JPS60180586A - Preparation of material containing enzyme immobilized with pva gel - Google Patents

Abstract

PURPOSE:To produce a material containing immobilized enzyme having high strength to endure the industrial use, by freezing a mixture of a specific aqueous solution of PVA, an enzyme-containing composition and boric acid in a vessel, and drying the formed mixture. CONSTITUTION:An aqueous solution of a polyvinyl alcohol having a saponification degree of >=95mol% and an average polymerization degree of >=1,000 is mixed homogeneously with an enzyme-containing material and boron or borax. The mixture is frozen in a vessel having an arbitrary form at <=-6 deg.C, and the obtained formed product is dried spontaneously or under aeration. The concentration of the aqueous solution of polyvinyl alcohol is 5-30W/W%, preferably 10-20W/W%. When the concentration is low, the mechanical strength of the obtained material containing immobilized enzyme becomes poor, and when the concentration is too high, the preparation is difficult because of the solubility of the polyvinyl alcohol. The enzyme-containing material means an enzymatic agent or an enzyme-producing microbial cell.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing immobilized enzymes or bacterial cells using polyvinyl alcohol (PVA) gel.

In recent years, technology related to the use of microorganisms and the enzymes produced by them has made significant progress, and many methods for immobilizing enzymes or microbial cells have been proposed and many inventions have been made in order to effectively utilize enzymatic reactions. However, very few technologies have been successfully commercialized. The reasons for this include, in addition to the production cost of the product, the immobilization of enzymes or bacterial cells requires extreme conditions, or the use of polyfunctional reagents. There is a problem in the expression of biological activity due to a decrease in the enzyme activity of the bacterial cells, the strength of the obtained immobilized enzyme or bacterial cells is so low that they cannot withstand industrial use, or they are deformed by the liquid pressure of the substrate. Immobilized enzymes or bacterial cells may cause clogging, problems with the physical durability of the immobilized enzymes or bacterial cells, and harmful monomers, cross-linking reagents, etc. are used for immobilization. This was due to various drawbacks, including problems with product safety.

Conventionally, various methods have been known for immobilizing enzymes or bacterial cells using PvA.

For example, an enzyme immobilization method has been proposed in which an aqueous solution containing PVA and an enzyme is gelled at a low temperature (Japanese Patent Laid-Open No. 50-52276).

The gel obtained by this method exhibits no elasticity and has extremely low mechanical strength. In addition, if air-drying is performed after solidification and thawing, only a soft material is obtained, and furthermore, even if vacuum dehydration is attempted instead of air-drying, even if it takes a long time to dehydrate, the product shows almost no elasticity. However, only a brittle gel can be obtained, and this cannot be said to be an industrially applicable method.

A method of immobilizing enzymes or bacterial cells by adding an aqueous solution of boric acid or porcelain to an aqueous solution of enzymes or bacterial cells and immediately gelling them (Japanese Patent Publication No. 55-51552-4, Japanese Patent Application Laid-open No. 135295-1982) However, the gel produced is soft and difficult to mold.

An immobilization method in which acid is added to a suspension containing PVA, tetraethyl silifute, and bacterial cells and air-dried (Special Publication No. 55
11311) has also been proposed, but this film is also weak. In this case, even if the membrane was freeze-dried after adding an acid, the mechanical strength of the resulting membrane actually decreased, and it was almost impossible to form the membrane.

A suspended aqueous solution containing a PVA aqueous solution, viable bacterial cells, and clay minerals is dried at -6°C to +40°C until a predetermined moisture content is reached, and the viable bacterial cells are immobilized in the resulting gel. A method has been proposed (Japanese Unexamined Patent Publication No. 57-138390), but this gelation is not low-temperature gelation, and the obtained immobilized bacterial cells are still soft and brittle, and cannot be strong enough to be used industrially. Moreover, since it contains a large amount of clay minerals, the bacterial cell activity per volume decreases, and the volume of the reaction vessel for the enzymatic reaction increases accordingly.

A method of immobilizing live bacteria in a gel produced by freezing and molding an aqueous suspension containing a PVA aqueous solution, live bacteria, and clay minerals, and then vacuum drying without thawing, so-called freeze-drying ( Japanese Patent Publication No. 57-141291, 1972
7-198088) has been proposed. Similarly, P
A method of immobilizing live bacteria in a gel produced by freezing and molding an aqueous suspension containing a VA aqueous solution and live bacteria, and then vacuum drying without thawing, so-called lyophilization. No. 57-141292, No. 57-19808
Publication No. 8) has been proposed.

These immobilized bacterial cells are relatively strong, but only a small amount of volume can be obtained, and when a large amount of clay minerals are added, the volume increases further, resulting in a decrease in bacterial cell activity per unit volume. In addition, the reaction volume of immobilized bacterial cells when used in enzyme reactions increases significantly. Industrially, increasing the reaction volume is extremely disadvantageous, and also has the drawback of increasing equipment costs and energy costs associated with freeze-drying.

The present inventors have devised a method to overcome the drawbacks of the prior art.
As a result of extensive research, we have found that a PVA aqueous solution, enzymes or bacterial cells, and boric acid or its aqueous solution are uniformly mixed at a pH of 5 to 7.5, and the mixture is frozen to form a low-temperature gel. We have found that very strong immobilized enzymes or bacterial cells can be obtained by air drying or ventilation drying.

In general, PvA gelation is known to be carried out by composite gelation using reagents such as boric acid, borax, tetraalkyl silicate, and organosilicon, and by low-temperature gelation by freezing, but both of these methods are based on weak fixation. Only sterilized bacterial cells were obtained. We used boric acid or borax to
Composite gelation is performed, followed by low-temperature gelation by freezing,
Furthermore, they discovered that dry gelation progresses even in a simple drying process, and learned that by performing three types of gelation, immobilized enzymes and bacterial cells with strength that can withstand industrial use can be obtained.

Furthermore, the composite gelation of PVA with boric acid or borax, depending on their quantitative relationship to PVA and the pH conditions during mixing, only greatly affects the strength of the final immobilized product. I learned that this also affects the ease of molding.

The present invention was completed based on the above findings, and consists of uniformly mixing a PVA aqueous solution with a saponification degree of 95 molti or more and an average polymerization degree of 100 or more, an enzyme-containing material, and boric acid or borax. , the mixture in a container of any shape.
A method for producing a strong immobilized enzyme-containing product, which is characterized by freezing at a temperature below ℃ and then air drying or ventilation drying the obtained molded product, and its purpose is for industrial use. The purpose of the present invention is to provide a method for producing strong immobilized enzymes or bacterial cells that can withstand harsh conditions.

The PVA used in the present invention has a saponification degree of 95 molt or more, preferably 9.7 molt or more.

If the degree of saponification is lower than this, for example below 85 mol, only a weak immobilized enzyme-containing material is obtained. The degree of polymerization used is one having an average viscosity of 1000 or more, preferably 1500 or more. As the degree of polymerization of PVA decreases, the mechanical strength of the obtained immobilized enzyme-containing product also decreases.
It is preferable to use products with a high polymerization degree of about 1,700 to 2,600, which are usually commercially available.

In the present invention, an aqueous solution of pVA is first prepared, and the concentration is 5 to 30 W/W, preferably 10
It is preferable to use it at ~20 W/W%. If the concentration is too low, the mechanical strength of the obtained immobilized enzyme-containing product will decrease, and if the concentration is too high, the solubility of PVA will make it difficult to prepare.

The enzyme-containing substance used in the present invention means an enzyme agent or an enzyme-producing bacterial cell. They may be used as such or as solutions or suspensions in aqueous media such as water, suitable buffers and the like.

The enzyme agent may be partially purified or fully purified, and the enzyme may be of any degree of purity. Further, the enzyme may be in a form in which it is physically adsorbed on an inorganic or organic carrier, or in a form in which it is mixed with the carrier. Here, the carrier is a water-insoluble inorganic or organic substance that does not inactivate the enzyme or the bacterial cells, and in some cases, a substance that physically adsorbs the enzyme or the bacterial cells. Examples include known organic polymers such as cane earth, silica gel, alumina, activated carbon, Sephalex, agarose, and cellulose.

The enzyme-containing material of the present invention includes (a) an enzyme;
(b) Mixture of enzyme and inorganic carrier: (C
) A combination of an enzyme and an inorganic or organic carrier;
or a suspension of (cl; (d) an enzyme solution; (0) a mixture of an enzyme solution and an inorganic or organic carrier; (f) a combination of an enzyme solution and an inorganic or organic carrier; and the above (el or (f) ).On the other hand, the enzyme-producing bacterial cells include live bacterial cells, dried bacterial cells, destroyed bacterial cells, (g) a mixture of the bacterial cells and an inorganic or organic carrier, and (h) the bacterial cells. A combination of microorganisms and an inorganic or organic carrier; suspensions of live microbial cells, dried microbial cells, and destroyed microbial cells; and WA suspensions of the above (gl or (hl) are included. The above enzymes include known enzymes, For example, glucose isomerase, fumarase, aspartase, amylase, glucoamylase, trypsin, chymotrypsin, pepsin, papain, pancreatin, aminoacylase, penicillin acylase, cephalosporin acylase, nuclease, ribonuclease, phytin, catalase, galactosidase, ATP -Deaminase, L-glutamate dehydrogenase, phosphatase, tyrosinase, invertase, flavokinase, streptokinase, avirase, ATP-creatine phosphotransferase, pectinase, carpoxypeptidase, L
- Asparaginase, maltase, lactase, urease, tannase, lipase, melipiase, aldolase, cellulase, anthocyanase, naringinase, hesperidinase, D-amino acids, oxidase, glucose oxidase, L-phenylalan/ammonium lyase, L-aspartic acid and L- Examples include enzymes that synthesize aspartame from phenylalanine methyl ester. In addition to the above, enzymes]
Of course, it is also possible to mention the known enzymes described in the issue published by Shokusen Shoten, December 1, 1982, supervised by Bunji Maruo and Nobuo Tamiya.

The enzyme-producing microbial cells may be bacteria, actinomycetes, filamentous fungi, or yeast as long as they produce the enzymes. The above-mentioned microbial cells may be live microbial cells, dried microbial cells, or partially or completely destroyed microbial cells as long as they have enzymatic activity. Furthermore, these bacterial cells may be in a form in which they are physically adsorbed on an inorganic or organic water-soluble carrier, or in a form in which they are mixed with the carrier.

The boric acid or borax used in the present invention may be used in a solid state per se, or the amount used in a solution dissolved in an aqueous medium, such as water or a suitable buffer, is sufficient to form a composite gel. It does not progress, has extremely high fluidity, and is unsuitable for molding. On the other hand, if too much is used, gelation progresses too much, making it difficult for subsequent low-temperature gelation and dry gelation to proceed. The final immobilized content can only be mechanically brittle. Although it depends on the concentration of the above PVA aqueous solution, it is preferable to use 1 to 4% by weight.

The PVA aqueous solution, the enzyme-containing material, and boric acid or borax are mixed at a pil of 5.5 to 7.5, preferably at a pH of 6 to 7. If the addition of the three substances deviates from the above p (2), the above p (2) may be adjusted with an appropriate acid or alkali and mixed uniformly. If the pH on the lower side of the above pH range is too low, it will not only damage the activity of enzymes and bacterial cells, but also prevent the composite gelation from proceeding and the viscosity suitable for molding. The strength of the enzyme-containing material is significantly inferior.

On the other hand, if pn is too high, composite gelation progresses too much, which not only makes molding difficult, but also extremely weakens the strength of the final immobilized enzyme-containing product.

The mixture mixed under the above mixing conditions in the present invention is
After becoming uniform, it is molded in a container of any shape, and cooled and frozen at -6° C. or below to proceed with low-temperature gelation. This low-temperature gelation greatly affects the mechanical strength of the final immobilized enzyme-containing product, and if this step is omitted, an immobilized enzyme-containing product with satisfactory mechanical strength will not be obtained, and if cooling is insufficient, Since the mechanical strength of the immobilized enzyme-containing material is poor, it is usually necessary to -
Preferably, it is cooled to around 20°C. Cooling time is -20
If the temperature is around 0.9°C, it is preferable to spend 5 hours or more.

The molded product thus obtained by low-temperature gelation is sufficiently gelled by composite gelation using boric acid or borax and low-temperature gelation by the freezing process. Drying or ventilation drying further progresses dry gelation, and it is possible to obtain an immobilized enzyme-containing material having sufficient mechanical strength to withstand industrial use. The above-mentioned natural drying and ventilation drying are industrially advantageous drying methods because their equipment costs and energy costs are extremely low compared to vacuum freeze drying. The above-mentioned drying temperature should be a temperature that does not deactivate the enzyme activity, so it is usually room temperature to 3 ℃.
It is preferable to carry out the reaction under conditions of 0 to 40°C. Of course, in the case of thermostable enzymes, such as enzymes that are stable even under heating at 60°C,
Needless to say, ventilation drying may be performed at about 0°C.

In the case of natural drying such as leaving at room temperature, a sufficiently dried immobilized enzyme-containing material can usually be obtained in about 48 hours, although this varies depending on the moisture content of the immobilized enzyme-containing material.

The thus obtained immobilized enzyme-containing material has mechanical strength that can withstand industrial use by complex gelation, low-temperature gelation, and dry gelation, and has a water content of 15 to 30%. can get.

Next, the present invention will be specifically explained with reference to Examples, Reference Examples, and Comparative Examples. The strength of the obtained nine-immobilized bacterial cells was measured by the following test method.

<Strength test method> Pour 300 immobilized bacterial cells to be tested and 10 m of water into an L-shaped test tube, shake in a constant temperature bath at 70°C, and rinse for 90 minutes.
The state of disintegration of the immobilized bacterial cells was investigated by measuring the transmittance at a wavelength of 0 nm. Immobilized bacterial cells with less disintegration have a higher permeability.

Examples 1 to 3 Commercially available PVA (saponification degree 99.45 molti, polymerization degree 170
0) Dissolve 20 (l in 800 g of water, 20 W/W%
A PVA aqueous solution was prepared.

10 (8.0% of the above PVA aqueous solution in a ml beaker)
Streptomyces alpus Y-T-N [L51 FERM-PNα, which produces g and glucose isomerase
463) moist bacterial cells (moisture content: 85 cm, JP-A-52-74)
80 (obtained by culturing according to the method described in Publication No. 3′) adjusted to 0,
25 W/W % diluted water (pH 6,5 and 7.0 adjusted with IN aqueous sodium hydroxide solution 1 20 m7
! After adding, the mixture was uniformly mixed for 5 minutes at room temperature to form a composite gel, and the resulting mixture was injected with a syringe into the molding groove of 2 x 2 squares to be molded, and then frozen at -20°C for 24 hours. and gelatinized at low temperature. The obtained molded products were left to stand at 20 to 25° C. for 48 hours and air-dried to obtain immobilized bacterial cells.

In addition to observing the difficulty of molding during the production of each immobilized bacterial cell, a strength test was conducted, and the possibility of dissolution after 90 minutes was observed and the transmittance was measured. The results are shown in Table 1, and the immobilized bacterial bodies of the present invention are easy to mold during production, and are shown in Reference Examples 1 to 3 and Comparative Examples 1 to 3 described later.
It has extremely high strength compared to the immobilized bacterial cells of No. 2, and has a strength of 90%
It was observed that the product did not dissolve even after a few minutes and was suitable for industrial use.

Reference Examples 1 to 2 In Examples 1 to 3, the pH was set to 7.85 (Reference Example 1).
) and 8.0 (Reference Example 2) to produce immobilized bacterial cells. The difficulty of molding during the production of each immobilized bacterial cell, the possibility of dissolution after 90 minutes, and the measurement results of transmittance are as shown in Table 1. In the case of Reference Examples 1 and 2, The product is difficult to mold and has extremely weak strength compared to the products of Examples 1 to 3. Particularly in the case of Reference Example 2, it cannot be used as an enzyme source for the glucose isomerization reaction.

Comparative Example 1 20 W used in Examples 1 to 3 in a 100 d beaker
/W ChiPVA aqueous solution 8F, Streptomyces alpus YT-NIL5 wet bacterial cells 10g and 0.25W/W
% boric acid solution + IN sodium hydroxide solution 6.
After adding 20- (adjusted to 5), the mixture was uniformly mixed at room temperature for 5 minutes to form a composite gel. The resulting mixture is 2x2mm
It was injected into the corner molding groove with a syringe and molded. The obtained molded product was left to stand at 20 to 25°C for 48 hours to air dry to obtain immobilized bacterial cells. The moisture content of the obtained immobilized bacterial cells was 23%.

The results of the measurement of the difficulty of molding during the production of the obtained immobilized car body, the possibility of dissolution after 90 minutes, and the transmittance are as shown in Table 1. The transformed bacterial cells have very low strength and are completely dissolved, so they cannot be used as an enzyme source for the glucose isomerization reaction.

Comparative Example 2 The molded product obtained in Comparative Example 1 was frozen at -20°C for 24 hours to form a low-temperature gel to obtain immobilized bacterial cells. The water content of the obtained immobilized bacterial cells was 92%.

The results of the measurement of the difficulty of molding during the production of the obtained immobilized bacterial cells, the possibility of dissolution after 90 minutes, and the transmittance are as shown in Table 1, and there was no dry gelation. The above-mentioned immobilized bacterial cells not only have very low strength but also completely dissolve, so they cannot be used as an enzyme source for the isomerization reaction of K and glucose.

(Margins below) 1st Table 10: Completely dissolved, -: Not dissolved Examples 4 to 7 20 W/W used in Examples 1 to 3 in a 100-capacity beaker
%PVA aqueous solution and 1°g of wet bacterial cells of Streptomyces alpus YT-N [L4], and
．． 04 (Example 4), 0.08 (Example 5), 0.15
(Example 6) and 0.25 (Example 7) w/w %
concentration of boric acid (J in sodium hydroxide solution)
After adding 6, OK adjusted) 20-, the mixture was uniformly mixed for 5 minutes at room temperature to form a composite gel. 2 of each of the resulting mixtures
×2- After injection into the corner molding groove with a syringe and molding, -
It was frozen at 20°C for 24 hours to form a low-temperature gel.

The obtained molded products were left to stand at 20 to 25° C. for 48 hours to air dry to obtain immobilized bacterial cells.

In addition to observing the degree of difficulty in molding during the production of each immobilized bacterial cell, a strength test was conducted, and the possibility of dissolution after 90 minutes was observed and the transmittance was measured. The results are shown in Table 2. The immobilized bacterial cells of the present invention are easy to mold during production, and the fixed bacterial cells of Reference Examples 4 to 9 and Comparative Example 3 described later are It was observed that the product had extremely high strength, did not dissolve even after 90 minutes, and was durable for industrial use.

Reference Examples 3 to 5 In Examples 4 to 7, the concentration of boric acid water was 0.01, respectively.
+Reference Example 4), 0.4 (Reference Example 5), and 1.0 (Reference Example 61 w/w%) to produce immobilized bacterial cells.

The difficulty of molding during the production of each immobilized enzyme,
The results of measurement of dissolution and transmittance after 90 minutes are shown in the second
As shown in the table, in the case of Reference Example 3 (the amount of boric acid is PVA
The amount of 0.25 cm) was lower than that of the immobilized bacterial cells of Examples 4 to 7, indicating that sufficient composite gelation was not progressing due to the too small amount of boric acid. . In addition, in the case of Reference Example 4 (boric acid amount is 10% of PVA1) and Reference Example 5 (boric acid amount is 25% of PVA amount), as the boric acid amount increases, the immobilized bacteria of Examples 4 to 7 The strength was significantly lower than that of the body, indicating that too much boric acid caused the composite gel to progress too much, making the gel extremely brittle.

Comparative Example 3 In Examples 4 to 7, immobilized bacterial cells were produced using water in place of the boric acid solution.

The results of the measurement of the difficulty of molding, whether or not it dissolves after 90 minutes, and the transmittance during the production of the obtained immobilized enzyme are shown in Table 2. Composite gelation with boric acid was performed. It is shown that by not having this material, only a material with extremely weak mechanical strength can be obtained.

Table 2 Example 8 In Examples 1 to 3, 0.25 W/
An immobilized enzyme with a water content of 2° was obtained using a W% borax aqueous solution.

The above-mentioned immobilized enzyme is easy to mold during molding during its production;
It was observed that the product did not dissolve even after shaking for 90 minutes and had a transmittance of 80 degrees, indicating that it was a product that could withstand industrial use.

Representative: Masao Miyake, Sen 1 person

Claims (14)

[Claims] (1) The degree of saponification is 95 molti or more, and the average degree of polymerization is 10
00 or higher polyvinyl alcohol aqueous solution, enzyme-containing material, and boric acid or borax are uniformly mixed, the mixture is frozen at a temperature of -6°C or less in a container of any shape, and then the obtained molded product is A method for producing a strong immobilized enzyme-containing product characterized by air drying or ventilation drying. (2) Polyvinyl alcohol aqueous solution is 5 to 30 w/w
%. (3) The production method according to claim 1, wherein the enzyme-containing substance is an enzyme preparation or an enzyme-producing bacterial cell. (4) The production method according to claim 3, wherein the enzyme agent is (a) an enzyme, (b) a mixture of an enzyme and an inorganic or organic carrier, or (C) a combination of an enzyme and an inorganic or organic carrier. . (5) The production method according to claim 3, wherein the enzyme preparation is a suspension of the above (b) or (C). (6) The enzyme agent according to claim 3, wherein the enzyme agent is (a) an enzyme solution, a mixture of an enzyme solution and an inorganic or organic carrier, or (f) a combination of an enzyme solution and an inorganic or organic carrier. Manufacturing method. (7) The production method according to claim 3, wherein the enzyme preparation is a suspension of (el or (f)). (8) Enzyme-producing bacterial cells may be live bacterial cells, dried bacterial cells, destroyed bacterial cells, (
gl A mixture of the bacterial cells and an inorganic or organic carrier, (h
3.) The production method according to claim 3, wherein the bacterial species is combined with an inorganic or organic carrier. (9) The production method according to claim 3, wherein the enzyme-producing microbial cells are live microbial cells, dried microbial cells, a suspension of destroyed microbial cells, or a suspension of the above (g) or (h). (10) The manufacturing method according to claim 1, wherein boric acid or borax is used in an amount of 0.5 to 7 by weight based on the polyvinyl alcohol. (11) The production method according to claim 10, wherein boric acid or borax is itself or an aqueous solution thereof. (12) The manufacturing method according to claim 1, wherein the mixing is carried out under conditions of a pH in the range of 5.5 to 7.5. (13) The manufacturing method according to claim 1, wherein the natural drying or ventilation drying is carried out at a temperature that does not deactivate the enzyme. (14) The production method according to claim 11, wherein natural drying or ventilation drying is performed until the moisture content of the immobilized enzyme-containing product falls within the range of 15 to 30%.