JPH0319687A - Production of immobilized enzyme - Google Patents

Abstract

PURPOSE:To obtain an immobilized enzyme having high activity and strength by impregnating an enzyme solution and a chitosan solution into a porous solid and reacting with a dialdehyde to crosslink the components. CONSTITUTION:A porous solid (A) having particle diameter of 0.2-3mm and a pore volume of >=0.2ml/g is prepared from celite, diatomaceous earth, etc. Chitin is deacetylated to obtain a chitosan solution (B) which is dissociated into cation and made to be water-soluble in the presence of HCl, etc. A solution (C) of an enzyme such as amylase is mixed with the solution B to obtain an impregnation liquid (D) having a prescribed pH and a viscosity of <=100cp. The solid A is evacuated to remove air from the voids, immersed in the liquid D and then in a dialdehyde solution (E) such as glutaraldehyde. The objective immobilized enzyme is produced by carrying out crosslinking reaction of the above product under a pH and temperature condition not to inactivate the enzyme.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing an immobilized enzyme that has high enzymatic activity and strong strength as a carrier, and can be advantageously used as a high-reactor.

When carrying out the Araikususei enzyme reaction on an industrial scale, conventionally it was carried out in a batch manner, and the enzyme was used only once, making it uneconomical. Many proposals have been made.

In particular, in the food industry, from the standpoint of safety, immobilization methods using chitosan, which is obtained by deacetylating the natural product chitin, are being investigated.

For example, JP-A No. 59-213390 discloses a method of adsorbing and immobilizing an enzyme on a carrier from a porous solid and a chitosan derivative, and JP-A No. 53-10150 discloses a method of adsorbing and immobilizing an enzyme on a carrier using a porous solid and a chitosan derivative. , a method is described in which an enzyme is added thereto by adsorption or peptide bonding. Furthermore, Japanese Patent Publication No. 16637/1983 discloses a method in which inorganic particles are coated with chitosan, which is activated or stabilized with glucuraldehyde, and then enzymes are immobilized.

However, since the method described in Japanese Patent Publication No. 53-10150 uses chitosan gel as a carrier, the mechanical strength of the obtained immobilized enzyme is weak, and the problem is that pressure loss increases or breakage easily occurs due to deformation. There is. Furthermore, the method disclosed in Japanese Patent Publication No. 62-16637 uses chitosan gel that has been crosslinked in advance, and since it is difficult for the enzyme to penetrate into the gel, there is a problem that the activity of the immobilized enzyme is low. be.

Incidentally, porous solids that have high mechanical strength and are water insoluble cannot immobilize enzymes because they do not have reactive groups that bind to enzymes.

The present invention has been made to solve the problems seen in the above-mentioned prior art, and has high mechanical strength, high activity, and is suitable for use in the food industry. The objective is to provide an immobilized enzyme that can be safely used in the future.

Means for Solving the Problems The present invention uses a porous solid as a carrier, impregnates it with an enzyme solution and a chitosan solution, and then crosslinks the impregnated porous solid by reacting with dialdehyde. It is characterized by

The porous solid used in the present invention is not particularly limited as long as it is a water-insoluble and inert substance, and examples thereof include coral, celite, diatomaceous earth, barley 1, shirasuharun, charcoal, and cork. These porous solids usually have a particle size of 0
．． It is used by filling a column as a granular material of about 2 to 3 mm, but it can be used in any shape such as a tube or a plate as long as it can have a similar specific surface area. Note that the porous solid referred to here refers to a porous solid with a diameter of at least 0.2 m.
R/B means a solid material having a pore volume of 2, preferably 0.5 ra/g or more.

Furthermore, the term "ki-sans" used in the present invention is a general term for deacetylated products of chitin, and refers to those that become water-soluble by dissociating into cations in the presence of monobasic acids such as acetic acid and hydrochloric acid. Incidentally, even if the above-mentioned Ki-1-zans are re-acetylated or have a lower molecular weight, as long as they exhibit the above-mentioned water solubility, they can be used in the present invention.
Included in Sans.

In the present invention, the above-mentioned porous solid is impregnated with the above-mentioned solution of the above compounds and an enzyme solution. At that time, the chitosan solution and the enzyme solution may be impregnated into the porous solid sequentially.
Alternatively, both solutions may be mixed in advance and impregnated. Impregnation is efficient if the air in the solid voids is removed by reduced pressure and then immersed in a liquid.

It is desirable that the impregnating liquid has a low viscosity, preferably 100 cp or less. The pH of the impregnating solution is limited to a range in which the enzyme is not deactivated and chitosan is not precipitated.

Since the enzyme dissociates into anions at a pH above its isoelectric point, it may form an ionic complex with the cations of chitosan and become cloudy, but the pores are usually smaller than the pore diameter of the porous solid, so this may hinder entrainment. Not only does it not occur, but a moderate amount of precipitation due to the cloudiness has a favorable influence on the crosslinking reaction of the porous solid impregnated with dialdehyde,
Good results for enzyme immobilization.

Even when suspended solids such as bacterial cells are present in the enzyme solution, this does not interfere with the implementation of the present invention as long as the suspended solids are smaller than the pore diameter of the porous solid.

The above crosslinking reaction is preferably carried out by immersing a porous solid impregnated with an enzyme solution and a xane solution in a dialdehyde solution.
It is necessary to carry out the reaction at a temperature of

Examples of the dialdehyde used here include glutaraldehyde, glyoxal, malonaldehyde, succinaldehyde, and azibaldehyde, with glutaraldehyde being particularly commonly used.

Enzymes that can be immobilized according to the present invention are not particularly limited, and almost all enzymes can be used.

For example, amylase, glucoagolase, trypsin,
Chymotrivcin, pepsin, papain, vancreatin, aminoacylase, nuclease, ribonuclease, ATP deanase, phosphatase, streptokinase, apyrase (ATP-dikisphatase), A
TP creatine phosphotransferase, pectinase, maltase, lactose, urease, tannase, lipase, glucose isomerase, melibiase, aldolase
enzyme, cellulase, an1-sinase, naringinase,
Glucose oxidase, asparaxidase, etc. can be mentioned, and any of these can be easily fixed according to the method of the present invention.

As stated in "Operations and Effects of the Invention", in the present invention, enzymes and stozans are impregnated in an aqueous solution state into a porous solid with good permeability, and then this impregnated porous solid is reacted with dialdehyde to crosslink it. Therefore, the enzyme is efficiently immobilized and insolubilized within the network structure of chitozan molecules, and is supported on a porous solid with increased mechanical strength.

Therefore, according to the present invention, an immobilized enzyme with high activity and high strength can be obtained.

Incidentally, if a porous solid was impregnated with only an enzyme solution, the enzyme would not be immobilized and would easily flow out, whereas if chitosan was crosslinked without using a porous solid, the chitosan gel would be mechanically It has low strength and is prone to increased pressure loss or damage due to deformation. Furthermore, when a porous solid is impregnated with only a ki1-san solution and a cross-linked substance with dialdehyde is impregnated with an enzyme solution, it is difficult for the enzyme to penetrate into the network structure of the ki1-san molecules. Therefore, an immobilized enzyme with high activity cannot be obtained.

EXAMPLES The present invention will be explained in more detail by examples below, but the scope of the present invention is not limited to the following examples unless the gist of the invention is changed.

Example 1 Low molecular weight chitosan [1% solution viscosity (in 1% acetic acid) = 8.
Ocp, degree of deacetylation 83%) 5.0g to 10%
After dissolving in 45 ml of acetic acid, the pH was adjusted with 2N-NaOH to make the total amount 100 mffi, and a 5% low molecular weight chitosan solution (pll=6.1) was prepared.

This 5% chitosan medicine & 10 mR and gnoreco-
Swiss isomerase solution (manufactured by Nagase Sangyo Co., Ltd., 1,400 G
IU/m0I Q mR were mixed to obtain an emulsified mixture.

Celite (Manville R-647, particle size:
14-30Mesh, pore volume 0.97mJ! /g)5.
After placing 0g under reduced pressure and impregnating it with the enzyme-chitosan mixture,
Filter, 1). 3 g of impregnated Serai 1. was obtained.

2.5% glutaraldehyde 4.
After mixing 2 ml and reacting at 4'C for 1 hour, 0.
After thorough washing with 05-phosphate buffer (pl+=6.5), 9.3 g of immobilized enzyme was obtained. Its activity is 162GI
The activity after washing four times with 50 ml of 10% NaC] solution was 162 GIU/g, indicating that the enzyme was covalently bound to xane and did not release. In this way, an immobilized enzyme with high activity and high strength was obtained.

The activity of the immobilized enzyme was measured using pl1 7.2 0.
．． An appropriate amount of the immobilized enzyme was added to 100 g of a 40% solution of glucose dissolved in 1 M'J acid buffer and reacted at 60°C for 1 hour. Determined by LC method, per 1 g of immobilized enzyme, ■
The amount of fructose converted per hour was determined by calculation.

Example 2 According to the same method as described in Example 1, an inverdase solution (enzyme activity 15.9 g-SUC/mj!) was solidified by stirring to obtain 9.6 g of immobilized inverdase. . Its enzyme activity is 2.93gSUC/g,
2. Even after washing with 1o% NaCl. Almost no enzyme detachment was observed at 85 g SUC/g.

In addition, cleaning after glutaraldehyde treatment is performed at pl14.2.
To measure the activity after immobilization, add an appropriate amount of the immobilized enzyme to pl+4.2
The immobilized enzyme was added to 1 liter of a 10% solution of sasocallose dissolved in 0.05 M phosphate/citrate buffer, reacted for 1 hour at 40'C, and the amount of reducing sugars recovered was determined by the methylene flue method. The amount of decomposed loin roast per 1 g and per hour was calculated.

Example 3 After dissolving 5.0 g of low molecular weight chitosan similar to that used in Example 1 in 45 m2 of 10% acetic acid, 3,0 g of acetic anhydride was added, and the mixture was reacted at 35"C for 18 hours, resulting in a degree of deacetylation of 43.
% chitosan was obtained. This was neutralized with NaOII and diluted to give a 5% solution with a pl+6.5.

5.0 g of Shirasu balloons (particle size: 14-30 Mesh, pore volume 0.98 mffi/g) were placed under reduced pressure, impregnated with 5 mf of glucose isomerase solution, and then
5 ml of % acetylated Chyl-san solution was added thereto, and after standing for 1 hour, the remaining liquid was filtered off.

This impregnated Shirasu balloon was mixed with 3% glutaraldehyde 5
Qmffi, reacted for 1 hour at room temperature with stirring, filtered, and added to 0.05M phosphate buffer (pH 6.5).
After thorough washing, 9.75 g of immobilized enzyme was obtained.

Its activity is 141 GTU/g and 10% NaC]?
The activity after washing with -3 liquid was 127 GIU/g.

Comparative Example 1 A 5% low molecular weight chitosan solution l Q mR was added to 5.0 g of Celite under reduced pressure to impregnate it, and then filtered. 2.5 for this
% glutaraldehyde and reacted for 1 hour at 4°C, glucose isomerase solution IQmE
was added and allowed to react overnight. Although 9.5 g of immobilized enzyme was obtained by thorough washing with 0.05 M phosphate buffer, its activity was as low as 21.3 GTU/g.

The Serai 1., low molecular weight K1-'J solution, and glucose isomerase solution used were the same as in Example 1.

Claims (2)

[Claims] (1) A method for producing an immobilized enzyme, which comprises impregnating a porous solid with an enzyme solution and a chitosan solution, and then reacting the impregnated porous solid with dialdehyde to crosslink it. (2) Claim (1) wherein the chitosan is selected from the group consisting of deacetylated chitin, low molecular weight products thereof, reacetylated products thereof, and mixtures thereof.
A method for producing an immobilized enzyme as described in .