JPH05168481A - Preparation method of immobilized gel including biocatalyst and its use - Google Patents

Abstract

PURPOSE:To enable to simultaneously achieve the formation of a gel and the addition of a strength by using a polymeric raw material comprising polyvinyl alcohol and carrageenan as a gel base material and also by employing an inorganic salt solution containing carbonate ions in a high concentration. CONSTITUTION:A polymeric raw material comprising polyvinyl alcohol and K-carrageenan is used as a gel base material, and an inorganic salt solution containing carbonate ions in a high concentration is employed on the formation of the gel to obtain the gel entrappingly immobilizing a biological catalyst such as a microorganism or enzyme. Thereby, the formation of the gel and the addition of a strength are simultaneously achieved.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for preparing an immobilized gel by a comprehensive method using polyvinyl alcohol and a water-soluble polymer polysaccharide as a gel substrate, and a method for using the same.

[0002] Using polyvinyl alcohol and water-soluble polymer polysaccharide as a gel base material, a granular molded product (so-called immobilized gel) in which a biocatalyst such as a microorganism or an enzyme is retained and stabilized (so-called immobilized gel) is prepared and included BACKGROUND ART Techniques for producing and decomposing various substances by utilizing biochemical reactions specific to catalysts have been conventionally explored. Here, the "immobilized gel" is simply referred to as a gel hereinafter. In addition, the "biochemical reaction specific to the entrapped biocatalyst" is hereinafter simply referred to as "reaction".

[0003]

2. Description of the Related Art Various techniques have already been proposed as a method for producing a gel.
In particular, in order to immobilize the microbial cells themselves, it is general to employ a so-called “entrapping method” gel preparation method. JP-A-2-211872, JP-A-63-
109779, JP-A-62-138193 and the like disclose a gel preparation method by such a comprehensive method.

That is, a polymer material (for example, polyvinyl alcohol and a water-soluble polymer polysaccharide such as κ-carrageenan) which is a gel base material and a biocatalyst (microorganism or enzyme) are uniformly mixed, and then an injection needle / sprayer. The gel is formed into a spherical shape by dropping it into an inorganic salt solution under appropriate conditions using a disc molding machine or the like. Generally, after this, a treatment for enhancing the strength, water resistance and stability of the molded gel is performed. For example, techniques such as freeze / thaw treatment and suspension in an aqueous solution of boric acid are used.

In order to make a gel that can be used industrially, the gel must maintain a certain strength and stability during the reaction. That is, in order to proceed the reaction, the gel is continuously stirred in the reaction vessel,
It becomes necessary to fill the inside of the reaction tank. Further, it is desirable that the gel can withstand continuous use for a long period of time or be reused several times. For this reason, in the conventional technique, the process of imparting strength to the gel during or after the gel molding is a particularly important process, and is also a feature thereof.

However, it goes without saying that such a strength adding process is industrially complicated in itself. For example, in order to add strength to the gel by freezing, it is necessary to remove the gel from this solution once. On the other hand, the gel which has not been subjected to the strength addition treatment is relatively stable in the suspended state in the inorganic salt solution used for molding, but in the non-suspended state, the mutual adhesion of the gel occurs,
It is easy to get into a "dumpling state". Therefore, it is necessary to efficiently collect the gel while preventing the gels from sticking to each other.

Further, the method of simultaneously forming a gel and adding strength in an aqueous solution containing boric acid, which is disclosed in Japanese Patent Laid-Open No. 62-138193, is a food / feed industry in consideration of the toxicity of boric acid itself to humans. There is a limit to the application to the above, and it is necessary to wash the gel after gel molding.

The present inventors considered that the ability to add strength to the gel at the same time as molding the gel is an industrially necessary gel preparation method, and made various studies.

[0009]

[Means for Solving the Problems] As a result of various studies on the type and concentration of an inorganic salt solution capable of achieving gel molding and strength addition at one time, the present inventors have found that a high concentration carbonate ion aqueous solution, preferably Completed the present invention by concluding that an aqueous solution of ammonium carbonate is the most optimal.

Further, the gel prepared in a high-concentration carbonate ion aqueous solution contains a high-concentration inorganic salt solution, preferably a high-concentration carbonate ion aqueous solution, and more preferably a high-concentration ammonium carbonate aqueous solution. They have found that they can retain their moldability and strength for a long time even under various operations such as continuous stirring and filling.

In particular, according to the present invention, not only the presence of cations as disclosed heretofore but also the simultaneous presence of a high concentration of carbonate ions is essential for adding strength to the gel simultaneously with molding. is there.

That is, according to the present invention, when a polymer material composed of polyvinyl alcohol and κ-carrageenan is used as a gel base material and a gel in which a biocatalyst such as a microorganism or an enzyme is comprehensively immobilized is prepared, gel molding is performed. An inorganic salt solution used at times is a method for preparing a gel under conditions containing a high concentration of carbonate ions, and a method of using this gel in a high concentration of an inorganic salt solution.

Specifically, an industrially effective gel can be produced by the following operations. First, a polymer material for immobilization consisting of polyvinyl alcohol and κ-carrageenan and a biocatalyst (microorganism or enzyme etc.)
Stir and mix uniformly at a constant temperature of 0 ° C to 50 ° C, preferably 15 ° C to 35 ° C. Hereinafter, this solution is referred to as a cell suspension. In this case, as the polyvinyl alcohol to be used, for example, one having a polymerization degree of 500 to 2000 and a saponification degree of 87.0 to 89.0 can be selected as a representative example.

Further, as the water-soluble polymer polysaccharide used as the gel base material, κ-carrageenan is a preferable example, and when κ-carrageenan is used, the moldability sufficient for the subsequent reaction is obtained. It is possible to produce a gel having strength.

Next, the bacterial cell suspension prepared by the above-mentioned method is successively added dropwise into a high-concentration carbonate ion aqueous solution in a stirring state by using an injection needle / sprayer, a rotary disk molding machine, or the like. , Molded into a spherical gel having a uniform particle size. This high-concentration carbonate ion aqueous solution has a temperature of 0 ° C to 50 ° C.
C., preferably 20 to 30.degree. In this way, it is possible to mold a spherical gel of any size having a diameter of at least 1 mm to 10 mm or less. Then, by continuing stirring for 30 minutes or more, a gel having sufficient strength can be prepared.

The high-concentration carbonate ion aqueous solution according to the present invention may specifically have a carbonate ion concentration of 1.5 M or more, preferably 2.0 M or more. As the cation to be contained, ammonium ion is most desirable, but other inorganic cations other than hydrogen ion can be selected. Representative examples thereof include monatomic cations such as sodium ions, potassium ions, magnesium ions, calcium ions, barium ions, and aluminum ions.

The gel which has been molded in a high-concentration carbonate ion aqueous solution and added with strength can be directly subjected to the reaction in a high-concentration inorganic salt solution. At this time, the type of the inorganic salt solution can be arbitrarily selected as long as it is maintained at a sufficiently high concentration to maintain the moldability and strength of the gel. Preferably, the following (a) and (b)
It is only necessary to satisfy the two conditions at the same time.

(A) The contained anion is most preferably a carbonate ion, but an inorganic anion excluding hydroxide ion can be selected. Representative examples thereof include monoatomic anions such as chloride ions and polyatomic anions such as sulfate ions. Further, the anion can be used alone or as a mixture of a plurality of the anions exemplified above. Furthermore, as the total anion concentration,
It is desired to be 1.5 M or more, preferably 2.0 M or more.

(B) Ammonium ions are most preferable as the contained cations, but inorganic cations other than hydrogen ions can be selected. For example, monatomic cations such as sodium ion, potassium ion, magnesium ion, calcium ion, barium ion, aluminum ion and the like can be mentioned. Also, the cation is
The cations exemplified above can be used alone or in a mixed state of plural cations. Further, the total cation concentration is desirably 1.5 M or higher, preferably 3.0 M or higher. Further, in the case of ammonium ion, 5.0M or more is desirable.

A wide variety of biochemical reactions, which are characteristic of biocatalysts such as microorganisms and enzymes, require a specific concentration of an inorganic salt, and the concentration ranges from an extremely low concentration to a high concentration. When this reaction specifically requires a high inorganic salt concentration, or when the reactivity is not sufficiently lost even under a high inorganic salt concentration, the biocatalyst is incorporated into a gel by the method disclosed in the present invention. Can be included.

Further, in some reactions, the equilibrium reaction may shift to one side at high salt concentration, and the present invention can be applied to such reactions. That is, the present invention is
Use not only for reactions that specifically require high salt concentrations and reactions that do not lose reactivity under high salt concentrations, but also for all reactions in which the equilibrium reaction shifts to either side under high salt concentrations Is possible.

For example, an example of a reaction in which the gel according to the present invention can be used is an L-phenylalanine-forming reaction with L-phenylalanine-ammonia-lyase. This enzyme decomposes L-phenylalanine to produce cinnamic acid and ammonia under low ammonium ion concentration, but in high-concentration ammonium ion solution, conversely L-phenylalanine is produced using cinnamic acid and ammonia as substrates. .. Therefore, the same enzyme or a microbial cell producing the same enzyme is fixed in a gel, and then L-containing cinnamic acid and ammonia in a high-concentration ammonium carbonate solution.
Phenylalanine can be produced and accumulated.

As the microorganisms that can be entrapped and immobilized in the gel according to the present invention, the following genera can be mentioned as typical examples. Escherichia Rhodosporidiu
m) Rhizopus Saccharomyces Saccharomyces Candida Bacillus

The following enzymes can be listed as typical examples of the enzyme that can be entrapped and immobilized on the gel according to the present invention. Phenylalanine ammonia lyase (EC 4.
3.1.5. ) Histidine ammonia lyase (EC.4.
3.1.3. ) Aspartic acid ammonia lyase (EC.4.
3.1.1. ) Α-amylase (EC.3.
2.1.1. )

[0025]

EXAMPLES Hereinafter, the method of the present invention will be described more specifically with reference to examples. Example 1 10 parts by weight of polyvinyl alcohol having a degree of polymerization of 1500 and a degree of saponification of 87.0 to 89.0 and 2 parts by weight of κ-carrageenan were mixed with 88 parts by weight of warm water at 50 ° C., and the mixture was stirred well to give a uniform solution. I chose 1% by weight of this mixture, 1% suspension of Escherichia coli cell suspension in terms of dry weight
Parts by weight were added and mixed with stirring. Hereinafter, such a mixed liquid of the bacterial cells and the immobilizing polymer material is referred to as an immobilizing bacterial cell suspension.

Here, the Escherichia coli bacterium has been deposited at the Institute of Biological Industry, Ministry of International Trade and Industry, and the deposit number is FERM-BP3569. Hereinafter, this strain is referred to as BP3569.

Further, the Escherichia coli cell (B
P3569) was prepared by culturing using a jar fermenter as in JP-A-63-317087, centrifuging and collecting the cells.

On the other hand, the ammonium ion concentration is 11.6.
M, various ammonium carbonate buffer solutions in which the carbonate ion concentration was changed from 1.6 M to 4.0 M were prepared.

The above-mentioned ammonium carbonate buffer solution having various carbonate ion concentrations is gently stirred, and then,
Immobilize the bacterial suspension for immobilization into the solution from the tip of the injection needle.
The mixture was added dropwise at 5 ° C to prepare a gel having a particle size of about 2.0 mm.

The results of measuring the relative strength of the gels prepared in the respective ammonium carbonate buffer solutions are shown in Table 1. Among them, the gel having a relative strength of 70 or more is used for a long time (20
Sufficient moldability and strength could be maintained under the stirring condition of 100 rpm in the ammonium carbonate buffer solution of the same composition for more than time). Here, as a method for measuring the strength of the gel, the method of left-handedness (Chemical Engineering, V
ol. 36, No. 6, 1991).

[0031]

[Table 1]

Example 2 10 parts by weight of polyvinyl alcohol having a degree of polymerization of 1500 and a saponification degree of 87.0 to 89.0 and 2 parts by weight of κ-carrageenan were mixed with 88 parts by weight of warm water at 50 ° C. and stirred well. Make a homogeneous solution. 1% by weight of this mixture, 1% suspension of Escherichia coli cell suspension in terms of dry weight
The parts by weight were well stirred and mixed. Here, this Escherichia coli cell (BP3569) was prepared by the same method as in Example 1.

On the other hand, the ammonium ion concentration is 5.0
M, various ammonium carbonate buffer solutions in which the carbonate ion concentration is changed from 0.5 M to 2.5 M are prepared.

The above-mentioned ammonium carbonate buffer solution having various carbonate ion concentrations is gently stirred, and then,
Immobilize the bacterial suspension for immobilization into the solution from the tip of the injection needle.
The mixture was added dropwise at 5 ° C to prepare a gel having a particle size of about 2.0 mm.

The results of measuring the relative strength of the gels prepared in the respective ammonium carbonate buffer solutions are shown in Table 2. Among them, the gel having a relative strength of 70 or more is used for a long time (20
Sufficient moldability and strength could be maintained under the stirring condition of 100 rpm in the ammonium carbonate buffer solution of the same composition for more than time).

[0036]

[Table 2]

Example 3 10 parts by weight of polyvinyl alcohol having a degree of polymerization of 1500 and a saponification degree of 87.0 to 89.0 and 2 parts by weight of κ-carrageenan were mixed with 88 parts by weight of warm water at 50 ° C. and stirred well. Make a homogeneous solution. 1% by weight of this mixture, 1% suspension of Escherichia coli cell suspension in terms of dry weight
The parts by weight were well stirred and mixed. Here, this Escherichia coli cell (BP3569) was prepared by the same method as in Example 1.

On the other hand, various ammonium carbonate buffer solutions having a carbonate ion concentration of 2.5 M and an ammonium ion concentration of 3.0 M to 11.0 M are prepared.

The above ammonium carbonate buffer solution having various ammonium ion concentrations is gently stirred,
Subsequently, the immobilizing bacterial cell suspension was dropped into this solution from the tip of the injection needle at 25 ° C. to prepare a gel having a particle size of about 2.0 mm.

The results of measuring the relative strength of gels prepared in the respective ammonium carbonate buffer solutions are shown in Table 3. Among them, the gel having a relative strength of 70 or more is used for a long time (20
Sufficient moldability and strength could be maintained under the stirring condition of 100 rpm in the ammonium carbonate buffer solution of the same composition for more than time).

[0041]

[Table 3]

Example 4 10 parts by weight of polyvinyl alcohol having a degree of polymerization of 1500 and a saponification degree of 87.0 to 89.0 and 2 parts by weight of κ-carrageenan were mixed with 88 parts by weight of warm water at 50 ° C. and stirred well. Make a homogeneous solution. 1% by weight of this mixture, 1% suspension of Escherichia coli cell suspension in terms of dry weight
The parts by weight were well stirred and mixed.

This Escherichia coli cell (BP35
69), a large amount of phenylalanine ammonia lyase derived from Rhodosporidium toruloides was expressed in the cells. Further, this phenylalanine-ammonia-lyase can generate L-phenylalanine from cinnamic acid and ammonium ions in an alkaline high-concentration ammonium carbonate solution.

Next, the ammonium ion concentration was 9.6.
A 2.5 M ammonium carbonate buffer solution (pH 9.6) having a concentration of M and a carbonate ion concentration was prepared and gently stirred. Subsequently, the suspension of immobilized cells was dropped into the ammonium carbonate buffer solution from the tip of an injection needle to prepare a gel having a particle size of about 2.0 mm. At this time, all the dropping was performed at 25 ° C.

Further, 1 part by weight of the obtained gel was added to 5 parts by weight of an ammonium carbonate buffer solution (ammonium ion concentration of 8.0 M, carbonate ion concentration of 1.6 M), and mixed and stirred. did. Here, this gel was able to stably maintain its morphology and strength in this ammonium carbonate buffer solution under stirring conditions of 100 rpm.

Subsequently, 12 parts by weight of cinnamic acid per 1 part by weight of the bacterial cells was charged while keeping the concentration between 0.3% and 0.7%, and the reaction was carried out for about 20 hours. Performed and L-phenylalanine was allowed to accumulate in the buffer. The reaction temperature was 30 ° C.

After the reaction was completed, the concentration of L-phenylalanine was 5.0%. The results of Example 4 and Comparative Example 1 are also shown in Table 4.

Comparative Example 1 L-phenylalanine was produced and accumulated in the same ammonium carbonate buffer solution as in Example 4 and under the reaction conditions, using the bacterial cells which were not included in the immobilizing polymer material at all. It was That is, using the same microbial cell having the same weight as in Example 4, the case where L-phenylalanine was accumulated was compared with the presence or absence of immobilization as the difference in the reaction conditions, and the both were compared.

The concentration of L-phenylalanine after the reaction was 5.2%. The results of Example 4 and Comparative Example 1 are also shown in Table 4.

[0050]

[Table 4]

[0051]

According to the present invention, as long as a gel is prepared under conditions containing a high concentration of carbonate ion, and this gel is used in a high concentration inorganic salt solution, the formed gel will be strong. Since it is possible to completely omit the process of adding the gel, it can be expected that the industrial use of the gel will be easier.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kiyoteru Nagahara 30 Asmuta-cho, Omuta City, Fukuoka Prefecture Mitsui Toatsu Chemical Co., Ltd. (72) Nobuhiro Fukuhara 30, Asamu-cho, Omuta City, Fukuoka Prefecture Within

Claims (7)

[Claims] 1. An inorganic material used at the time of gel formation in the case of producing a gel in which a biocatalyst such as a microorganism or an enzyme is comprehensively immobilized by using a polymer material composed of polyvinyl alcohol and κ-carrageenan as a gel substrate. A method for preparing an immobilized gel under conditions in which a salt solution contains a high concentration of carbonate ions. 2. A method for producing an immobilized gel, wherein the carbonate ion concentration of the inorganic salt solution according to claim 1 is 1.5 M or more. 3. A method for preparing an immobilized gel, wherein the inorganic salt solution of claim 2 contains 5.0 M or more ammonium ions. 4. Use of the immobilized gel prepared by the method according to claim 1, 2 or 3 in a high-concentration inorganic salt solution to obtain specific biocatalyst such as microorganisms or enzymes. Of using an immobilized gel characterized by advancing various biochemical reactions. 5. The high-concentration inorganic salt solution according to claim 4,
Method of using immobilized gel when it contains M or more carbonate ions. 6. A method of using an immobilized gel, wherein the high-concentration inorganic salt solution of claim 4 is a high-concentration ammonium carbonate solution. 7. A method of using an immobilized gel, wherein the high-concentration ammonium carbonate solution according to claim 6 has a carbonate ion concentration of 1.5 M or more and an ammonium ion concentration of 5.0 M or more.