JPH01228472A - Method for immobilizing coenzyme - Google Patents

Abstract

PURPOSE:To prevent lowering of function of a coenzyme by including the coenzyme with a fibroin film. CONSTITUTION:A coenzyme is included with a fibroin film obtained by casting an aqueous solution of fibroin collected from cocoon produced by a silkworm on a substrate and drying the resultant film and dipping the film into ethanol.

Description

[Detailed description of the invention] (Industrial application field) The present invention relates to a method for immobilizing coenzymes.

More specifically, the present invention relates to a method for immobilizing coenzymes, which is characterized by enclosing the coenzymes in a fibroin membrane.

(Prior art) NADH, NADPH, NAD,
Coenzymes such as NADP and ATP are required. Since these coenzymes are expensive, it is desirable to collect and reuse them; however, coenzymes have a smaller molecular weight than enzymes, which are proteins, and it has been difficult to immobilize them using the entrapment method. From this point of view, conventionally proposed methods for immobilizing coenzymes include chemically modifying coenzymes with polymeric compounds such as polyethylene glycol, converting them into polymers, and immobilizing them. Although this method has been put into practical use in some areas, chemical modification of the coenzyme lowers its affinity with the enzyme, leading to problems such as a decrease in the enzyme reaction rate.

(Problems to be Solved by the Invention) In order to solve the above-mentioned conventional problems, the present inventor has conducted various studies in an attempt to provide a highly functional and economical immobilization method for coenzymes. discovered that fibroin membranes can be used to immobilize coenzymes, and have now completed the present invention.

(Means for Solving the Problems) That is, the present invention is a method for immobilizing a coenzyme, which is characterized by enclosing the coenzyme in a fibroin membrane.

The details of the present invention will be explained below.

Fibroin membrane fibroin is collected from cocoons produced by silkworms. Fibroin membranes are not limited to the manufacturing method; for example, a fibroin aqueous solution is dried and immersed in ethanol to make it insolubilized, or a fibroin aqueous solution is mixed with polyols such as glycerin, ethylene glycol, and polyethylene glycol and dried. Those manufactured by insolubilization treatment can be used.

(Example) and (effect) Examples and effects of the present invention will be described below.

Example 1 4g of cocoon layer was thoroughly loosened in hot water and 0.5% (w/v))
It was immersed in 200 ml of Brizier 35 (polyoxyethylene lauryl alcohol ether) aqueous solution. This was placed in a hot water bath in boiling water and stirred for 40 minutes. The disassembled cocoon layer was placed in a fresh Bridgie 35 aqueous solution and the same operation was repeated. Thereafter, it was washed with 3% sodium carbonate, warm water, water, and distilled water in this order. As a result, about 3.5 g of pure fibroin was obtained.

3.0 g of the obtained pure fibroin was added to 200 ml of 9.3
It was immersed in M lithium bromide aqueous solution and incubated at 40°C. After 12 hours, it was confirmed that the fibroin was completely dissolved, and then dialyzed against distilled water for 48 hours at room temperature.

After dialysis, the mixture was centrifuged at 18,000 g for 30 minutes to remove the precipitate, and the supernatant was used as liquid fibroin. This liquid fibroin was poured onto a glass plate and dried at 40°C. The obtained membrane was immersed in 95% ethanol for 4 hours to obtain an insolubilized fibroin membrane. The film thickness was approximately 70 μm in a dry state.

A hole with a diameter of 2 cm was made in the bottom of a plastic cylindrical container with a height of 4.5 cm and a diameter of 3 cm, and a fibroin membrane was pasted therein. After confirming that there was no water leakage, 5 ml of 50 mM potassium phosphate buffer (pH 7.2) containing 1 mM of various coenzymes shown in Table 1 was placed inside. Next, the entire container was floated in a beaker containing 20 ml of pure water, and the liquid levels of the inner and outer liquids were adjusted to the same level. While stirring the inner solution with a stirrer, the coenzyme concentration in the outer solution was measured every 1, 2, 3, 4, 5, 6, and 24 hours.

The concentration was measured by measuring absorbance at 260 mm. Experiments were conducted at 28°C. Table 1 shows the results.

The unit is μM Leakage rate = Number of moles of each coenzyme in the external solution / Number of moles of coenzyme initially added to the internal solution As shown in Table 1, the leakage rate of coenzymes is 3 to 5% after 24 hours. , indicating that the coenzyme was immobilized by the method of the present invention. Note that the results using a fibroin membrane prepared by mixing polyols in an aqueous fibroin solution and drying the mixture are similar to those shown in Table 1, and it is clear that the fibroin membrane that can be applied to the method of this invention is not limited to its manufacturing method. became.

Example 2 A bioreactor was prepared in which a coenzyme and an enzyme were simultaneously immobilized on a fibroin membrane to convert pyruvate and ammonia into alanine. 3mM inside the device used in Example 1;
NADH, Sigma alanine dehydrogenase (A-765)
3,58 units/mg) 0.1 mg, Sigma alcohol dehydrogenase (A-7011, 300 units
/mg) 1mg, 0.1 containing 1mg of sodium azide
5 ml of M Tris-HCl buffer (pH 8.5) was added. This container was floated on 20 ml of 0.7 M Tris-HCl buffer (pH 8.5) containing 4 mg of sodium azide, and the liquid levels of the internal and external solutions were measured.

While stirring the internal solution at 28°C, add 0.5M every 12 hours.
Potassium pyruvate, 1M with 1M ammonium chloride
200 μl of trizphosphate buffer (pH 8.5) and 9
16.6 μl of 8% ethyl alcohol was added to the internal solution. In addition, the external solution was exchanged every 12 hours, and the amount of alanine was determined every 12 hours using a Shimadzu LC-6A high performance liquid chromatograph. The results are shown in Table 2.

Amount of alanine: Cumulative amount of alanine produced up to that time Conversion effect: Amount of alanine / Cumulative amount of potassium pyruvate added up to that time As shown in Table 2, the conversion efficiency from pyruvate to alanine is 72 By time, little had changed. That is, in the application of the present invention to a bioreactor, no leakage of enzymes or coenzymes that would cause a decrease in reaction efficiency was observed.

(Effects of the Invention) The present invention has the structure as explained above, and the fibroin membrane encloses and immobilizes the coenzyme.Since the coenzyme itself is not modified, there is no reduction in the function of the coenzyme. , simple and economical.

In addition, the present invention can achieve good results when applied to a bioreactor for enzymatic reactions, with no leakage of enzymes or coenzymes that would cause a decrease in reaction effectiveness.

Claims (1)

[Claims] (1) A method for immobilizing a coenzyme, which is characterized by enclosing the coenzyme in a fibroin membrane.