JPS6231912B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides CAH containing creatine amidinohydrolase (hereinafter abbreviated as CAH) in a stable state.
It is related to formulations. Creatine is an amino acid that appears in the urine during starvation, fever, kidney disease, muscle disease, hyperthyroidism, etc., or during pregnancy. The daily emissions are considered to be almost constant. Therefore, measuring the body fluid concentration of creatine and creatinine is considered to provide important indicators in clinical tests, and CAH preparations used for these quantitative analyzes have a position as important diagnostic enzyme agents. is occupying. Conventionally, CAH has been analyzed by conjugation with other enzymes or in combination with chemical measurement methods. Measurement methods that use a combination of the following are attracting attention. That is, the latter method is

By the reaction in [Table], creatinine is converted to creatine, and creatine is further decomposed into sarcosine and urea, and the resulting sarcosine and urea can be measured by combining them with other enzymes or by combining them with chemical methods. However, the target creatinine and creatine are quantified, and CAH plays an important role in this method. However, when CAH is stored in a solution state, significant denaturation progresses during storage, sometimes resulting in turbidity, which has a serious negative effect on measurement accuracy. Concerned about this situation, the present inventors conducted various studies on stabilizing enzyme preparations containing CAH, and found that if the enzyme preparation has the composition described below, CAH can be stored in solution. It was discovered that the above-mentioned denaturation and turbidity did not occur, and the present invention was completed. That is, the CHA preparation of the present invention is
In addition to containing CAH as an essential component, one or more nonionic surfactants with an HLB of 15 or higher, or one or more surfactants selected from the group consisting of chelating agents and/or preservatives (hereinafter collectively referred to as these) The gist lies in the inclusion of a stabilizer (referred to as a stabilizer). All CAHs referred to in this specification are subject to stabilization regardless of their origin, but CAHs originating from microorganisms belonging to the genus Pseudomonas, Flavobacterium, etc. are particularly frequently used. The CHA preparation in the present invention is not limited to one containing CAH alone as described above, but also contains other substances, such as enzymes and coenzymes necessary for quantifying creatinine and creatine, as well as substrates and their stability. A general term for those containing stabilizer components necessary for chemical conversion.
Note that CAH preparations include those provided in any form such as a solution or a dry powder. By the way, the most typical stabilizer used in the present invention is a nonionic surfactant with an HLB of 15 or higher (hereinafter referred to as AA agent).
As the AA agent, as long as it satisfies the two conditions of HLB being 15 or more and being nonionic, there are no restrictions on the structural formula or other aspects, and various agents can be used. However, examples of typical AA agents include polyoxyethylene alkyl phenyl ether, polyoxyethylene alkyl phenyl ether, polyoxyethylene acyl ester, etc. The alkyl in these includes alkyl having 7 or more carbon atoms, Examples include octyl, nonyl, lauryl, and the like.
As a chelating agent, a general metal chelating agent is used, and the type is not restricted as long as it can capture and inactivate metal ions, but typical examples include ethylenediaminetetraacetic acid and its An example is salt. In addition, the preservative only needs to have the function of preventing the deterioration of CAH, etc. caused by microorganisms mixed in the enzyme preparation, and may have a bacteriostatic or bactericidal effect on microorganisms, or one that inactivates the activity of microorganisms. Any substance may be used as long as it is suitable, but some typical examples include azides, antibiotics, sulfur drugs, boric acid,
Examples include organic acids. The mixing ratio of the above-mentioned stabilizers may be determined by taking into account the strength of the stabilizing effect, the chemical stability of the stabilizer itself, and the combined use of each of the above-mentioned stabilizers. As a general guideline, certain effects can be obtained by selecting from the range of 0.01 to 1000 mg per 100 units of CAH. In order to obtain a CAH enzyme preparation containing the above-mentioned stabilizer, there is a step of preparing a solution by combining CAH alone or CAH with other enzymes, coenzymes, substrates, etc., and adding the above-mentioned stabilizer to the solution. The process of adding and mixing the agent and, if necessary, drying the solution is combined. When preparing the CAH solution mentioned at the beginning, select an appropriate buffer solution, adjust the concentration to 1 to 500mM, and adjust the pH to 500mM. It is desirable to set it to about 6.5 to 9.0. Next, when adding and mixing the stabilizer, either directly mix the stabilizer powder with the above CAH solution, or add the powder after dispersing or dissolving it in water or a buffer solution, and then add it according to the usual method. Stir. Finally, if you want to dry this mixture,
Any conventional means such as freeze drying or spray drying may be used. Since the CAH preparation of the present invention has the above-mentioned structure, it can be used regardless of whether it is in powder form or solution form.
It has become possible to maintain the activity of CAH over a long period of time, and to maintain a high degree of reliability in the measurement accuracy of creatine and creatinine, thus providing an extremely useful diagnostic enzyme preparation. Next, examples of the present invention will be described. In the examples, the enzyme activity was measured in accordance with the following. (A) Reagent composition (1) Substrate solution: 0.1M creatine solution [creatine dissolved in 0.05M phosphate buffer (PH7.5)] (2) NaOH solution: 1N sodium hydroxide aqueous solution (3) Coloring solution: 1% Picric acid solution (B) Measurement method Add 0.1 ml of enzyme solution to 0.9 ml of substrate solution and heat at 37℃.
The reaction was allowed to proceed for 10 minutes. 0.1 ml of the reaction solution was collected and added to 0.9 ml of cold water to stop the reaction. next
Add 1 ml of NaOH solution and 1 ml of coloring solution and leave at room temperature.
The sample was left for 20 minutes to allow the creatine produced by the enzymatic reaction to develop color. This color development was measured by absorbance at 520 nm to determine the enzyme titer. The enzyme titer is expressed as one unit, which is the amount of enzyme that produces 1 μmol of creatinine per minute under the above conditions. Example 1 Obtained from a microorganism belonging to the genus Pseudomonas
Using CAH, add 20% to 0.05M phosphate buffer (PH7.5).
It was dissolved in units/ml. Add the first
Each compound listed in the table was added, and the pH was readjusted to 7.5 if necessary. The obtained enzyme preparation solution was stored under the conditions shown in Table 1, and the occurrence of turbidity was measured by absorbance at 660 nm, and the results shown in Table 1 were obtained. As is clear from Table 1, AA agents with HLB15 or higher,
Or, those containing the AA agent, chelating agent, and/or preservative had less turbidity, but the other ones had significant turbidity.

【table】

[Table] Example 2 An enzyme preparation having the following composition was prepared. CAH (Pseudomonas origin) 200 units Sarcosine oxidase (Corynebacterium origin)
30 units Formaldehyde dehydrogenase (originated from Pseudomonas sp.) 3 units NAD 7.2mg Polyexyethylene oleyl ether (HLB16.2) 10mg Sodium azide 10mg EDTA・2Na 10mg Dissolve the above preparation in 0.05M phosphate buffer (PH7.5). , the total volume was made 10 ml. On the other hand, from the above formulation, polyoxyethylene oleyl ether, sodium azide and EDTA・2Na
A comparative formulation was prepared except for the following: and dissolved in 0.05M phosphate buffer (PH7.5) in the same manner to make the total volume 10 ml. Immediately after preparation and after storage at 25°C for 5 days, 1 ml of each was sampled and subjected to a quantitative test. In other words, 1 ml of enzyme preparation contains a sample containing creatine.
50μ was added and reacted at 37°C for 10 minutes. And NAD changed depending on the amount of creatine in the sample.
The amount of creatine in the sample was determined by measuring the change in absorbance at 340 nm (NADH). On the other hand, the above enzyme preparation minus CAH was operated in the same manner as a control preparation, a blank value of the sample was obtained, and the true amount of creatine was determined from the difference from the above measured value (Table 2).

[Table] As shown in Table 2, the examples in which a stabilizer was added gave almost the same results immediately after preparation and after storage, but the comparative examples in which no stabilizer was added gave almost the same results after storage. was virtually impossible to measure, confirming the effect of adding a stabilizer (storage stability).

Claims (1)

[Claims] 1. Contains creatinamidinohydrolase,
A stable creatine amidinohydrolase preparation, further comprising a nonionic surfactant with an HLB of 15 or higher, or the surfactant, and a chelating agent and/or preservative.