medium (PH5,0) from the bottom of the column.
It was run continuously using a tube pump at V=Q, 5 hr'. The outer tube was kept warm by circulating water at 80°C. Ethanol fermentation was carried out by continuous column reaction at a constant S for 20 days, and the ethanol concentration and the number of viable bacteria in the effluent at the beginning of the 20th day and the number of viable bacteria in the gel of immobilized yeast were measured. The ethanol concentration in the effluent was determined by gas chromatography (PEG-1000/Simalite-F, 1
The number of viable bacteria in the cell was measured using a plate coating method after crushing the beads in a mortar and thoroughly mixing and suspending them in an appropriate amount of physiological saline for dilution. The results are shown in Table 1. During the continuous column reaction, there was no collapse or dissolution of the immobilized yeast, and the mechanical strength was sufficiently high.

Example 2 The immobilized yeast 16- obtained in Example 1 was suspended in roughly sterilized physiological saline 100-, and a 25 (W/V)% glutaraldehyde aqueous solution 11j was added to this. 2- was added and slowly stirred at 25°C,
The reaction is carried out for 10 minutes. After the reaction, the mixture was filtered and further washed with 300 ml of sterilized water to obtain immobilized yeast whose surface was treated by glutaraldehyde crosslinking reaction.

Example 1 10 m/m of the immobilized yeast thus obtained was
20 as immobilized propagated yeast by the same operation and method as
Ethanol fermentation was carried out by continuous column reaction for days.

The results are shown in Table 1. No collapse or dissolution of the immobilized yeast was observed during the continuous column reaction, and the mechanical strength was sufficiently high.

Table-1 Example 8 Sodium alginate < 800 CI) S) 15
4 and carboxymethyl cellulose (CMC) 0.6F were dissolved in distilled water 25-, and then heated at 120°C, IK9/c++.
12 for 20 minutes.

Egg albumin 0°5, sterilized by ultraviolet light after cooling. Add
Further, 0.7% of a polyethyleneimine aqueous solution (80 (vV)%) is added and mixed well.

Medium containing 1% glucose, 0.5% peptone, 0.8% yeast extract, 0.3% malt extract, pus, o
A mixed solution was prepared by adding 5 ml of culture solution (wet weight of bacterial cells: 0.8 P) of Satucharomyces cerevisi (Sake Yeast Percentage Association No. 7) cultured at 30°C for 24 hours at 30°C. This mixed solution was added dropwise with a pipette to a previously sterilized 0.1M 9°C calcium chloride aqueous solution, and stirred at 30°C for 1 hour to complete gelation. Thereafter, the immobilized yeast 34- was obtained.

The thus obtained immobilized yeast 10- was packed in a column with a jacket tube and used as a medium containing 10% glucose, 0.2% yeast extract, 0.25% ammonium chloride, 0.1% sodium chloride, and phosphorus. Potassium dibasic acid 0.55%, magnesium chloride/71El120 0.025%, citric acid 0
．． A liquid medium containing 3% (pH 5,0) was continuously flowed from the bottom of the column at SV = Q, 5 hr-' using a tube pump, and water at 30°C was circulated in the mantle to keep it warm. Ethanol fermentation was carried out by continuous column reaction at a constant S for 9 and 20 days, and the ethanol concentration and the number of viable bacteria in the effluent at the beginning of the 20th day and the number of viable bacteria in the gel of the immobilized yeast were measured. The ethanol concentration in the effluent was determined by gas chromatography (PEG-10007 Simalai 1-
-F, 1m column, 9 C1"C,), and the number of viable bacteria in the gel was determined by crushing the beads in a mortar, mixing and suspending them in an appropriate amount of physiological saline for dilution, and then measuring by plate coating method. The results are shown in Table 2. During the continuous column reaction, no collapse or dissolution of the immobilized yeast was observed, and the mechanical strength was sufficiently high. Sterilized and suspended in 100 d of physiological saline, add 25 ml of
(v'v)% concentration of glutaraldehyde aqueous solution 0.2
After adding mlf, let the reaction proceed for 10 minutes at 25°C with slow stirring. The reaction solution was filtered and further washed with 300 ml of sterilized water to obtain immobilized yeast whose surface was treated by a curcurulaldehyde crosslinking reaction. The thus obtained immobilized yeast 10mZ was subjected to ethanol fermentation by column reaction for 20 days as an immobilized propagated yeast using the same operations and methods as in Example 8. The results are shown in Table 2. No collapse or dissolution of the immobilized yeast was observed during the continuous column reaction, and the mechanical strength was sufficiently high.

Table 2 Example 5 Sodium alginate (B 00 Cps ) 1.2
2 and carboxymethyl cellulose o, 61 to distilled water 80
- Lactase obtained by culturing Aspergillus olise in this solution
"Activity at c 82.000 ullit/y-)
Q. Prepare a mixed solution by adding 8f and 80 (w/v%) polyethyleneimine aqueous solution. Add this mixed solution dropwise to a 0.1y concentration calcium chloride aqueous solution using a high-pressure injection pump to prepare Kelchis. After that, the mixture was stirred at 80° C. for 2 hours to complete gelatinization. After that, filtration and washing with water were repeated to obtain immobilized compound 9-Se82-. The activity of the identified lactase was determined by measuring the amount of glucose produced using 18.8 (W/'V)% lactose as a substrate at pH 4, 5, and 80''c.
The activity of immobilized lactase was shown. Activity yield is 54.1
%Met. Note that the enzyme activity shown here is expressed as the amount of enzyme that produces 1 μmole of glucose per minute.
nit. Moreover, the activity yield was calculated by the following formula.

Example 6) Sodium alginate (300cps) 1
．． Lactase obtained by dissolving 0.6 y of ovalbumin and 0.6 y of ovalbumin in distilled water and culturing Aspergillus olise in this solution (Shin Nippon Chemical Co., Ltd. pH 4, 580° C. Niol Lt activity 82,000 units 15 L) 0.
BP and an aqueous polyethyleneimine solution of 80 (w/v) 96 are added and mixed well to prepare a mixed solution g. This mixed solution was added dropwise to a calcium chloride aqueous solution with a concentration of 0.1y using a high-pressure injection pump to prepare a gel piece, and the mixture was stirred at 30° C. for 2 hours to complete gelation. After that, 82ml of immobilized lactase was obtained by repeating filtration and washing with water.
I got it. Immobilized lactase' 0) Activity 18.8
(w/v) PIli4 using 4 concentrations of lactose as substrate
．． 5. As a result of measuring the amount of glucose produced at 30°C,
370 units/m7! - Showed the activity of immobilized rafters. The activity yield was 48.1%. Example 7 Sodium alginate (300Cll8) 2P and carboxymethyl pullulan 11 were dissolved in 40-g of water.
Kg/c+++, autoclave at 120°C for 20 minutes. After cooling, 1.2rnt of a 30 (W/V)% polyethyleneimine aqueous solution was added to this solution and mixed, and then to this mixed solution was added 1% glucose, 05% yeast extract, 1% peptone, 0.5% meat extract, and mixed minerals. Solution (water I
J to (NH4)2F04180 f! -, KH2PO4
701i', BlySO4-7111208y-1Z
++ 804-7 H200, 5f/-11”esO+
-7H200,9f-, Cu804・5820 0.
05y-1MnSO4・4H200, IP, Na0r
Pseudomonas flagii IFo 8 obtained by culturing in a liquid medium (pH 7,0) containing 5% (including L9-)
458 culture solution 10- (wet bacterial weight: 0.2 F) was added and mixed until uniform. This mixed solution was dropped into a sterilized 0.1 M calcium chloride aqueous solution using a pressurized automatic injection pump to prepare gel beads.
child. After the dropwise addition, the solution was stirred at 25°C for 1 hour, and then 25 (w/v
)% glutaraltehyde aqueous solution 0.6 - plus 30
After the reaction was carried out with stirring at ℃ for 10 minutes, the gel heath was collected by filtration, and then heated for 300 rn1. 0 of
．． IM phosphorus 14 buffer pu 7.0 and 13QQ
The cells were washed with sterile water to obtain 58 ml of heath-like immobilized bacterial cells with a diameter of 12 to 1.5 D. This immobilized bacterial cell 20- was added to 100 ml of the above medium and cultured in a 500 m7 flask for 48 hours at 30°C and 220 rpm. The number of viable bacteria after culturing was 6 x 10 cells/--gel. Methyl phenylglyoxylate was reduced using 20 WLt of the stabilized and proliferated bacterial cells. For the reduction reaction, the above-mentioned immobilized and grown bacterial cells were newly suspended in the above-mentioned medium 100°C, 1 tnt of methyl phenylglyoxylate was added thereto, and the shaking reaction was carried out at 30°C and 22 Orpm for 20 hours. The production yield of methyl mandelate, which is a reaction product, was determined by gas chromatography analysis (5% DEG 8.1 m
, '150℃). As a result, the yield of methyl mandelate was 91%. In addition, almost no detached bacteria were observed in the reaction solution, and almost no disintegration or dissolution of Kelbeads was observed.

Example 8 Sodium alginate (800 cps) 0.61 and carboxymethylcellulose (CMC) 0.6 P were dissolved in distilled water 30-30 cm, and Aspergillus oryzae was cultured in this solution. Manufactured; p
H4, 5, activity at 30℃ is 82.000 uni
t/ji'-)9,259- and chitosan (Yamato Yushi ■ product) 031 are added and mixed to prepare a mixed solution. This mixed solution was added to a calcium chloride aqueous solution with a concentration of 0.1M.
After preparing a gel piece by dropping it into m1 using a high-pressure injection pump, it was stirred at 25° C. for 2 hours to complete gelation. Thereafter, 0.15% of 25 (w/v) glutaraldehyde was added to this solution, and the gel surface was cross-linked with glutaraldehyde at 80° C. for 10 minutes. After the reaction, filtration and washing with water were repeated to obtain immobilized lactase 88-. As a result of measuring and calculating the activity and activity yield of immobilized lactase using the same operations and methods as in Example 5, the activity was 385.
unit ~ - activity yield is 61.9% with immobilized lactase
Met.

Example 9 Sodium alginate (800 cps) 1.21 and carboxymethylcellulose (CMC) 0.6z were mixed with distilled water 2
5- and autoclaved at 120°C for 20 minutes in IK-2. After cooling, an aqueous solution of polyethyleneimine having a concentration of 85 (W/V) 0.7- is added and mixed. This mixture contains 1% glucose, 0.2% yeast extract,
Meat extract 0.2%, peptone 0.4%, NaC10.5
%, My-804-7H200,025N, pH7.0
Streptomyces griseus IFO12875 cells (wet weight 1.8P) obtained by culturing in a medium for 48 hours
) in 5- physiological saline is added and mixed well. This mixed solution was added dropwise to a 0.1 M Ca Oz 2 aqueous solution using a high-pressure injection pump to prepare Kelbeads, and stirring was continued at 25° C. for 1 hour.

Claims (1)

[Claims] In an aqueous solution consisting of (LHI) sodium alginate, (II) a polysaccharide having a carboxymethyl group in the molecule and/or a water-soluble protein, an enzyme or a microbial cell and a water-soluble polycationic polymer are added. After adding and mixing the compounds,
1. A method for producing an immobilized enzyme or an immobilized microbial cell, which comprises immobilizing the enzyme or microbial cell by contacting with an aqueous solution containing calcium ions or aluminum ions and causing gelation. (2+(+) Sodium alginate, (I) Add and mix enzymes or microbial cells and a water-soluble polycationic polymer compound to an aqueous solution consisting of a polysaccharide having a carboxymethyl group in the molecule and/or a water-soluble protein. After that, the enzyme or microbial cells are immobilized by contacting with an aqueous solution containing calcium ions or aluminum ions to form a gel, and then the surface is treated by reacting with a polyfunctional crosslinking agent. A method for producing an immobilized enzyme or an immobilized microbial cell.