JPS6117467B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to stable urease compositions. Urease, which has been known for a long time and is widely used, is currently in increasing demand in the field of clinical testing and requires highly purified products. However, highly purified urease is extremely unstable, and various attempts have been made to stabilize it. One of the attempts is to add thiol compounds such as cysteine, mercaptoethanol, EDTA, maleic acid, etc.
Additions such as citrate have been used. However, even when each of these compounds was added alone, satisfactory results could not be obtained. Also, a preparation containing a mixture of glutathione, EDTA and citrate as a stabilizer (JP-A-52-117488
(No. 2) are being considered, but they have not been sufficiently effective. Taking these circumstances into consideration, the present inventors conducted various intensive studies to obtain a more stable urease. As a result, the addition of a mixture of a thiol compound, a chelating reagent, and a dibasic acid or its salt could eliminate the conventional stabilization method. The present invention has been achieved based on the discovery that it is significantly more stable than the conventional method. That is, the present invention is a stable urease composition characterized by containing a mixture of urease, a thiol compound, a chelating reagent, and an organic dibasic acid or a salt thereof. The present invention not only achieves remarkable stabilization compared to individual stabilizers used alone, but also compared to the method using a mixture of tribasic acids such as citric acid (Japanese Patent Application Laid-Open No. 117488/1983). , the stabilizing effect is clearly superior. Furthermore, the hygroscopicity of the resulting enzyme powder is clearly superior to the method of adding tribasic acid. Furthermore, according to the present invention, the amount of expensive glutathione and the like added can be kept low, and not only a highly stable enzyme powder can be obtained, but also a standard product with low hygroscopicity and a highly pure enzyme preparation can be obtained. Urease in the present invention refers to urease solution or urease powder, for example, by crushing beans,
Grinding, extracting the crude enzyme solution, salting out with ammonium sulfate, etc., solvent precipitation with organic solvents, etc., adsorption fractionation using an ion exchanger, gel filtration, etc., resulting in a solution, vacuum-dried powder, or freeze-drying. There are powders etc. The thiol compound used in the present invention refers to a reducing substance having a thiol group, such as glutathione, dithiothreitol, dithioerythritol, cysteine, and mercaptoethanol. Particularly preferred are glutathione, dithiothreitol, and dithioerythritol. The chelating reagent used in the present invention refers to compounds that are widely used, such as ethylenediaminetetraacetic acid (EDTA) and its salts, nitrilotriacetic acid and its salts, and cyclohexanediaminetetraacetic acid and its salts. Particularly preferred are EDTA and nitrilotriacetic acid. Organic dibasic acids and their salts used in the present invention include saturated dicarboxylic acids and their salts such as oxalic acid, malonic acid, succinic acid, and glutaric acid, hydroxydicarboxylic acids and their salts such as malic acid, tartaric acid, and isotartaric acid, and mylene. acids, fumaric acid, itaconic acid,
Refers to unsaturated dicarboxylic acids such as glutaconic acid and their salts. Particularly preferred are oxalic acid, malic acid, and fumaric acid. Salts include alkali metal salts and alkaline earth metal salts. In the present invention, 1.0 parts by weight of urease, 0.0001 to 0.5 parts by weight of a thiol compound, and 0.01 to 0.01 parts by weight of a chelate compound.
5.0 parts by weight, and 0.01 organic dibasic acids and their salts
Add ~100 parts by weight. In particular, the thiol compound is preferably 0.02 to 0.05 parts by weight per 1.0 parts by weight of urease, the chelate compound is preferably 0.1 to 1.0 parts by weight per 1.0 parts by weight of urease, and the organic dibasic acid and its salt is preferably added to 1.0 parts by weight of urease. for
0.2 to 50 parts by weight is preferred. In addition to the stabilizers of the invention, thiol compounds, chelating compounds and organic dibasic acids and their salts, serum albumin, especially bovine serum albumin (BSA)
The addition of urease further improves the stabilization of urease. The amount of serum albumin added is preferably 0 to 10 parts by weight, particularly 0.5 to 5 parts by weight, per 1.0 part by weight of urease. In the present invention, there are no particular limitations on the method of adding a stabilizer to urease, but for example, EDTA is dissolved in a urease solution, the pH is adjusted to 7.5, glutathione is added and dissolved, and then a dibasic acid or its salt is added. Alternatively, if necessary, bovine serum albumin is added and dissolved, the pH is adjusted to 7.5 again, and the resulting solution is freeze-dried. Next, the present invention will be explained by examples. The urease activity measurement method uses the indophenol method, and when the enzyme is allowed to act on a 0.1M urea solution (PH7.0) for 5 minutes at 30°C, 1 mg per 5 minutes is measured.
The enzymatic power to liberate ammonia nitrogen was defined as one unit. The indophenol method is a method using the following reagents and performing the following operations. Reagents Phenol reagent solution: phenol 10.0g, sodium nitroprusside 50mg/500ml Alkaline hypochlorite solution: Sodium hydroxide 5
g, Sodium hypochlorite 2.5ml/500ml 0.1M urea substrate solution: urea 3.0g/0.01M phosphate buffer (PH) 500ml Enzyme dilution solution: 0.01M phosphate buffer (PH7.0)
Dilute the urease enzyme powder. Procedure: Place 2 ml of the urea substrate solution in a test tube, leave it at 30°C for 5 minutes, and when the temperature becomes constant, add 0.5 ml of the diluted enzyme solution to start the reaction. After reacting at 30°C for 15 minutes, add 1.0 ml of phenol reagent solution and 1.0 ml of alkaline hypochlorite solution. After standing at 30°C for 30 minutes, absorbance is measured at 530nm. As a control, use a diluted enzyme solution added after adding an alkaline hypochlorite solution. Example 1 Urease 400 units/ml (protein content 10 mg/ml)
A composition was prepared by adding 1mM glutathione, 10mM EDTA, and 20mM of the organic acid shown in Table 1, and after freeze-drying, the residual activity of urease at 50°C was measured. The results are shown in Table 1.

【table】

[Table] Example 2 The following composition was prepared in the same manner as in Example 1, and after freeze-drying, the persistence of urease activity at 50°C was measured. The results are shown in Table 2. Urease 400 units/ml (Protein content 10mg/ml) Glutathione 1mM EDTA 10mM BSA 5mg/ml Each organic acid 20mM

[Table] Example 3 100 mg of the enzyme powder prepared in Example 2 was heated to 25
We investigated the moisture absorption rate at ℃ and 70% humidity. Moisture absorption rate was measured by weight increase over time. The results are shown in Table 3.

【table】

Claims (1)

[Scope of Claims] 1. A stable urease composition comprising urease, a thiol compound, a chelating reagent, and an organic dibasic acid or a salt thereof. 2. The stable urease composition according to claim 1, which contains urease, a thiol compound, a chelating reagent, serum albumin, and an organic dibasic acid or a salt thereof.