JPS596884A - Preparation of immobilized enzyme - Google Patents

Abstract

PURPOSE:To prepare an immobilized enzyme without applying a thermal load to an enzyme or microorganism, by using a sodium type carrageenan as a main gel agent. CONSTITUTION:An enzymic active substance, e.g. an enzyme or microorganism, is dissolved in an aqueous solution containing a fully sterilized sodium type carrageenan and if desired thannin or glutaraldehyde, etc. is added thereto to aggregate the enzymic active substance. The resultant mixture solution is then dropped to an aqueous solution containing a fully sterilized gelling agent.

Description

[Detailed description of the invention] The present invention relates to a novel method for producing immobilized enzymes.

Enzyme reactions have traditionally been used to produce useful substances and to decompose substances harmful to humans and animals, and in recent years, technology has been developed to use enzymes as catalysts in continuous reactions. With the development of microorganisms, various methods have been proposed for immobilizing enzymes and microbial cells.

Typical examples include a method in which microbial cells are immobilized by being encased in gels such as polyacrylamide gel, polyvinyl alcohol gel, agar gel, carrageenan gel, and farselan gel.

Among these, when using natural sulfate polysaccharides such as carrageenan, agar, and farcellan, these polysaccharides generally maintain their gel state in the temperature range where enzymes or the microorganisms that produce them tend to act. It is used as an entrapping agent, but for this reason, it is necessary to raise the gelling temperature, and when dissolving enzymes or microorganisms into the gel body, the gel is dissolved at a temperature higher than the desired growth temperature for the microorganisms, and this gel dissolution is performed. Even if it is an immobilized enzyme body in which microorganisms are dissolved in a liquid, it has the disadvantage that the desired effect cannot be expected. For this reason, it is necessary to select compatible microorganisms and enzymes that are relatively thermally stable, which currently hinders a wide range of applications.

In view of the current situation, the present inventors devised a method of completely dissolving the enzyme, maintaining the dissolved state even at room temperature, and then gelling it. It has been reached.

Carrageenan is a sulfated galactan contained in red algae, and is divided into three types: kappa (6), iota (i), and lambda (λ), depending on the sulfate group content and the content of 3.6 anhydrogalactone units. exists and has different gelling ability, protein reactivity, and viscosity behavior *As a true natural thickener,
Widely used in the food industry, etc.

Among these, kappa-type carrageenan has the highest gelling ability and has all the desirable properties as a gel agent for gelling-type immobilized enzymes, which is the object of the present invention. Furthermore, it is well known that carrageenan has different properties depending on the cation bonded to the sulfate ester, and sodium carrageenan is soluble in cold water and generally has no gelling ability at room temperature. Not worth it. On the other hand, potassium-type carrageenan, in which the bound cation is potassium, is characterized by having gelling ability. The present inventors focused on the non-gelling ability of sodium-type carrageenan, learned from research, and devised the present invention.

That is, the present invention involves dissolving an enzyme active substance in a sodium-type carrageenan solution, and then contacting the solution with a gelling agent such as a potassium salt to replace the sodium ions bound to the carrageenan with potassium ions, etc. A method for producing an immobilized enzyme characterized by gelation.

The enzymes and enzyme-producing microorganisms that can be used in the present invention are not particularly limited and any enzymes can be used, but examples include the following.

'7' ° Amino acid oxidase, catalase, gelose oxidase, peroxidase, tyrosinase, aspartate acetiketofunsferase, fructokinase, hexokinase, leucine aminopeptidase, asparaginase, arginase, trypsin, chymotrypsin, lactase , phosphatase, lipase, ribonuclease, pectinase, invertase, lyases, isomerase enzymes such as glycose isomerase, ligases, and microorganisms such as bacteria, fungi, Bacillus subtilis, lichens, and protozoa that produce these enzymes.

A granular enzyme-containing gel is obtained by dropping the above enzyme or microorganism into an aqueous solution that has been thoroughly sterilized and contains all of the sterilizing agent. By fully adjusting the contact time with the gelling agent,
It is possible to gel only the surface layer of the gel, while maintaining the entire fluid state inside the gel, and the mobility of the reaction solution used for enzymatic reaction can be maintained within the gel, making it practically possible to increase reaction efficiency. . In addition, if the enzyme-containing sodium carrageenan gelled with a gelling agent is dehydrated or dried, it can be treated with potassium salts such as lamb, rubidium salts such as rubidium chloride, or combinations of alkali metal salts such as sodium chloride (salt)/potassium chloride mixture. , any combination is acceptable. Furthermore, if tannins, glutaraldehyde, etc. are added to the mixed solution before gelling treatment with a gelling agent solution, enzymes and microorganisms will aggregate, so it is difficult to prevent these enzyme-active substances from leaking out of the gel lattice. It is possible to prevent a decrease in enzyme reaction efficiency.

Hereinafter, the present invention will be fully explained in detail based on Examples.

Example 1 N. coli ATCC11303'i nutrient medium (
Curucose 0.5%, yeast extract 1.25%, peptone 1.0%, meat extract 0.5%, sodium chloride 0.5%
(pH 7,0) 5-, and cultured with shaking at 37°C for 16 hours. This culture solution was sterilized 1.
5% sodium carrageenan was added and mixed.

This mixed solution is completely sterilized dipotassium chloride aqueous solution 20
A spherical gel with a diameter of 3 cranes was obtained by dropping the gel from a nozzle into the gel.The gel thus obtained was translucent, and the viable bacterial cells contained within the gel were impossible to observe with the naked eye. However, the amount of bacterial cells determined by the plate dilution method after gel collapse was equivalent to 1.3 platinum loops per 1002 gels. A reducing effect was observed.

Example 2 To the sodium carrageenan/bacterial cell mixture prepared in 1,
Add all 3 of the sterilized 1.5% tannin aqueous solutions and mix thoroughly, then drop into the sterilized 2% sodium chloride/potassium chloride/potassium chloride mixed solution. Spherical gel' (r%. Gel Loot
The number of viable bacteria contained therein corresponds to 1.3 platinum loops.

The immobilized bacterial cells were filled into a column of 2Of total volume t4o*,
Substrate solution containing IM ammonium fumarate and 1 mM magnesium chloride (1) H8,5) 1 smz/hr
The amount of L-aspartic acid produced in the effluent was 130 iv/d, and there was no change in the amount of L-aspartic acid produced even after continuous operation for 20 days. .

Example 3 After dropping the sodium carrageenan/bacterial cell mixture prepared in 1 into a gelling agent solution consisting of a sterilized 2-thiodium chloride-potassium chloride 1:1 mixed solution containing 0.2% total tannin, A spherical gel f was obtained. The internal structure of the gel was compared by changing the residence time of the gel in the gelling agent solution.

This was packed into a column in the same manner as in Example 2, and a substrate solution containing IM ammonium fumarate and 1 mM magnesium chloride was passed through the column to generate L-aspartic acid by enzymatic reaction. The results at this time are shown in Table 1.

Even if only the surface layer is covered with gel and the inside is in a sol state, there is no gel breakdown during the reaction process, and the amount of L-aspartic acid produced remains unchanged even after 20 days of continuous operation, and the entire sphere is gelled, as in sample B. The reaction rate is also higher than that of the
The effect of fluidization within the gel body is observed.

Claims (1)

[Claims] 1. A method for producing an immobilized enzyme, which comprises dissolving an enzyme active substance in a sodium-type carrageenan solution and then contacting the solution with a total gelling agent "Kiosehana" to gel it.