JPH02286085A - Immobilized enzyme - Google Patents

Abstract

PURPOSE:To obtain an immobilized enzyme, having high activity for polymer substrates, stable even in organic solvents and useful as an optically active catalyst, etc., by immobilizing an enzyme in a carrier through covalent bonds using polyethylene glycol as a spacer between the carrier and the enzyme. CONSTITUTION:The objective immobilized enzyme, obtained by dissolving polyethylene glycol in benzene, etc., adding cellular silica glass and gamma- isocyanatopropyltriethoxysilane to the resultant solution, sufficiently stirring the mixture solution, then vacuum drying the stirred solution at 105 deg.C for 2hr to provide a cellular silica glass-polyethylene glycol complex, subsequently mixing bromoacetic acid therewith, shaking the obtained mixture for 16hr, adding and reacting bromoacethyl bromide therewith and adding and reacting the resultant solution with an aqueous solution of an enzyme (e.g. glucoamylase) and having a spacer consisting of the polyethylene glycol between the carrier and the enzyme.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to an enzyme immobilized product by a carrier binding method, and in particular,
This invention relates to an enzyme immobilized product with a spacer interposed between the carrier and the enzyme.

[Prior Art] Unlike ordinary chemical catalysts, enzymes are characterized by their ability to efficiently catalytically promote chemical reactions under mild conditions such as room temperature and pressure, and their catalytic specificity is extremely high. It is used in a wide range of fields including the food industry, pharmaceutical industry, and chemical industry. However, enzymes are generally unstable to heat, strong acids, strong alkalis, organic solvents, etc., and their use is extremely limited. In addition, since enzymatic reactions are carried out using the so-called batch method (batch method) in which enzymes are dissolved in water and act on the substrate, it is technically extremely difficult to collect the enzymes from the reaction solution and reuse them after the reaction is complete. This method is difficult to perform, and the enzyme is discarded after each reaction, making it an uneconomical method of use.

Therefore, attempts have been made to take advantage of the inherent properties of enzymes and to artificially improve the properties of enzymes to make them more suitable for the purpose of use. As one means for achieving this, studies have begun to consider making enzymes insoluble in water while retaining their catalytic activity, that is, immobilizing enzymes on carriers.

The advantages of immobilizing enzymes include stabilization of the enzyme, the ability to use the same enzyme multiple times in reactions, the ability to increase the density of the enzyme, and the ability to perform enzyme reactions even in organic solvents.

Many enzyme immobilization methods have already been reported.
It can be broadly classified into the following three methods.

■Carrier binding method: A method of binding enzymes to water-insoluble carriers.

■Crosslinking method: A method in which a reagent with two or more functional groups acts on an enzyme to crosslink enzyme molecules to immobilize them.

■Inclusion method; A method in which enzymes are encapsulated in a polymer gel matrix lattice or in microcapsules made of a semipermeable polymer film.

However, the enzyme in the enzyme immobilized product by these methods is
None of them had high activity against polymer substrates, and
Although stability has increased in reactions in organic solvents, sufficient stability is still not achieved.

Furthermore, as methods to solve the above-mentioned problems, a method using a spacer between the enzyme and the carrier, and a method using a photocrosslinkable resin prepolymer or a urethane prepolymer have been developed.

Among these methods, the method of interposing a spacer between the enzyme and the carrier uses acrylic acid as the spacer, and this enzyme immobilization method is described in "Functional Materials" September issue (1985).
It is described on page 13 of . According to this, using bonded cellulose grafted with acrylic acid as a carrier and water-soluble carbodiimide [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride] as a condensing agent,
This method immobilizes the enzyme by bonding the enzyme to a spacer, and the method using a photocrosslinkable resin prepolymer or urethane prepolymer involves mixing a hydrophilic prepolymer and a hydrophobic prepolymer. This is a method of immobilizing enzymes on a gel with a hydrophilic-hydrophobic balance adjusted to an arbitrary ratio, Fermentation Engineering Vol. 61, No. 3
No. 153 (1983).

In the method using a photocrosslinkable resin prepolymer, ENT (trade name manufactured by Nikansai Paint) synthesized from polyethylene glycol, hydroxyethyl acrylate, and inphorone diisocyanate, and polypropylene glycol, hydroxyethyl acrylate, and inphorone diisocyanate are synthesized. Synthesized ENTP (Product name: Manufactured by Ni Kansai Paint)
etc., a photosensitizer (benzoin ethyl ether, benzoin isobutyl ether, etc.), add an enzyme aqueous solution,
The enzyme is immobilized by irradiation with near ultraviolet light of 300 to 400 Na+.

In addition, in the method using a urethane prepolymer, a urethane prepolymer in which toluene diisocyanate is bonded to both ends of the main chain consisting of polyethylene glycol or polyethylene glycol and polypropylene glycol is mixed in an arbitrary ratio, and an enzyme aqueous solution is added. The enzyme is immobilized by

[Problems to be solved by the invention] Enzyme immobilization products using acrylic acid as a spacer between the enzyme and the carrier, enzyme immobilization products using photocrosslinkable resin prepolymers or urethane prepolymers are different from conventional enzyme immobilization products. In comparison, when acrylic acid is interposed between the enzyme and the carrier, there is less steric hindrance by the carrier to the substrate that is catalyzed by the enzyme, and high activity can be obtained even for polymeric substrates. No studies have been conducted on the reaction in organic solvents, and the enzyme is unstable in organic solvents and its activity remains low.

In addition, when a photocrosslinkable resin prepolymer or urethane prepolymer is used, stability in organic solvents increases due to the gel's protective effect against organic solvents, permeation through the gel, and adjustment of diffusion, resulting in high activity. However, with polymeric substrates, the enzyme is present in the gel network, so the substrate cannot reach close to the enzyme, so the activity of the enzyme was not improved.

[Means for Solving the Problems] The present invention provides an enzyme that has high activity against a polymeric substrate that is catalyzed by the enzyme, and that the enzyme is stable and maintains high activity even in an organic solvent. As a result of extensive research into immobilized enzymes that can be used for polymeric substrates, we found that when a spacer is interposed between the carrier and the enzyme in the carrier binding method, by covalently interposing polyethylene glycol as a spacer, the enzyme can be attached to the polymeric substrate as a carrier. It has high activity without being sterically hindered by polyethylene glycol, and is used to retain water even in organic solvents.
It was discovered that the enzyme is stable and can maintain high activity.

That is, the present invention provides an enzyme immobilized product in which a spacer is interposed between a carrier and an enzyme, and the spacer is polyethylene glycol.

[Function] The enzyme immobilized product of the present invention has high activity against polymer substrates by covalently interposing polyethylene glycol as a spacer between the carrier and the enzyme, and also has high activity in organic solvents. It is stable and can maintain high activity.

The enzyme immobilized material is porous glass, porous ceramic,
Either polyethylene glycol is covalently bonded to a carrier such as cellulose using a coupling agent, and then an enzyme is covalently bonded to the tip of the polyethylene glycol chain using a coupling agent, or the enzyme is covalently bonded to the carrier. It can be obtained by covalently bonding the ends of polyethylene glycol chains using a coupling agent, or by covalently bonding a carrier and an enzyme to polyethylene glycol having an active group.

The carrier that can be used in the enzyme immobilization product of the present invention preferably has at least one functional group that participates in covalent bonding with polyethylene glycol, and for example, porous glass, porous ceramics, cellulose, etc. can be used.

Polyethylene glycol has a molecular weight of 200 to 20,000
Various products are commercially available, and any of these can be used.

The coupling agent for covalently bonding the carrier and polyethylene glycol must be selected depending on the type of carrier. For example, when porous glass is used as the carrier,
A silane coupling agent such as γ-incyanatepropyltriethoxysilane or γ-glycidoxypropyltrimethoxysilane can be used.

A carrier-polyethylene glycol complex can be obtained by combining the above-mentioned carrier, coupling agent, and polyethylene glycol according to a conventional method.

In addition, the coupling agent for covalently bonding polyethylene glycol and the enzyme is selected depending on the desired functional group in the enzyme, but when covalently bonding it to the amino group in glucoamylase, an enzyme derived from Rhizopus niveus, For example, bromoacetyl bromide or trichloro-8-triazine can be used as a coupling agent.

An enzyme immobilized product via polyethylene glycol can be obtained by covalently bonding the enzyme, coupling agent, and carrier-polyethylene glycol complex according to a conventional method.

The enzyme to be immobilized in the enzyme immobilized product of the present invention is not particularly limited, and any enzyme can be immobilized, such as lipase, which is an enzyme originating from bacteria, yeast, filamentous fungi, and porcine pancreas. , esterase, an enzyme derived from pig liver, etc. can be immobilized.

[Example] The enzyme immobilized product of the present invention will be further explained with reference to Examples below.

Example 1 Polyethylene glycol 2000 (molecular weight 2000) 0
．． 1y was dissolved in 2.7 liters of benzene, and porous silica glass (manufactured by Electro-Nucleonics: CPG-10, surface area 6.8 m"/f, pore volume 0.73 ce/#, Average pore size: 2734, particle size: 12
0 to 200 mesh) 1000B, γ-isocyanatepropyltriethoxysilane (manufactured by Nippon Unicar: Y-9
030>After adding 0.3 vhl and stirring thoroughly, 25
The mixture was allowed to stand at ℃ for 4 hours. This mixture was vacuum dried at 105°C for 2 hours, and after the drying was completed, acetone was used at 150 emf! using a glass filter (IO2). The mixture was washed and filtered, and dried under vacuum again to obtain a porous silica glass-polyethylene glycol composite (1029 mg).

This porous silica glass-polyethylene glycol composite 500B was mixed with 3 ml of dioxane and bromoacetic acid to
into the mixed solution and heated to 30°C using a rotary shaker.
After shaking at 100 rps+ for 166 hours, 7.5 mN of bromoacetyl bromide was added, and the mixture was further shaken for 7 hours to react. After one reaction, the reaction product was poured into 1 liter of distilled water and allowed to stand still. After that, the supernatant was removed. This operation was repeated twice.

The obtained precipitate was mixed with 150 N of 0.1 N sodium carbonate and 150 A of distilled water using a glass filter (IO2).
Washed and filtered with #.

Next, the obtained activated porous silica glass-polyethylene glycol complex was mixed with 200 u of glucoamylase [glucoamylase (manufactured by Nagase Seikagaku Kogyo Co., Ltd.: Rhizopus niveus origin, specific activity 5 units/-9, molecular weight 70000] and ammonium sulfate 2 .01 was dissolved in 10 ml of 0.2 molar phosphate buffer at pH 8.5, and added to a solution 1'zal containing 2.4 B of enzyme as enzyme protein per 1 ml, using a rotating shaker. 2 at 7°C, 100 rpm
The mixture was shaken for 44 hours to immobilize glucoamylase.

After the reaction is complete, use a glass filter (IO2) to remove the
9% sodium chloride aqueous solution 150mj! , washed and filtered with 0.055 molar McIlvaine buffer 15 (1+1') at pH 5.0, and immobilized glucoamylase (enzyme immobilized product).
I got it.

Next, this immobilized glucoamylase (enzyme immobilized product) was added to 50 ml of a 2% soluble starch (polymer substrate) aqueous solution and reacted for 10 minutes at 25°C and 100 rpm using a rotary shaker. After completion, the amount of glucose produced was measured using a glucose determination reagent (Glucinet reagent, manufactured by Technicon), and the activity of the immobilized glucoamylase was determined.

In addition, glucoamylase activated porous silica glass
The amount of immobilization on the polyethylene glycol complex was determined by measuring the amount of remaining protein in the residual solution of the immobilization reaction using the method of Lowry et al., and determining the difference from the amount of added protein. The results are shown in Table 1.

From the results in Table 1, the activity retention rate with the polymer substrate is 5 to 20% in the case of the conventional enzyme immobilized product in which the enzyme is directly covalently bonded to the carrier, and in the case of the enzyme immobilized product using acrylic acid as a spacer and grafted cotton cellulose as the carrier. 20-65 for enzyme immobilized product
% (the enzyme in both cases is glucoamylase) [80% compared to Reference Bifunctional Materials September issue (1985) p. 13
It can be seen that the effect of the present invention is very large.

Example 2 Porous silica glass obtained by the same method as Example 1
Benzene 2 to polyethylene glycol complex 500eH
0mff, anhydrous sodium carbonate 1.3g, trichloro-
8-Triazine 75. Calorie 6 wheels 9 and add 1 while refluxing.
After 1 reaction of 20 hours, petroleum ether 15
0g+# and benzene:acetone (1:1) solution 150ml
1 was used for washing. This operation was repeated three times. After the washing was completed, drying was performed at 60° C. for 2 hours using a vacuum dryer. This activated porous silica glass-polyethylene glycol complex was dissolved in a glucoamylase aqueous solution (200 mg of glucoamylase was dissolved in 10 0.4 molar borate buffers with pH 10. .3ml solution containing 11) 5s+l! The glucoamylase was immobilized by shaking at 37°C and 1100 rpm for 1 hour using a large rotating shaker, and the pH was adjusted to 8.
, 0 of 0.1 molar borate buffer 100+at! The reaction was terminated by adding . After the reaction is complete, use a glass filter (IO2) to remove McIlvaine M at pH 5.0.
The mixture was washed and filtered with 50 ml of 8V liquid L to obtain immobilized glucoamylase (enzyme immobilized product). The activity and amount of immobilized glucoamylase were measured in the same manner as in Example 1. The results are shown in Table 2.

Round 1 glucoamylase 4. ')') 1.
22 <) 8.0 From the results in Table 2, the activity retention rate is lower than that of Example 1, but it is higher than that of the enzyme immobilized product using acrylic acid as a spacer by the conventional method, and the effect of the present invention is large. It turned out that.

[Effects of the Invention 1] As described above, by covalently interposing polyethylene glycol, which is a feature of the present invention, as a spacer between the carrier and the enzyme, the enzyme has high activity toward polymer substrates,
In addition, it is possible to obtain an immobilized enzyme that is stable even in organic solvents and maintains high activity, and can be effectively used industrially.

For example, by using enzymes such as bacteria, yeast, filamentous fungi, lipase, an enzyme originating from pig pancreas, and esterase, an enzyme originating from pig liver, it is possible to hydrolyze or synthesize various polyesters. In the enzyme immobilized product of the present invention, which has high activity against polyester resin, is stable even in organic solvents, and can maintain high activity, when lipase, esterase, etc. are used as the enzyme, it can be used to decompose discarded polyester resin or to recover from various monomers. Polyester synthesis can be performed efficiently, making industrialization possible. The polyester synthesis at this time is the synthesis of a biodegradable (microbially degradable) polymer. In addition, by using substances that are insoluble in water and soluble in organic solvents as substrates and using optically selective enzymatic reactions, we can efficiently produce fine chemicals such as highly pure fragrances and pigments that cannot be obtained by chemical synthesis methods. It can be synthesized and industrialized.

Patent applicant: Japan Steel Works, Ltd.

Claims (1)

[Scope of Claims] 1. An enzyme immobilized product in which a spacer is interposed between a carrier and an enzyme, wherein the spacer is polyethylene glycol. 2. The enzyme immobilized product according to claim 1, wherein the carrier and the polyethylene glycol are chemically bonded. 3. The enzyme immobilized product according to claim 1, wherein the polyethylene glycol and the enzyme are chemically bonded.