JPS6112675B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for stabilizing ascorbic acid oxidase [EC1.1033] (hereinafter abbreviated as ASO). ASO has long been known to be present in various higher plants, and much research has been carried out using the fruit peels of Cucurbitaceae plants, such as cucumber and pumpkin, which have a high content of the enzyme. The properties of ASO have been discussed and reported in detail. recent years,
The purified product is used in the field of clinical testing to quantify ascorbic acid, or when ascorbic acid in a sample affects the measurement system.
A commonly used method is to provide ASO and oxidize and decompose ascorbic acid to eliminate its harmful effects. However, since ASO is unstable in a dry formulation state, there is a strong desire to develop a stable commercial formulation of this enzyme. The present inventors have conducted various studies in order to solve these difficulties and obtain a stabilized ASO formulation that is sufficiently storage stable. As a result, the present inventors have discovered that stability can be significantly improved by incorporating one or more compounds selected from arginine, lysine, histidine, and borate into an ASO formulation, and have completed the present invention. I reached it. That is, the present invention is a method for stabilizing ASO, which is characterized in that the ASO preparation contains one or more compounds selected from the group consisting of arginine, lysine, histidine, and borate. The ASO preparation in the present invention is a solid product separated from cucumber or pumpkin and dried.
means ASO. This ASO preparation is not limited to highly purified products. The stabilizer of the present invention is selected from the group consisting of arginine, lysine, histidine and borate.
It is a species or a compound of two or more kinds, and borate refers to an alkali metal salt such as sodium or potassium boric acid. The blending ratio of stabilizer is per 100 parts by weight of ASO formulation.
10 to 1000 parts by weight. To obtain ASO formulations containing stabilizers,
It consists of a step of blending the ASO solution and a stabilizer, and a step of drying this mixture. The ASO solution is
ASO and dissolved in buffer, preferably at a concentration of 0.5
-5% by weight, pH 6.5-9.5 solution. The stabilizer is added to the ASO solution directly or dissolved in a buffer or water. The method for drying this mixture to form a formulation follows a conventional method. For example, freeze drying or spray drying is performed. According to the stabilization method of the present invention, the stabilization when formulating ASO is extremely large, and the obtained
ASO formulations are stable over long periods of time. The enzymatic activity of ASO used in the present invention is measured by the following method. (1) Composition of reaction solution 1mM ascorbic acid solution (contains 0.2M monopotassium phosphate and 1mM ethylenediaminetetraacetic acid disodium salt) 0.5m1 0.01M disodium phosphate solution 0.5m1 Enzyme solution (0.04-0.3 units/ml) (diluted with 0.01M disodium phosphate solution containing 0.05% bovine serum albumin) 0.1ml (2) Reaction conditions and enzyme titer React at 30℃ for 5 minutes. The reaction is stopped by adding 3.0ml of 0.2N hydrochloric acid, and the amount of decomposed ascorbic acid is determined from the decrease in absorbance at 245nm.
That is, under the above conditions of ascorbic acid
As the molecular extinction coefficient at 245 nm (ε−10×
10 ^-3 ), determine the amount of decomposed ascorbic acid from the decrease in absorbance at 245 nm during the reaction, and calculate the enzyme titer in the sample based on this. The enzyme titer is displayed at 1 minute per minute under the above conditions.
The amount of enzyme that decomposes μmol of ascorbic acid is taken as one unit. The present invention will be explained in detail below with reference to Examples. Example 1 ASO derived from cucumber and pumpkin [derived from cucumber (specific activity (unit/OD ₂₈₀ ): 910) and derived from pumpkin (specific activity (unit/OD ₂₈₀ ): 500)] were each added to 0.01 M phosphate buffer (PH8). .0) with a solid content of 20mg per ml.
I adjusted it so that A 2% (W/V) solution of the compounds shown in Table 1 in the same buffer was added to the above enzyme solution in 1 ml portions and freeze-dried in a 5 ml vial. The residual rate of enzyme activity was measured immediately after drying and when the dried product was further stored at 40°C for 4 days. The results are shown in Table 1. As is clear from Table 1, both the freeze-drying yield and the subsequent storage stability were significantly improved by the addition of arginine, lysine, histidine, or sodium borate compared to the case where no ASO preparations were added.

[Table] Example 2 ASO derived from cucumber [specific activity (unit/OD ₂₈₀ ):
910] and further operated in the same manner as in Example 1 to prepare a lyophilized product, and the concentration levels of arginine, lysine, histidine, or borate and the stabilizing effects of their combinations were investigated. . The results are shown in Table 2. As is clear from Table 2, all additives exhibited a significant stabilizing effect when added at least 10 to 90% by weight. Also, when these additives were used in combination, a significant improvement in storage stability was observed as in the case of each additive alone.

【table】

[Table] Freeze-dried product weight

Claims (1)

[Claims] 1 Arginine in ascorbic acid oxidase preparation,
1. A method for stabilizing ascorbic acid oxidase, which comprises containing one or more compounds selected from the group consisting of lysine, histidine, and borate.