JPS645877B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Background and Problems of the Invention The present invention relates to a method for producing immobilized enzymes. Several methods are known for making enzymes insoluble in order to eliminate the instability of enzymes in aqueous solutions and to ensure continuous utilization. One such method is the entrapment method, in which the enzyme is encapsulated in a polymer gel lattice or coated with a polymer film. Polyacrylamide gel, carrageenan potassium gel, calcium alginate, aluminum, and the like have been known as polymer gels that can be used in conventional entrapment methods, but these have the drawbacks of weak membrane strength and large leakage of enzymes.

The present inventor attempted to immobilize an enzyme using a polyurethane gel produced by the reaction between a urethane prepolymer and a chain extender containing water, but there are various difficulties when using a normal urethane prepolymer. I found out. In other words, because the reaction is fast, the gel time is short and operation is difficult, and the generated gas cannot escape and is easily engulfed by the gel body.The resulting gel body has a large leakage of fragile enzymes, making it difficult to use continuously for long periods of time. Intolerable. Furthermore, the stability of the enzyme is low and the activity decreases over time. Furthermore, it has poor substrate permeability. Furthermore, there are also drawbacks such as the fact that the free isocyanate groups of the urethane prepolymer react with the enzyme protein itself, reducing enzyme activity.

The present invention provides a new enzyme immobilization method that can overcome these difficulties.

Solution Method The present invention is directed to the production of an immobilized enzyme characterized by enclosing the enzyme or its source microbial cells in a gel produced by the reaction of a water-soluble urethane prepolymer bisulfite adduct with a chain extender. Regarding the law.

The use of water-soluble urethane prepolymer bisulfite adducts, i.e., prepolymers in which the terminal free isocyanate groups of the urethane prepolymer are blocked with bisulfite, increases the gel time and traps gas (CO ₂ ) inside the gel body. , and the enzyme immobilization rate also improves. It also has a good enzyme activity residual rate and is highly stable even during continuous long-term use. The resulting membrane is strong and has excellent substrate permeability. Furthermore, there is little decrease in enzyme activity due to the reaction between the enzyme protein and the isocyanate group.

Preferred Embodiment Water-soluble urethane prepolymer bisulfite adducts are commercially available and well known as agents used for anti-wrinkle treatment of textiles. They are generally prepared by reacting a polyol component and a polyisocyanate in conventional manner to form a urethane prepolymer, the terminal free isocyanate groups of which are blocked by reaction with a bisulfite, such as a sodium salt. For example, a bisulfite adduct of a urethane prepolymer prepared from a polyether polyol consisting of a block copolymer of ethylene oxide (EO) and propylene oxide (PO) and a diisocyanate is represented by the following formula.

NaO ₃ SOCHN−R−OCHN−(EO) _y −(PO) _x −
(EO) _y -NHCO-R-NHCOSO ₃ Na This compound is water-soluble and stable at room temperature, but the isocyanate functionality can be regenerated by heating or by active hydrogen-containing compounds, leading to chain extension of water, polyamines, polyols, etc. The chain can be extended by a chemical agent.

As the polyol component of the prepolymer, known polyether polyols, polyester polyols, polydimethylsiloxane-containing polyols, etc. can be used, and alkylene oxides can be added to starting compounds containing three or more active hydrogen atoms such as glycerin and trimethylolpropane. Polyether polyols or polyester polyols obtained by addition can also be used. Preferably, these polyol components contain at least 10% by weight of ethylene oxide to impart adequate hydrophilicity to the product.

Usable polyisocyanates include common polyisocyanates, such as tolylene diisocyanate, xylylene diisocyanate, hexamethylene diisocyanate, isophorone diisocyanate, and the like.

Water-soluble urethane prepolymer bisulfite adducts are commercially available, for example, from Applicant Company under the trade name Elastron.

As chain extenders, water and polyamines, such as hexamethylene diamine, are preferred in this case;
In particular, it is most preferable to use both in combination.

Any enzyme can be immobilized, such as amylase, protease, lipase, glucose oxidase, cellulase, glucose isomerase, etc., and the degree of purification of these enzymes is arbitrary. In addition, cells of source microorganisms of these enzymes, such as bacteria such as Bacillus layer, molds such as Aspergillus sp., Rhizopus sp., and Penicillium sp., yeasts such as Satucharomyces sp., and actinomycetes such as Streptomyces sp. can also be immobilized. .

Immobilization means include enveloping it in a polymer gel lattice. In this method, the urethane prepolymer bisulfite adduct is dissolved in water or a buffer solution with an appropriate pH range, preferably pH 9 or higher, and an enzyme and, if necessary, a polyamine such as hexamethylene diamine are added thereto, and the mixture is thoroughly stirred. All you have to do is pour it into a suitably sized vat and let the whole thing gel into a film. Although the reaction proceeds at room temperature, it is accelerated by heating to a temperature at which the enzyme is not inactivated, for example up to 50°C.

Example: A polyether polyol obtained by adding 9.5 mol of ethylene oxide and 9.5 mol of propylene oxide per hydroxyl group in the form of a block to glycerin in that order, and an OH/NCO ratio of 1/2,
35% aqueous solution of sodium bisulfite adduct of urethane prepolymer obtained by reacting 2,5-dimethylbenzene-1,4-diisocyanate 100
1 part was dissolved in 250 parts of pH9 buffer solution at room temperature, 100 mg of lipase (manufactured by Meito Sangyo Co., Ltd.) was added thereto, and the mixture was stirred well. Next, 45 parts of hexamethylene diamine was added and heated to 40°C. It was poured into a vat and formed into a film. It took 20 minutes to form a film, and there were almost no bubbles. After gelation is complete, cool, cut into pellets with scissors, transfer to a beaker,
It was washed with cold water at 5°C until the washing liquid became neutral.

When the lipase activity of this pellet was measured, it contained 50% of the enzyme activity used. still,
After this immobilized product was used continuously for one month through a substrate, the enzyme activity was measured, and it was found that 90% of the activity after immobilization was retained.

Comparative Example Lipase was immobilized using a urethane prepolymer that was exactly the same as in the example but was not blocked with NaHSO ₃ . Film formation occurred in 2 to 3 minutes, leaving bubbles and resulting in a brittle film. When the lipase activity at this time was measured, it was found that only 10% of the enzyme activity used was retained. Furthermore, when the enzyme activity was measured after using this immobilized product for 3 consecutive days through a substrate, it was found that only 10% of the activity after immobilization was retained. Furthermore, after 5 days, the membrane collapsed and almost no enzyme remained.

Claims (1)

[Claims] 1. A method for producing an immobilized enzyme, which comprises enclosing the enzyme or its source microbial cells in a gel produced by the reaction of a water-soluble urethane prepolymer bisulfite adduct and a chain extender.