JPH0517835B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing an immobilized enzyme, and more particularly to a method for producing an immobilized enzyme that provides a chitin molded article in the form of particles, fibers, or films having enzyme activity. be.

In recent years, in the enzyme utilization industry, research has progressed to make the enzyme reaction process continuous and repeatedly use enzymes by immobilizing water-soluble enzymes on water-insoluble polymer carriers, etc., and some have already been put into practical use. There is also. Methods for producing this insoluble enzyme include a carrier binding method in which the enzyme is bound to a water-insoluble carrier, a crosslinking method in which the enzyme is made insolubilized by reacting with a functional reagent, and a method in which the enzyme is wrapped in a gel lattice or coated with a polymer film. Comprehensive methods are known.

When immobilizing enzymes on an industrial scale, the immobilization procedure must be simple, the immobilized enzyme must remain stable for a long time, and the carrier used must be porous, robust, and inexpensive. be.

Chitin is an abundant unused natural resource, and its use as a carrier for enzyme immobilization has been proposed. For example, Japanese Patent Publication No. 56-35 describes a method for immobilizing enzymes through the ion exchange group by introducing diethylaminoethyl groups into chitin.
No. 10150 describes a method of immobilizing enzymes by adsorption or peptide bonding to chitosan obtained from chitin, and JP-A-52-3892 describes a method of crosslinking to deacetylated chitin and a method of immobilizing enzymes by diazo method. Immobilization methods have been proposed. however,
The immobilized enzyme obtained by these methods is in the form of powder or flakes, so when packing the immobilized enzyme into a column and making the enzyme reaction process continuous, the fluid resistance is large, making it difficult to obtain a sufficient flow rate. Moreover, it also had major practical drawbacks such as column clogging. In other words, when trying to put immobilized enzymes using chitin as a carrier into practical use, there was a major problem with the morphology of chitin, and it is therefore desirable to develop immobilized enzymes that do not restrict operational conditions. Ta.

The present inventors have made extensive efforts to provide an immobilized enzyme that has improved the above-mentioned drawbacks, and as a result,
The present invention was achieved by successfully developing a method in which chitin is pre-molded into a preferred form as an immobilized enzyme, and then an enzyme is immobilized on this molded body.

That is, the present invention provides an immobilized enzyme, which is characterized in that pre-shaped chitin is treated with an alkali to obtain a swollen chitin molded body, the obtained swollen chitin molded body is immersed in an enzyme solution, and then treated with glutaraldehyde. This is the manufacturing method.

Chitin as used in the present invention refers to poly(N-acetyl-D-glucosamine) itself and its derivatives. Such chitin can be obtained by, for example, treating the exoskeletons of shellfish and insects with hydrochloric acid and caustic soda to separate and purify protein and calcium components, or by etherifying them,
It can be prepared by esterification or the like. Examples of chitin derivatives preferably used in the present invention include etherified chitins such as carboxymethylated chitin and hydroxyethylated chitin, and esterified chitins such as acetylated chitin and sulfonated chitin. Examples of esterified products include carboxylic acids such as formic acid, acetic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, benzoic acid, cinnamic acid, salicylic acid, anthranilic acid, and phthalic acid, sulfuric acid, toluenesulfonic acid, and sulfophanilic acid. Examples include sulfonic acids, carbonates, and esters of their anhydrides.

Molding in the present invention refers to molding chitin or its derivatives in a suitable solvent, such as methylene chloride containing trichloroacetic acid, N-methylpyrrolidone containing lithium chloride, dimethylacetamide containing lithium chloride, trifluoroacetone, or hexafluoroisopropyl alcohol. After melting, it is formed and solidified. To coagulate a chitin solution, it is sufficient to bring the chitin solution into contact with a coagulating liquid, and the coagulating liquid may be any liquid in which chitin or its derivatives are not dissolved, such as methanol, ethanol, isopropyl alcohol, isobutanol, and acetone. , water, etc. can be used. The shape of the molded body may be any shape suitable for use as an immobilized enzyme, such as granules, fibers, films, etc.
Examples include tube-like and spongy shapes.

The alkali treatment in the present invention includes any method in which chitin that has been shaped in advance is brought into contact with an alkaline solution. As the alkaline solution, a sodium hydroxide aqueous solution is practical, and its concentration is preferably 0.1 w/v% or more, more preferably 10 w/v% or more, and optimally in the range of 30 to 60 w/v%, and the preferable treatment is The temperature may be 10°C or higher, more preferably 40°C or higher, and most preferably in the range of 60 to 120°C. The treatment time varies depending on the alkali concentration and treatment temperature, but is preferably 1 minute to 24 hours, more preferably 15 minutes to 12 hours, and most preferably 1 minute to 24 hours.
It is sufficient if the time is about 6 hours. The bath ratio of the alkaline solution to 1 part by weight of the chitin molded article is preferably 25 parts by weight or more, more preferably 50 parts by weight or more, and most preferably 100 parts by weight or more. The alkaline solution may be stirred if necessary, and operations such as neutralization and water washing may be performed to remove the alkali after treatment.

The chitin molded body treated with alkali and washed with water is swollen by water, and the swollen chitin molded body obtained in this way may be immersed in an enzyme solution as it is, but before that, it should be immersed in a buffer solution. It is desirable to immerse the enzyme solution in advance for sufficient equilibration, and it is preferable that the type, concentration, pH, etc. of the buffer used be the same as that of the enzyme solution.

The enzyme solution used in the present invention is a liquid having enzyme activity, and the enzyme type, purity, concentration, origin, etc. may be of any kind, and the enzyme solution may be a liquid containing a plurality of enzymes.

In the present invention, for example, the following types of enzymes can be used.

Oxidoreductase: ascorbate oxidase,
Alanine dehydrogenase, amino acid oxidase, uricase, catalase, xanthine oxidase, glucose oxidase, glucose-
6-phosphate dehydrogenase, glutamate dehydrogenase, diaphorase, cytochrome C oxidase, tyrosinase, lactate dehydrogenase, peroxidase, 6-phosphogluconate dehydrogenase, leucine dehydrogenase,
NADH oxidase etc.

Transferase: aspartate acetyltransferase, amino acid transferase, acetate kinase, adenylate kinase, creatine phosphokinase, glucokinase, hexokinase,
phosphoacetyl kinase, pyruvate kinase,
fructokinase etc.

Hydrolytic enzymes: amylase, asparaginase, acetylcholinesterase, aminoacylase, arginase, invertase, urease, uricase, urokinase, esterase,
kallikrein, chymotrypsin, trypsin, thrombin, naringinase, nucleotidase,
papain, hyauronidase, plasmin, pectinase, hesperidinase, pepsin, penicillinase, penicillin amidase, phospholipase, phosphatase, lactase, lipase,
Ribonuclease, rennin, keratinase, dehalogenase, etc.

Lyase: aspartate decarboxylase, aspartase, citrate lyase, glutamate decarboxylase, threonine aldolase, histidine ammonia lyase, phenylalanine ammonia lyase, fumarase, fumarate hydrolase, malate synthetase, methioninase, etc.

Isomerase: Alanine racemase, glucose isomerase, glucose phosphate isomerase, glutamate racemase, lactate racemase, superoxide dismutase, etc.

Ligase: Amino acid activating enzyme, asparagine synthetase, glutathione synthetase, glutamine synthetase, pyruvate synthetase, etc.

A buffer is suitable as a solvent for dissolving the above enzyme, and its type, concentration, and pH are determined based on the stability of the enzyme used and the rate of enzyme immobilization on the chitin molded body, so they cannot be uniformly determined. Although it cannot be specified, the generally preferred concentration is 1mM to 0.5M, more preferably 5mM to 0.2M, and optimally 10mM.
~0.1M range. Preferred PH or PH2~
11, more preferably in the range of 4 to 9.

In carrying out the method of the present invention, the swollen chitin molded body is immersed in an enzyme solution, but the conditions are not particularly limited. However, from the viewpoint of enzyme stability and improvement of immobilization yield, the preferred temperature is 0.
-30°C, more preferably 0-20°C, optimally 0
~10℃, and the soaking time is preferably 10 minutes or more.
More preferably, the heating time is at least 1 hour, and most preferably from 2 to 12 hours.

After the swollen chitin molded body is immersed in the enzyme solution as described above, the glutaraldehyde treatment is performed; however, in carrying out the process, glutaraldehyde may be added to the enzyme solution in which the swollen chitin molded body is immersed, or The swollen chitin molded article taken out from the enzyme solution may be immersed in a buffer solution containing glutaraldehyde. In any case, the concentration of glutaraldehyde is preferably 0.01 to 20w/
v%, more preferably 0.02 to 10 w/v%, optimally 0.05 to 5 w/v%. Further, the temperature and time for the glutaraldehyde treatment cannot be absolutely specified, but for example, it may be about 10 hours when the treatment is performed at 4°C, and 2 to 5 hours when the treatment is performed at 20°C.

It is desirable that the immobilized enzyme obtained by the glutaraldehyde treatment be thoroughly washed with water or a buffer to remove excess glutaraldehyde, free enzyme, and the like.

The feature of the immobilized enzyme obtained by the method of the present invention described above is that it is shaped into a granular or fibrous form, so it has very good operability when actually used. According to the method described above, since granular chitin is particularly porous, an immobilized enzyme with high enzyme activity per carrier weight can be easily obtained. In addition, since chitin has excellent biocompatibility, the immobilized enzyme obtained by the method of the present invention can also be applied to applications that come into direct contact with the human body, such as cosmetics and pharmaceuticals.

The present invention will be explained in more detail with reference to Examples below.

Example 1 5 g of chitin powder was dissolved in 995 g of dimethylacetamide containing 8 w/v% LiCl to obtain a chitin solution.
This solution was dropped into methanol through a nozzle with a diameter of 0.2 mm to coagulate into particles, and then washed with water to obtain 350 ml of chitin beads with a diameter of about 0.8 mm. Add these chitin beads and water to 400g of sodium hydroxide.
The volume was adjusted to 1000 ml, kept at 85°C for 30 minutes, cooled, and washed with water repeatedly. 10ml of the resulting swollen chitin beads
was immersed in 25 mM phosphate buffer (PH7.2) for equilibration, and then immersed in 15 ml of an enzyme solution containing 130 units of glucokinase per ml of the same buffer. 4℃
After gently stirring for 5 hours, 0.1 ml of 25% glutaraldehyde solution was added, and the mixture was gently stirred at 4°C for 8 hours, and then washed with 50 mM Tris-HCl buffer (PH8.5).

The activity of the glucokinase-immobilized chitin beads thus obtained was determined by measuring the amount of glucose-6-phosphate generated from glucose and ATP.
As a result of measurement using 6-phosphate dehydrogenase, it was 70 units per ml of beads, and the immobilization rate was approximately
It was 36%.

Example 2 A chitin solution was obtained by dissolving 30 g of chitin powder in 970 g of dimethylacetamide containing 8 w/w% LiCl. This solution was transported under pressure using a gear pump and extruded into water through a 150-hole nozzle with a diameter of 0.07 mm to form fibers, which were then wound up in water. The obtained fibers were immersed for 2 hours in a 40w/V% NaOH aqueous solution heated to 80°C, thoroughly washed with water, and immersed in 20mM phosphate buffer (PH5.5). 5 g of the fibrous swollen chitin thus obtained was immersed in 20 ml of the same buffer containing 260 units of protease.
After being left at ℃ for 6 hours, it was immersed in 50 ml of the same buffer containing 0.2% glutaraldehyde for 2 hours at room temperature. The protease activity measured after thorough washing with a 0.5M NaCl aqueous solution was 1.8 units per 50 mg of chitin fibers, and the immobilization rate was 69%.

Reference Example 1 The following experiment was conducted to compare the amount of enzyme immobilized on chitin powder and swollen chitin molded body.

Chitin powder (100 mesh) 200mg and water-swollen chitin beads (15ml) with a dry weight of 200mg
was immersed in a 40w/v% NaOH aqueous solution at 80°C for 30 minutes, thoroughly washed with water, and equilibrated with 200mM phosphate buffer (PH6). Each chitin was immersed in 30 ml of the same buffer containing 250 units of glucose oxidase, and after 3 hours at 4°C, 0.2
ml of 25% glutaraldehyde solution was added and left in the refrigerator overnight. 50mM phosphate buffer (PH5.6)
After washing, the enzyme activity immobilized on each chitin was determined from the oxygen absorption rate at 30°C, and it was found to be 95 units for chitin powder and 170 units for chitin beads.

From the above results, it can be seen that the swollen chitin molded body is far superior to chitin powder as a carrier for enzyme immobilization.

Reference Example 2 The operability of molded chitin-immobilized enzyme was investigated when the immobilized enzyme was packed in a column and continuous reactions were performed.

The glucokinase-immobilized beads obtained in Example 1 were packed into a column with a diameter of 10 mm and a length of 5 cm.
M Tris-HCl buffer (PH8.5) was passed through. The liquid flow rate was gradually increased and no compression of the bead layer occurred even after 6 hours at SV=30.

The above results show that the bead-shaped chitin-immobilized enzyme can be used under a wide range of operating conditions.

Claims (1)

[Claims] 1. A method for producing an immobilized enzyme, which comprises treating preformed chitin with an alkali to obtain a swollen chitin molded body, immersing the obtained swollen chitin molded body in an enzyme solution, and then treating it with glutaraldehyde.