JPH0353911B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel immobilized microorganism, plant cell, or enzyme. 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. Concerning enhanced immobilized microorganisms, plant cells and/or enzymes. Alginate metal salt gels are natural polysaccharides that have been used as gels for immobilizing microorganisms, plant cells, or enzymes. Generally, an aqueous sodium alginate solution containing microorganisms, plant cells or enzymes is dropped as droplets into an aqueous solution containing a metal salt, such as a calcium salt.
A method of obtaining a granular alginate metal salt gel body has been adopted. However, only the alginate metal salt gel body,
When used as a gel for immobilizing microorganisms, 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 causes loss of these enzymes. It was hot. 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 containing the microorganism, plant cell, or 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, produced according to a conventional method,
Immobilization that significantly increases the immobilization rate of enzymes or enzymes generated through the inclusion, secretion, and autolysis of plant cells while maintaining the activity of the enzymes or enzymes generated through the inclusion, secretion, and autolysis of microorganisms and plant cells. microorganisms, plant cells and/or enzymes can be obtained. Furthermore, the immobilized microorganisms, plant cells and/or enzymes of the present invention have the advantage of not being dissolved 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 polyalkyleneimine quaternary ammonium compounds, polyamide polyamine quaternary ammonium compounds, polyoxyalkylene polyamine quaternary ammonium compounds, and epihalohydrin-alkylene polyamine quaternary ammonium compounds. compounds, polyvinylamine quaternary ammonium compounds, etc., 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 [3] Reacting polyethyleneimines with alkylene oxide Then, the reaction between polyethyleneimine and halogenated hydrocarbon is carried out at a temperature of room temperature to 200°C, preferably 40 to 150°C, and normal pressure to 20Kg/cm ² G, preferably normal pressure. ~Ten
It can be carried out within a pressure range of Kg/cm ² G. The reaction between polyethyleneimines and protonic acids can be carried out at room temperature to 100°C and under normal pressure. The reaction between polyethyleneimine and alkylene oxide is carried out at a temperature of 20 to 200°C, preferably 30 to 100°C, normal pressure to 20Kg/cm ² G, preferably normal pressure to 10Kg/cm 2 G.
It can be carried out in the pressure range of cm ² G. Polyethyleneimines polyethyleneimine, polypropyleneimine, polybutyleneimine or modified polyethyleneimines are used. The polyethyleneimine used here has a molecular weight of 100~
1,000,000, preferably 300-200,000. Also,
Modified polyethyleneimine includes polyethyleneimine partially modified with a compound such as an aldehyde, a halogenated hydrocarbon, an isocyanate, urea, or an acid. The alkylene oxide may be one or more of ethylene oxide, propylene oxide, isobutylene oxide, 1,2-butylene oxide, 2,3-butylene oxide, hexene oxide, styrene oxide, etc., and the arrangement thereof may be a graft type or a block type. and/or random type. Particularly preferred is a copolymer of ethylene oxide and proprene oxide. The molecular weight of the polyether obtained ranges from 600 to 5,000,000. Protonic acids include hydrochloric acid, perchloric acid, sulfuric acid,
Sulfite, phosphoric acid, phosphorous acid, hypophosphorous acid, nitric acid,
Inorganic acids such as boric acid and hydroiodic acid, formic acid, acetic acid,
Propionic acid, oxalic acid, lactic acid, malonic acid, citric acid, phthalic acid, maleic acid, fumaric acid, succinic acid, p-toluenesulfonic acid, adipic acid, sorbic acid, trimellitic acid, pyromellitic acid, acrylic acid, methacrylic acid , glutaric acid, sebacic acid, terephthalic acid, and other organic acids. Examples of halogenated hydrocarbons include methyl chloride, ethyl chloride, methyl bromide, ethyl bromide, methyl iodide, ethyl iodide, ethylene chlorohydrin, epichlorohydrin, ethylene bromohydrin, monochloroacetic acid, benzyl chloride, 1,2 -dichloroethane, 1,3-dichloropropane, 1,2-dichlorobutane and the like. There are no particular limitations on the microorganisms and plant cells that can be immobilized with the alginate metal salt gel used in the present invention, as long as they are capable of enzymatic action. Enzymes include, although there are no particular limitations, oxidoreductases such as ascorbate oxidase and glucose oxidase, transferases such as hexokinase and cyclosphosphorylase, and hydrolases such as isoamylase, α-glucosidase and glucoamylase. It will be done. A metal alginate gel body containing microorganisms, plant cells, or enzymes can be prepared by adding microorganisms, plant cells, or enzymes to a sodium alginate aqueous solution, making it homogeneous, and adding this to calcium chloride or aluminum chloride, etc. to form a gel containing microorganisms, plant cells, or enzymes. A granular alginate metal salt gel body can be obtained. 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 starts with 0.1 to 20% by weight of the polyamine quaternary ammonium compound.
Prepare an aqueous solution, add granular alginate metal salt gel containing microorganisms, plant cells, or enzymes to it, and keep at a temperature of 0 to 50°C for at least 1 minute, preferably for 1 minute or more.
This is carried out by stirring and mixing for 60 minutes, filtering to recover the granular alginate metal salt gel, and then thoroughly washing with water, and it is possible to easily obtain a gel with immobilized microorganisms, plant cells, or enzymes. As is clear from the Examples and Comparative Examples described below, the gel body of the present invention in which microorganisms, plant cells, or enzymes are immobilized is a group of enzymes produced by the inclusion, secretion, and autolysis of microorganisms or plant cells, or the immobilization of enzymes. The 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 Epomin P-1000 (Nippon Shokubai Kagaku Kogyo Co., Ltd.) was placed in a stainless steel autoclave equipped with a stirrer.
100 parts by weight of a 30% aqueous solution of polyethyleneimine (molecular weight 70,000) was added, and the temperature was 90 to 100°C.
70 parts by weight of methyl chloride was supplied at a pressure of 1 to 5 kg/cm ² G, and the reaction was carried out for 5 hours to obtain a polyethyleneimine quaternary ammonium compound. [2] Preparation of glucoamylase-containing calcium alginate gel body 2% by weight sodium alginate aqueous solution 100%
1 part by weight of glucoamylase was added to the part by weight to make a homogeneous solution. This solution was dropped into a 100 milmol calcium chloride aqueous solution from above using an extruder so as 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 granular gel formed in the solution. Then, 3 to 4 mm granular gel bodies of calcium alginate containing glucoamylase were collected by filtration and thoroughly washed with water. [3] Treatment of glucoamylase-containing calcium alginate gel body Into the 1% by weight aqueous solution of the polyethyleneimine quaternary ammonium compound obtained in [1] above,
The glucoamylase-containing calcium alginate gel obtained in [2] above was added, and after gently stirring for about 10 minutes, the granular gel was collected by filtration, and the gel was thoroughly washed with water. A 4 mm granular calcium alginate gel body was obtained. [4] Measurement of the immobilization rate of glucoamylase Place the granular calcium alginate gel with immobilized glucoamylase obtained in [3] above in a cylindrical reactor containing 50 ml of 0.2 molar acetate buffer (PH5.0). Approximately 500 pieces were added and 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, samples were taken,
The glucoamylase activity in each solution was measured, and the difference was taken from the glucoamylase activity in the granular gel body at the initial stage, and the ratio was taken as the immobilization rate. The activation rate of glucoamylase was determined by shaking the samples collected at each time above, a mixture of 0.2M acetate buffer (PH5.0) and 0.5% maltose at 40°C for 2 hours. Glucose concentration was measured and determined. As shown in Table 1, no loss of glucoamylase was observed. Comparative Example 1 The glucoamylase-containing calcium alginate gel body obtained in [2] above was not treated with a polyethyleneimine quaternary ammonium compound, and the immobilization rate of glucoamylase in [4] above was measured. As shown in Table 1, all of the glucoamylase was distilled into the liquid after 8 hours.

[Table] Example 2 <Solubility test in phosphoric acid> 25 ml of 0.025 mmol dipotassium phosphate aqueous solution
500 granular calcium alginate gel bodies with immobilized glucoamylase treated with the polyethyleneimide quaternary ammonium compound obtained in [3] above were added into a mixed aqueous solution of 25 ml of 0.025 mmol sodium phosphate, and the mixture was heated at a temperature of 30 ml. Soaked at ℃.
After 24 hours, the granular calcium alginate gel body on which glucoamylase was immobilized was observed, and it was found that it had not been dissolved at all. Comparative Example 2 <Solubility test in phosphoric acid> 25 ml of 0.025 mmol dipotassium phosphate aqueous solution
500 pieces of the glucoamylase-containing calcium alginate gel body obtained in [2] above were placed in a mixed aqueous solution with 25 ml of 0.025 mmol sodium phosphate and immersed at a temperature of 30°C. After 24 hours, the glucoamylase-containing calcium alginate gel was observed and found to have completely dissolved.

Claims (1)

[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.