JPS5914788A - Stabilization of l-alpha-glycerol-3-phosphate oxidase - Google Patents

Abstract

PURPOSE:To improve the stability of L-alpha-glycerol-3-phosphate oxidase in a buffer solution, remarkably, by adding flavin adenine dinucleotide and/or a chelating agent. CONSTITUTION:A buffer solution containing L-alpha-glycerol-3-phosphate is incorporated with flavin adenine dinucleotide or a chelating agent such as ethylenediaminetetraacetic acid, ethylene glycol ether diamine tetraacetic acid, etc. or their mixture, to obtain a buffer solution having a pH of 5.0-9.0, flavine adenine dinucleotide concentration of 0.01-50mM/1 and chelating agent concentration of 0.1-200mM/1.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for stabilizing L-α-glycerol-3-phosphate oxidase (hereinafter abbreviated as GPO); (hereinafter abbreviated as FAD) or a chelating agent,
This relates to a method of stabilizing GP□0 by adding each one alone or as a mixture of the two. That is, neutral fats are hydrolyzed by riboprotein lipase or the like, and the resulting glycerol is treated with glycerol kinase in the presence of adenosine tertiary acid (ATP) and Mg2+ ions.
Adenosine diphosphoric acid (ADP) and chrysero-ru-3-phosphate are obtained. GP to this glycerol-3゜phosphate
The hydrogen peroxide generated by the action of O is introduced into the peroxidase-4-aminoantipyrine brocade system, and neutral fats are determined by colorimetric determination of the quinone pigment to be removed. The reaction route (reaction route) is shown as follows.

Glycerol + 3 fatty acids゛ Glycerol kinase glycerol + ATP-Mg2+ Glycerol-6゛-phosphate + ADP-Mg”H2O2
+ dihydroxyacetone phosphate Hw (h + 4-7
□Senoantibilin + phenol, etc. 2! The bottom is 7. .

In the conventional method, for example, glycerol obtained by alkaline saponification of neutral fats is oxidized with periodic acid, and the resulting formaldehyde is colored with chromotropic acid or acetylacetone, which is then used for colorimetric determination. However, this method requires preliminary treatment such as solvent extraction to avoid the influence of contaminants, and suffers from the drawbacks of complicated operations and long measurement times.

This drawback was temporarily solved by using an enzyme reaction. That is, the enzyme reaction has high substrate specificity, and ordinary contaminants do not act as a deterrent, so rapid measurement is possible without the need for pretreatment such as sample purification. □
However, on the other hand, it has a major drawback in that the enzymes used are extremely unstable in solution, and new solutions are needed.

Like general enzymes, GPO lacks stability, and when used in an automatic analyzer, the reagent solution needs to be replaced frequently, leading to wasted reagents and the automatic analyzer's functions cannot be fully utilized.

As a result of intensive studies to overcome such difficulties, the inventors of the present invention found that the stability of GPO solutions can be significantly improved by adding FAD or a chelating agent, either alone or as a mixture of the two. The present invention was completed based on this discovery. That is, the present invention is characterized in that FAD or Ichimon Yotata is added to a buffer containing GPO, either alone or as a mixture of the two.
This is related to the stabilization of O.
.

The fact that FAD is a coenzyme of G'PO has been confirmed in the literature (for example, Li, L., Koditcheck, W.
W., Umbreit.

J, of Bacteriology, 98 1063 (
This is a well-known fact described in 1969)), but FA
It was unexpected that the addition of D would stabilize an aqueous solution of GPO over a wide temperature range for a long period of time. Furthermore, it was completely unexpected that the addition of a chelating agent would be effective in stabilizing GPO.

If the method of the present invention is applied to an automatic analyzer, enzymes can be used continuously over a long period of time, and significant improvements can be expected in terms of efficiency and cost.

The present invention consists of 1. Applicable to GPOs of any origin. Furthermore, the FAD used in the present invention may be of any origin.
Examples thereof include ethylenediaminetetraacetic acid (EDTA), ethylene glycol ether diaminetetraacetic acid (EGTA), and the like.

The preferred pH range of the buffer used in the present invention is 5,
Ov9.0.・Also, the concentration of FAD is 9.01~
50mM/11 preferably 0.05-5mM/l-.

The concentration of the chelating agent is 0.1 to 200 mM/l, preferably 110.5 to 10 mM/l.

However, the enzymatic activity of GPO used in the present invention can be measured, for example, by the method described below.

Veroxidase 1-ase 0. , 58 u/d, 4.
-A, containing 0.25 mM minoantipyrine, 25 mM phenol and 1.5 mM magnesium acetate.
pH7.5 0.1M/l) Liss-HCl buffer 0.5dK
Add sample 3CVpt, mix and preheat at 37°C for 1 to 2 minutes. Then, 8
Add 100 pt of 0mM/l glycerol-3-phosphate solution and react at 37°C for 1 to 3 minutes. The enzyme activity is determined by measuring the rate of increase in absorbance at 500 tm due to the quinone dye produced.

Next, the present invention will be explained in more detail by way of example.

Example 1 0.1M/l Heves buffer (1) H5.0-9.0)
Aerococcus viride 77 (Aerococc)
G P Q derived from us Viri-dans)
' k 2-3 u/-, and FAD (final concentration 0.1 mM/1) was added alone.
In addition, a test solution is prepared by mixing and adding EDTA (final concentration 21 mM/l). The solution was heated to 27℃! It was stored for 7 days and the residual activity was measured. Results first. Shown in the table.

Example, 2.

Aerococcus e viridides was added to 0.1 M/l Hepes buffer (pH 5.0-9.0).
Viri-dans) derived GPO from 2-3u/-
To this, FAD (final concentration 0
．． 1 mM/l) and EDTA (final concentration 32 m
M/L) are added individually or as a mixture of both to prepare a test solution. The solution was stored at 27°C for 6 days and the residual activity was measured. The results are shown in Table 2.

(Leaving space below) Table 1 *Test solution composition l is 0.1M/l Hepes buffer only ■ is F
When AD is added alone, FAD and EDTA are added. At pH 5.0 and pH 9.0, the pH value is somewhat unstable because the buffering effect of Hepes is weak.

Table 2 *Test solution composition 1jo, IM/l Heves buffer only ■ is F
AD added alone (■) EDTA added alone mV ll1 FAD and EDTA added pH 5
, 0 and pH 9, 0, the buffering effect of Hepes is weak, so the pH value is somewhat unstable.

Claims (1)

[Claims] L-α-L-α-IJ cello-3-phosphate oxidase-containing buffer solution containing flavin adenine dinucleotide or a chelating agent, respectively (or a mixture of both) - A method for stabilizing chrysero-ru-3-phosphate oxidase.