JPS5849233B2 - Enzyme or bacterial cell immobilized product and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an immobilized product of an enzyme or a microbial cell and a method for producing the same, and more specifically, an immobilized product comprising an enzyme or a microbial cell enclosed in a semipermeable resin membrane and the production thereof. Regarding the method.

Conventionally, one of the methods for immobilizing enzymes and making them insoluble is
There is a so-called microcapsule method that uses a semipermeable membrane.

This method is a method in which enzymes are coated and immobilized with a film of translucent resin, and the resulting enzyme microcapsules are usually spherical with a diameter of several to several hundred microns.

However, the microencapsulation method has a major drawback in that it is difficult to control the pores of the membrane, which severely limits the types of enzymes that can be used.

Furthermore, enzyme deactivation may occur during microcapsule preparation.

The purpose of the present invention is to provide a desired enzyme immobilized product by a method that eliminates the drawbacks of such enzyme immobilization methods, that is, by a novel comprehensive method that reduces activity reduction and can be easily carried out under a wide range of conditions. It is in.

As a result of intensive research to achieve this objective, the inventors of the present invention have developed a composition that consists of: ■ semipermeable membrane resin, ■ hydrophilic solvent, ■ fine cellulose powder, ■ water, ■ aqueous protein solution, and ■ enzyme. It was found that a dried film retains high enzyme activity for a long period of time.

Furthermore, we have discovered that not only enzymes but also microbial cells can be immobilized in a similar manner.

The present invention has been developed based on the above-mentioned findings, and its gist is: (1) A semipermeable resin membrane is characterized by a mixture of enzymes or microbial cells together with cellulose fine powder and protein. enzyme or bacterial cell immobilized product,
and ■ Dissolve the semipermeable membrane resin in a hydrophilic solvent, mix it with fine cellulose powder and water, mix it with an aqueous protein solution, and finally mix it with an aqueous dispersion of enzymes or microorganisms. The present invention resides in the above-mentioned method for producing an immobilized product, which comprises forming a film into a film and then drying it.

The enzymes or microorganisms targeted by the present invention are not particularly limited in type; examples of enzymes include hydrolytic enzymes (amylase, protease, lipase, pectinase, invertase, etc.), oxidoreductases (glucose oxidase, catalase, etc.) ), isomerase (glucose isomerase, etc.), and the like.

As a microbial cell, Escherichia coli (Escherichia coli)
richia coli), Streptomyces P.
haeochromogenes) and the like.

The semipermeable membrane resin used in the present invention may be any resin that has semipermeable membrane performance and is soluble in a hydrophilic solvent, such as cellulose resin (collodion, ethyl cellulose resin, etc.), polyvinyl alcohol, etc. Examples include resins such as butyral resins, vinyl chloride-vinyl acetate copolymer resins, etc., and these resins may be used alone or as a mixture of two or more thereof.

Examples of the hydrophilic solvent used in the present invention include methanol, ethanol, isopropyl alcohol, butanol, ethylene glycol, polyethylene glycol, glycerin,
Examples include acetone, methyl ethyl ketone, etc.
It can be used as a species or as a mixture of two or more species.

The fine cellulose powder used in the present invention may be any ordinary commercially available cellulose powder, and if necessary, a mixture containing cellulose powder (but without any enzymes, etc.) may be dispersed using a homogenizer or the like to make it fine. You can also use it even if it has been squeezed.

The cellulose fine powder has a particle size of 50μ or less, preferably 2
It may be 0 μ or less.

By blending this fine cellulose powder, the water permeability of the target immobilized product and the permeability of the substance to be reacted are improved, and as a result, the reaction efficiency of enzymes and the like is also improved.

The protein used in the present invention may be a water-soluble protein such as gelatin or casein, and by using this, it is possible to stabilize enzymes, etc. in the target immobilized product.

The composition of each of the above-mentioned precepts for producing the immobilized product of the present invention is preferably composed of the following proportions.

■Semi-permeable membrane resin 3~10w//■Hydrophilic solvent 20~60〃■Cellulose fine powder 1~20//■Water
2-15 〃■10 weight protein aqueous solution 5-30 〃■Enzyme (or microbial cells) 0
．． 01-5 In the present invention, the above-mentioned components (1) to (2) are mixed in order according to the numbers with sufficient stirring.

If the order is incorrect, the semipermeable membrane resin, protein, enzyme, etc. will aggregate and separate, making it impossible to obtain the desired immobilized product.

Note that the order of blending the cellulose fine powder and water may be changed.

The composite thus obtained is applied to a substrate such as metal or plastic by an appropriate method such as brush coating, flow coating, spatula coating, etc. to form a film, which is then force-dried at a temperature of 60°C or less. The desired immobilized product can be obtained by drying at room temperature or low temperature.

Since this immobilized product is highly elastic and has good physical strength, it can be packed into a column and subjected to continuous enzyme (or microbial cell) reactions.

According to the present invention having the above-described structure, it is possible to obtain a desired immobilized product that has a small reduction in activity and can be used over a wide range of applications, and has the following advantages.

■ Enzyme immobilization is simple, and the activity decreases less during the immobilization procedure than with conventional methods.

■ There are almost no restrictions on the type of enzyme to be used, and even crude enzyme products can be used without any problems.

(2) By being able to prevent enzyme leakage and appropriately controlling the permeability of the semipermeable membrane, it is possible to provide an immobilized product that matches the size of the substance to be reacted.

■ Not only enzymes but also microbial cells can be immobilized in the same way.

Next, the present invention will be specifically explained with reference to Examples and Comparative Examples.

Example 1 Prepare the following precepts.

■Ethyl cellulose powder 5fI■Ethanol 20g Ethyl acetate
20gisopropyl alcohol
51■ Cellulose fine powder
51■Wednesday
101 ■ 10 weight multi-gelatin aqueous solution 51
■Glucose isomerase 0.11
The above steps (1) to (2) are sequentially blended.

That is, after sufficiently dissolving component (2) in a certain amount of (2), component (2) is added thereto and thoroughly dispersed.

Dispersion is carried out using a homogenizer until the particle size of component (1) becomes 20 μm or less.

Next, add component (2) for a certain amount and stir, then add component (2) and mix thoroughly.

At this stage, add a certain amount of water to obtain a uniform composition.

This composite is poured onto a tin plate and forcedly dried at room temperature to obtain a fixed film.

The above-mentioned immobilized membrane was cut into pieces of 5 to 10 mmH, and reacted with a solution having the composition shown below at 60°C for 30 minutes with slow stirring, and then 420 ml of 1/2M-HC10 was added to stop the reaction.

The amount of fructose produced by the reaction is measured by the cysteine-rubasol method.

Composition 30 weight glucose solution 10I1ll1
/IOM-phosphate buffer (pH 7.5) 6
orrti1/IOM-Magnesium sulfate solution
10Ill Pure Water 101rLl On the other hand, a similar reaction is carried out using 0.1f of glucose isomerase at the same concentration, and the amount of fructose produced is measured.

From these two measurement results, the activity of the insolubilized enzyme is 41.8 times higher than that of the non-insolubilized enzyme.

Comparative Examples 1 and 2 An immobilized membrane is obtained in the same manner as in Example 1, except that the aqueous solution of zecitin (component (2)) is not blended in the composition of the composition for producing an immobilized membrane in Example 1 (Comparative Example 1).

Further, an immobilized membrane was obtained in the same manner as in Example 1 except that the cellulose fine powder of component (①) was not blended (Comparative Example 2).
.

When the resulting immobilized membranes of both Comparative Examples were subjected to an enzymatic reaction in the same manner as in Example 1, the activity of the insolubilized enzyme was 10.3% in Comparative Example 1 compared to that without insolubilization. , Comparative Example 2 shows an activity of 16.8%.

Example 2 Prepare the following ingredients.

■Butyral resin powder 51 ■Ethanol 20? acetone
20g polyethylene glycol
5g■ Cellulose fine powder
5t water
ioy■10 weight steamed gelatin aqueous solution 51■
Glucose isomerase 0.1? An immobilized membrane was obtained in the same manner as in Example 1 using the above precepts ① to ②.

As a result of measuring its activity in the same manner as in Example 1, the activity of the insolubilized enzyme was 38.9% compared to that of the non-insolubilized enzyme.
activity.

Example 3 Prepare the following ingredients.

■Vinyl chloride-vinyl acetate copolymer resin 5g ■Ethanol 2o Guacetone
20? isoprobyl alcohol
5L? ■Cellulose fine powder
51■Wednesday
1og ■10 weight gelatin aqueous solution 5
10 ■ Invertase 0.1f
An immobilized membrane was obtained in the same manner as in Example 1 using the weight ratios (1) to (2) of t or more.

Cut this into 5-10mm wide pieces and mix with the following solution.
React at 0° C. for 30 minutes with slow stirring.

The amount of reducing sugar produced by the reaction is measured by the Wilstadter-Schudel method.

Kumikai 10 weight dosucrose solution 101'fLl
1/2 5 M citric acid/phosphate buffer (pH 3.8
) 80rrLl On the other hand, a similar reaction is carried out using invertase 0.11 at the same concentration, and the amount of reducing sugar produced is measured.

From these two measurement results, the activity of the insolubilized enzyme is 49.2% compared to that of the non-insolubilized enzyme.

Example 4 Prepare the following.

■Butyral resin powder 51 ■Ethanol 20r acetone
20g polyethylene glycol
51■ Cellulose fine powder
8t ■ 10 weight gelatin aqueous solution
6f? ■α-amylase 0.11
An immobilized membrane was obtained in the same manner as in Example 1 using the above precepts ① to ②.

This was cut into 5 to 10 square pieces, and 40
The reaction is allowed to proceed at 0.degree. C. for 30 minutes with slow stirring.

Soluble starch Is're'5
0 M phosphate buffer (pH 6.9)
9 0 1'rLl 50rLl of 3,5-dinitrosalicylic acid solution was added to the above reaction solution to stop the reaction.

This reaction solution was heated to 10 m. Take one, soak it in a boiling bath for 5 minutes,
Next, after cooling with running water, 10 rrLl of pure water was added, and the amount of reducing sugar produced was measured using a photoelectric colorimeter.

On the other hand, a similar reaction is carried out using α-amylase 0.11 at the same concentration, and the amount of reducing sugar produced is measured.

From these two measurement results, the activity of the insolubilized enzyme is 46.7% compared to that of the non-insolubilized enzyme.

Example 5 Prepare the following ingredients.

■Butyral resin powder 81 ■Ethanol 20f acetone
20f glycerin
51 ■Cellulose fine powder 8g■10
Aqueous gelatin solution by weight: 6 t Streptomyces faeokuchi mozenes (microbial cells lyophilized, thawed, and dehydrated with acetone) An immobilized membrane was obtained in the same manner as in Example 1 using the above components 1 to 2.

This was cut into a width of 5 to 10 ml and reacted with the following solution at 60° C. for 30 minutes with slow stirring, and then 1/2M-HC 10420 rrtl was added to stop the reaction.

The amount of fructose produced by the reaction is measured by the cysteine-carbazole method.

Composition 30 weight ratio Glucose solution 10 vertical 1/IO
M-phosphate buffer (pI{7.5) 6011
Ll1/IOM-Magnesium sulfate solution 1om
1 771 liters of pure water On the other hand, the same concentration of bacterial cells 11 was used to react in the same manner, and the amount of fructose produced was measured.

From these two measurement results, the activity of the insolubilized bacterial cells is 4.3 compared to that of non-insolubilized cells. 1 Indicates q6 activity.

Claims (1)

[Scope of Claims] 1. An enzyme or microbial cell immobilized product characterized in that enzymes or microbial cells are mixed together with cellulose fine powder and protein in a semipermeable resin membrane. 2. The product according to item 1 above, wherein the semipermeable membrane resin is at least one selected from cellulose resins, polyvinyl alcohol resins, and vinyl chloride-vinino acetate v copolymer resins. 3. The product according to item 1 above, wherein the protein is at least one selected from gelatin and casein. 4 Dissolve the semipermeable membrane resin in a hydrophilic solvent, mix it with fine cellulose powder and water, mix it with an aqueous protein solution, and finally mix it with an aqueous dispersion of enzymes or microorganisms. A method for producing an immobilized enzyme or bacterial cell, which comprises forming the material into a film and then drying it. 5. The combination of semipermeable membrane resin 3 to 10 weight, hydrophilic solvent 20 to 60 weight, cellulose fine powder 1 to 20 weight,
Water 2-15% by weight, 10-30% protein aqueous solution 5-30% by weight, and enzyme or microbial cells 0.0%
5. The method of claim 4, comprising a ratio of 1 to 5 parts by weight. 6. The method according to item 4 or 5 above, wherein at least one selected from cellulose resin, polyvinyl alcohol resin, and vinyl chloride-vinyl acetate copolymer resin is used as the semipermeable membrane resin. 7. Hydrophilic solvents include methanol, ethanol, isopropyl alcohol, butanol, ethylene glycol,
The method according to item 4 or 5 above, wherein at least one selected from polyethylene glycol, glycerin, acetone and methyl ethyl ketone is used. 8. The method according to item 4 or 5 above, wherein at least one kind selected from gelatin and casein is used as the protein. 9. The method according to item 4 above, wherein the cellulose fine powder and water are mixed into the hydrophilic solvent solution of the semipermeable membrane resin by mixing either one first and then the other. 10. The method according to item 4 above, wherein brush coating, flow coating, or spatula coating is employed to form a film on the 10 sets. 11. The method according to item 4 above, wherein the drying is performed by forced drying at a temperature of 60° C. or lower, normal temperature drying, or low temperature drying.