JP3219181B2 - Stabilization method of cholesterol oxidase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for stabilizing cholesterol oxidase. It relates to the stabilization method.

[0002]

2. Description of the Related Art Cholesterol oxidase is an enzyme used clinically for measuring cholesterol in body fluids, which is used as an index for diagnosis of endocrine diseases and metabolic disorders. Conventional methods for stabilizing cholesterol oxidase include the addition of alkali metal salts and alkaline earth metal salts (Japanese Patent Publication No. 57-13272), the addition of bovine serum albumin, dextran, and polyethylene glycol (JP-A-6-6284).
No. 6) is known. However, the addition of these substances is not sufficient for stabilization, and there are problems such as a decrease in the activity of the enzyme agent during storage and generation of turbidity.
In particular, there was no description for turbidity, and no fundamental solution had been made.

[0003]

SUMMARY OF THE INVENTION The present invention relates to a method for stabilizing cholesterol oxidase, and an object of the present invention is to provide a stable enzyme agent which does not decrease its enzymatic activity even after long-term storage and does not produce turbidity. Is to do. The term “stabilization” as used herein refers to two points, namely, retention of enzyme activity and suppression of turbidity generation from the enzyme agent due to denaturation.

[0004]

According to the present invention, a solution containing cholesterol oxidase is added with at least one compound selected from the group consisting of (1) bovine serum albumin and (2) saccharides and amino acids. Is a method for stabilizing cholesterol oxidase.

[0005] The present invention is also a cholesterol oxidase preparation characterized by containing at least one compound selected from the group consisting of cholesterol oxidase, (1) bovine serum albumin and (2) saccharides and amino acids.

The present invention further provides a cholesterol esterase preparation, a cholesterol oxidase preparation containing (1) bovine serum albumin and (2) at least one compound selected from the group consisting of saccharides and amino acids, peroxidase, 4-aminoantipyrine and This is a reagent kit for measuring cholesterol comprising an aniline derivative or a phenol derivative and a buffer.

Further, the present invention is a method for stabilizing cholesterol oxidase, which comprises adding lysine to a solution containing cholesterol oxidase.

[0008] The present invention is also a cholesterol oxidase preparation containing cholesterol oxidase and lysine.

Further, the present invention is a reagent kit for measuring cholesterol comprising a cholesterol esterase preparation, a cholesterol oxidase preparation containing cholesterol oxidase and lysine, peroxidase, 4-aminoantipyrine and an aniline derivative or a phenol derivative and a buffer.

The cholesterol oxidase used in the present invention may be of any origin. For example, cholesterol oxidase produced by microorganisms such as Streptomyces, Corynebacterium, Brevibacterium and Nocardia is used. is there. A cholesterol oxidase-containing culture obtained by culturing these microorganisms, a crude enzyme obtained by extracting the culture by a conventional method,
Alternatively, a purified enzyme obtained by purifying the crude enzyme by an ordinary method is suitably used. Furthermore, enzymes obtained by genetically modifying these enzymes or chemically and physically are also included.

The method of compounding the stabilizer of the present invention is not particularly limited. For example, there are a method of adding a stabilizer to a buffer containing cholesterol oxidase, a method of adding cholesterol oxidase to a buffer containing a stabilizer, and a method of adding cholesterol oxidase and a stabilizer simultaneously to the buffer. In the present invention, it is preferable to freeze-dry the enzyme solution containing the stabilizer. The lyophilization conditions are not particularly limited.

The buffer used in the present invention may be any of a phosphate buffer, a good buffer and other buffers used in biochemistry, but the pH is 4 to 10, preferably 6.0 to 8.0. is there.

The concentration of bovine serum albumin used in the present invention is 0.1% based on the cholesterol oxidase solution.
~ 20%. However, bovine serum albumin alone,
As shown in Table 1 below, after storage at 37 ° C. for one week, only 88% residual activity was observed, and a sufficient stabilizing effect was not obtained. Also, the formation of turbidity was remarkable.

The saccharides used in the present invention include monosaccharides such as glucose, galactose, xylose and fructose, disaccharides such as lactose and maltose, and polysaccharides such as trehalose, sucrose and gluconic acid.
Sucrose, glucose, trehalose, lactose, sodium gluconate and mannitol are particularly preferred. The concentration is 0.1 to 20% based on the cholesterol oxidase solution. As shown in Table 1 below, the saccharide alone could not obtain a sufficient stabilizing effect when stored at 37 ° C. for one week, and was remarkable in the formation of turbidity. The saccharides may be used alone or in combination of two or more.

The amino acids or salts thereof used in the present invention include hydrophilic amino acids such as glycine, glutamic acid and lysine, and salts, acetates and hydrochlorides thereof such as sodium, potassium and ammonium. Salts and lysine hydrochloride are preferred. The concentration is 0.1 to 20% based on the cholesterol oxidase solution. As shown in Table 1 below, when amino acids other than lysine alone were stored at 37 ° C. for one week, a sufficient stabilizing effect was not obtained, and formation of turbidity was remarkable. Lysine alone is excellent in stabilizing effect and turbidity formation. One or two amino acids
A combination of more than one species may be used.

In a preferred embodiment of the present invention, a solution containing cholesterol oxidase is added with at least one compound selected from the group consisting of (1) bovine serum albumin and (2) sugars and amino acids, and further lyophilized. This is a method for stabilizing cholesterol oxidase.

In another preferred embodiment of the present invention, lysine is added to a solution containing cholesterol oxidase,
Further, it is a method for stabilizing cholesterol oxidase which is freeze-dried.

In another preferred embodiment of the present invention,
A cholesterol oxidase solid preparation containing at least one compound selected from the group consisting of cholesterol oxidase, (1) bovine serum albumin, and (2) sugars and amino acids.

Still another preferred embodiment of the present invention is:
It is a cholesterol oxidase solid preparation containing cholesterol oxidase and lysine.

The reagent kit for measuring cholesterol of the present invention measures hydrogen peroxide generated by the action of cholesterol oxidase by various known methods. The most commonly used method is the method of measuring peroxidase. In the presence, a method of oxidizing a chromogen, condensing it with a coupler such as 4-aminoantipyrine to form a dye, and performing colorimetric determination. Chromogens used in this method include phenol, 2-chlorophenol, and 4-chlorophenol.
Phenol derivatives such as chlorophenol and 2,4-dichlorophenol, or aniline, N, N-dimethylaniline, N, N-diethylaniline, N, N-diethyl-m-toluidine, N, N-diethyl-m-anisidine , N-ethyl-N- (3-methylphenyl) -N '
-Acetylethylenediamine, N-ethyl-N- (2-
(Hydroxy-3-sulfopropyl) -m-anisidine;
Aniline derivatives such as N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine, N-ethyl-N-sulfopropyl-m-toluidine, N-ethyl-N-sulfopropyl-m-anisidine Is mentioned.
The reagent for measuring hydrogen peroxide is not limited to the above reagent.

[0021]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail with reference to embodiments. Example 1 Cholesterol oxidase solution (2,000 U / ml)
17.2 mg of bovine serum albumin (hereinafter abbreviated as BSA; manufactured by Organotechnica) and one kind selected from the group consisting of sucrose, glucose, trehalose, lactose, sodium gluconate, mannitol, sodium glutamate, and glycine,
Each 14.7 mg was added, mixed and freeze-dried to obtain 50 mg of a freeze-dried powder. Similarly, a lyophilized powder was obtained with or without the addition of each additive alone.

The cholesterol oxidase freeze-dried powder to which bovine serum albumin and sugars or amino acids obtained by the above-mentioned method are added, and BSA 1
A freeze-dried powder to which only 7.2 mg was added and 14.7 mg of only a saccharide or an amino acid was added, and a freeze-dried powder and a freeze-dried cholesterol oxidase powder without additives were compared as follows. The freeze-dried powder was stored at 37 ° C. for one week, and the residual activity thereafter was measured. Moreover, after saving the powder, as excessive testing, so that the powder in 100 U / ml, dissolved in 50 mM PIPES buffer (pH 7.0), The solution was maintained 37 ° C., the formation of turbidity OD ₆₆₀ And turbidity was visually observed. Table 1 and Table 2 show the results.
Shown in

[0023]

[Table 1]

[0024]

[Table 2]

The freeze-dried stabilized cholesterol oxidase powder of the present invention, when stored at 37 ° C., maintains a sufficient activity for one week and improves the formation of turbidity to a practically acceptable level. I confirmed that it was done. On the other hand, freeze-dried cholesterol oxidase powder without additives has a high activity after storage at 37 ° C for one week.
Only 60% remained, and significant turbidity was observed. The lyophilized powder to which only BSA was added had a residual activity of 88%, but marked turbidity was observed.

Example 2 1 ml of a cholesterol oxidase solution (2,000 U / ml)
Lysine was added and mixed with 14.7 mg of lysine, and lyophilized to obtain 50 mg of lyophilized powder. Similarly, 17.2 mg of BSA and 14.7 mg of lysine were added to 1 ml of a cholesterol oxidase solution (2,000 U / ml), and the mixture was lyophilized to obtain 50 mg of a lyophilized powder. These freeze-dried powders were stored at 37 ° C. for one week, and the residual activity thereafter was measured. Moreover, after saving the powder, as excessive testing, so that the powder in 100 U / ml, dissolved in 50 mM PIPES buffer (pH 7.0), The solution was maintained 37 ° C., the formation of turbidity OD ₆₆₀ And turbidity was visually observed. Table 3 shows the results.

[0027]

[Table 3]

[0028]Example 3 1 ml of cholesterol oxidase solution (2,000 U / ml)
21.2 mg of BSA (manufactured by Organotechnica) and 16.8 mg of lysine
(Lysine 1) or 33.6 mg (lysine 2) is added and mixed,
Lyophilization was performed to obtain a lyophilized powder. The choles obtained
Keep freeze dried terol oxidase powder at 37 ° C for 2 weeks
And the remaining activity thereafter was measured. Also save the powder
After that, 50mMPIPES buffer (pH7.0) to 100U / ml
 , And the solution is kept at 37 ° C.
₆₆₀Was measured. Table 4 shows the results.

[0029]

[Table 4]

The cholesterol oxidase stabilized according to the present invention, when stored at 37 ° C., maintains sufficient activity for two weeks, and the formation of turbidity has been improved to a practically acceptable level. It was confirmed.

Example 4 1 ml of cholesterol oxidase solution (2,000 U / ml)
BSA 18.6mg and lysine 14.8mg or sucrose 14.
8 mg was added and mixed, and 18.6 mg of BSA and 14.8 mg of lysine and sucrose were added and mixed, respectively, and lyophilized to obtain a lyophilized powder.

The resulting freeze-dried cholesterol oxidase powder was stored at 37 ° C. for one week, and the residual activity thereafter was measured. After storing the powder, the powder was dissolved in 50 mM PIPES buffer (pH 7.0) to a concentration of 100 U / ml.
It was kept at 37 ° C. and the formation of turbidity was measured at OD ₆₆₀ . Table 5 shows the results.

[0033]

[Table 5]

The cholesterol oxidase stabilized according to the present invention, when stored at 37 ° C., maintains a sufficient activity for one week, and its turbidity has been improved to a practically acceptable level. It was confirmed.

Comparative Example 1 1 ml of cholesterol oxidase solution (2,000 U / ml)
29 mg (0.5 M) of sodium chloride, 17.5 mg (0.3 M),
8.8 mg (0.15 M) was added, mixed and freeze-dried to obtain a freeze-dried powder. The obtained lyophilized powder was stored at 37 ° C. for two weeks, and the remaining activity thereafter was measured. After storing the powder, add 50 mM PIPES buffer (pH 7.
0), the solution was kept at 37 ° C., and the formation of turbidity was visually judged. Table 6 shows the results.

[0036]

[Table 6] As described above, formation of turbidity is remarkable in alkali metals.

[0037]

According to the present invention, by adding lysine, a cholesterol oxidase preparation excellent in turbidity as well as stabilizing effect can be obtained. Bovine serum albumin, saccharides, and amino acids are conventionally known as stabilizers for various enzymes. However, in the present invention, by combining them, more excellent stability against heat than when used alone is obtained. The effect is seen. Further, in the present invention, a stable enzyme preparation having a very high effect of suppressing the generation of turbidity can be obtained by the combination.
Therefore, it has an advantage that it can be used as a biochemical reagent or the like without pretreatment.

Continued on the front page (72) Inventor Shigenori Aizumi 10-24, Toyo-cho, Tsuruga-shi, Fukui Prefecture Toyobo Co., Ltd. Examiner at Tsuruga Bio-Research Laboratory Natsuko Homma JP-A-55-340001 (JP, A) JP-A-60-224499 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12Q 1/00-3/00

Claims (6)

(57) [Claims] 1. A method for stabilizing cholesterol oxidase, comprising adding (1) bovine serum albumin and (2) lysine to a solution containing cholesterol oxidase. 2. A solution containing cholesterol oxidase, comprising adding (1) bovine serum albumin and (2) lysine,
The method for stabilizing cholesterol oxidase according to claim 1, further comprising freeze-drying. 3. The method according to claim 1, wherein 0.1 to 20% of bovine serum albumin and 0.1 to 20% of lysine are added to the cholesterol oxidase solution. 4. A cholesterol oxidase preparation comprising cholesterol oxidase, (1) bovine serum albumin and (2) lysine. 5. The cholesterol oxidase preparation according to claim 4, which is a solid preparation. 6. A cholesterol esterase preparation, (1)
A cholesterol measurement reagent kit comprising a cholesterol oxidase preparation containing bovine serum albumin and (2) lysine, peroxidase, 4-aminoantipyrine and an aniline derivative or a phenol derivative, and a buffer.