JPH0632622B2 - Method for producing immobilized cells - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing immobilized cells. The immobilized bacterial cells are useful for producing various substances such as amino acids by enzymatic reaction.

BACKGROUND ART It is known to immobilize an enzyme with glutaraldehyde or a polymer substance and immobilize a coenzyme with hexamethylenediamine. [Edited by Ichiro Chibata; Immobilized Enzyme, Inc.]
Kodansha (1975)]. It is known that an enzyme or microbial cell is fixed with carrageenan and then treated with glutaraldehyde and hexamethylenediamine [Fukui, Chibata, Suzuki, Enzyme Engineering, Tokyo Kagaku Dojin (1981)].

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, an immobilized bacterium that can be used repeatedly for a long period of time by treating cells with a diamine and dialdehyde and then immobilizing the treated product with a polymer substance. You can get the body.

The diamine used in the present invention is a compound having two amino groups and having a molecular weight of 200 or less, for example, ethylenediamine, trimethylenediamine, tetramethylenediamine, pentamethylenediamine, hexamethylenediamine, triethylenetetramine, 1,7-diamino. Heplan, 1,8-diaminooctane, 1,10-diaminodecane and the like can be mentioned.

Examples of the dialdehyde are aliphatic or aromatic aldehydes having 10 or less carbon atoms, such as glyoxal, malonaldehyde, succinaldehyde, glutaraldehyde, adipine aldehyde, pimelin aldehyde, suberin aldehyde, azelain aldehyde, sebacin aldehyde, malein aldehyde, and fumaraldehyde. Examples thereof include aldehyde, phthalaldehyde, isophthalaldehyde, terephthalaldehyde and the like.

Examples of the polymer substance include polyvinyl alcohol gel, polyacrylamide gel, carrageenan, sodium alginate and the like.

Examples of the bacterial cells used in the present invention include bacterial cells such as bacteria, actinomycetes, filamentous fungi, yeasts, molds and algae. The method of the present invention is particularly effective for those in which enzyme leakage from bacterial cells is remarkable. Specific examples include cells of microorganisms belonging to the genus Salmonella, the genus Citrobacter and the like. As a typical example of the microorganism, Salmonella typhimurium (Salmonella typhim
urium) ATCC 19585 and Citrobacter Freundi (Ci
trobacter freundi) ATCC 6750.

In the present invention, in order to treat microbial cells with diamine and dialdehyde, diamine and dialdehyde are added to the culture solution of the microorganism, or after separating the microbial cells from the culture solution, the microbial cells with diamine. It may be suspended in a solution containing dialdehyde. The weights of the diamine and dialdehyde used are both 1/100 to 5 times, preferably 1/10 to 1/2 times the dry weight of the microbial cells. The concentration of diamine and dialdehyde in the liquid for treating microbial cells is both 0.1 to 5
%, Preferably 1 to 3%. Treatment is temperature 1
It is carried out at 40 ° C, preferably 4 to 25 ° C, 0.5 to 3 hours, preferably 1 to 2 hours, and pH 5 to 8, preferably 6 to 7.

A treated bacterial cell product is obtained as described above.

When the treated bacterial cell product is immobilized with a polymeric substance, it is carried out, for example, as follows.

When polyvinyl alcohol is used, the treated cells are added to an aqueous polyvinyl alcohol solution, mixed, and then frozen and thawed.

When a polyacrylamide gel is used, the treated cells are suspended in a buffer solution in which acrylamide and a cross-linking agent (for example, N, N'-methylenebisacrylamide) are dissolved, and a polymerization accelerator (for example, β-dimethylaminoproton) is suspended. Polymerization is performed by adding pionitrile and a polymerization initiator (eg, potassium persulfate).

When using carrageenan and sodium alginate, the treated cells are added to and mixed with the carrageenan or sodium alginate aqueous solution, and then the mixed solution is added dropwise to an aqueous potassium chloride (calcium chloride in the case of sodium alginate) aqueous solution.

As a method for separating the immobilized cells from the reaction solution, there are a centrifugation method, an excess method and the like.

Examples will be shown below.

Example 1 Salmonella typhimurium was added to a medium (pH 7) containing 5% glucose, 2% corn steep liquor, 1% ammonium sulfate, 0.05% monopotassium phosphate, 0.05% dipotassium phosphate and 0.025% magnesium sulfate heptahydrate. ATCC 19
50 g of wet cells obtained by culturing 585 (dry weight: 12
g) was suspended in 150 ml of 0.2 M phosphate buffer (pH 7) containing 2% of adipic aldehyde and 2% of triethylenetetramine, and the suspension was left at 4 ° C. for 3 hours. Then, the bacterial cells were centrifuged from the suspension, and the bacterial cells were washed with 150 ml of physiological saline.
It was washed twice with. The washed cells were treated with Gohsenol NH-
26 [Polyvinyl alcohol, manufactured by Nippon Synthetic Industry Co., Ltd.]
After mixing with 50 g of a 20% aqueous solution of the above, the mixture was left standing at -20 ° C. for 18 hours, thawed and shredded into 3 to 4 mm square pieces to obtain immobilized cells. The immobilized cells were treated with phenylpyruvic acid 0.2M, sodium aspartate 0.24M and pyridoxal phosphate.
Suspend in 100 ml of 0.01 mM substrate solution (pH 7) and
The reaction was carried out at 5 ° C. for 24 hours to obtain 2.9 g of phenylalanine. After the reaction, the immobilized bacterial cells were collected by a wire net, and the recovered immobilized bacterial cells were suspended in a new substrate solution to carry out a second reaction. When the reaction was repeated in this way,
The amount of phenylalanine produced in the 15th reaction was 2.3.
It was g.

On the other hand, as Control 1, the cells of Salmonella typhimurium ATCC 19585 cultured in the same manner as the above procedure were immobilized with Gohsenol NH-26 without pretreatment with adipinaldehyde and triethylenetetramine, and the immobilized cells were Phenylalanine was obtained by the same procedure as above except that it was used for the reaction for producing phenylalanine. As a result, the amounts of phenylalanine produced in the first and fifteenth reactions were 2.9 g and 0.1 g, respectively.

As a control 2, Salmonella cultured in the same manner as the above operation
Gysenol NH-
The mixture was mixed with 50 g of a 20% aqueous solution of 26, left standing at -20 ° C for 18 hours, then thawed to obtain immobilized cells which were shredded into 3 to 4 mm square pieces. Hexamethylenediamine 2% with the immobilized cells
And 0.2M phosphate buffer containing 2% glutaraldehyde
The suspension was suspended in 300 ml (pH 7) and left at 4 ° C. for 3 hours.
The immobilized cells were recovered, and the recovered immobilized cells were washed twice with 300 ml of physiological saline. Using the immobilized cells that had been used for the reaction for producing phenylalanine, phenylalanine was obtained by the same procedure as described above. As a result, the amounts of phenylalanine produced in the 1st and 15th cycles were 2.9 g and 1.7 g, respectively.

Examples 2 and 3 The results shown in Table 1 were obtained in the same manner as in Example 1 except that the diamine and dialdehyde shown in Table 1 were used.

Example 4 50 g (12 g dry weight) of wet bacterial cells obtained by culturing Citrobacter freundii ATCC 6750 in a medium having the same composition as in Example 1 was adjusted to pH 7 with a 2% hexamethylenediamine aqueous solution (hydrochloric acid). ) Suspend in 150 ml and stir at 25 ° C. while stirring 25% glutaraldehyde aqueous solution 12 m
1 was added dropwise over 15 minutes. During the dropping, the pH of the liquid was maintained at 6 to 7 with 5% aqueous ammonia. After further stirring for 30 minutes, the bacterial cells were centrifuged from the suspension, and the bacterial cells were washed twice with 200 ml of physiological saline. The washed cells were suspended in 50 g of a 3% aqueous solution of Soagina MV-101 [carrageenan, manufactured by Mitsubishi Acetate Co., Ltd.], and the suspension was added dropwise to a 5% potassium chloride aqueous solution to form a spherical particle having a diameter of about 5 mm. Immobilized cells were obtained. The immobilized cells were treated with sodium phenylpyruvate 0.2 M, diammonium fumarate 0.3 M, pyridoxal phosphate 0.01 mM, sodium sulfite heptahydrate.
Substrate solution (pH 7) consisting of 05% and magnesium chloride 0.005%
It was suspended in 50 ml and reacted at 30 ° C. for 24 hours to obtain 1.5 g of phenylalanine.

After the reaction, the immobilized bacterial cells were collected by a wire net, and the recovered immobilized bacterial cells were suspended in a new substrate solution to carry out a second reaction. When the reaction was repeated in this manner, reaction 6
The amount of phenylalanine produced in the 0th cycle was 1.5 g.

On the other hand, as Control 1, phenylalanine was produced in the same manner as described above except that hexamethylenediamine was used instead of hexamethylenediamine and glutaraldehyde in the above operation. As a result, the first time and 10
The amount of phenylalanine produced for the first time was 1.5 g and
It was 0.1 g.

As Control 2, phenylalanine was produced in the same manner as above except that hexamethylenediamine and glutaraldehyde were not used in the above operation. as a result,
The amounts of phenylalanine produced in the 1st and 20th cycles were 1.5 g and 0.2 g, respectively.

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 method for producing an immobilized microbial cell, which comprises treating the microbial cell with a diamine and a dialdehyde and then immobilizing the treated product with a polymer substance.