JPS5837836B2 - Method for producing immobilized uricase membrane - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing an immobilized uricase membrane.

In obtaining an immobilized enzyme, it has been known to use chitosan, which is obtained by deacetylating chitin with an alkali, as a carrier.

For example, a method in which a polyfunctional compound such as a dialdehyde compound or a diincyanate compound is used in a chitosan solution of enzymes or microbial cells to carry out a crosslinking reaction (Japanese Patent Laid-Open No. 52
-120182, JP-A-52130-979), a covalent bonding method in which chitosan powder or flakes are treated with a dialdehyde compound or diazo compound, and then an enzyme is attached (see JP-A-52-3892). , JP-A No. 52-12849), and a method in which chitosan and enzyme are covalently bonded in an organic acid aqueous solution using a carbodiimide reagent, and then made alkaline to coagulate and precipitate (see JP-A-51-76482). (see).

However, these conventional methods have not yielded membrane-like immobilized enzymes.

In addition, when uricase, among other enzymes, is used in these methods, the resulting immobilized uricase in the form of powder, flakes, or granules exhibits no activity since uricase is extremely unstable to these chemical reagents. It disappears.

On the other hand, methods for producing immobilized enzyme membranes include a method in which a cross-linking agent such as glutaraldehyde is added to a mixed solution of an enzyme and an inactive protein and molded into a membrane, and a method in which the enzyme is enclosed in a collagen membrane. There is.

However, when uricase is immobilized using these methods, the enzyme activity disappears when glutaraldehyde is used, and when it is enclosed in a collagen membrane, the enzyme gradually flows out of the membrane, and the collagen membrane swells in water. Mechanical strength is low or dimensional stability is very poor.

As described above, the current situation is that a method for immobilizing uricase, particularly a suitable method for producing an immobilized uricase membrane, has not been found.

The present inventors took into consideration the characteristics of uricase, and also
As a result of extensive research in order to obtain an immobilized uricase membrane, the inventors discovered that the desired objective could be achieved by selecting chitosan as a carrier and specifying the conditions for membrane formation, and thus arrived at the present invention.

That is, the present invention is a method for producing an immobilized uricase membrane, which is characterized in that uricase is dissolved in an acidic aqueous solution of chitosan, then cast, and the dried membrane is immersed in an aqueous solution having a pH of 9 to 13.

Chitosan in the present invention is obtained by deacetylating chitin with an alkali, and is conceptualized to include partially deacetylated chitin.

The acidic aqueous solution of chitosan in the present invention is a solution having a pH of 1 to 6, preferably 2 to 5, in which the chitosan is dissolved.

As the acidic substance, organic acids such as acetic acid and formic acid are generally used, and the concentration thereof is 0.5 to 2%.

The concentration of chitosan in the acidic aqueous solution is preferably 0.5 to 2%.

In the present invention, uricase is first dissolved in the acidic aqueous solution of chitosan, and then cast onto a horizontal plate such as a glass plate to obtain a film-like product.

The obtained film-like material is dried at 0 to 20°C to a moisture content of 15% or less, and then dried to a pH of 9 to 13, preferably pH 10.
~110 Immerse in a weakly alkaline aqueous solution for 10 to 60 minutes to solidify.

The thickness of the film-like material after drying is not particularly limited, but is preferably 10 to 100 microns.

In the present invention, if the chitosan acidic aqueous solution of uricase is immersed in an alkaline aqueous solution in a wet state, the chitosan aqueous solution will solidify while diffusing toward the alkaline aqueous solution, and no film-like material will be obtained.

Drying conditions are not particularly limited as long as uricase is not deactivated, and air drying is preferably performed.

Examples of the aqueous solution having a pH of 9 to 13 in which the dried film-like material is immersed include a phosphate buffer, a borate buffer, and a sodium carbonate/bicarbonate buffer.

If the pH of the alkaline aqueous solution is lower than 9, even if the optimum pH for uricase is weakly alkaline, 8.5, the chitosan film will swell, partially dissolve, and diffuse into the aqueous solution, retaining its shape as a film. I can't.

Moreover, if the pH of the alkaline aqueous solution is higher than 13, the activity of uricase will be lost within a short time.

The immobilized uricase membrane of the present invention, which is immersed and coagulated in an alkaline aqueous solution with a pH of 9 to 13, retains the shape of a membrane in a buffer solution with a pH of 8.5, which is the optimum pH for uricase.
Uricase has sufficient activity without leaching into the buffer.

In the method for producing an immobilized uricase membrane of the present invention, since uricase is stable on the relatively alkaline side, there is little loss of enzyme activity even when immersed in an alkaline aqueous solution with a pH of 9 to 13.
Furthermore, the method is compatible with the characteristics of uricase and chitosan membranes, such that the immobilized uricase membrane does not dissolve even at the optimum pH of 8.5.

Enzymes whose optimum pH is on the acidic side, such as glucose oxidase, are extremely unstable in alkaline conditions, and at pH 10
In addition to rapidly losing enzyme activity even in
If the immobilized glucose oxidase membrane is immersed in the phosphate buffer solution in step 5, it will dissolve in the buffer solution.

The immobilized uricase membrane obtained by the method of the present invention has (1
) Since uricase is included in the chitosan membrane, it can be easily produced without using special reagents.
1) Since no reagents that directly react with the enzyme are used, there is little deactivation of the enzyme. (ri) The amount of enzyme in the membrane can be freely increased or decreased, so a product with the desired enzyme activity can be obtained. Furthermore, 6V
) It has many excellent characteristics, such as being extremely stable and showing no decrease in activity even when stored in a dry state at room temperature for several months.

When such an immobilized uricase membrane is attached to an electrode and used as an analysis device, it is possible to repeatedly measure the amount of uric acid contained in blood, urine, etc. in a short time and with high precision using a small amount of membrane.

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 6.5f of acetic acid aqueous solution in which 1% of chitosan 100cm was dissolved.
20 m9 of uricase (manufactured by Toyobo Co., Ltd., 3.5 units/■) was dissolved in and uniformly cast onto a Teflon plate using a knife coater with a thickness of 1000 μm.

The film was then air-dried at 5°C to obtain a chitosan film containing uricase with a moisture content of 10.3%.

This chitosan membrane was placed in a boric acid buffer solution of pH 10.0 for 3 hours.
It was immersed for 0 minutes, further immersed in a boric acid buffer solution of pH 8.5 for 15 minutes, and air-dried again to a thickness of 27μ and an area of 275.
An immobilized uricase membrane of ci was obtained.

M/20 borate buffer (p
4 ml of M/20 borate buffer (pH 8.5)
．． 5) Add the above immobilized uricase membrane I to the mixture with 2 ml of
CrrL×ICrrL was soaked and shaken at 25° C. for 10 minutes.

After that, the absorbance (OD29a) of the mixed solution was measured, and the OD of the mixed solution without immersing the immobilized uricase membrane was measured.
The activity of the immobilized uricase membrane was determined by calculating the amount of uric acid decomposed from the difference between 290 and 0.08 units/cr.
It was A.

Claims (1)

[Claims] 1. A method for producing an immobilized uricase membrane, which comprises dissolving uricase in an acidic aqueous solution of chitosan, then casting the membrane, and immersing the dried membrane in an aqueous solution having a pH of 9 to 13.