JP2008206491A - METHOD FOR STABILIZING p-HYDROXYBENZOATE HYDROXYLASE - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To improve the storage stability of p-hydroxybenzoate hydroxylase and to enable long-term stability of enzyme product by selecting a stabilizer. <P>SOLUTION: The method for stabilizing p-hydroxybenzoate hydroxylase comprises the addition of at least one kind of α-cyclodextrin, inositol, sucrose, sorbitose, citric acid, sodium gluconate, BSA, arabitol and trisodium EDTA as a stabilizer. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to the stabilization of p-hydroxybenzoate hydroxylase.

In vivo cholinesterase is known to inhibit its activity when exposed to drugs such as agricultural chemicals and insecticides, and is a useful marker for diagnosis of drug addiction.
Several methods for measuring cholinesterase are known, but the enzymatic method is superior in terms of accuracy, rapidity, and economic efficiency, especially measurement using p-hydroxybenzoate hydroxylase and protocatechuate dioxygenase. The method is excellent, and there are many reports on the production method of these enzymes and the analysis method using the enzymes.
JP 58-129999 Patent registration number 2756800 JP 2004-33029 A

Stabilizers for p-hydroxybenzoate hydroxylase have been studied in the past, but the level required for clinical diagnostic applications has not been reached, and a method for further improving the stability has been desired.
JP 59-25684

  The poor stability of enzyme products means that more management and analysis work is derived.

  In addition, when preparing a measurement reagent by the enzyme method, the amount of activity to be added must be accurately weighed. However, when handling enzyme powders with large activity fluctuations, the activity at that time should be measured each time preparation is performed. Must be recalculated. In order to accurately evaluate the titer of the enzyme powder, it takes half a day even for a skilled analytical worker including weighing and dissolution of the powder and adjustment of the measuring reagent, and the work load is large.

  Decreasing enzyme activity not only reduces the value of enzyme products evaluated for activity, but also causes the formation of turbidity due to precipitation of inactivated enzyme protein, and the performance of analytical reagents using these as raw materials. Give a fatal defect.

  The problem to be solved by the present invention is to improve the storage stability of p-hydroxybenzoic acid hydroxylase by selecting a stabilizer and to enable long-term stability of the enzyme product, as well as management work derived from activity fluctuations. -To reduce analytical work and avoid problems caused by precipitation of inactivated enzyme protein in a cholinesterase measurement reagent.

  In order to improve the stability of p-hydroxybenzoic acid hydroxylase, the present inventor has studied various substances that can be used as stabilizers for the enzyme, and those having a stabilizing effect on p-hydroxybenzoic acid hydroxylase. The headline and the present invention have been completed.

That is, the present invention has the following configuration.
[Claim 1]
A method for stabilizing p-hydroxybenzoic acid hydroxylase, comprising adding at least one of the compounds described in (A) below as a stabilizer.
(A) α-cyclodextrin, inositol, sucrose, sorbitol, citric acid, sodium gluconate, BSA, arabitol, EDTA.3 sodium [section 2]
Item 2. The method according to Item 1, wherein the following (A) and mannitol and / or flavin adenine dinucleotide are added.
(A) α-cyclodextrin, inositol, sucrose, sorbitol, citric acid, sodium gluconate, BSA, arabitol, EDTA.3 sodium [section 3]
A method for producing a stabilized lyophilized product of p-hydroxybenzoic acid hydroxylase, comprising freeze-drying an aqueous p-hydroxybenzoic acid hydroxylase solution containing at least one kind described in (A) below .
(A) α-cyclodextrin, inositol, sucrose, sorbitol, citric acid, sodium gluconate, BSA, arabitol, EDTA.3 sodium [section 4]
Item 4. The method according to Item 3, wherein the following (A) and mannitol and / or flavin adenine dinucleotide are added.
[Section 5]
A stabilized p-hydroxybenzoic acid hydroxylase lyophilized composition, wherein the aqueous p-hydroxybenzoic acid hydroxylase solution containing at least one of the following (A) is lyophilized.
[Claim 6]
Item 6. The method according to Item 5, wherein the following (A) and mannitol and / or flavin adenine dinucleotide are added.

  According to the present invention, the stability of p-hydroxybenzoate hydroxylase is improved.

Hereinafter, the present invention will be described in detail.

P-hydroxybenzoate hydroxylase is an enzyme classified as EC 1.14.13.2.

In the present invention, the origin of P-hydroxybenzoate hydroxylase to be stabilized is not particularly limited. For example, Pseudomonas desmoterica, Pseudomonas dacuune, Pseudomonas putida, Pseudomonas fluorescens, etc. are produced.

The present invention is selected from the group consisting of α-cyclodextrin, inositol, sucrose, sorbitol, citric acid, sodium gluconate, BSA, arabitol, EDTA trisodium as a stabilizer for P-hydroxybenzoate hydroxylase At least one kind may be used and may further contain mannitol and / or flavin adenine dinucleotide (hereinafter abbreviated as FAD).

The form of the P-hydroxybenzoic acid hydroxylase composition referred to in the present invention may be any shape such as a dry powder product, a freeze-dried product, a suspension, or a solution. In general, dried products (especially freeze-dried products) are superior from the standpoint of stability. From the viewpoint of handling, liquid products (particularly solutions) are excellent.
In another aspect, the composition of the present invention can be commercialized as, for example, an enzyme preparation for an in vitro diagnostic drug or an in vitro diagnostic drug for measuring cholinesterase. These product forms may be kits obtained by further combining necessary products.

The P-hydroxybenzoate hydroxylase composition of the present invention may further contain other components as necessary. Alternatively, it may be preferable to limit the content of other components as necessary.

When this enzyme is commercialized, various buffers can be added. The type of the buffer solution is not particularly limited, and those that are common in the field of biochemistry can be used, but those having a buffer capacity in the stable pH range of the enzyme are preferred. For example, various phosphate buffers such as potassium phosphate (K-phosphate), sodium phosphate, tris salt, organic acid salts such as acetic acid, malic acid, maleic acid, PIPES, TES, MOPS, HEPES, BisTris, Bicine, etc. Can be used. According to the study by the present inventors, for example, in the case of P-hydroxybenzoate hydroxylase derived from Comonas testosteroni, the lower limit upon commercialization from the pH stable range of this enzyme is preferably 5.0, more preferably 5 .5, and most preferably 6.0. The upper limit of pH is preferably 7.5, more preferably 7.0, and still more preferably 6.5.

If the buffer solution concentration is too dilute, the buffer capacity is weak, and therefore the pH of the enzyme solution tends to fluctuate, which is not preferable. Conversely, when lyophilizing with a high concentration buffer, part of the enzyme solution becomes antifreeze during the process of freezing, leading to inactivation of the enzyme in the product and poor powder shape and solubility. The use of concentrated buffers should be avoided. Therefore, the lower limit of the buffer concentration is 5 mM or more, preferably 10 mM or more. The upper limit of the buffer concentration is 1000 mM or less, preferably 800 mM or less, more preferably 600 mM or less.

If the amount of the stabilizer used in the present invention is too small relative to the enzyme, there will be no effect. Conversely, if the amount is too large, the enzyme protein in the product may be diluted, leading to inactivation. Even if the ratio of the addition amount of the stabilizer and the addition amount of the enzyme is the same, the stabilization effect further varies depending on the concentration of the enzyme protein. In general, it is considered that when the enzyme concentration is high, the range of the necessary stabilizer addition concentration tends to decrease.

When lyophilizing P-hydroxybenzoate hydroxylase, if the protein concentration of the enzyme solution is too dilute, the amount of liquid at the time of lyophilization increases, and it takes a long time to dry. Since it becomes large, it may cause a decrease in activity during drying. Conversely, if the protein concentration is too high, it will take a long time to dissolve when it is made into a dry product, and the protein will easily precipitate to form turbidity. When the amount of turbidity produced is large, the activity recovery rate is reduced.

In the present invention, when producing a freeze-dried product, the lower limit of the added weight of the stabilizer is 0.02, preferably 0.05, and more preferably 0.1 with respect to the dry weight 1 of the dry enzyme protein. The upper limit of the added weight is 50, preferably 30, and more preferably 10.
However, the concentration ratio between the enzyme protein and the stabilizer is not all to bring out the effects of the invention. The solid content concentration including the enzyme protein, stabilizer, and buffer component of the solution to be lyophilized should be taken into consideration, and it is important that the solid content is set to a concentration suitable for lyophilization. The lower limit of the solid content concentration is 3 mg / ml, preferably 5 mg / ml, more preferably 10 mg / ml. The upper limit of the solid concentration is 350 mg / ml, preferably 250 mg / ml, more preferably 150 mg / ml.
In order to commercialize an enzyme, it is natural to set optimum concentration conditions based on the concentration of the enzyme protein, the concentration of the stabilizer to be added, the solubility of the selected stabilizer, and the like.

For liquid products, the lower limit of stabilizer addition is preferably 0.05% (w / w), more preferably 0.1% (w / w), most preferably 1% (w / W). The upper limit of the amount added is preferably 20% (w / w), more preferably 10% (w / w), and most preferably 5% (w / w) with respect to the enzyme protein weight.
Of course, since the solubility in water varies depending on the substance, it is important to consider the solubility of the substance selected as the stabilizer and select an appropriate combination of protein concentration and stabilizer concentration that has a high enzyme stabilizing effect. It is.

Since an enzyme is a protein, it gradually deteriorates and denatures during storage and its activity decreases, and stabilization of the product does not mean complete retention of the initial activity. The stabilization referred to in the present invention means that the activity remaining rate after product storage is high. Specifically, the activity remaining rate when stored at 37 ° C. for 1 week is 70% or more, preferably 80% or more, more preferably Means holding 90% or more. In the case of a dry product, a product having an enzyme protein content of 25% (w / w) to 75% (w / w) in the dry product is preferably stored under dry conditions. In the case of a liquid product, it is preferable to store a product in which the enzyme protein content of P-hydroxybenzoic acid hydroxylase in the solution is 0.1 mg / ml to 100 mg / ml.

Commercially available products can be used as the above-mentioned reagents as stabilizers used in the present invention or the above-mentioned reagents added to the present invention as necessary.

In order to use it as an enzyme stabilizer, it is generally desirable that it is commercially available and can be easily obtained. However, it is unavoidable that a commercially available product is unavoidably obtained, or it is synthesized by various known means depending on circumstances such as cost reduction. You may do it.

EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to the examples.

P-hydroxybenzoate hydroxylase was manufactured by Toyobo Co., Ltd .: product code HBH-301.

The enzyme activity of P-hydroxybenzoic acid hydroxylase was measured by measuring the disappearance of NADPH, which is a coenzyme, by measuring the change in absorbance at 340 nm using P-hydroxybenzoic acid as a substrate. A specific method is shown below.
Prepare 5.0 mM P-hydroxybenzoic acid solution (condition adjustment), 0.2 mM FAD solution (condition adjustment), 3 mM NADPH solution (condition adjustment), 50 mM tris maleate buffer (pH 8.2 (25 ° C.)). .
Next, the reaction mixture is adjusted using these as follows.
21 ml Trismaleic acid buffer 3 ml P-hydroxybenzoic acid solution 3 ml FAD solution 3 ml NADPH solution In addition, a measurement sample (enzyme solution) was 50 mM K-phosphate buffer (pH 6.0 (25) containing 0.2% BSA previously cooled on ice. At 1 ° C.) and was diluted to 0.2 to 0.6 U / ml with the same buffer immediately before the analysis.
For each reaction, take 3.0 ml of the reaction mixture, preheat at 37 ° C. for about 5 minutes, add 0.05 ml of the measurement sample (enzyme solution), mix, and then use a spectrophotometer controlled at 37 ° C. The absorbance at 340 nm was recorded for 3 to 4 minutes, and the change in absorbance per minute was determined from the initial linear portion (ΔODtest). In the blank test, 0.05 ml of water was added instead of the enzyme solution, and the same operation as described above was performed to determine the amount of change in absorbance per minute (ΔOD blank).
The enzyme activity of P-hydroxybenzoate hydroxylase was calculated from the obtained change in absorbance based on the following formula. The amount of enzyme that oxidizes 1 micromole of NADPH per minute under the above conditions is defined as 1 unit (1 U).
Formula activity value (U / ml) = {ΔOD / min (ΔODtest−ΔODblank) × 3.05 (ml) × dilution factor} / {6.22 × 1.0 (cm) × 0.05 (ml)}
3.05 ml = reaction mixture liquid volume 3.8 = NADPH millimolecular absorption coefficient 1.0 cm = cell optical path length 0.05 ml = enzyme sample liquid volume

[Example-1]
P-hydroxybenzoate hydroxylase was prepared in 50 mM K-phosphate buffer pH 6.0 to a concentration of 30 mg-protein / ml, and the additives were added and dissolved to 20 mg / ml each. Powdered by drying.
The obtained freeze-dried powder was stored at 37 ° C. for 1 week under dry conditions, and the residual activity rate was determined with the initial activity as 100%. The results are shown in (Table 1).

As can be seen from this result, when freeze-dried with the above additives, the stabilizing effect of P-hydroxybenzoate hydroxylase is observed.

The present invention can be used in a cholinesterase measurement method using p-hydroxybenzoate hydroxylase and protocatechuate dioxygenase. Cholinesterase is known to inhibit its activity when exposed to drugs such as pesticides and insecticides, and is a useful marker for the diagnosis of drug addiction.

Claims (6)

A method for stabilizing p-hydroxybenzoic acid hydroxylase, comprising adding at least one of the compounds described in (A) below as a stabilizer.
(A) α-cyclodextrin, inositol, sucrose, sorbitol, citric acid, sodium gluconate, BSA, arabitol, EDTA.3 sodium The method according to claim 1, wherein the following (A) and mannitol and / or flavin adenine dinucleotide are added.
(A) α-cyclodextrin, inositol, sucrose, sorbitol, citric acid, sodium gluconate, BSA, arabitol, EDTA.3 sodium A method for producing a stabilized lyophilized product of p-hydroxybenzoic acid hydroxylase, comprising freeze-drying an aqueous p-hydroxybenzoic acid hydroxylase solution containing at least one kind described in (A) below .
(A) α-cyclodextrin, inositol, sucrose, sorbitol, citric acid, sodium gluconate, BSA, arabitol, EDTA.3 sodium   The method according to claim 3, wherein the following (A) and mannitol and / or flavin adenine dinucleotide are added.   A stabilized p-hydroxybenzoic acid hydroxylase lyophilized composition, wherein the aqueous p-hydroxybenzoic acid hydroxylase solution containing at least one of the following (A) is lyophilized. 6. The method according to claim 5, wherein the following (A) and mannitol and / or flavin adenine dinucleotide are added.