JPH06284886A - Stabilization of enzyme in solution - Google Patents

Abstract

PURPOSE:To stabilize an enzyme in a solution. CONSTITUTION:An enzyme comprising peroxidase, cholesterol esterase, lipoprotein lipase, glycerol kinase, malate dehydrogenase, bilirubin oxidase, glucose dehydrogenase, lactate dehydrogenase and glucose oxidase is combined with a stabilizing agent comprising arginine, aspartic acid, tryptophan, sodium chloride, glycine, sodium sulfate, mannitol, sorbitol, phosphoric acid salt, calcium salt, magnesium salt, succinic acid salt, malic acid salt, tartaric acid salt and glycolic acid salt.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for stabilizing an enzyme in a solution. More specifically, it relates to a method for stabilizing an enzyme in a solution by using a specific stabilizer for various enzymes.
In particular, it relates to a method for stabilizing an enzyme with various liquid reagents used for biochemical measurement in the field of clinical examination.

[0002]

2. Description of the Related Art Since an enzyme can carry out a catalytic reaction under mild conditions, the enzyme has been actively used in recent years in the fields of clinical examination, biochemistry, food industry and the like. Particularly in various biological component measurement kits used in the field of clinical testing, conventionally, a lyophilized reagent containing an enzyme is dissolved in an appropriate buffer solution to form an enzyme solution, and the biological component is measured by a manual method or an autoanalyzer. Was there. In order to enhance the stability of the enzyme in the enzyme-containing reagent obtained by the dissolution, various additives have been studied so far.

For example, a method of adding amino acids or proteins such as sodium glutamate, albumin and skim milk, sugars such as sucrose and maltose, reducing agents such as glutathione and mercaptoethanol, and polyhydric alcohols such as glycerol and sorbitol to an enzyme solution, Alternatively, there is a stabilization method (Japanese Patent Laid-Open No. 64-60380) in which a water-soluble polymer such as dextran is oxidized and then converted into an aldehyde with a reducing agent, and an enzyme is covalently immobilized on the aldehyde.

[0004]

The biochemical test kits used in the field of clinical testing in recent years enable rapid measurement because of the conventional method in which a freeze-dried enzyme reagent is dissolved before use. Thus, liquid supply has been desired. However, there is a problem that the enzyme used for clinical examination has a high degree of purification and its stability is extremely low in a dilute state such as used for measurement.

Various enzymes are used as the enzymes used in clinical tests depending on the intended test items, and it has been desired to provide preferable stabilizers for each enzyme.

[0006]

[Means for Solving the Problems] Therefore, the present inventors examined various stabilizers for stabilizing various enzymes in a solution, and screened a stabilizer suitable for each enzyme. The present invention has been completed by solving the problems by combining each specific enzyme with each specific stabilizer.

That is, the present invention provides a method for stabilizing an enzyme in a solution using a stabilizing agent, which is a combination of the enzyme and the stabilizing agent shown below, in the solution. It is a thing.

The enzyme is peroxidase or cholesterol esterase and the stabilizer is magnesium salt. The enzyme is glycerol kinase, and the stabilizing agent is at least one of mannitol, sorbitol, sodium sulfate and glycine. The enzyme is malate dehydrogenase and the stabilizer is arginine and / or magnesium salt. The enzyme is bilirubin oxidase and the stabilizer is aspartic acid and / or tryptophan. The enzyme is glucose dehydrogenase and the stabilizer is at least one of sodium chloride, glycine, arginine, and sodium sulfate. The enzyme is lactate dehydrogenase and the stabilizer is any one of sodium sulfate, phosphate, magnesium salt, succinate, malate, tartrate and glycolate 1
More than seeds. The enzyme is glucose oxidase and the stabilizer is at least one of arginine, sodium sulfate, calcium salt, and aspartic acid. The enzyme is lipoprotein lipase and the stabilizer is magnesium salt and / or calcium salt.

Next, each enzyme and stabilizer will be described in detail. Each of the enzymes mentioned above is an International Union of Biochemistry (IU
B) According to the classification of the enzyme committee, the classification is shown after the following enzyme names. That is, peroxidase (EC 1.11.1.
7), cholesterol esterase (EC 3.1.1.13), lipoprotein lipase (EC 3.1.1.34), glycerol kinase (EC 2.7.1.30), malate dehydrogenase (EC 1.1.1.38), bilirubin oxidase (EC 1.3.3.
5), glucose dehydrogenase (EC 1.1.1.47), lactate dehydrogenase (EC 1.1.1.27) and glucose oxidase (EC 1.1.3.4).

These enzymes need to be purified to the extent that they do not interfere with the measurement in order to be used in clinical tests, but the method of the present invention is applied only to the field of clinical tests. Instead, it need only be refined to the extent required for the field.

As the stabilizer, arginine, aspartic acid, tryptophan, sodium chloride, glycine,
Mannitol, sorbitol, sodium sulfate, phosphate (eg, monopotassium phosphate, etc.), calcium salt (eg, calcium chloride etc.), magnesium salt (eg,
Magnesium chloride etc.), succinate (eg sodium succinate etc.), malate (eg sodium malate etc.), tartrate (eg calcium tartrate etc.),
Glycolate (for example, sodium glycolate)
And commercially available reagents can be used for each.

[0012] The amount of each stabilizer added to various enzymes varies depending on the degree of purification of the target enzyme and other components, but is usually 0.01% to 10% in the solution.

By adding a stabilizer to each enzyme as described above, the enzyme solution is stabilized and can be used for clinical examination for a long period of time. In order to stably store the stabilized enzyme solution for a longer period of time, it is preferable that the temperature is low enough not to freeze, but the enzyme solution stabilized by the present invention can be stored at room temperature and It is also possible to provide an enzyme solution that is stable for several months at room temperature. Hereinafter, the present invention will be described in detail with reference to Experimental Examples and Examples.
The present invention is not limited to these.

[0014]

【Example】

Example 1 The stabilizing effect of addition of magnesium salt on peroxidase and cholesterol esterase was measured. Buffer solution 50 mM PIPES buffer solution (pH 6.5) 0.1% Triton X-100 0.02% Sodium dehydroacetate 0.02% Sodium azide 1 mM EDTA enzyme peroxidase (PO "Amano" III: Amano Pharmaceutical Co., Ltd.) (hereinafter referred to as PO) added Unit 2 u / ml Cholesterol esterase (CHE "Amano" II: manufactured by Amano Pharmaceutical Co., Ltd.) (hereinafter referred to as CHE) Addition unit 1 u / ml Storage temperature 10 ° C and 37 ° C Storage period 90 days or more The results are shown in Table 1.

[0015]

[Table 1]

As is clear from Table 1, addition of magnesium chloride increases the solution stability of each enzyme.

Example 2 The stabilizing effects of mannitol, sorbitol, sodium sulfate and glycine on glycerol kinase were measured. Buffer solution 50 mM PIPES buffer solution (pH 6.5) 0.1% Triton X-100 0.02% Sodium dehydroacetate 0.02% Sodium azide 1 mM EDTA 1 mM ATP enzyme Glycerol kinase (GK "Amano" II: Amano Pharmaceutical Co., Ltd.) (hereinafter referred to as GK ) Addition unit 0.5 u / ml Storage temperature 10 ℃ and 30 ℃ Storage period 90 days or more results are shown in Table 2.

[0018]

[Table 2]

As is clear from Table 2, mannitol,
The solution stability of GK is increased by the addition of sorbitol, sodium sulfate and glycine.

Example 3 The stabilizing effect of the addition of arginine and magnesium salts on malate dehydrogenase was measured. Buffer 80 mM Tris buffer (pH 7.8) 0.1% Triton X-100 0.02% Sodium dehydroacetate 0.02% Sodium azide 1 mM EDTA 200 mM L-Aspartic acid 0.16 mM β-NADH enzyme malate dehydrogenase (MDH "Amano" III: Amano Pharmaceutical Co., Ltd. (hereinafter referred to as MDH) Addition unit 1 u / ml Storage temperature 10 ° C and 37 ° C Storage period 90 days or more The results are shown in Table 3.

[0021]

[Table 3]

As is clear from Table 3, the solution stability of MDH is increased by the addition of arginine and magnesium chloride.

Example 4 The stabilizing effect of addition of aspartic acid and tryptophan on bilirubin oxidase was measured. Buffer solution 50 mM BES buffer solution (pH 7.0) 0.1% Triton X-100 0.02% Sodium dehydroacetate 0.02% Sodium azide 1 mM EDTA enzyme Bilirubin oxidase (BO "Amano" II: Amano Pharmaceutical Co., Ltd.) (hereinafter referred to as BO) added Unit 3 u / ml Storage temperature 10 ℃ Storage period 20 days or more The results are shown in Table 4.

[0024]

[Table 4]

As is clear from Table 4, the solution stability of BO is increased by the addition of aspartic acid and tryptophan.

Example 5 The stabilizing effect of glucose dehydrogenase by the addition of sodium chloride, glycine, arginine and sodium sulfate was measured. Buffer 100 mM HEPES buffer (pH 7.8) 0.1% Triton X-100 0.02% Sodium dehydroacetate 0.02% Sodium azide 1 mM EDTA Enzyme Glucose dehydrogenase (GLUCDH "Amano" II: Amano Pharmaceutical Co., Ltd.) (hereinafter referred to as GLUCDH) Added Unit 30 u / ml Storage temperature 37 ℃ Storage period 60 days or more is shown in Table 5.

[0027]

[Table 5]

As is apparent from Table 5, the solution stability of GLUCDH is increased by the addition of sodium chloride, glycine, arginine and sodium sulfate.

Example 6 The stabilizing effect of lactate dehydrogenase by adding sodium sulfate, phosphate, magnesium salt, succinate, malate, tartrate and glycolate was measured.

Buffer solution B-1 100 mM Tris buffer solution (pH 7.5) 0.02% Triton X-100 0.02% Sodium dehydroacetate 0.02% Sodium azide 1 mM EDTA 500 mM L-alanine 0.16 mM β-NADH buffer solution B-2 100 mM Tris buffer (pH 8.5) 0.02% Triton X-100 0.02% Sodium dehydroacetate 0.02% Sodium azide 1 mM EDTA 500 mM L-alanine 0.16 mM β-NADH enzyme lactate dehydrogenase (LDH "Amano" III: Amano Pharmaceuticals) ( Hereinafter, referred to as LDH) Addition unit 1 u / ml (buffer solution A) Addition unit 5 u / ml (buffer solution B) Storage temperature 10 ° C and 30 ° C Storage period 90 days or more The results are shown in Tables 6 and 7. Buffer A-1 (prescription for GOT measurement)

[0031]

[Table 6]

Buffer solution B-1 (prescription for GPT measurement)

[0033]

[Table 7]

Buffer A-2 (prescription for GOT measurement)

[0035]

[Table 8]

Buffer B-2 (prescription for GPT measurement)

[0037]

[Table 9]

As is clear from Tables 6, 7, 8 and 9, LDH solution stabilization by addition of sodium sulfate, phosphate, magnesium salt, succinate, malate, tartrate and glycolate. Sex is increasing.

Example 7 The stabilizing effect of glucose oxidase by the addition of arginine, sodium sulfate, calcium salt and aspartic acid was measured. Buffer solution 50 mM PIPES buffer solution (pH 6.5) 0.1% Triton X-100 0.02% Sodium dehydroacetate 0.02% Sodium azide 1 mM EDTA enzyme Glucose oxidase (GO "Amano" II: Amano Pharmaceutical Co., Ltd.) (hereinafter referred to as GO) added Unit: 10 u / ml Storage temperature: 10 ℃ and 37 ℃: Storage period: 90 days or more The results are shown in Table 8.

[0040]

[Table 10]

As is clear from Table 10, arginine,
The solution stability of GO is increased by the addition of sodium sulfate, calcium salt and aspartic acid.

Example 8 The stabilizing effect of the addition of magnesium and calcium salts on lipoprotein lipase was measured. Buffer solution 50 mM PIPES buffer solution (pH 6.5) 0.1% Triton X-100 0.02% Sodium dehydroacetate 0.02% Sodium azide 1 mM EDTA enzyme lipoprotein lipase (LPL "Amano" VI: Amano Pharmaceutical Co., Ltd.) (hereinafter referred to as LPL) Addition unit 20 u / ml Storage temperature 10 ℃ and 37 ℃ Storage period 90 days or more results are shown in Table 1.

[0043]

[Table 11]

As is clear from Table 11, the solution stability of LPL is increased by adding magnesium chloride and calcium chloride.

INDUSTRIAL APPLICABILITY According to the present invention, an efficient method for stabilizing various enzymes in a solution becomes possible, and it can be expected that the use of enzymes will be expanded not only in clinical tests but also in biochemistry and food industry.

Claims (1)

[Claims] 1. A method for stabilizing an enzyme in a solution using a stabilizer, which is a combination of the enzyme and the stabilizer shown below. The enzyme is peroxidase or cholesterol esterase and the stabilizer is magnesium salt. The enzyme is glycerol kinase, and the stabilizing agent is at least one of mannitol, sorbitol, sodium sulfate and glycine. The enzyme is malate dehydrogenase and the stabilizer is arginine and / or magnesium salt. The enzyme is bilirubin oxidase and the stabilizer is aspartic acid and / or tryptophan. The enzyme is glucose dehydrogenase and the stabilizer is at least one of sodium chloride, glycine, arginine, and sodium sulfate. The enzyme is lactate dehydrogenase and the stabilizing agent is sodium sulfate, phosphate, magnesium salt, succinate, malate, tartrate or glycolate 1
More than seeds. The enzyme is glucose oxidase and the stabilizer is at least one of arginine, sodium sulfate, calcium salt, and aspartic acid. The enzyme is lipoprotein lipase and the stabilizer is magnesium salt and / or calcium salt.