JPS5925684A - Stable p-hydroxybenzoic acid hydroxylase composition - Google Patents

Abstract

PURPOSE:To stabilize p-hydroxybenzoic acid hydroxylase (HBH), by adding flavine-adenine dinucleotide and/or one or more compounds selected from sugars, sugar alcohols, amino acids or their salts to HBH. CONSTITUTION:HBH is stabilized by adding flavine-adenine dinucleotide and/or one or more compounds selected from sugars, sugar alcohols, amino acids and their salts to HBH. HBH is p-hydroxybenzoate hydroxylase belonging to flavine- participating atomic oxygen-addition enzymes requiring external electron donor, and is obtained from bacterial especially belonging to Pseudomonas genus. The reaction of the enzyme is shown in the formula.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides power hydroxybenzoic acid hydroxylase (hereinafter referred to as I
I B II). More specifically, HBHK fufuvin adenine dinuclemeted (hereinafter abbreviated as FAD) and/-makase sugar, sugar alcohol,
This reagent composition stabilizes HBH by containing one or more compounds selected from the group consisting of amino acids or salts thereof.

The IIBH described here is a power hydroxybenzoate hydroxylase (fW% 4th grade E C
, 1, 14, 13, 2). This enzyme is bacteria,
It is especially obtained from the genus Pseudomonas.

In the literature, Pseudomonas desmolitii [Yano.

K, + Higash i, N-+ Arima
, K, + :Biochem, Biophys,
Res.

Co+nmun1341 (1969)) Pseudomonas furmeretse:ysu(I(owelL L,
G, +5pector+i', , R(asse
y+V, :J.

Biol, Chem1247+4340 (1972
):] Pseudomonas buchida [)Iosokawa
+ K,, 5tanier + I also Y, :J, Biol
, Chem, +241.2453 (1966))
and so on.

The reaction of this enzyme is as follows.

Recently, in the field of clinical diagnostic reagents, there have been expectations for the development of a measuring reagent for measuring serum 1-phosphate esterase using this enzyme. In other words, the measurement values of conventional choline interase vary depending on the measurement method, and there is no standard method, so measurement methods using this enzyme are attracting attention as meeting these expectations.

This enzyme also exhibits interesting properties such as the formation of an enzyme-substrate complex and the accompanying activation of reduction of enzyme-bound FAD by NADPH, making it a useful enzyme for research. Therefore, in order to meet these news demands, it has become necessary to provide stable and highly purified 118H.

The present inventors have arrived at the present invention after conducting various intensive studies on stabilizers for HB II (HB II).That is, the present invention has been developed by combining FAD and/or sugar, sugar alcohol,
This is a stable HI3H composition characterized by containing one or more compounds selected from the group consisting of amino acids or salts thereof.

The amount of FAD used in the present invention is HBHiooo
It is at least 0.001 kg per unit, preferably at least 0.1. There is no particular upper limit, but from an economic standpoint it is 100.
~ or less is sufficient.

Sugars used in the present invention include monosaccharides such as glucose, fructose, galoctose, xylose, ribose, and ribulose, and diamoses such as sucrose, lactose, and maltose. Sugar and lactose are particularly good.

Sugar alcohols include xylitol, mannitol,
Sorbitol etc. Mannitol is particularly preferred.

Examples of amino acids or their salts include hydrophilic temino acids and their salts such as glutamic acid, amepawagic acid, glutamine, asbawogin, lysine, histidine, arginine, glycine, and γ-aminobutyric acid; salts or hydrochlorides of IJum, potassium, ammonium, etc. There is. Particularly preferred are glutamic acid, amepawagic acid, or salts thereof.

The amount of sugar, monosaccharide alcohol, amino acid, or salt thereof can be selected from a wide range, but is between 1 and 10 per 1000 units of IIBH.
00~. A suitable freezing ratio is 5 to 100.

The method for measuring the activity of Buff Hydroquine benzoate hydroxylase is as follows.

Tris, Mat buffer 50mM pH8
, 2 rose hydroxybenzoic acid 0.5mMNAD
PHO, 3mM FAD O, 02mM reaction solution 3.0ml (37℃) and enzyme dilution solution 50μ! is added and the decrease in absorbance at +340 nm is measured. The amount of enzyme that changes the NADPH of lμtnole per minute is defined as 1 unit. (Molecular extinction coefficient of N A D P H 6.226 A/micromole) There is no particular restriction on the method of blending the stabilizer of the present invention.

For example, there are methods such as adding a stabilizer to a cargo solution containing II B H, adding HB H to a buffer solution containing a stabilizer, or adding HB H and a stabilizer to a buffer solution at the same time. be. The buffer may be phosphate buffer, Tris buffer, or any other buffer used in biochemistry.

In the present invention, one or more compounds selected from the group consisting of FAD and/or sugar, sugar alcohol, or a salt thereof are blended.

Enzyme stability, especially enzyme stability after freeze-drying, is significantly improved compared to the case without additives. Moreover, the appearance after freeze-drying becomes even more excellent. Furthermore, with the stabilizer of the present invention, hygroscopicity is significantly promoted even if the enzyme is stabilized using other compounds (potassium gluconate, tripotassium citrate), whereas hygroscopicity remains unchanged or is improved. Ru.

The stabilizer of the present invention includes a compound selected from the group consisting of sugar, sugar alcohol, amino acid or salt thereof, and FAD.
When used in combination, long-term stabilization is achieved.

The present invention will be explained below using a specific example.

Phosphate buffer 50mM pH 6.0 Each stabilizer 10~/ls1 or more compositions were prepared and freeze-dried in duplicate. After freeze-drying, 2 wl of distilled water was added once again, and the residual activity was measured. The results are shown in Table IK.

Table 1 (The φ mark indicates a comparative example.) Example 2 The same operation as in Example 1 was carried out to examine the temperature stability at 45° C. after freeze-drying. The results are shown in Table 2.

Table 2 Residual activity (%) at 45°C Rose hydroxybenzoic acid hydroxylase 20~/
Solution: Fiobin adenine dinucleotide (FAD) 0
．． 1~/1gl phosphate buffer 50mM pH
6.0 each stabilizer 1
Compositions of 0 to /yx1 or more were prepared and 2 ml of each was freeze-dried. After freeze-drying, temperature stability at 45°C was examined. The results are shown in Table 3.

Table 3 Patent applicant: Toyobo Co., Ltd.

Claims (1)

[Claims] Buff-hydroxybenzoic acid hydroxylase. fibrobin adenine dinucleotide and/or sugar,
A stable rose-hydroxybenzoic acid hydroxylase composition comprising one or more compounds selected from the group consisting of sugar alcohols, amino acids, or salts thereof.