JP2006101777A - Method for stabilizing protocatechuate dioxygenase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To improve the preservation stability of protocatechuate dioxygenase and to preserve an enzymic product over a long period. <P>SOLUTION: A method for stabilizing the protocatechuate dioxygenase is characterized as adding at least either one of the following (a) and (b) as a stabilizer. (a) at least one kind selected from an L-amino acid, an L-imino acid, a derivative of the L-amino acid, a D-amino acid, a D-imino acid, a derivative of the D-amino acid, betaines, an oligopeptide, a polypeptide and a water-soluble protein and (b) an organic acid and/or an organic acid salt. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to the stabilization of protocatechuate dioxygenase.

  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 economy, and in particular, the measurement method using p-hydroxybenzoate hydroxylase and protocatechuate dioxygenase. It is excellent, and there are many reports on the production method of these enzymes and the analysis method using the enzymes.

As described above, protocatechuate dioxygenase is an enzyme necessary for measuring cholinesterase together with p-hydroxybenzoate hydroxylase, but the conventional product has poor stability of the enzyme product powder, and is stored at room temperature (25 ° C) in a dry state. In this case, 40% of the initial activity is deactivated in 2 weeks, and even when stored frozen in a dry state, it is deactivated by about 20% in one year.

The poor stability of the enzyme product means that more management and analysis operations are derived. For example, enzyme product powder is usually stored frozen, but when opened during use, it is necessary to raise the product temperature to room temperature in advance to avoid moisture absorption. It is known that the activity of protocatechuate dioxygenase currently on the market decreases even when the temperature rises for such a short time, and it is necessary to manage the temperature history of each container in which the enzyme is packaged.

Furthermore, 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 the preparation is added. 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 preparation of a reagent for measurement.

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

In this way, ensuring the stability of enzyme powder is an important issue, but there is no description in the prior patent literature and non-patent analysis regarding the commercialization conditions of protocatechuate dioxygenase, and stabilization of enzyme activity The technical information about was very poor. (For example, see Patent Documents 1 to 3 and Non-Patent Documents 1 and 2.)
Patent 2756800 JP-B-2-36237 JP-A-60-153793 Hygiene inspection, vol. 34, no. 4,729-733, 1985 Biochemistry, vol36, no. 33, 10052-10066, 1997

The problem to be solved by the present invention is to improve the storage stability of protocatechuate dioxygenase by selecting a stabilizer and to enable long-term storage of enzyme products, and to reduce management and analysis operations derived from activity fluctuations. And avoiding problems caused by precipitation of the deactivated enzyme protein in the cholinesterase measurement reagent.

In order to improve the stability of the protocatechuate dioxygenase product, the present inventor has studied various substances that can be used as stabilizers of the enzyme, and L-amino acids, L-imino acids, L-amino acid derivatives, D-amino acids, D-imino acids, D-amino acid derivatives, betaines, oligopeptides, polypeptides, water-soluble proteins, organic acids and / or organic acid salts that have a stabilizing effect on protocatechuate dioxygenase have been found and the present invention It came to complete.

The present invention relates to the following methods for stabilizing protocatechuate dioxygenase and methods for producing stabilized products and / or compositions. That is,
Item 1.
A method for stabilizing protocatechuate dioxygenase, comprising adding at least one of the following (a) and (b) as a stabilizer.
(A) at least one selected from L-amino acids, L-imino acids, derivatives of L-amino acids, D-amino acids, D-imino acids, derivatives of D-amino acids, betaines, oligopeptides, polypeptides, water-soluble proteins Species (b) Organic acid and / or organic acid salt
From L-amino acids, L-imino acids, L-amino acid derivatives, D-amino acids, D-imino acids, D-amino acid derivatives, betaines, oligopeptides, polypeptides, water-soluble proteins, organic acids, organic acid salts A stabilized protocatechuate dioxygenase dry powder composition comprising at least one member selected from the group consisting of:
Item 3.
From L-amino acids, L-imino acids, L-amino acid derivatives, D-amino acids, D-imino acids, D-amino acid derivatives, betaines, oligopeptides, polypeptides, water-soluble proteins, organic acids, organic acid salts A stabilized protocatechuate dioxygenase liquid composition comprising at least one member selected from the group consisting of:
Item 4.
From L-amino acids, L-imino acids, L-amino acid derivatives, D-amino acids, D-imino acids, D-amino acid derivatives, betaines, oligopeptides, polypeptides, water-soluble proteins, organic acids, organic acid salts A method for producing a stabilized freeze-dried protocatechuate dioxygenase product comprising lyophilizing an aqueous protocatechuate dioxygenase solution containing at least one selected from the group consisting of
It is.

  According to the present invention, the stability of protocatechuate dioxygenase is improved.

  In the present invention, the origin of protocatechuate dioxygenase to be stabilized is not particularly limited. For example, a product derived from Pseudomonas bacteria can be used.

In the present invention, at least one of the following (a) and (b) is used for stabilizing protocatechuate dioxygenase.
(A) at least one selected from L-amino acids, L-imino acids, derivatives of L-amino acids, D-amino acids, D-imino acids, derivatives of D-amino acids, betaines, oligopeptides, polypeptides, water-soluble proteins Species (b) Organic acids and / or organic acid salts

Since α-amino acid has an asymmetric carbon at the α-position, it has an enantiomer and has optical activity. These are identified as D-form and L-form, and only L-form is used for protein synthesis. There are 19 L-amino acids and 1 L-imino acid in natural products. In the present invention, the mixing ratio of D-form and L-form is not limited. The mixing ratio of the two is arbitrary from 1 to 0 to 0 to 1. Of course, racemates can also be used as stabilizers.
β-amino acids and γ-amino acids whose side chains are bonded to carbon atoms other than the α-position carbon atom, and amino acid modifications and derivatives such as betaine (quaternary ammonium salts), ornithine, citrulline, creatine, creatinine, Amino acid metabolites such as sarcosine (N-methylglycine) can also be used in the present invention.

Oligopeptides / polypeptides are conveniently classified by the number of amino acids to be bound, with about 30 as a boundary, but any of oligopeptides, polypeptides, and proteins can be used.

The form of the protocatechuate dioxygenase 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 protocatechuate dioxygenase 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 commonly used in the field of biochemistry can be used, but those having a buffer capacity in the stable pH range of the enzyme are preferable. 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 protocatechuate dioxygenase derived from Pseudomonas genus, the lower limit upon commercialization from the pH stable range of this enzyme is preferably 6.0, more preferably 6.5, most preferably Preferably it is 7.0. The upper limit of the pH is preferably 10.5, more preferably 10.0, and still more preferably 9.5.

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.

  Moreover, since protocatechuate dioxygenase is a metal enzyme, it is desirable not to add a metal chelating agent such as EDTA or o-Phenanthroline or a reducing agent such as nitrite. On the other hand, it may be preferable to add these metal chelating agents for the purpose of preserving or the like, but in such a case, it is desirable to keep the addition concentration to a minimum so as not to impair the stability of the enzyme.

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 protocatechuate dioxygenase is freeze-dried, if the protein concentration of the enzyme solution is too dilute, the amount of liquid at the time of freeze-drying will increase and it will take a long time to dry, and the crystals of ice to be purified will also become large. May cause a decrease in activity. On the other hand, 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 produce 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. 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 25 ° C. for 2 weeks 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 protocatechuate dioxygenase 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.

As such reagents, amino acids include standard amino acids such as alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, selenocysteine, Valine, tryptophan, tyrosine, and non-standard amino acids include 2-aminoadipic acid, 3-aminoadipic acid, 2-aminobutanoic acid, 2,4-diaminobutanoic acid, 2-aminohexanoic acid, 6-aminohexanoic acid, β -Alanine, 2-aminopentanoic acid, 2,3-diaminopentanoic acid, 2,3-diaminopropanoic acid, 2-aminopimelic acid, citrulline, cysteic acid, 2,4-diaminobutanoic acid, 2,6-diaminopimelic acid, 2, 3 Diaminopropanoic acid, 4-carboxyglutamic acid, 5-oxoproline (pyroglutamic acid), homocysteine, homoserine, homoserine lactone, 5-hydroxylysine, allohydroxylysine, 3-hydroxyproline, 4-hydroxyproline, alloisoleucine, norleucine, Examples include norvaline, ornithine, sarcosine, allothreonine, and thyroxine, and L-form or D-form of amino acids excluding glycine, and a mixture of D-form and L-form. Examples of oligopeptides include glycylglycine, glycylglycylglycine, glutamylglycine, glutamylglutamic acid, glycylleucine, glycylproline, glycylglycylhistidine, and leucylglycylglycine, and proteins include BSA, casein, and ovalbumin. Examples of the organic acid include gluconic acid, maleic acid, succinic acid, malic acid, citric acid, acetic acid, and lactic acid.

Organic acids, certain amino acids and oligopeptides, and preferred examples include glycylglycine, but there are those that themselves become buffers when they form salts in aqueous solutions. In this case, for example, the buffer solution itself can be used as a stabilizer without adding another stabilizer to the glycylglycine buffer solution.

  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.

  Protocatechuate dioxygenase was a product derived from Pseudomonas bacteria (manufactured by Toyobo).

The enzymatic activity of protocatechuic acid dioxygenase was measured by measuring the disappearance of protocatechuic acid as a substrate and the change in absorbance at 290 nm. A specific method is shown below.
0.4 mM, protocatechuic acid solution, 50 mM, Tris acetate buffer (pH 7.5 (25 ° C.)) was prepared and used as a stock solution for the following measurement. In addition, a measurement sample (enzyme solution) was dissolved in a concentration of 1.0 mg / ml or more in 50 mM Tris acetate buffer (pH 7.5 (25 ° C.)) that had been ice-cooled in advance. Diluted to 0.8 U / ml.
In each reaction, 3.0 ml of 0.4 mM protocatechuic acid solution was taken, pre-warmed at 37 ° C. for about 5 minutes, 0.05 ml of a measurement sample (enzyme solution) was added, mixed, and then controlled at 37 ° C. The absorbance at 290 nm was recorded for 3 to 4 minutes using a spectrophotometer, and the change in absorbance per minute was determined from the initial linear portion (ΔODtest). In the blank test, 0.05 ml of 50 mM Tris acetate buffer (pH 7.5 (25 ° C.)) was added instead of the enzyme solution and the same operation was performed to determine the amount of change in absorbance per minute (ΔOD blank). ).
The enzyme activity of protocatechuate dioxygenase was calculated from the obtained change in absorbance based on the following formula. The amount of enzyme that oxidizes 1 micromol of protocatechuic acid 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} / {3.8 × 1.0 (cm) × 0.05 (ml)}
3.05 ml = reaction mixture liquid volume 3.8 = protocolecic acid millimolar extinction coefficient 1.0 cm = cell optical path length 0.05 ml = enzyme sample liquid volume

[Example-1]
Prepare protocatechuate dioxygenase at a concentration of 15 mg-protein / ml in 10 mM potassium phosphate buffer pH 7.5 so that the amino acid, amino acid derivative, oligopeptide, water-soluble protein, and organic acid salt are 20 mg / ml each. And dissolved in powder, and pulverized by freeze-drying.
The obtained freeze-dried powder was stored under dry conditions at 25 ° C. for 2 weeks, 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 lyophilized with amino acids, amino acid derivatives, oligopeptides, water-soluble proteins, and organic acid salts, the stabilizing effect of protocatechuate dioxygenase is observed.
The stabilizing effect is not biased to either L-form amino acids or D-form amino acids, and it is clear that either can be used in the present invention.

[Example-2]
Protocatechuate dioxygenase was prepared to be 15 mg-protein / ml in 10 mM potassium phosphate buffer pH 7.5, and the addition concentration of glycine per enzyme protein was changed in the range of 0: 1 to 20: 1. Powdered by lyophilization. The obtained lyophilized powder was stored under dry conditions at 25 ° C. for 2 weeks, and the residual activity was determined with the initial activity as 100. The results are shown in (Table 2).

This result shows that glycine stabilizes protocatechuate dioxygenase in a wide concentration range. When freeze-dried under the above conditions, the addition range is preferably a glycine: protein ratio of 0.2: 1 or more, more preferably 0.5: 1 to 20: 1, and 1.5: 1 to 10 It is more preferable that the ratio is 1.

[Example-3]
Protocatechuate dioxygenase was prepared from 3.5 mg-protein / ml to 20 mg-protein / ml with 20 mM potassium phosphate buffer, pH 7.5, and glycine was added and dissolved at a concentration of 20 mg / ml and lyophilized. Powdered. The obtained lyophilized powder was stored under dry conditions at 25 ° C. for 2 weeks, and the residual activity was determined with the initial activity as 100. The results are shown in (Table 3).

When the enzyme protein concentration at the time of lyophilization is low, the activity tends to decrease, but the stabilization effect of glycine was shown even under dilute conditions such as a protein concentration of 3.5 mg / ml.

[Example-4]
Protocatechuate dioxygenase was adjusted to 15 mg-protein / ml with 10 mM to 1000 mM potassium phosphate buffer pH 7.5, respectively, and glycine was added and dissolved at a concentration of 20 mg / ml to prepare a freeze-dried powder. The obtained lyophilized powder was stored under dry conditions at 25 ° C. for 2 weeks, and the residual activity was determined with the initial activity as 100. The results are shown in (Table 4).

As can be seen from this result, the protocatechuate dioxygenase lyophilized powder can be used at a buffer concentration of up to 1000 mM, but considering the solubility of the product powder, it is preferably 800 mM or less, and more preferably 600 mM or less. preferable.

[Example-5]
Protocatechuate dioxygenase was prepared to be 15 mg-protein / ml using various buffers, and glycine was added and dissolved to a concentration of 20 mg / ml, and lyophilized powder. The obtained lyophilized powder was stored under dry conditions at 25 ° C. for 2 weeks, and the residual activity was determined with the initial activity as 100. The results are shown in (Table 5).

As can be seen from this result, it is possible to select and use a buffer species from a wide range of options in the production of protocatechuate dioxygenase using glycine as a stabilizer.

  By improving the stability of protocatechuate dioxygenase according to the present invention, practicality in clinical examinations and the like is improved, and a great industrial effect is brought about.

Claims (4)

A method for stabilizing protocatechuate dioxygenase, comprising adding at least one of the following (a) and (b) as a stabilizer.
(A) at least one selected from L-amino acids, L-imino acids, derivatives of L-amino acids, D-amino acids, D-imino acids, derivatives of D-amino acids, betaines, oligopeptides, polypeptides, water-soluble proteins Species (b) Organic acids and / or organic acid salts   From L-amino acids, L-imino acids, L-amino acid derivatives, D-amino acids, D-imino acids, D-amino acid derivatives, betaines, oligopeptides, polypeptides, water-soluble proteins, organic acids, organic acid salts A stabilized protocatechuate dioxygenase dry powder composition comprising at least one member selected from the group consisting of:   From L-amino acids, L-imino acids, L-amino acid derivatives, D-amino acids, D-imino acids, D-amino acid derivatives, betaines, oligopeptides, polypeptides, water-soluble proteins, organic acids, organic acid salts A stabilized protocatechuate dioxygenase liquid composition comprising at least one member selected from the group consisting of:   From L-amino acids, L-imino acids, L-amino acid derivatives, D-amino acids, D-imino acids, D-amino acid derivatives, betaines, oligopeptides, polypeptides, water-soluble proteins, organic acids, organic acid salts A method for producing a stabilized freeze-dried protocatechuate dioxygenase product comprising lyophilizing an aqueous protocatechuate dioxygenase solution containing at least one selected from the group consisting of