JPS62138193A - Production of immobilization granules of enzyme or microorganism - Google Patents

Abstract

PURPOSE:Inexpensive polyvinyl alcohol is used as an immobilization carrier and it is combined with a water-soluble polysaccharide which forms gell by contacting with polyvalent metal ions to produce granular immobilization product of high strength with enzyme loss reduced. CONSTITUTION:Polyvinyl alcohol, a water-soluble polysaccharide which is capable of forming gel by contacting with polyvalent metal ions and an enzyme or cell bodies of a microorganism are mixed and the liquid composition is added dropwise in an aqueous medium containing polyvalent metal ions and boric acid to convert the composition into granular gel. The water-soluble polysaccharide is preferably an alkali metal salt of alginic acid, carrageenan, mannan or chitosan.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the immobilization of enzymes or microbial cells, and more particularly to a method for immobilizing enzymes or microbial cells in the form of granular molded products.

Conventionally, methods for immobilizing enzymes or microorganisms include comprehensive method,
Many methods are known, such as physical adsorption methods and covalent bonding methods. When a lump or sheet-like immobilized product obtained by these methods is used for a microbial reaction or an enzymatic reaction, it is usually cut into small pieces or crushed and then packed into a rear ram. However, in this case, the immobilized substances often come into close contact with each other, which reduces the efficiency of microbial reactions and enzymatic reactions, and also has drawbacks such as frequent channeling phenomena that clog the column.

The present inventors believe that if enzymes or microbial cells can be immobilized as a granular molded product, it will be easier to fill the fluidized column and the contact area between particles will be reduced, increasing the efficiency of microbial reactions and enzymatic reactions. Considering that it is possible to do so, we have previously proposed a method for preparing a granular gel of enzymes or microorganisms using a photocurable resin and a water-soluble polymeric polysaccharide (see JP-A-59-11182).

However, photocurable resins are expensive, and since it is necessary to uniformly irradiate actinic rays onto granular materials, there are problems in that suitable irradiation equipment is difficult to obtain, and improvements have been desired. Therefore, the present inventors conducted extensive research on a method of immobilizing enzymes or microbial cells as granular molded products at low cost and easily, and found that using inexpensive polyvinyl alcohol as an immobilization carrier, this and polyvalent By using a combination of water-soluble polymeric polysaccharides that have the ability to gel on contact with metal ions, it is extremely easy to remove enzymes or microbial cells from aqueous media containing boric acid and polyvalent metal ions. The present inventors have discovered that it is possible to produce a granular immobilized product with high mechanical strength without using any of the above methods, and have completed the present invention. Therefore, according to the present invention, (a) polyvinyl alcohol, (b) a water-soluble polymeric polysaccharide capable of gelling upon contact with polyvalent metal ions, and (c) an enzyme or a microbial cell. A granular immobilized molded product of enzymes or microorganisms, characterized in that a liquid composition comprising the above composition is dropped into an aqueous medium containing polyvalent metal ions and boric acid to gel the composition into granules. A manufacturing method is provided. The method of the present invention will be explained in more detail below.

(a) Polyvinyl alcohol Polyvinyl alcohol is inexpensive, has little toxicity, and has excellent mechanical strength. The polyvinyl alcohol generally has a degree of saponification of ioo to 75 and a degree of polymerization of 3.
The saponification degree is preferably 90 to 3,000, but considering handling viscosity and mechanical strength, saponification degree is preferably 90 to 3,000.
80 and a degree of polymerization of 500 to 2.500.

(b) Water-soluble polymeric polysaccharide In order to achieve gelation and granularity of an enzyme or microbial immobilization carrier in an aqueous medium, the method of the present invention uses the above-mentioned (a) as an immobilization carrier. There is one major feature in using a water-soluble polymeric polysaccharide in combination with polyvinyl alcohol as described above.

The water-soluble polymeric polysaccharide used in the present invention is a polymeric polysaccharide that is water-soluble and has the ability to change into a gel that is insoluble or poorly soluble in water when it comes into contact with polyvalent metal ions in an aqueous medium. , - generally about 3,000 to about 2,000,0
00 and typically has a molecular weight of at least about log/, u(
Those exhibiting a solubility of 2s° C.) are preferably used.

Specific examples of water-soluble polymeric polysaccharides having such characteristics include alkali metal salts of alginic acid, carrageenan, mannan, chitosan, and the like.

The metal salt of alginic acid, which is one of these water-soluble polymeric polysaccharides, is dissolved in an aqueous medium and contains polyvalent metal ions,
For example, when it comes into contact with at least one polyvalent metal ion selected from alkaline earth metal ions such as magnesium ions, calcium ions, strontium ions, and barium ions, or other polyvalent metal ions such as aluminum ions, cerium ions, and nickel ions. Can be gelled. In addition, the water-soluble polymeric polysaccharides that can be used in the present invention are:
It is not necessary to have gelation ability upon contact with all polyvalent metal ions; at least one type of polyvalent metal ion,
Preferably, it is sufficient that it has the ability to gel when contacted with alkaline earth metal ions. The concentration of polyvalent metal ions at which gelation occurs varies depending on the type of water-soluble polymer polysaccharide, etc., but is generally at least 0.01 mo/! l
It is.

(d) Enzymes or microbial cells There are no particular restrictions on the types of enzymes or microbial cells that can be immobilized by the method of the present invention, and according to the method of the present invention, any type of enzyme or microbial cells can be immobilized. It can be immobilized without substantially deactivating its enzyme activity.

Representative examples of enzymes and microbial cells that can be immobilized by the method of the present invention are as follows.

(b) Examples of enzymes Lactate dehydrogenase (1, 1, 2, 3) Lactate oxidase (1, 1, 3, 2) Glucose oxidase (1, 1, 3, 4) Formate dehydrogenase (1, 2, 1, 2 ) aldehyde dehydrogenase (1,
2,1,3) Aldehyde oxidase (1,2,3,1
) xanthine oxidase (1,2,3,2) pyruvate oxidase (1,2,3,3) pyruvate reductase (1,2,4,1) cortisone-α-reductase (1,3,1,4) Acyl-CoA-dehydrogenase (13,99,3) 3-ketosteroid Δl-dehydrogenase (1,3,
99,4) 3-ketosteroid Δ4-dehydrogenase (1,3,
99,5) L-alanine dehydrogenase (1,4,1,1) L-glutamate dehydrogenase (1,4,1,3) L-amino acid oxidase (1,4,3,2) D-amino acid oxidase (1, 4,3,3) Catalase (1,
11,1,6) Catechol methyltransferase (2,1,1,e) Carnitine acetyltransferase (2,3,1,
7) Acetyl-CoA acetyltransferase (2,3,
1,9) Asbertate aminotransferase (2,6,1
, 1) Alanine aminotransferase (2,6,1,2) Pyridoxamine pyruvate transferase (2,6
, 1) Hexokinase (2,7,11) Glucokinase (2,7,1,2) Fructokinase (2,7,1,4) Phosphoglucokinase (2,7,1,10) Phosphofructokinase Kinase (2,7,1,11) Pyruvate Kinase (2,7,1,40) Carboxylesterase (3,1
, 1, 1) Aryl esterase (3, 1, 1, 2) Lipase (3, 1, 1, 3) Phospholipase A (3, 1, 1, 4) Acetyl esterase (3, 1, 1, 6) Cholesterol esterase (3,1,1,13) Glucoamylase (3,2,1,3) Cellulase (3,2,1,4) Inulase (3,2,1,7) α-Glucosidase (3,2,1, 20) β-glucosidase (3,2,1,21) α-galactosidase (3
, 2, 1, 22) β-galactosidase (3, 2, 1,
23) Invertase (3,2,1,26) Pepsin (3,4,4,1) Trypsin (3,4,4,4) Chymotrypsin A (3,4,4,5) Cathepsin A (3,4) Papain (3,4,4,10) Thrombin (3,4,,4,13) Amidase (3,5,1,4) Urease (3,5,1,5) Penicillin acidugase (3,5, 1,11) Aminoacylase (3,5,1,14) Adenine deaminase (3
, 5, 4, 2) A, T, P,ase (3, 6, 1, 3)
Pyruvé i decarboxylase (4,1,1,1) aldolase (4,1,2,13) malatestase (4,1,3,2) tryptophan synthase (4,2,1,20) aldolase (4,3
, 11,) Lysine racemase (5, 1, 1, 5) Glucose-6-phosphate imerase (5, 3, 1, 9) Steroid Δ-imerase (5, 3, 3, 1) Maxinyl-CoA synthetase (6, 2, 1, 5) etc. (Note) Numbers in parentheses represent enzyme numbers.

(b) Examples of microbial cells: Lactobacillus bulgaricus
) Aerobacter aerogenes Bacillus subtilis
) Azotobacter vinelandii Proteus vulgaris
is) Arthrobacter simplex Escherichia coli
i) Pseudomonas putida
putida) Achromobacter liquidum (Achromobacter liquidum) Curvularia lunata (curvularia 1un
ata) Corynebacterium glutamicum
um) Nocardia War Rodoclas (Nocardia
rhodcrous) Methanosarcina berkerii
Methanobacterium formicicum (Methanobacterium IIlformici)
cum) etc.

1) Each of the components (a), (b) and (c) described in 2 below can be made into a liquid composition by thoroughly mixing them with phase Q2 in an aqueous medium. . The aqueous medium that can be used is preferably water or an aqueous buffer solution, but in some cases, a mixture of water-soluble alcohols and water or an aqueous buffer solution, a mixture of water-soluble ketones and water or an aqueous buffer solution may be used. It is also possible to use an ester solvent solution that can be uniformly mixed with water or an aqueous buffer solution.

The mutual usage ratio of each component (a), (b), and (c) above is not strictly limited and can be varied over a wide range depending on the type of each component. It is appropriate to use the following ratios based on 100 parts by weight of polyvinyl alcohol as component (a) (the preferred range is in parentheses).

(b) Water-soluble polymeric polysaccharide 20.5 to 15 parts (1 to 15 parts)
81 fence (c) Enzyme or microbial cell: O, OO1 to 50 volumes
In addition, the aqueous medium can be used in the range of 10 to 1,500 parts by weight (50 to 90 parts by weight) based on the total of (a) to (c) above. .

Gelation: The liquid composition prepared as described above is then dropped into an aqueous medium containing polyvalent metal ions and boric acid to form a gel into particles.

Both polyvalent metal ions and boric acid are essential components in order to gel the liquid composition described above into granular form, and the gelation cannot be achieved even if either one is missing. That is, if polyvalent metal ions are missing, the liquid composition described in - will gel, but will not become granular. On the other hand, when boric acid is lacking, the above liquid composition becomes
Although it becomes something that looks like granules, this gelatinized material has almost no mechanical strength and cannot be put to practical use. The liquid composition described in item 1- is very easily gelled into granules due to the synergistic action of both the polyvalent metal ions and boric acid.

In addition, gelation with boric acid takes time even if it can be made into granules, so it is difficult to continuously produce granular fixed molded products. On the other hand, if polyvalent metal ions and boric acid are used at the same time, a granular fixed molded product is generated instantly, and there is no adsorption to each other or change in the granular state, making continuous production possible. . This significantly reduces manufacturing costs.

The polyvalent metal ions that can be included in the aqueous medium include:
One is selected that has the ability to gel the water-soluble polymeric polysaccharide in the liquid composition.

Preparation of an aqueous medium containing selected polyvalent metal ions and boric acid involves adding water-soluble compounds of the polyvalent metal, such as halides, carbonates, bicarbonates, sulfates of the polyvalent metal, to the aqueous medium. This can be carried out by dissolving boric acid after dissolving nitrate or the like. The concentration of polyvalent metal ions in the aqueous medium at that time is generally 0.01 to 5 mo, u/! ;
1. Preferably, it can be within the range of 0.1 to 2 mou/u. On the other hand, the concentration of boric acid is generally 0.01 to 10
mon/l, preferably 0, 1 to 4.11 O,Q,
/, il'.

Further, the pH of the aqueous medium containing the selected polyvalent metal ion and boric acid is in the acidic range. If you want this to be in the neutral or alkaline range, use a monovalent alkali metal,
It may coexist with polyvalent metal ions and boric acid. The concentration of this alkali metal can be arbitrarily determined depending on the pH to be set.

Dropping of the liquid composition into the aqueous medium containing polyvalent metal ions and boric acid can be carried out, for example, by dropping the liquid composition from the tip of a thin tube such as a syringe needle, or by using centrifugal force. This can be carried out by methods such as a method in which the liquid composition is dispersed in the form of particles, or a method in which the liquid composition is atomized into particles and dropped from the tip of a spray nozzle.

The size of the droplets to be dropped can be freely changed depending on the particle size desired for the final granular immobilized product, but usually the diameter is about 0.1 to about 5 mm, preferably about 0.5 to about 311 mm.
Advantageously, it is applied as 1 va droplets.

Gelation of the dropped liquid M+ product in the aqueous medium is thought to occur in two ways: by the water-soluble polymeric polysaccharide and polyvalent metal ions, and by polyvinyl alcohol and boric acid.

Gelation between the water-soluble polymeric polysaccharide and the polyvalent metal ion occurs immediately, and although the mechanism of gelation at that time is not completely clear, the water-soluble polymeric polysaccharide aggregates with the halide or salt solution. It is assumed that the anionic groups contained in the water-soluble polymeric polysaccharide then form ionic bonds via polyvalent metal ions, resulting in three-dimensional crosslinking and gelation. On the other hand, it is presumed that gelation between polyvinyl alcohol and boric acid is caused by chemical bonding between the hydroxyl group of polyvinyl alcohol and the boron of boric acid in a monodiol type. This gelation occurs from the surface of the granular molded product to the inside, and is thought to completely gel within 1 to 24 hours, depending on the particle size. Furthermore, as the number of residual acetate groups in polyvinyl alcohol increases, the concentration of boric acid required for gelation decreases in a more or less linear manner, and gelation becomes easier.

Room temperature is usually sufficient for the gelation, but if necessary, the gelation may be carried out under heating to a degree that does not deactivate the enzyme or microbial cells, or may be carried out under cooling.

The granular gel thus produced can be washed with water or an aqueous buffer solution and stored as is, or the granular gel can be lyophilized and stored.

Thus, according to the present invention, granular immobilized enzymes or microorganisms having a particle size of about 0.5 to about 5 mm can be produced by extremely simple operations, and continuous production is also possible.

According to the method of the present invention, the granular immobilization of enzymes or microbial cells using a synthetic carrier is facilitated, and the use of water-soluble polymeric polysaccharides provides a protective effect on the activity of enzymes and microbial cells. Furthermore, by using inexpensive polyvinyl alcohol as the immobilization carrier, advantages such as a wider range of applications can be obtained from the viewpoint of cost.

These granular immobilized products can be easily used particularly in reactors whose scale is not too large, fluidized bed type reactors, and the like.

Next, the present invention will be further explained by examples.

The degree of polymerization carried out is 1500. 80 parts by weight of distilled water is added to 20 parts by weight of polyvinyl alcohol having a saponification degree of 87.0 to 89.0, heated to about 50°C, and thoroughly mixed to form a uniform aqueous solution. Add to this mixture 20 parts by weight of a 3% sodium alginate aqueous solution, 60 parts by weight of distilled water, and the enzyme invertase (0,
Add 20 parts by weight of 1% concentration) and mix well, and mix the resulting polyvinyl alcohol-enzyme mixture with 3% boric acid and 0.1% boric acid.
An aqueous solution containing 1M calcium chloride (NaOH)
When the solution was dropped from a liquid level height of 10c+o from a syringe at the tip of the syringe into a syringe (prepared in No. 6), granules having a particle size of 2II11 were obtained. When this granular material was immersed as it was for 10 hours, a granular immobilized enzyme with good mechanical strength was obtained.

When the invertase activity of these particles was measured using sucrose as a substrate, the specific activity relative to non-immobilized invertase was 60%.

Example? Degree of polymerization: 1500. Add 90 parts by weight of distilled water to 10 parts by weight of polyvinyl alcohol with a saponification degree of 100, heat to about 50°C, and mix thoroughly to form a uniform aqueous solution. A homogeneous mixed solution was prepared by adding 10 parts by weight of a 3% gyanan aqueous solution and 3 parts by weight of a 2% glucose isomerase cell enzyme solution (sodium bicarbonate buffer pH=8) to this mixed solution. 3% boric acid and 5% boric acid from the tip of the syringe needle.
When dropped into an aqueous solution containing % potassium chloride (adjusted to pH=6 with NaOH) from a position 15 cm from the liquid surface, granules with a particle size of 2.5 mm were obtained. When this granular material was immersed as it was for 15 hours, a granular immobilized enzyme with good mechanical strength was obtained.

When the activity of this granular glucose isomerase was measured at 60° C. in a sodium bicarbonate buffer at pH 8 using glucose as a substrate, the specific activity was 70% relative to non-immobilized glucose isomerase.

Example 3 Degree of polymerization 1100. Saponification degree 87. Add 80 parts by weight of distilled water to 20 parts by weight of polyvinyl alcohol of O ~ 89.0,
Heat to about 50°C and mix well to make a homogeneous aqueous solution. Add 20 g of 3% sodium alginate aqueous solution to this mixture.
60 parts by weight of distilled water and 0.0 parts by weight of acetone-treated bacterial cells of Arthrobafter One Simplex (ATCC6946).
2 parts by weight were uniformly mixed and dispersed. After that, the dispersion was dropped from the tip of a syringe into an aqueous solution containing 3% boric acid and 0.2M aluminum chloride (prepared with NaOH to a pH of 6), whereupon it gelled into a spherical shape. When this granular material was immersed as it was for 16 hours, spherical immobilized microorganisms with a diameter of 2.8+1 m and good mechanical strength were obtained.

Example 4 Degree of polymerization 500. Saponification degree 87. 75 parts by weight of distilled water are added to 25 parts by weight of polyvinyl alcohol having a molecular weight of 0 to 89.0, heated to about 50°C and mixed well to form a uniform aqueous solution. To this mixture, 25 parts by weight of 3% sodium alginate aqueous solution,
and 0 dissolved in 0.1 M acetate buffer (pH 5, 6).
, 25 parts by weight of a 5% glucose oxidase aqueous solution were mixed uniformly, and the resulting mixture was injected into the tip of a syringe for 3 minutes.
% boric acid and 0.1M calcium chloride (Na
When added dropwise into the solution (adjusted to pH=6 with OH), it turned into a spherical gel. When this granular material was immersed as it was for 15 hours, spherical immobilized microorganisms with a diameter of 2.5 square meters and good mechanical strength were obtained.

Example 5 Degree of polymerization 1500. Add 80 parts by weight of distilled water to 20 parts by weight of polyvinyl alcohol with a saponification degree of 87.0 to 89.0, heat to about 50' (1!) and mix well to make a homogeneous aqueous solution.To this mixture, add 3% 20 parts by weight of an aqueous Uginan solution, 60 parts by weight of distilled water, and 10 parts by weight of an Eracelicia coli cell suspension were uniformly mixed and dispersed. The mixed solution is scattered by the centrifugal force, and the scattered particles are dropped into an aqueous solution (adjusted to pH = 6 with N'aOH) containing 3% boric acid and 0.3M potassium to form particles. When the granules were immersed in this state for 15 hours, they had a diameter of 3.0 m with good mechanical strength.
+w immobilized bacterial cells could be produced.

Example 6 Polymerization degree was 1500 in an argon gas atmosphere. Saponification degree 87
．． 80 parts by weight of distilled water is added to 20 parts by weight of polyvinyl alcohol having a molecular weight of 0 to 89.0, heated to about 50°C and mixed thoroughly to form a uniform aqueous solution. To this mixture, 20 parts by weight of a 3% sodium alginate aqueous solution, 70 parts by weight of distilled water, and 10 parts by weight of Mesanosarcina e. Berkelii cell suspension were uniformly mixed and dispersed. When the resulting mixture of polyvinyl alcohol and bacterial cells was dropped from the tip of a syringe into an aqueous solution containing 3% boric acid and 0.1M calcium chloride (adjusted to pH=8 with NaOH), a granular gel formed. It became.

When this granular material was immersed as it was for 10 hours, granular immobilized microorganisms with a diameter of about 2 mm and good mechanical strength were obtained.

Claims (1)

Scope of Claims: (a) polyvinyl alcohol, (b) a water-soluble polymeric polysaccharide capable of gelling upon contact with at least one type of polyvalent metal ion, and (c) an enzyme or a microbial cell. A granular immobilized molded product of enzymes or microorganisms, characterized in that a liquid composition comprising the above composition is dropped into an aqueous medium containing polyvalent metal ions and boric acid to gel the composition into granules. Production method.