JPH036792B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing an immobilized enzyme used in a non-aqueous reaction. Several non-aqueous enzyme reactions are known in which the enzyme reaction is carried out in an organic solvent in the presence of a trace amount of water. One of the excellent immobilization resin polymers used for enzyme immobilization is photopolymerizable resin (hereinafter referred to as "photopolymerizable resin"), which is made by bonding a compound having a photopolymerizable ethylenically unsaturated group to both ends of polyalkylene glycol. Fixed polymers obtained by photopolymerizing a resin by irradiating it with light are known. ( European J.Appl.Microbiol.Biotechnol.6
325, (1979)). The present inventors have begun research to produce immobilized enzymes for use in non-aqueous reactions using such photopolymerizable resins. The above-mentioned immobilized resin polymer preferably has higher permeability to organic solvents, so the photocurable resin should be
Generally, those soluble in organic solvents are used.
Therefore, polymerization reactions for enzyme immobilization are often carried out in organic solvents. However, when an enzyme aqueous solution and an organic solvent are mixed, if the organic solvent is hydrophilic, the enzyme will precipitate, and if the organic solvent is lipophilic, it will not mix uniformly with the enzyme aqueous solution, and in either case, the enzyme will not be mixed uniformly with the enzyme aqueous solution, and in either case, the enzyme will not be mixed uniformly with the enzyme aqueous solution. It is not possible to obtain an immobilized enzyme. In order to uniformly mix the lipophilic organic solvent and the enzyme aqueous solution, a method using an emulsifier has been considered, but when this method was tested, the desired results were not necessarily obtained. Therefore, as a result of further research, the present inventors discovered that the enzyme aqueous solution was brought into contact with a carrier capable of adsorbing water in advance, and was adsorbed onto the carrier, and the obtained enzyme aqueous solution adsorbed carrier was photopolymerized in an organic solvent. They learned that enzymes can be efficiently immobilized by mixing them with resin and allowing a photopolymerization reaction to occur, and based on this knowledge they completed this discovery. Examples of the photopolymerizable resin used in the present invention include the following. (1) Both terminal hydroxyl groups of 1 mole of polyethylene glycol with a molecular weight of 400 to 6000 are converted to (meth)acrylic acid 2
Mol esterified polyethylene glycol di(meth)acrylates. (2) Polypropylene glycol di(meth)acrylates obtained by esterifying both terminal hydroxyl groups of 1 mol of polypropylene glycol with a molecular weight of 200 to 4,000 with 2 mol of (meth)acrylic acid. (3) Both terminal hydroxyl groups of 1 mol of polyethylene glycol having a molecular weight of 400 to 6,000 are urethanized with 2 mol of a diisocyanate compound such as tolylene diisocyanate, xylylene diisocyanate, or isophorone diisocyanate, and then a non-terminal hydroxyl group such as 2-hydroxyethyl (meth)acrylate is used. An unsaturated polyethylene glycol urethane added with 2 moles of a saturated monohydroxyethyl compound. (4) Both terminal hydroxyl groups of 1 mole of polypropylene glycol having a molecular weight of 200 to 4000 are urethanized with 2 moles of a diisocyanate compound such as tolylene diisocyanate, xylylene diisocyanate, isophorone diisocyanate, etc. Unsaturated polypropylene glycol urethane added with 2 moles of a saturated monohydroxy compound, etc. Examples of the photocurable resin of the present invention include those having the following structural formula. (European
J. Appl. Microbiol. Biotechnol. 6 , 325 (1979)). As seen in the above examples, the photopolymerizable resin of the present invention has a photopolymerizable ethylenically unsaturated group at both ends of polyalkylene glycol. When trying to produce an immobilized enzyme for use in the non-aqueous reaction of the present invention, such a photopolymerizable resin is
An enzyme aqueous solution adsorption carrier obtained by contacting an enzyme aqueous solution with a carrier capable of adsorbing water in advance may be mixed in an organic solvent, and this mixture may be irradiated with light to polymerize the photopolymerizable resin. Examples of carriers capable of adsorbing water include celite, quartz sand, clay, activated carbon, glass fibers or beads, silicic acid gel, florisil, alumina, potassium carbonate, potassium sulfate, glucose,
These include β-cyclodextrin, cellulose, casein, starch, and agar. All of these carrier shapes are insoluble in organic solvents. A larger surface area is desirable so that more water can be adsorbed. In order to make the carrier adsorb more enzyme protein, the enzyme aqueous solution preferably has a higher concentration.
The solvent for the aqueous enzyme solution may be a buffer solution, or an aqueous solution containing an inorganic salt, an antioxidant effective for stabilizing the enzyme, and the like. As an organic solvent, it dissolves photopolymerizable resin, but
A solvent that does not dissolve the immobilized polymer, regardless of whether it is polar or non-polar. For example: Petroleum benzine, hexane, huptan, benzene, petroleum ether, toluene, ethyl acetate, acetone, alcohol, dioxane ethylene glycol, formamide, pyridine, tetrahydrofuran, dimethyl sulfoxide, dimethylformamide. It is more economical to mix the photopolymerizable resin and the enzyme aqueous solution adsorption carrier, or to set the ratio such that the minimum amount of photopolymerizable resin necessary to generate the photopolymerized product is more economical. It is set within a more preferable range, taking into consideration the strength of the object. The light beam to carry out the photopolymerization reaction is 250
The most desirable range is from 600 nm to 600 nm. The immobilized polymer may be in the form of a thin film, beads, or other shapes. In order to carry out a non-aqueous enzyme reaction using such an immobilized enzyme, the substrate dissolved in an organic solvent and the immobilized enzyme may be brought into contact for a period of time at a preferred temperature range. As a non-aqueous enzymatic reaction, a transesterification method using lipase (JP-A-52-104506, JP-A-55
-843976 publication), oxidation, reduction, hydroxylation, or dehydrogenation reactions of steroids, reactions that use physiologically active substances such as fat-soluble vitamins and prostaglandins as substrates, or reactions that produce these physiologically active substances, etc. and enzymatic reactions whose products are fat-soluble substances. Example 1 0.2ml of 0.3M in which 0.25g of Celite was dissolved in 5mg of lipase produced by Rhizopus deremer (Seikagaku reagent, fine grade, 600 units)
Add to TES buffer (PH6.5) and mix well. In experimental area 1, this was used as ENTP (average molecular weight 2000).
In experimental group 2, 0.5 g of benzoin ethyl ether and 5 mg of benzoin ethyl ether were dissolved in 0.4 ml of hexane.
A solution of 0.5 g of ENT (average molecular weight 4000) and 5 mg of benzoin ethyl ether dissolved in 0.4 ml of acetone was charged and mixed well. The mixture was then poured onto a transparent film and irradiated with near ultraviolet light of 300 to 400 nm for 3 minutes on both sides to cause a polymerization reaction. The immobilized lipase was prepared by cutting the polymer into a size of 3 x 3 mm. This immobilized lipase was mixed with 2.5 g of olive oil and 2.5 g of stearic acid in 10 ml of hexane prepared in advance.
The mixture was poured into a reaction solution containing g and shaken at 40°C for 1 hour. Table 1 shows the amount of stearic acid introduced into triglycerides in olive oil.

[Table] Example 2 The stability of the immobilized lipase of Experiment Group 1 shown in Example 1 was investigated. In the method shown in Example 1, the reaction solution was replaced with a new one every hour and the reaction was repeated. The results are shown in Table 2. The lipase adsorbed on Celite lost about half its activity after the fourth use, whereas the immobilized lipase was completely stable.

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Claims (1)

[Claims] 1. (a) A photopolymerizable resin formed by bonding a compound having a photopolymerizable ethylenically unsaturated group to both ends of a polyalkylene glycol, and (b) a carrier capable of adsorbing water in advance and an enzyme aqueous solution. Production of an immobilized enzyme used in a non-aqueous reaction characterized by mixing an enzyme aqueous solution adsorbing carrier obtained by cleavage in an organic solvent and irradiating the mixture with light to polymerize a photopolymerizable resin. Law.