JPH09313178A - Stabilization of enzyme and enzyme composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enable the extreme increasing of the stability of an enzyme and accordingly the preparation of a liquid stable agent from the enzyme by adding a glycoside to a cholesterol dehydrogenase and obtain an enzyme composition useful for clinical examination, etc., such as determining cholesterol in blood. SOLUTION: This liquid-type enzyme composition is obtained by dissolving a cholesterol dehydrogenase in an N-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer solution of 50mM and pH7.0 and adding n-dodecyl-β-D- maltoside, n-heptyl-β-D-thioglycoside, n-octyl-β-D-glycoside, n-octyl-β-D- thioglycoside, digitonin, sucrose monocaprate, 2-ethylhexylglycoside, etc., or cholic acid and/or its derivative as a glycoside to the resultant enzyme solution. The enzyme composition, whose enzyme component is stabilized, can be used for determining cholesterol in blood, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for stabilizing cholesterol dehydrogenase and a cholesterol dehydrogenase composition. More particularly, the present invention relates to a method for stabilizing cholesterol dehydrogenase and a stable cholesterol dehydrogenase composition, which are mainly intended for use in the field of clinical examination.

[0002]

2. Description of the Related Art Conventionally, many measuring methods using enzymes have been developed because of their excellent reaction specificity, reproducibility, and simple operation. Particularly in the field of clinical examination, many methods are known for measuring components in blood samples. But,
In general, enzymes have poor stability when used as reagents, and therefore various measures have been required. For example, it has been reported that the protein is modified to stabilize the enzyme α-glucosidase, and that glycerol is added to LDH as an organic solvent and acetone is added to benzyl alcohol dehydrogenase to stabilize the protein. . It is also known that urease is stabilized with an SH protecting agent such as dithiothreitol, and that lipase is stabilized by adding a metal ion such as Ca. However, these methods are often ineffective when different enzymes are used, and individual stabilization methods have been studied for each enzyme. In particular, in recent years, in order to improve the efficiency of daily work of clinical tests, the reagents used are often changed from conventional freeze-dried form to liquid form, and it is becoming more important to stabilize the enzyme in the reagents. . By the way, in the field of clinical examinations, lipid examinations have recently been increasing, and particularly, cholesterol has been attracting attention as a risk factor for adult diseases, and cholesterol examinations are increasing, including examination of fractionated components thereof. This cholesterol measurement is currently
A method using an enzyme is generally used, and a colorimetric measurement method in the visible part using cholesterol oxidase and an absorbance measurement method in the ultraviolet using cholesterol dehydrogenase are known. The former method requires a step of reacting hydrogen peroxide generated by an enzymatic reaction with a chromogenic substrate in the presence of peroxidase to lead to a quinone dye, which is complicated in operation. Further, there is a drawback that an error may occur due to bilirubin, ascorbic acid, etc. in the sample depending on the measurement conditions. On the other hand, the latter method produces N produced by enzymatic reaction of cholesterol with cholesterol dehydrogenase in the presence of NAD (P).
It measures the amount of AD (P) H, and has the advantage that it can be performed simply by measuring the change in the absorbance in the ultraviolet region, and is simple, and that the influence of impurities in the sample is small.

[0003]

As described above, the method of using cholesterol dehydrogenase is preferable as the method for measuring cholesterol, but the cholesterol dehydrogenase used in this method has been more difficult to measure in solution than before. Known as a stable enzyme. To stabilize the cholesterol dehydrogenase-containing solution, albumin, cholic acid or its derivatives have been added, but both have a slight effect on storage at low temperature, but are sufficiently stable as reagents. Has not been obtained. In addition, although there is a report of stabilizing crystallin by including it in a solution of a physiologically active protein (Japanese Patent Laid-Open No. 7-236483), when it is applied to cholesterol dehydrogenase, the effect of preventing cloudiness is recognized, but the activity of the enzyme is There was a problem maintaining. The present invention solves the above problems, and an object of the present invention is to stabilize cholesterol dehydrogenase and to contribute usefully to the field of clinical examination as a reagent.

[0004]

Means for Solving the Problems As a result of intensive studies, the present inventors have found that glycosides contribute to stabilization of cholesterol dehydrogenase, and further studies have shown that cholesterol dehydrogenase. It was found that the object of the present invention can be achieved by adding glycosides to the present invention, thus completing the present invention. That is, the gist of the present invention is a method for stabilizing a cholesterol dehydrogenase, which comprises adding a glycoside to the cholesterol dehydrogenase; and the cholesterol dehydrogenase described above, wherein cholate and / or a derivative thereof is added. A method for stabilizing cholesterol dehydrogenase; and a cholesterol dehydrogenase composition characterized by containing a cholesterol dehydrogenase and a glycoside;

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION Cholesterol dehydrogenases stabilized by the present invention are those which depend on nicotinamide adenine dinucleotide (NAD) as coenzymes and those which depend on nicotinamide adenine dinucleotide phosphate (NADP). Both can be targeted.

As the glycoside added to the cholesterol dehydrogenase in the present invention, any glycoside that meets the purpose of the present invention can be used. Particularly, the sugar moiety is preferably selected from glucose, fructose, mannose, galactose, sucrose, maltose and the like. Further, the non-sugar portion preferably has a hydrophobic structure, and examples thereof include expensive alcohols, fatty acids, steroids, terpenoids and the like. Specific examples of such glycoside include, for example, n-dodecyl-β-D-maltoside, n-heptyl-β-D-thioglucoside, n-octyl-β-D-glucoside, n-
Octyl-β-D-thioglucoside, digitonin, sucrose monocaprate, sucrose monolaurate, 2-ethyl-hexyl glucoside, n-octanoyl-N-methylglucamide, n-methylglucamide,
Examples thereof include n-nonanoyl-N-methylglucamide and n-decanoyl-N-methylglucamide, and two or more kinds may be used in combination.

In the present invention, the stability of cholesterol dehydrogenase can be further improved by adding cholic acid and / or its derivative. Any such cholic acid derivative can be used as long as it can achieve the object of the present invention. In particular, salts of cholic acid (eg sodium salt), deoxycholic acid or salts thereof (eg sodium salt), 3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonate (CHAPS) ), 3-[(3-cholamidopropyl)
Dimethylammonio] -2-hydroxy-1-propane sulfonate (CHAPSO), N, N-bis (3-D-gluconamidopropyl) cholamide (deoxy-BIGCHAP) are preferred. Cholic acid and its derivative can be used in combination.

The stabilizing effect of the glycosyl cholesterol dehydrogenase of the present invention has a remarkable effect in the liquid reagent of cholesterol dehydrogenase, but can also exert the effect in the freeze-dried reagent. In the case of a liquid reagent, it can be prepared by adding a glycoside to a cholesterol dehydrogenase solution dissolved in an appropriate buffer solution (Hepes buffer solution or the like). In the case of a freeze-dried reagent, it can be prepared by adding a glycoside to a cholesterol dehydrogenase solution and then freeze-drying it according to a conventional method. However, a glycoside may be added to the freeze-dried product. The glycoside may be added to both the solution before freeze-drying and the freeze-dried product.

The amount of the glycoside added to the cholesterol dehydrogenase in the present invention can be appropriately set depending on the type of glycoside, the cholesterol dehydrogenase content in the reagent, the storage condition of the reagent and the like. In the liquid reagent, for example, when n-dodecyl-β-D-maltoside is used, 0.01 to
50 mM is preferred, more preferably 0.1-20 m
M. When sucrose monolaurate is used, it is preferably 0.05 to 100 mM, more preferably 0.5 mM to 50 mM. The amount of glycoside added to the freeze-dried reagent can be set according to the above amount. The addition amount of cholic acid and / or its derivative is preferably 0.01 to 10%, more preferably 0.05 to 5%.

INDUSTRIAL APPLICABILITY The method and composition of the present invention are useful for preparing a cholesterol dehydrogenase-containing reagent used for measurement of blood cholesterol and the like. Conventionally, when cholesterol dehydrogenase is dissolved in a buffer solution, The residual activity disappeared after 1 month even when stored under refrigerated conditions, but the residual activity can be remarkably increased by adding a glycoside by the method of the present invention. The residual activity value varies depending on the type of glycoside to be added, but it can be made into a reagent by using the amount of enzyme predicted in advance from the residual activity value as the reagent amount.

[0011]

EFFECTS OF THE INVENTION According to the present invention, cholesterol dehydrogenase can be stabilized and can be made into a reagent, and in particular, a liquid reagent can be supplied. Therefore, conventionally,
Compared to the product that was freeze-dried because it could not be stabilized, a liquid reagent can be supplied, so that usability is significantly improved and it can contribute to the field of clinical testing.

[0012]

EXAMPLES Examples will be given below, but the present invention is not limited thereto. Example 1 unit / ml of cholesterol dehydrogenase was added to 50 mM,
N-2-hydroxyethylpiperazine having a pH of 7.0
Dissolve in N'-2-ethanesulfonic acid (HEPES) buffer. In addition, as a glycoside, n-dodecyl-β-D-
Maltoside, n-heptyl-β-D-thioglucoside,
n-octyl-β-D-glucoside, n-octyl-β
10 mM each of -D-thioglucoside, digitonin, sucrose monocaprate, sucrose monolaurate and 2-ethyl-hexyl glucoside are added and dissolved to obtain a sample. After refrigerated and left at 25 ° C. for a predetermined period of time, the residual activity of cholesterol dehydrogenase was determined by the following method. Table 1 shows the results. Table 1
As shown in, the residual activity is high when glycosides are added.

Method for measuring cholesterol dehydrogenase activity 1. Reagents Reagents having the following compositions were used. Substrate solution Cholesterol 0.1% Triton X-100 0.2% Coenzyme solution Trishydroxymethylaminomethane 300 mmol / l NAD 2 mmol / l 2. Assay method Mix 1.0 ml of coenzyme solution and 2.0 ml of substrate solution at 25 ° C.
To constant temperature. 50 μl of the sample is added to the constant temperature mixture and mixed well, and the amount of change in absorbance per minute at 340 nm is determined using purified water as a control. Reagent blank is determined using saline instead of sample. The enzyme activity immediately after the sample preparation was set to 100%, and the residual enzyme activity after storage was expressed as a relative value.

[0014]

[Table 1]

Claims (4)

[Claims] 1. A method for stabilizing cholesterol dehydrogenase, which comprises adding a glycoside to cholesterol dehydrogenase. 2. The method for stabilizing cholesterol dehydrogenase according to claim 1, wherein cholate and / or its derivative is added. 3. A cholesterol dehydrogenase composition comprising a cholesterol dehydrogenase and a glycoside. 4. The cholesterol dehydrogenase composition according to claim 3, wherein the composition is liquid.