JPH0632619B2 - Method for producing immobilized cells - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing immobilized cells.

The immobilized cells are useful for producing various substances (for example, amino acids) by enzymatic reaction.

2. Description of the Related Art Conventionally, an enzyme or a microbial cell was aggregated in an aqueous solution of a poly (2-methacryloxyethyltrialkylammonium) type cationic synthetic polymer flocculant, and then reacted with glutaraldehyde to give an enzyme or Method for immobilizing microbial cells (Japanese Patent Laid-Open No. 58-60987), immobilization by treating microbial cells containing an enzyme with casein, a binding agent such as serum albumin, a polycationic and polyanionic flocculant, and glutaraldehyde A method for producing an enzyme (Japanese Patent Laid-Open No. 59-159781) is known.

Problems to be Solved by the Invention There is a demand for the development of immobilized cells that can be repeatedly used for a long period of time.

Means for Solving the Problems According to the method of the present invention, cells are treated with the general formula (I). (Wherein X represents an amino group or a saturated hydrocarbon group having 1 to 4 carbon atoms and having 1 to 4 carbon atoms, Y represents a hydrogen atom or a lower alkyl group, and n represents an integer of 100 to 5000). By treating with the polyamine and dialdehyde, it is possible to obtain an immobilized microbial cell that can be reused for a long period of time.

In the general formula (I), the number of carbon atoms having one amino group is 1
Examples of the saturated hydrocarbon group of ~ 4 include aminomethyl,
2-aminoethyl, 1-aminoethyl, 3-aminopropyl, 2-aminopropyl, 1-aminopropyl, 2-
Amino-1-methylethyl, 1-amino-1-methylethyl, 4-aminobutyl, 3-aminobutyl, 2-aminobutyl, 1-aminobutyl, 3-amino-2-methylpropyl, 2-amino-2 -Methylpropyl, 1-amino-2-methylpropyl, 3-amino-1-methylpropyl, 2-amino-1-methylpropyl, 1-amino-
1-methylpropyl, 1- (aminomethyl) propyl,
1-amino-1,1-dimethylethyl and the like can be mentioned.

The lower alkyl group has a carbon number of 1 to 4, and examples thereof include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl and the like.

Specific examples of the compound represented by the general formula (I) include polyallylamine, polyvinylamine, poly (4-amino-).
1-butene), poly (3-amino-1-butene), poly (1-amino-2-butene), poly (5-amino-1-)
Pentene), poly (4-amino-1-pentene), poly (3-amino-1-pentene), poly (5-amino-2)
-Pentene), poly (4-amino-2-pentene), poly (1-amino-3-pentene) and the like.

As the dialdehyde, an aliphatic or aromatic aldehyde having 10 or less carbon atoms is used, and specifically, glyoxal, malonaldehyde, succinaldehyde, glutaraldehyde, adipine aldehyde, pimerine aldehyde, suberin aldehyde, azelaine aldehyde, Sebacin aldehyde, maleic aldehyde, fumaraldehyde, phthal aldehyde, isophthal aldehyde, terephthal aldehyde, etc. are used.

Examples of the bacterial cells used in the present invention include bacterial cells such as bacteria, actinomycetes, filamentous fungi, yeasts, molds and algae.

Specific examples include cells of microorganisms belonging to the genus Salmonella, genus Citrobacter, genus Escherichia, and the like. As a typical example of the microorganism, Salmonella typhimurium (Sal
monella typhimurium) ATCC 19585, Citrobacter freundi ATCC 6750, Escherichia coli ATCC 11303 and the like are used.

In the present invention, in order to treat microbial cells with polyamine and dialdehyde, polyamine and dialdehyde are added to the culture solution of the microorganism, or after the microbial cells are separated from the culture solution, the microbial cells are treated with polyamine. And may be suspended in a solution containing dialdehyde. The weight of both polyamine and dialdehyde used is 1/100 of the dry weight of microbial cells.
-5 times, preferably 1/10 to 1/2 times.

The concentration of both polyamine and dialdehyde in the liquid for treating microbial cells is in the range of 0.1 to 5%, preferably 1 to 3%.

The treatment temperature is 1 to 40 ° C, preferably 4 to 25 ° C, 0.5 to
It is carried out for 3 hours, preferably 1 to 2 hours, at pH 5 to 8, preferably 6 to 7.

As the pH adjuster, buffers such as acetic acid, phosphoric acid and citric acid, acids such as sulfuric acid, hydrochloric acid and phosphoric acid, and alkalis such as caustic soda, caustic potash and ammonia are used.

As a method for separating the immobilized microbial cells from the treatment liquid,
The treatment liquid is 1000 to 5000 G, preferably 2000
Centrifuge at ~ 4000 G (G is gravitational acceleration) for 5-30 minutes, preferably 10-20 minutes, or 1-100
A method of using a filter material (paper, cloth, membrane filter, etc.) having a particle size of μm, preferably 5 to 50 μm is used.

Examples will be shown below.

Example 1 Salmonella in a medium (pH 7.0) consisting of 5% glucose, 2% corn steep liquor, 1% ammonium sulfate, 0.05% monopotassium phosphate, 0.05% dipotassium phosphate and 0.025% magnesium sulfate heptahydrate.・ Cyphymurium ATC
C19585 was inoculated and cultured at 28 ° C. for 17 hours. 5.0 g (dry weight 1.2 g) of the obtained wet bacterial cells was added to PAA-HCl-1.
The suspension was suspended in 20 ml of a 0.2 M acetate buffer (pH 7) containing 2% of 0S (polyallylamine hydrochloride having a polymerization degree of 530 to 890: manufactured by Nitto Boseki Co., Ltd.). Then, while stirring at 25 ° C, add 25% glutaraldehyde aqueous solution to this suspension.
After adding 1.6 ml, the mixture was stirred at pH 6.5 ± 0.5 for 1 hour.

The suspension was then centrifuged at 2000G for 10 minutes.
The obtained precipitate was washed twice with 30 ml of physiological saline to obtain microbial cells. Sodium phenylpyruvate 0.28M, sodium aspartate 0.
It was suspended in 10 ml of a substrate solution (pH 7) consisting of 42 M and pyridoxal phosphate 0.01 mM and reacted at 35 ° C. for 24 hours to obtain 0.41 g of phenylalanine.

After the reaction, the cells were centrifuged at 2000 G for 10 minutes and suspended in a substrate solution having the same composition as above to carry out a second reaction. When the reaction was repeated in this manner, the amounts of phenylalanine produced in the 10th and 15th reactions were 0.41 g and 0.39 g, respectively.

Example 2 In Example 1, Salmonella typhimurium ATC
Citrobacter Freundi ATCC 6750 was used instead of C19585, and cultured in the same manner as in Example 1.
10 g (2.4 g dry weight) of the wet cells thus obtained were treated with PAA-H.
It was suspended in 40 ml of a 1% aqueous solution of Cl-10S (adjusted to pH 7.0 with caustic soda), and 1.6 ml of a 25% glutaraldehyde aqueous solution was added dropwise thereto at 25 ° C. for 30 minutes while stirring. During this time, adjust the pH of the solution to 6.5 with diluted ammonia water.
Adjusted to ~ 7.0. Then, stirring was continued for another 30 minutes at 25 ° C., and then the cells were centrifuged at 4000 G for 5 minutes. The obtained precipitate was washed 3 times with 40 ml of physiological saline to obtain immobilized cells. A substrate solution (pH 7) consisting of sodium phenylpyrivicate 0.2M, diammonium fumarate 0.3M, pyridoxal phosphate 0.01mM and magnesium chloride hexahydrate 0.005%.
It was suspended in 20 ml and reacted at 30 ° C. for 24 hours to obtain 0.30 g of phenylalanine. After the reaction, the immobilized cells were precipitated, so the supernatant of the reaction solution was extracted. A new substrate solution was added to and suspended in the cells remaining in the reactor to carry out the second reaction. When the reaction was repeated in this manner, the amount of phenylalanine produced in the 30th reaction was 0.30 g, which was the same as in the first reaction.

Example 3 Escherichia coli ATCC11303 was used as an inoculum, and 10 g of wet cells obtained by culturing in a medium having the same composition as in Example 1
The same operation as in Example 2 was performed on (dry weight 2.4 g),
Immobilized cells were obtained. The bacterial cells were treated with diammonium fumarate.
Suspended in 200 ml of a substrate solution (pH 7) consisting of 4M and 0.005% magnesium chloride hexahydrate, and reacted at 30 ° C. for 24 hours to obtain 10.1 g of aspartic acid. After the reaction, the immobilized cells were precipitated, so the supernatant of the reaction solution was extracted. A new substrate solution having the above composition was added to the immobilized bacterial cells remaining in the reactor to suspend them, and the second reaction was performed. When the reaction was repeated in this manner, the amount of aspartic acid produced in the 10th reaction was the same as in the 1st reaction.
It was 0.1 g.

Effect of the Invention According to the method of the present invention, it is possible to obtain immobilized cells that can be repeatedly used for a long period of time.

Claims (1)

[Claims] 1. A bacterial cell of the general formula (I) (In the formula, X represents an amino group or a saturated hydrocarbon group having 1 to 4 carbon atoms and having 1 to 4 carbon atoms, Y represents a hydrogen atom or a lower alkyl group, and n represents an integer of 100 to 5000). A method for producing an immobilized microbial cell, which comprises treating with the represented polyamine and dialdehyde.