JPH06169772A - Immobilized biocatalyst and its production - Google Patents

Abstract

PURPOSE:To obtain the subject catalyst, useful for a bioreactor, etc., without any deterioration in enzymic activity by subjecting a cultured microbial cell of Pseudomonas putida MR-2068 strain or Escherichia coli MR-2103 strain to the cross-linking treatment with glutaraldehyde. CONSTITUTION:Pseudomonas putida MR-2068 strain (FERM P-3846) or Escherichia coli MR-2103 strain (FERM P-3835) is inoculated into a liquid culture medium and cultured at 30 deg.C for 1 day under shaking and the resultant culture solution is subsequently centrifuged to provide a cultured microbial cell, which is then suspended in a 0.05M phosphoric acid buffer solution. A solution of glutaraldehyde is subsequently added to the microbial cell suspension so as to afford 0-5.0% concentration thereof based on the dry weight of the microbial cell. The temperature is increased to 60 deg.C to carry out reaction for about 1 hr. Thereby, cross-linking treatment is performed to provide the objective immobilized biocatalyst utilizable for producing useful substances by the use thereof in a bioreactor, etc., without causing the deterioration in enzymic activity.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an immobilized biocatalyst used in a reaction for asymmetrically hydrolyzing a carboxylic acid ester.

[0002]

[Prior Art] Pseudomonas
putida ) MR-2068 strain and Escherichia
coli ) MR-2103 strain has been proposed as a microorganism that produces an enzyme that asymmetrically hydrolyzes a carboxylic acid ester to produce an optically active carboxylic acid with high optical purity (JP-A 1-222).
(See Japanese Patent No. 2798 and Japanese Patent Application No. 3-249923). The enzymes produced by these microorganisms have excellent thermostability and are expected to be applied in a wide range of industries.

On the other hand, the technology for applying immobilized biocatalysts is advancing rapidly, and various immobilization methods are possible depending on the purpose. For example, 1) a method in which an enzyme is bound to a water-insoluble carrier, 2) a cross-linking method in which an enzyme is cross-linked with a reagent having two or more functional groups, and 3) an enzyme in a fine lattice of a polymer gel. The encapsulation method includes wrapping in or coating with a semipermeable polymer film. Immobilization of microorganisms, organelles, etc. is essentially the same as the method of immobilizing enzymes.

When a useful substance is produced in a bioreactor, solvent extraction, recrystallization, purification by column chromatography and the like are usually carried out in the purification of the useful substance from the enzyme reaction completed liquid. The presence of denatured proteins eluted from the cells during these steps is often a problem. For example, when the target substance is extracted from the reaction-terminated liquid with a solvent, an emulsion is often formed, which makes extraction impossible. In order to avoid these problems, it is often effective to use an immobilized biocatalyst that prevents protein elution.

[0005]

DISCLOSURE OF THE INVENTION The present invention relates to an immobilized biocatalyst used for the production of a useful substance using Pseudomonas putida MR-2068 strain or Escherichia coli MR-2103 strain and its production. The purpose is to provide the law.

[0006]

Means for Solving the Problems] Immobilized biocatalyst of the present invention, Pseudomonas putida (Pseudomonas putida) MR-
2068 strain or Escherichia coli MR-
It is characterized in that the cultured bacterial cells of strain 2103 are crosslinked. Furthermore, the method for producing an immobilized biocatalyst of the present invention is a method for producing Pseudomonas putida MR.
-2068 strain or Escherichia coli MR
-2103 strain cultured cells are cross-linked with glutaraldehyde.

Pseudomonas putida MR-2068 strain and Escherichia used in the present invention
Escherichia coli MR-2103 strains were distributed to the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology, respectively.
No. 3 and No. 3835 of the National Institute of Micro-Technology.
Pseudomonas putida for use in the present invention (Pseudomo
nas putida) MR-2068 strain and Eserikia coli (Escheri
The chia coli MR-2103 strain can be cultivated in either liquid culture or solid culture. As the medium, a medium in which components such as a carbon source, a nitrogen source, vitamins and minerals that can normally be assimilated by microorganisms are appropriately mixed is used. The culturing is carried out at a temperature and pH at which the microorganism can grow, but it is usually carried out at a temperature of 50 ° C. or lower and a pH range of 2-11. Aeration and agitation may be performed to promote the growth of microorganisms.

On the other hand, the cross-linking reagent which can be used in the present invention is not particularly limited in its kind, and glutaraldehyde which forms a Schiff base, an isocyanic acid derivative which forms a peptide bond, N, N'-ethylene bismaleimide, diazo. Coupling bisdiazobenzidine, alkylating N, N′-polymethylenebisiodoacetamide and the like can be used.

The form of the biocatalyst used in the production of the immobilized biocatalyst of the present invention is a culture-finished solution, wet cells, dry cells (eg freeze-dried cells), spray-dried cells or an organic solvent (eg acetone, Cells treated with toluene or the like) can be used. As the production method, a usual method can be applied, for example, the cultured cells are suspended in a pH buffer solution or physiological saline,
It is possible to use a method in which a crosslinking reagent is added to this to be crosslinked to obtain an immobilized biocatalyst.

When glutaraldehyde is used as the cross-linking reagent, the addition amount thereof is usually 0.1 to the dry weight of the cells.
It can be added in the range of 10.0% (wt%),
It is desirable to add in the range of 5 to 5.0% (wt%). The cross-linking treatment temperature is usually within the range of 5 to 70 ° C., but it is possible to simultaneously sterilize the cells by treating at a temperature of 60 ° C. or higher, and it is also possible to deactivate the non-thermostable enzyme. .

The cross-linking treatment time can be usually in the range of 10 minutes to 5 hours, but it is desirable to perform the treatment for 1 hour or more. The immobilized biocatalyst can be used in a solution state or a dried state such as freeze-dried. As described above, the solid-state biocatalyst of the present invention is extremely easy to prepare, and when this immobilized biocatalyst is used for the production of useful substances by a bioreactor, it is necessary to minimize the outflow of the enzyme into the reaction solution. Therefore, the operability in the purification process can be expected to be significantly improved.

[0012]

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to these examples.

[0013]

Example 1 (1) Preparation of immobilized biocatalyst Pseudomonas putida MR-206
8 strains (Mikoken Kenjoyori No. 3846) were inoculated into 100 ml of liquid medium (pH 6.8) consisting of 0.5% meat extract, 0.75% peptone, 0.25% NaCl, 0.5% glucose and 0.15% malt extract, and 30 Shaking culture was carried out at 0 ° C for 1 day. After the completion of the culture, the culture solution was centrifuged, the whole amount of the obtained bacterial cells was washed with ion-exchanged water, and then suspended in about 10 ml of M / 20 phosphate buffer.
A glutaraldehyde solution was added to this cell suspension to a concentration of 0 to 5.0% based on the dry weight of the cells, and 60%
After the temperature was raised to ° C, the mixture was stirred for about 1 hour. The obtained bacterial cell suspension was used as an immobilized bacterial cell solution. (2) Measurement of residual activity and protein elution concentration Immobilized cell solution containing 16 mg of immobilized cells was added to 20 ml of M / 20 phosphate buffer, and the hydrolysis activity of carboxylic acid ester was measured. In addition, the concentration of eluted protein in the supernatant
-Measured by the Lowry method. The results are shown in Table 1. In each case, the untreated one was set to 100%.

[0014]

[Table 1]

[0015]

Example 2 (1) Preparation of Immobilized Biocatalyst Escherichia coli MR-2103 strain (Mikoken Kenjoyori No. 3835) was prepared with 0.5% meat extract and 1.
100 ml of a liquid medium (pH 7.0) containing 0% and 0.5% of NaCl was inoculated and shake culture was carried out at 37 ° C for 1 day. After the completion of the culture, the culture solution was centrifuged, the whole amount of the obtained bacterial cells was washed with ion-exchanged water, and then suspended in about 10 ml of M / 20 phosphate buffer.
A glutaraldehyde solution was added to this cell suspension to a concentration of 0 to 5.0% based on the dry weight of the cells, and 60%
After the temperature was raised to ° C, the mixture was stirred for about 1 hour. The obtained bacterial cell suspension was used as an immobilized bacterial cell solution. (2) Measurement of residual activity and protein elution concentration Immobilized bacterial cell solution containing 16 mg of immobilized bacterial cells was added to 20 ml of M / 20 phosphate buffer solution, and the hydrolysis activity of carboxylic acid ester was measured. In addition, the concentration of eluted protein in the supernatant
-Measured by the Lowry method. The results are shown in Table 2. In each case, the untreated one was set to 100%.

[0016]

[Table 2]

[0017]

EFFECT OF THE INVENTION Since the immobilized biocatalyst of the present invention is subjected to a cross-linking treatment on the surface of bacterial cells, the enzyme activity does not decrease. In addition, the amount of protein eluted from microbial cells can be reduced to 1/3 or less compared to untreated proteins. Therefore, when the immobilized biocatalyst of the present invention is used for the production of a useful substance by a bioreactor, it is expected that the operability in the purification step will be greatly improved.

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Claims (2)

[Claims] 1. Pseudomonas
putida ) MR-2068 strain or Escherichi coli ( Escherichi
a coli ) An immobilized biocatalyst characterized in that cultured cells of MR-2103 strain are cross-linked. 2. Pseudomonas
putida ) MR-2068 strain or Escherichi coli ( Escherichi
The method for producing an immobilized biocatalyst according to claim 1, wherein the cultured cells of the a coli ) MR-2103 strain are crosslinked with glutaraldehyde.