JPS60160885A - Immobilized microorganism, plant cell and/or enzyme - Google Patents

Abstract

PURPOSE:To immobilize a microorganism, plant cell and/or enzyme in high immobilization ratio of microorganism, enzyme group obtained by addition, secretion, and self digestion of plant cell, or enzyme, by treating a metal alginate gel containing a microorganism, etc. with a polyamine quaternary ammonium compound. CONSTITUTION:A granular metal alginate gel containing a microorganism, plant cell or enzyme is added to 0.1-20wt% aqueous solution of a polyamine quaternary ammonium compound such as polyethyleneimine quaternary ammonium compound, etc., and blended at 0-50 deg.C for 1-60min while stirring. A granular metal aliginate gel is recovered and washed thoroughly.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel immobilized microorganisms, plant cells and/or enzymes in which microorganisms, plant cells or acid systems are immobilized.

Specifically, an alginate metal salt gel containing microorganisms, plant cells, or enzymes is treated with a polyamine quaternary ammonium compound to significantly increase the immobilization rate of enzymes or enzymes produced by the inclusion, secretion, and autolysis of microorganisms and plant cells. It concerns immobilized microorganisms, plant cells and/or enzymes.

Alginate metal salt gels are natural polysaccharides that have been used conventionally as gels for immobilizing microorganisms, plant cells, or enzymes.

Generally, a method is employed in which a sodium alginate aqueous solution containing microorganisms, plant cells, or enzymes is dropped as droplets into an aqueous solution containing a metal salt, such as a calcium salt, to obtain a granular alginate metal salt gel body.

However, only alginate metal salt gel forms, microorganisms,
When used as a gel body for immobilizing plant cells or enzymes, the immobilization rate of enzymes or enzymes generated by inclusion, secretion, and autolysis of microorganisms and plant cells is low, and there is a problem that the loss of 1U of enzymes occurs. Ta. Furthermore, when a metal alginate gel alone is used as a gel for immobilizing microorganisms, plant cells, or enzymes, there is a problem that it dissolves in a substrate or medium containing phosphoric acid.

In order to solve the above-mentioned problems, the present inventors conducted intensive research and arrived at the present invention.

The present invention is an immobilized microorganism, a plant cell, and/or an enzyme, which is characterized in that an alginate metal salt gel body containing a microorganism, a 4f1 cell, or an enzyme is treated with a polyamine quaternary ammonium compound.

According to the present invention, by immersing an alginate metal salt gel body containing microorganisms, plant cells and/or enzymes in an aqueous solution of a polyamine quaternary ammonium compound, microorganisms and plant cells are produced according to a conventional method. containing,
Immobilized microorganisms and plants that have been known to improve the immobilization rate of enzymes or enzymes produced through secretion and autolysis in plant cells while maintaining the activity of the enzymes or enzymes produced through secretion and autolysis. cells and/or enzymes can be obtained. Furthermore, the immobilized microorganisms, plant cells and/or enzymes of the present invention have the advantage that they do not dissolve in substrates or media containing phosphoric acid.

The present invention is thought to be because the polyamine quaternary ammonium compound causes a crosslinking reaction with the alginate metal salt gel body to form a strong bond, and the present invention can be achieved without the need for a new crosslinking agent. It is.

The polyamine quaternary ammonium compounds used in the present invention include polyalkylene imine quaternary ammonium compounds, polyno I midopolyamine quaternary ammonium compounds, polyoxyalkylene polyamine quaternary ammonium compounds, shrimp herrohydrin-alkylene Examples include polyamine quaternary ammonium compounds, polyvinylamine quaternary ammonium compounds, and polyethyleneimine quaternary ammonium compounds are particularly preferred.

The polyethyleneimine quaternary ammonium compound used in the present invention is produced, for example, by the following method.

[1] Method of reacting polyethyleneimines with a halogenated hydrocarbon [2] Method of reacting polyethyleneimines with protonic acid and then reacting with alkylene oxide Method 0] After reacting polyethyleneimines with alkylene oxide , A method for reacting halogenated hydrocarbons The reaction between polyethyleneimines and halogenated hydrocarbons is carried out at a temperature of room temperature to 200°C, preferably 40 to 150°C,
Normal pressure to 20 to 9/aKG, preferably normal pressure to 10Kt/
It can be carried out within the pressure range of ly#G.

The reaction between polyethyleneimines and protonic acid occurs at room temperature ~
This can be carried out at 100°C and normal pressure.

The reaction between polyethyleneimines and alkylene oxide is carried out at a temperature of 20 to 200 °C, preferably 30 to 100 °C, and at normal pressure to 20 g/c/g, preferably at normal pressure to
It can be carried out in a pressure range of 10 to 9/ajG.

As the polyethyleneimine, polyethyleneimine, polypropyleneimine, polybutyleneimine, or modified polyethyleneimine is used. The polyethyleneimine used here has a molecular weight of 100 to i, ooo, ooo, preferably 300 to 200,000.

In addition, polyethyleneimine modified products can be modified by converting polyethyleneimine into aldehydes, halogenated hydrocarbons, isocyanates, etc.
, urea, acids, and other compounds partially modified.

As alkylene oxide, ethylene oxide, /1
One or more types of pyrene A oxide, isostyrene oxide, 1゜2-styrene oxide, 2,3-butylene oxide, hexylene oxide, styrene oxide, etc. may be used, and the arrangement thereof may be graft type, block type, and/or Either random type is fine. Particularly preferred is a combination of ethylene oxide and propylene oxide. The molecular weight of the polyether obtained ranges from 600 to 5.000.000.

Examples of protonic acids include hydrochloric acid, perchloric acid, and sulfuric acid! 11!
Inorganic acids such as W, i acid, phosphoric acid, phosphorous acid, hypophosphorous acid, nitric acid, boric acid, hydroiodic acid, formic acid, acetic acid, propionic acid, oxalic acid, lactic acid, malonic acid, citric acid, phthalic acid Acid, maleic acid, fumaric acid, succinic acid, p-toluenesulfonic acid, adipic acid, sorbic acid, trimellitic acid,
Examples include pyromellitic acid, acrylic acid, methacrylic acid, glutaric acid, basic acid, and terephthalic acid.

Examples of halogenated mercuric hydrocarbons include methyl chloride, ethyl chloride, methyl bromide, ethyl bromide, methyl iodide, conityl iodide, ethylene chlorohydrin, epichlorohydrin,
Examples include ethylene bromohydrin, monochloroacetic acid, benzyl chloride, 1,2-dichloroethane, 1,3-dichloropropane, and 1,2-dichlorobutane.

There are no particular limitations on the microorganisms and plant cells that can be immobilized with the norginate metal salt gel used in the present invention, as long as they are capable of enzymatic action.

There are no particular restrictions on the enzyme, but ascorbine 1iUJ
Enzymes include oxidoreductases such as oxidase and glucose oxidase, transferases such as hexokinase and cy:L cross small sphosphorylase, and hydrolytic enzymes such as isoamylase, α-glucosidase and glucoamylase.

For alginate metal salt gel bodies containing microorganisms, plant cells, or enzymes, microorganisms,
A granular alginate metal salt gel body containing microorganisms, plant cells or enzymes can be obtained by adding plant cells or enzymes to make the mixture homogeneous, and then adding this to calcium chloride or aluminum chloride or the like.

In the present invention, the method for treating an alginate metal salt gel containing microorganisms, plant cells, or enzymes with a polyamine quaternary ammonium compound involves first preparing a 0.1 to 20% by weight aqueous solution of a polyamine quaternary ammonium compound; The granular alginate metal salt gel containing microorganisms, plant cells, or enzymes is added to the solution, stirred and mixed at a temperature of 0 to 50°C for 1 minute or more, preferably 1 minute to 60 minutes, and filtered to obtain the granular alginate metal salt gel. The gel is collected and then thoroughly washed with water to easily obtain a gel containing immobilized microorganisms, plant cells, or enzymes.

As is clear from the Examples and Comparative Examples described later, the gel body on which microorganisms, plant cells, or enzymes of the present invention are immobilized contains microorganisms, plant cells, and immobilization of enzyme groups or enzymes produced by autolysis. The conversion rate is extremely high.

Hereinafter, the present invention will be explained in detail with reference to Examples and Comparative Examples.

Example 1 [1] Preparation of polyethyleneimine quaternary ammonium compound In a stainless steel 1-t-crepe equipped with a stirrer, Evomin P-1000 (manufactured by Nippon Shokubai Chemical Co., Ltd., 30ml 1% aqueous solution of polyethyleneimine, molecule 1117) 10,000) 1
00 m m M wo (i included, warm Ji90~'lO
O℃, pressure 1-5 K'J/aK C+ -c l
1 part of (Chill 70!i!) was supplied and a 5 genital reaction was carried out to obtain a polyethyleneimine quaternary ammonium compound.

[2] Preparation of calcium nofulgate gel containing glucoamylase 1 part by weight of glucoamylase was added to 100 parts by weight of a 2% by weight aqueous sodium alginate solution to form a homogeneous solution. This solution was dropped into a 100 mmol calcium chloride aqueous solution from above using an extruder to form droplets to obtain a granular gel body. Stirring was continued in the calcium chloride aqueous solution for an additional 2 hours to strengthen the lζ granular gel.

Then, it was filtered to collect 3-4n1m granular gel bodies of calcium alginate containing glucoamylamine and thoroughly washed with water.

0] Treatment of glucoamylase-containing calcium alginate gluform In the aqueous solution of polyethyleneimine quaternary ammonium compound 1% aqueous solution obtained in [1] above, gluni]7-mylase obtained in [2] above was added. of! Calcium alginate glue was added, and after gently stirring for about 10 minutes, the granular gel was collected by filtration, and thoroughly washed with water to obtain 3-4 mm granular calcium alginate glue with immobilized glucoamylase.1C0 (b) Measurement of immobilization rate of glucoamylase 50mj1
Approximately 500 granular calcium alginate glu bodies immobilized with glucoamylase obtained in the above mortar were charged into a cylindrical reactor containing 0.2 molar acetate buffer (pH 5,0),
The mixture was stirred and mixed at a temperature of 30°C. After stirring for 2 hours, 4 hours, 6 hours, 8 hours, 24 hours, and 48 hours, each sample was sampled, and the glucoamylase activity in each solution was measured, and the differences in each were determined from the glucoamylase activity in the granular gel at the initial stage. The ratio was taken as the immobilization rate.

In addition, the activation rate of glucoamylase is based on the samples collected at each time above, 0.2 molar vinegar ATSJ solution < D 1.
5. O) ;13 ci:tyo, 5%'? Jl
The Dn mixture was shaken at 40°C for 2 hours and the glucose concentration was measured. As shown in Table 1, no loss of glucoamylase was observed.

Comparative Example 1 The immobilization rate of gluco-1 amylase was measured without treating the glucoamylamine-containing calcium alginate gel body obtained in [2] above with a polyethyleneimine quaternary ammonium compound.

As shown in Table 1, all of the glucoamylase was distilled into the liquid after 8 hours.

Table 1 Example 2 <Solubility test in phosphoric acid> 0.025 mmol dipotassium phosphate aqueous solution 25 mN and 0.025 mmol sodium phosphate 25o+, l!
Into the mixed aqueous solution, 500 granular calcium alginate gel bodies on which glucoamylase was immobilized and treated with the polyethyleneimine quaternary ammonium compound obtained in 0) above were placed and immersed at a temperature of 30°C. After 24 hours, observation of the immobilized granular calcium alginate glubodies of glucoamylase revealed that they had not dissolved at all.

Comparative Example 2 Solubility test for phosphoric acid> 0.025 mmol dipotassium phosphate aqueous solution 251 and 0
．． 500 pieces of the glucoamylase-containing calcium alginate gel body obtained in [2] above were put into a mixed aqueous solution with 0.25 mmol of sodium phosphate, and the mixture was heated at a temperature of 3.
Immersed at 0°C. After 24 hours, the glucoamylase-containing lucium alginate gel was observed and found to have completely dissolved.

Hourly wage applicant: Azama Kasei Co., Ltd. Nippon Shokubai Chemical Co., Ltd.

Claims (1)

[Scope of Claims] [1] An immobilized microorganism, plant cell, and/or enzyme, which is obtained by treating an alginate metal salt gel containing the microorganism, plant cell, or enzyme with a polyamine quaternary ammonium compound.