JPS6343080B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for stabilizing acyl-coenzyme A synthetase [EC6,2,1,3] (hereinafter abbreviated as ACS). ACS has long been known to be present in animal sources such as rat liver and chicken liver, as well as in microbial cells such as yeast and bacteria. In recent years, purified products have been used in the field of clinical testing to quantify free fatty acids using coupled reactions with enzymes of other species. However, since purified products of ACS are extremely unstable, especially in a solution state, there is a strong desire to develop a method for stabilizing the enzyme. The present inventors have conducted various studies in order to solve these difficulties and find a stable ACS preparation that can be used satisfactorily under the intended conditions, and as a result, they have established the following stabilization method. In other words, ACS
5′-adenosine triphosphate, 5′-adenosine diphosphate, 5′-guanosine triphosphate, 5′-quanosine diphosphate, and salts thereof. 0.1mM of one or more compounds
This is a method for stabilizing ACS, characterized by containing ~0.1M or freeze-drying the solution. According to the method of the present invention, unstable ACS can be sufficiently stabilized, and as a result, it can be widely used in free fatty acid determination and other reaction systems using ACS. The ACS in the present invention is of microbial origin, and includes those produced from bacteria such as Pseudomonas and Escherichia, filamentous fungi such as Aspergillus, and yeast such as Candida.
The ACS preparations mentioned here are either solid ACS separated from the above sources and dried, or liquid ACS.
I'm talking about ACS. Examples of the salts of 5'-adenosine triphosphate, 5'-adenosine diphosphate, 5'-guanosine triphosphate, and 5'-guanosine diphosphate used in the present invention include lithium, sodium, potassium, etc. There are alkali metal salts of The content of these purine base nucleotide tri(or di)phosphates or salts thereof is about 0.001mM to 0.1M in the case of ACS solution, preferably about 1mM. Also, solid
For ACS preparations, 0.1mg to 100mg of ACS powder
The amount is about 1 g, preferably about 10 to 20 mg. When the amount is below the lower limit, there is no stabilizing effect, and when the amount exceeds the upper limit, ACS becomes inactivated or insoluble. Examples of polyhydric alcohols used in the present invention include monosaccharides such as sucrose, sorbitol, lactose, mannose, glucose, xylose, and fructose, oligosaccharides, ethylene glycol, 1,2-propylene glycol, and 1,3-propylene glycol. , 1,4-butanediol,
Examples include dihydric alcohols such as diethylene glycol, and trihydric or higher alcohols such as glycerin and pentaerythritol. The content of polyhydric alcohol is preferably about 0.5 to 50% by weight based on the enzyme preparation. In the present invention, as a method for incorporating the above-mentioned stabilizer into a preparation containing ACS, for example, purine base nucleotide tri(or di)phosphate or its salt is dissolved in 50 mM phosphate buffer (PH7.5), and the solution is dissolved in a caustic soda solution. Add and dissolve sucrose to the mixed solution, and add ACS to this prepared solution.
There is a method of dissolving the powder. The above-mentioned ACS preparation may be used as it is as an enzyme preparation, or it may be freeze-dried to form a solid (powder) and used as an enzyme preparation. In addition to the above stabilizers, formulations containing ACS may contain
Chelating agents such as EDTA, preservatives such as sodium nitride, magnesium chloride, etc. may be added. By following the method of the present invention, significantly improved stability is obtained compared to the addition of conventional stabilizers. In the present invention, the ACS activity measurement method is performed as shown below. The abbreviations are as follows. EDTA: Ethylenediamine tetraacetate salt ATP・Mg: 5′ adenosine triphosphate and magnesium salt ATP.Na ₂ : Adenosine triphosphate sodium salt CoA: Coenzyme A CoA・Na ₂ : Coenzyme A sodium salt ACO: Acyl coenzyme A Oxidase AHMT: 4-amino-3-hydrazino-5-
Mercapto-1,2,4-triazole O.Dtest: Absorbance (Test) O.Dblank: Absorbance (Blank) (1) Reagent Buffer: 0.1M phosphate buffer (PH7.5) 8% methanol catalase (1600U/ ml),
10mMEDTA ATP.Mg solution: ATP-Na ₂ 50mg/ml, MgCl ₂ .
6H ₂ O 20mg/ml CoA solution: CoA・Na ₂ 6mg/ml ACO solution: Acyl coenzyme A oxidase
2U/ml Stop solution: 2N-KOH Color development solution A: 0.6% AHMT (0.5N HC1) Color development solution B: 0.75% KIO ₄ (0.2N KOH) Substrate solution: Sodium palmitate (1% Triton X-100) (2 ) Procedure 1 Prepare the following reaction mixture and prewarm it at 37°C for 5 minutes. Buffer solution: 0.25ml ATP/Mg solution: 0.55ml CoA solution: 0.05ml ACO solution: 0.05ml Enzyme solution: 0.05ml 2 Add 0.1ml of substrate solution and react for 10 minutes at 37℃, then add 0.5ml of stop solution and coloring solution. Add 0.5 ml of A to stop the reaction. 3 After leaving at 37℃ for 5 minutes, add coloring solution B to develop color. 4 After 10 minutes at room temperature, absorbance at O.D550 (O.
Dtest). 5. Repeat the same procedure using 1% Triton x 100 solution instead of the substrate solution as a blank, and measure the absorbance (OD blank). (3) Calculation formula U/ml=△OD(O.Dtest−O.Dblank)×2.05/36.7×
10 x 0.05 x dilution factor = △OD Unit. Next, the present invention will be explained using an example. Example 1 1 mM each of the following nucleotides or nucleosides, bases, and sugars were added to a 50 mM phosphate buffer (PH7.5) containing ACS (originating from Pseudomonas). After treating these solutions at 45°C for 30 minutes, ACS activity was determined. The results are shown in Table 1.

[Table] As is clear from Table 1, the inclusion of the stabilizer of the present invention significantly stabilizes the ACS activity. Example 2 50mM phosphate buffer (PH
7.5) to which various amounts of 5'-ATP were added. After treating these solutions at 25°C for 19 hours, ACS activity was measured. The results are shown in Table 2.

[Table] As is clear from Table 2, 5′- of 0.1mM or more
Addition of ATP significantly stabilizes ACS. Example 3 The following nucleotides, nucleosides, bases, and sugars were added to a 50 mM phosphate buffer PH7.5 containing ACS (originating from Pseudomonas) and 20% sucrose. After processing these solutions at 45℃ for 30 minutes,
ACS activity was measured. The results are shown in Table 3.

[Table] Abbreviations are the same as above.
As is clear from Table 3, when (nucu)reotide and polyhydric alcohol are used together, even more improved stabilization of ACS activity can be seen. Example 4 1% sucrose and
1mMATP was added to adjust the solid content to approximately 3%, and the mixture was freeze-dried. Next, storage stability was examined at 40°C.

[Table] Comparative Example 1 Various CoA was added in the same manner as in Example 2, and the temperature was heated at 25°C.
After 19 hours of treatment, ACS activity was measured. The results are shown in Table 5.

[Table] As is clear from Table 5, CoA has no stabilizing effect regardless of its concentration. Example 5 1mM 5′- in 50mM phosphate buffer PH7.5 containing ACS (originating from Pseudomonas) and 20% of various sugars.
ATP was added. After treating these solutions at 45°C for 30 minutes, ACS activity was measured. The results are shown in Table 6.

【table】

Claims (1)

[Claims] 1. A solution containing acyl coenzyme A synthetase containing 5'-adenosine triphosphate, 5'-adenosine diphosphate, 5'-guanosine triphosphate, 5'-guanosine diphosphate, and salts thereof. 1. A method for stabilizing acyl coenzyme A synthetase, which comprises containing 0.1 mM to 0.1 M of one or more compounds selected from the group, or freeze-drying the solution.