JP3125610B2 - Method for stabilizing L-methionine γ-lyase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for stabilizing L-methionine γ-lyase useful for detecting and quantifying amino acids such as L-methionine and L-cysteine.

[0002]

BACKGROUND OF THE INVENTION L-methionine γ-lyase [EC 4.4.1.1
1] is an enzyme derived from microorganisms of the genus Pseudomonas, Aeromonas or the like. As shown in the following formulas 1 and 2, pyridoxal 5′-phosphate (PLP) is used as a coenzyme to produce L-methionine and its derivatives. It is a multifunctional pyridoxal enzyme that catalyzes α, γ-elimination reaction and γ-substitution reaction and also catalyzes α, β-elimination reaction and β-substitution reaction of L-cysteine analogs.

(Equation 1)

(Equation 2)

[0003] L-methionine γ-lyase has excellent substrate specificity.
Since it is possible to detect and quantify amino acids such as cysteine, the enzyme is expected to be used in fields such as clinical tests. L-methionine γ-lyase is an enzyme having an SH group derived from a cysteine residue of a protein in its active center, and this SH group is one of the so-called SH enzymes in which the SH group is essential for the expression of enzyme activity. is there. In general, SH enzymes are heavy metals such as Hg ²⁺ , p-mercury benzoic acid (PM
B), it has the property of being deactivated by an SH reagent such as N-ethylmaleimide or iodoacetic acid or an oxidizing agent such as ferricyanide.
The activity is generally protected by coexistence in a solution containing a chelating agent such as A) or a thiol compound such as 2-mercaptoethanol (2-ME) or dithiothreitol (DTT). It is also widely practiced to coexist a coenzyme such as PLP to protect the enzyme activity.

However, in the case of L-methionine γ-lyase, the enzymatic activity is significantly reduced at room temperature even when stored in a solution state by using such a method, and the enzyme activity is gradually decreased even at a low temperature. After long-term storage of 50 days or more, the enzyme activity will be 30% or less of the initial value. Further, even when L-methionine γ-lyase is freeze-dried and stored at a low temperature in the co-presence of a chelating agent, a coenzyme, DTT and the like, there is a problem that the enzyme activity is considerably reduced in long-term storage. As a method for preserving L-methionine γ-lyase, E-
After adding and dissolving the enzyme in a 0.1 M or more potassium phosphate buffer (pH 7.2) containing DTA, PLP, and 2-ME, the enzyme is frozen and stored at −20 ° C. and contains 50 mM DTT. 0.0
A method of using a 1M potassium phosphate solution by dialysis at 0 ° C. for 1 hour has also been reported (Analytical B)
iochemistry 138 , 421-424 (1984)).
-It is difficult to store methionine γ-lyase stably for a long time. Therefore, it is presently desired to develop a method capable of preserving L-methionine γ-lyase in a stable state for a long period of time.

[0005]

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned circumstances, and provides a method for stably stabilizing L-methionine γ-lyase for a long period of time.

[0006]

The present invention relates to L-methionine γ-lyase, (1) oligosaccharide, (2) water-soluble polysaccharide, (3) sugar alcohol, (4) protein, (5) acidic amino acid,
A method for stabilizing L-methionine γ-lyase, comprising freeze-drying a solution in which one or more substances selected from the group consisting of (6) glutathione and (7) N-acetylcysteine are co-existed. And L-methionine γ-lyase stabilized by the method. Further, the present invention provides an L-methionine γ-lyase, an oligosaccharide and one or two selected from the group consisting of (1) protein, (2) amino acid, (3) glutathione, and (4) N-acetylcysteine. An invention of a method for stabilizing L-methionine γ-lyase, characterized by freeze-drying a solution coexisting with at least one kind of substance, and an invention of L-methionine γ-lyase stabilized by the method.

That is, the present inventors have conducted intensive studies to find a method for stably stabilizing L-methionine γ-lyase, and as a result, L-methionine γ-lyase was obtained.
(1) oligosaccharide, (2) water-soluble polysaccharide, (3) sugar alcohol, (4) protein, (5) acidic amino acid, (6)
A solution containing one or more substances selected from the group consisting of glutathione and (7) N-acetylcysteine is freeze-dried, or L-methionine γ-lyase, oligosaccharide and ( The solution obtained by freeze-drying a solution in which one or more substances selected from the group consisting of 1) protein, (2) amino acid, (3) glutathione and (4) N-acetylcysteine are co-existed is freeze-dried. The inventors have found that the object has been achieved, and have completed the present invention.

Preferred oligosaccharides according to the present invention include disaccharides such as saccharose, maltose, lactose and trehalose, and trisaccharides such as raffinose and maltotriose. Among them, trehalose and raffinose are particularly preferred. And so on. As the water-soluble polysaccharide, for example, dextran and the like are preferably mentioned. As the addition ratio of the oligosaccharide or the water-soluble polysaccharide,
Usually 0.1 to 20 as the concentration in the enzyme solution before lyophilization
(W / v)%, preferably in the range of 1 to 10 (w / v)%. As the sugar alcohol according to the present invention, for example, sorbitol, mannitol, xylitol and the like are preferably mentioned, and the addition ratio thereof is usually 0.1 to 20 (w / v)%, preferably 0.1 to 20 (w / v)%, as the concentration in the enzyme solution before freeze-drying. Is in the range of 1 to 10 (w / v)%. As the protein according to the present invention, for example, a gelatin hydrolyzate is preferably mentioned, and its addition ratio is usually 0.1 to 20 (w / v)%, preferably 1 to 1 as a concentration in an enzyme solution before freeze-drying. A range of 10 (w / v)% is exemplified. The acidic amino acids according to the present invention preferably include, for example, glutamic acid and aspartic acid and salts thereof (eg, alkali metal salts), and the other amino acids preferably include, for example, glutamine and asparagine. The addition ratio of these amino acids is usually 0.1 to 20 (w / v)%, preferably 1 to 1 as a concentration in the enzyme solution before freeze-drying.
0 (w / v)% range. The addition ratio of glutathione or N-acetylcysteine according to the present invention is usually 1 to 20 mM as the concentration in the enzyme solution before lyophilization,
Preferably, the range is 5 to 10 mM.

The above-mentioned various additives (excluding amino acids such as glutamine and asparagine) according to the present invention may be used alone or in combination of two or more. When used in combination,
One is an oligosaccharide and the other is one or more selected from the group consisting of proteins, amino acids (including amino acids other than acidic amino acids such as glutamine and asparagine), glutathione and N-acetylcysteine. If
Even higher effects can be obtained. In this case, the lyophilized L-methionine γ-lyase obtained does not change its enzyme activity even after storage at 37 ° C. for 3 months.

The L-methionine γ-lyase used in the present invention can be produced by a conventional method (Analytical Biochemistry 138 , 421-424 (Analytical Biochemistry 138 , 421-424). 1984)
It suffices to use the one obtained by. An example may be performed as follows. That is, Pseudomonas
L-methionine of the genus, Aeromonas genus etc.L-methionine, urea, glycerin, phosphate, a suitable medium containing yeast extract, etc.
Incubate with shaking for ~ 30 hours. The cultured cells are collected, crushed by a suitable method, for example, trituration with aluminum oxide, and then extracted with a suitable buffer or the like to obtain a crude enzyme solution. If this is purified by a conventional method using ion exchange column chromatography or the like, L-methionine γ-lyase can be obtained. The obtained solution containing L-methionine γ-lyase may be further subjected to concentration treatment by ultrafiltration, dialysis treatment, and the like. The present invention may be implemented, for example, as follows. That is, L-methionine γ-lyase obtained by the method as described above,
(1) oligosaccharide, (2) water-soluble polysaccharide, (3) sugar alcohol, (4) protein, (5) acidic amino acid, (6)
A solution in which one or more compounds selected from the group consisting of glutathione and (7) N-acetylcysteine are present, or L-methionine γ-lyase obtained by the method as described above, and an oligosaccharide And (1) protein, (2) amino acid, (3) glutathione and (4) N-
One or two selected from the group consisting of acetylcysteine
Solutions coexisting with at least one kind of compound [These solutions may optionally contain a buffer such as phosphate, tris (hydroxymethyl) aminomethane, Good's, etc., for example, ethylenediaminetetraacetic acid (EDTA), etc. The metal masking agent, PLP which is a coenzyme of the present enzyme, and the like, which are appropriately added to the L-methionine γ-lyase solution, may be contained within the concentration range usually added in this field. ]
May be freeze-dried by a conventional method. The obtained freeze-dried product can be stored at an appropriate temperature as it is, but a lower storage temperature can be stored for a longer period, so that it is usually -40 to 30.
° C, preferably -40 to 10 ° C, more preferably -40 to -20 ° C
It is desirable to save with.

When the freeze-dried L-methionine γ-lyase obtained by the method of the present invention is used for actual measurement, etc., it is usually sufficient to redissolve it in water and use it. Considering EDTA, PLP, 2-ME and D
It is desirable to redissolve in an appropriate buffer containing TT or the like. Hereinafter, the present invention will be described in more detail with reference to Reference Examples, Examples, and Comparative Examples, but the present invention is not limited by these.

[0012]

EXAMPLES In the following Reference Examples, Examples and Comparative Examples,
The activity of L-methionine γ-lyase was measured by the following method.・ L-methionine γ-lyase activity measurement method (reagent solution) ・ Enzyme lysis solution 0.1 M phosphate buffer (pH 7.2) containing 1 mM EDTA, 0.02 mM PLP and 0.01% 2-ME was used as the enzyme lysis solution. did. -Substrate solution A 0.1 M potassium phosphate buffer (pH 8.0) containing 25 mM L-methionine and 0.01 mM PLP was used as the substrate solution. (Operation method) 2.0 ml of the substrate solution is heated at 37 ° C for 5 minutes.
To this is added 0.02 ml of an enzyme solution containing an appropriate amount of L-methionine γ-lyase, and the mixture is allowed to react at 37 ° C. for exactly 10 minutes. Then, 0.25 ml of a 50% trichloroacetic acid solution is added to stop the reaction. Next, 1.0 ml of this and 1.0 M acetate buffer (pH 5.0)
2.0 ml and 0.8 ml of a 0.1% 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) solution, and mix at 50 ° C. for 30 minutes.
After heating for one minute, cool and measure the absorbance At at 320 nm. As a blank test, 0.25 ml of a 50% trichloroacetic acid solution previously added to 2.0 ml of the substrate solution is heated at 37 ° C. for 5 minutes, then 0.02 ml of the enzyme lysis solution is added, and the mixture is further reacted at 37 ° C. for 10 minutes. Thereafter, the absorbance Ab is measured in the same manner as above. The enzyme activity value is determined by the following equation. L-methionine γ-lyase activity value (u / ml) = (At-A
b) x 2.74

Reference Example 1 Purification of L-methionine γ-lyase 0.25% L-methionine, 0.1% urea, 0.1% glycerin, 0.1%
1% KH ₂ PO ₄ , 0.1% K ₂ HPO ₄ , 0.01% MgSO _4.
600 ml of medium containing 7H ₂ O and 0.025% yeast extract (pH 7.
2) Transfer to a 2 liter Sakaguchi flask and add Pseudomon
As putida was inoculated with a platinum loop and shake-cultured at 28 ° C. and 120 rpm for 18 hours. Thereafter, the culture solution was centrifuged to collect the cells, and the collected cells were crushed by grinding with aluminum oxide in a mortar. A 10 mM potassium phosphate buffer solution (pH 7.2, containing 10 mM EDTA) was added to the disrupted cells, suspended, and then centrifuged to obtain a crude enzyme solution. Equilibrated with 10 mM potassium phosphate buffer (pH 7.2, containing 10 mM EDTA)
After adsorbing the above crude enzyme solution on a DEAE-Toyopearl 650M column (manufactured by Tosoh Corporation), 0.04M KCl and 10mM ED were used.
The column was washed thoroughly with 10 mM phosphate buffer (pH 7.2) containing TA. Then 0.08 M KCl and 10 mM EDTA
The target enzyme was eluted with 10 mM phosphate buffer (pH 7.2) containing The resulting enzyme solution contains 0.12M KCl containing 0.12M KCl.
M sodium phosphate buffer (pH 8.3)
After adjusting to 8.2 ± 0.1, containing 0.12M KCl
The column was adsorbed on a DEAE-Sephadex A-50 column (Pharmacia Biotech) equilibrated with a 0.02 M sodium pyrophosphate buffer (pH 8.3). After sufficiently washing the column with a 0.02 M sodium pyrophosphate buffer (pH 8.3, containing 0.12 M KCl), the target enzyme was eluted with a 0.02 M sodium pyrophosphate buffer (pH 8.3) containing 0.25 M KCl. The obtained purified enzyme solution was subjected to ultrafiltration and concentration using a PM-30 membrane manufactured by ADVANTEC, and 0.02 mM PLP, 0.01% 2-ME
Was dialyzed against a 0.1 M potassium phosphate buffer (pH 7.2) (enzyme solution) containing L-methionine γ-lyase.

Example 1 L-methionine γ-lyase obtained in Reference Example 1 was used in an amount of about 0.24 u / ml.
Predetermined monosaccharide, oligosaccharide or water-soluble polysaccharide is added to the enzyme solution containing 5% (w / v)%, and the solution is subdivided into 1.0 ml aliquots in vials and frozen by a conventional method. Dried. After storing the freeze-dried product at 37 ° C for 2 weeks,
After redissolving with 1.0 ml of the enzyme solution, the enzyme activity was measured. The obtained results are shown in Table 1 as the residual activity rate (%) of the enzyme after lyophilization, where the enzyme activity of the lyophilized stock solution is defined as 100%.
Shown in

[Table 1] From the results in Table 1, it was found that the lyophilized product without additives was
The residual activity of L-methionine γ-lyase after storage for 2 weeks is 19%, while the freeze-dried product to which a monosaccharide is added has a residual activity of 0%, that is, the monosaccharide is L-
It turns out that it promotes the inactivation of methionine γ-lyase. On the other hand, the freeze-dried product to which the disaccharides such as saccharose, maltose, lactose and trehalose, the trisaccharides such as raffinose and maltotriose, and the water-soluble polysaccharides such as dextran according to the present invention have a residual activity of 80% or more. Shows that L-methionine γ-
It turns out that the lyase can be stably stored. The lyophilized product prepared above was stored at 5 ° C. for 50 days, then redissolved and the enzyme activity was measured. The residual activity of the enzyme after lyophilization was as follows. Then
In contrast to 55%, the lyophilized product to which the oligosaccharide or water-soluble polysaccharide according to the present invention was added was 100% in all cases. From the above, it can be understood that L-methionine γ-lyase can be stored extremely stably by using the stabilization method of the present invention.

Example 2 L-methionine γ-lyase obtained in Reference Example 1 was used in an amount of about 0.24 u / ml.
Sorbitol, a sugar alcohol, in the enzyme solution containing
Xylitol or mannitol was added and dissolved at a concentration of 5 (w / v)%, and 1.0 ml of the resulting solution was divided into vials and freeze-dried by a conventional method. 37 of the freeze-dried product obtained
After storage at 2 ° C. for 2 weeks, the mixture was redissolved in 1.0 ml of an enzyme solution and the enzyme activity was measured. The obtained results are shown in Table 2 as the residual activity rate (%) of the enzyme after lyophilization, where the enzyme activity of the lyophilized stock solution is 100%.

[Table 2] From the results shown in Table 2, the lyophilized product to which no additive was added had a residual activity ratio of L-methionine γ-lyase of 19% after storage at 37 ° C. for 2 weeks. All the freeze-dried products show a residual activity rate of around 60%, indicating that the method of the present invention can stably store L-methionine γ-lyase. The lyophilized product prepared above was stored at 5 ° C. for 50 days, then redissolved and the enzyme activity was measured. The residual activity of the enzyme after lyophilization was as follows. Was 55%, whereas the freeze-dried product to which the sugar alcohol according to the present invention was added was 100% in all cases. From the above, it can be understood that L-methionine γ-lyase can be stored extremely stably by using the stabilization method of the present invention.

Example 3 L-methionine γ-lyase obtained in Reference Example 1 was used in an amount of about 0.26 u / ml.
To the enzyme solution containing the solution, gelatin hydrolyzate (NP-2000, manufactured by Nippi Co., Ltd.) or bovine serum albumin (BSA) was added to a concentration of 5 (w / v)%, and the solution was dissolved in 1.0 ml vials. And freeze-dried by a conventional method. After storing the freeze-dried product at 37 ° C for 3 weeks,
After redissolving with 1.0 ml, the enzyme activity was measured. The obtained results are shown in Table 3 as the residual activity rate (%) of the enzyme after lyophilization, where the enzyme activity of the lyophilized stock solution is 100%.

[Table 3] From the results in Table 3, it is found that the lyophilized product to which no additive was added had a residual activity of 15% of L-methionine γ-lyase after storage at 37 ° C. for 3 weeks, whereas the protein according to the present invention was added. It can be seen that the residual activity rate of the freeze-dried product thus obtained is higher than this. In particular, the freeze-dried product to which the gelatin hydrolyzate was added exhibited a residual activity rate of 85%. When the lyophilized product prepared above was stored at 5 ° C. for 50 days and then redissolved and the enzyme activity was measured, the residual activity of the enzyme after lyophilization was as follows:
The freeze-dried product without additives was 55%, while the freeze-dried product with gelatin hydrolyzate was 100%
Met. From the above, it can be understood that L-methionine γ-lyase can be stored extremely stably by using the stabilization method of the present invention.

Example 4 L-methionine γ-lyase obtained in Reference Example 1 was added to about 0.15
In the enzyme solution obtained by dissolving u / ml, 5 (w /
v) The solution added and dissolved so as to be% was divided into 1.0 ml aliquots in vials and freeze-dried by a conventional method. After storing the freeze-dried product at 37 ° C. for 4 weeks, 1.0 ml of enzyme solution
And the enzyme activity was measured. The obtained results are shown in Table 4 as the residual activity rate (%) of the enzyme after lyophilization, where the enzyme activity of the lyophilized stock solution is 100%.

[Table 4] From the results shown in Table 4, the residual activity of L-methionine γ-lyase after storage at 37 ° C. for 4 weeks of the freeze-dried product without the addition of the additive is 14%, whereas the amino acid according to the present invention among the amino acids For freeze-dried products to which sodium glutamate or sodium aspartate, which are acidic amino acids, are added,
It can be seen that the activity is maintained at 70% or more. It is also found that other amino acids have no stabilizing effect on L-methionine γ-lyase. The lyophilized product prepared above was stored at 5 ° C. for 50 days, then redissolved and the enzyme activity was measured. The residual activity of the enzyme after lyophilization was as follows. Then 55%
On the other hand, the lyophilized products to which the acidic amino acid according to the present invention was added were all 100%. From the above, it can be understood that L-methionine γ-lyase can be stored extremely stably by using the stabilization method of the present invention.

Example 5 L-methionine γ-lyase obtained in Reference Example 1 was used in an amount of about 0.52 u / ml.
A gelatin hydrolyzate and saccharose (or trehalose) were each added to the enzyme solution containing 5% (w / v)%, and the resulting solution was divided into 1.0 ml aliquots in vials and frozen by a conventional method. Dried. The obtained freeze-dried product
After storage at 37 ° C. for 3 months, the mixture was redissolved in 1.0 ml of the enzyme solution, and the enzyme activity was measured. The obtained results are shown in Table 5 as the residual activity rate (%) of the enzyme after lyophilization, where the enzyme activity of the lyophilized stock solution is 100%.

[Table 5] From the results shown in Table 5, the lyophilized product containing no additives had a residual activity ratio of L-methionine γ-lyase of 8% after storage at 37 ° C. for 3 months, whereas gelatin hydrolyzate and saccharose (Or trehalose) and the freeze-dried products added in combination all show 100% residual activity. Comparison of this result with the results of Example 1 and Example 3 indicates that the combination use of the oligosaccharide and the protein according to the present invention enables more stable storage of L-methionine γ-lyase.

Comparative Example 1 L-methionine γ-lyase obtained in Reference Example 1 was used in an amount of about 2.84 u / ml.
The enzyme solution containing the enzyme was stored at 5 ° C. for 50 days, and then the enzyme activity was measured. As a result, it was found that the enzyme activity after low-temperature storage for 50 days was reduced to 28% of the enzyme activity before storage.

Comparative Example 2 About 0.26 u / ml of L-methionine γ-lyase obtained in Reference Example 1
Dithiothreitol (DTT) was added to the enzyme solution containing 1.
The solution added to a concentration of 2 mM was subdivided into vials (1.0 ml each) and freeze-dried by a conventional method. After the obtained freeze-dried product was stored at 37 ° C. for one week, it was redissolved in 1.0 ml of an enzyme solution, and the enzyme activity was measured. The obtained results are shown in Table 6 as the residual activity rate (%) of the enzyme after lyophilization, where the enzyme activity of the lyophilized stock solution is 100%.

[Table 6] From the results in Table 6, it can be seen that the lyophilized product to which DTT was added was 32% while the residual activity of L-methionine γ-lyase was 32% after storage at 37 ° C. for 1 week. The residual activity ratio is 5%, which means that DTT has no effect of stabilizing L-methionine γ-lyase.

Comparative Example 3 L-methionine γ-lyase obtained in Reference Example 1 was used at about 1.67 u / ml.
After storing the enzyme solution at -20 ° C for 50 days,
The enzyme activity was measured for the redissolved dialysis for 1 hour in a 0.01 M potassium phosphate solution containing TT. As a result, it was found that the enzyme activity after cryopreservation for 70 days was reduced to 30% before storage. Example 6 L- obtained in Reference Example 1
Glutathione or N-acetylcysteine is added to the enzyme solution containing about 2.72 u / ml of methionine γ-lyase to a concentration of 10 mM, and the resulting solution is divided into 1.0 ml aliquots in vials and frozen by a standard method. Dried. After the obtained freeze-dried product was stored at 37 ° C. for 2 months, it was redissolved in 1.0 ml of an enzyme solution, and the enzyme activity was measured. The obtained results are shown in Table 7 as the residual activity rate (%) of the enzyme after lyophilization, where the enzyme activity of the lyophilized stock solution is 100%.

[Table 7] From the results shown in Table 7, the residual activity of L-methionine γ-lyase after storage at 37 ° C. for 2 months was 10% for the freeze-dried product to which no additive was added, whereas glutathione or N-acetylcysteine was used. In the freeze-dried products to which
It can be seen that the residual activity ratio is around 5%, that is, glutathione and N-acetylcysteine have an effect of preventing the inactivation of L-methionine γ-lyase during freeze-drying. From the results of Comparative Example 2, it was found that the thiol compound 1
It has been found that DTT, a species, has no effect in preventing the inactivation of L-methion γ-lyase during lyophilization, so it is surprising that glutathione and N-acetylcysteine have such an effect. Was that.

Example 7 L-methionine γ-lyase obtained in Reference Example 1 was used in an amount of about 0.52 u / ml.
Trehalose (or saccharose) in the enzyme solution containing
5% (w / v), or 5% trehalose (or saccharose) and 5% glutamine (or sodium glutamate)
(W / V)%, which was added and dissolved in 1.0 ml aliquots into vials and freeze-dried by a conventional method. After the obtained freeze-dried product was stored at 37 ° C. for 16 months, it was redissolved in 1.0 ml of an enzyme solution, and the enzyme activity was measured. The results obtained are shown in Table 8 as the residual activity ratio (%) of the enzyme after freeze-drying, where the enzyme activity of the freeze-dried stock solution is 100%.

[Table 8] From the results in Table 8, it is found that the lyophilized product to which no additive was added had a residual activity of 0% of L-methionine γ-lyase after storage at 37 ° C. for 2 months, whereas only trehalose (or saccharose) was used. The freeze-dried product added with shows a residual activity of 0%, and the freeze-dried product added with a combination of trehalose (or saccharose) and glutamine (or Na glutamate) shows a residual activity of 37 to 62%. You can see that From these facts, by using trehalose (or saccharose) in combination with glutamine (or sodium glutamate), the stability of L-methionine γ-lyase at the time of lyophilization is more than that of trehalose (or saccharose) alone. It can be seen that it can be improved.

Example 8 The amount of L-methionine γ-lyase obtained in Reference Example 1 was about 2.72 u / ml.
5% (w / v) trehalose or 5% (w / v) trehalose and glutathione (or N-
(Acetylcysteine) was added and dissolved to a concentration of 10 mM, and the resulting solution was divided into 1.0 ml vials, and freeze-dried by a conventional method. After the obtained freeze-dried product was stored at 37 ° C. for 2 months, it was redissolved in 1.0 ml of an enzyme solution, and the enzyme activity was measured. The obtained results were compared with the enzyme activity of the lyophilized stock solution of 100%.
Table 9 shows the residual activity ratio (%) of the enzyme after freeze-drying in the case of%.

[Table 9] From the results in Table 9, the lyophilized product containing no additive had a residual activity ratio of L-methionine γ-lyase of 10% after storage at 37 ° C. for 2 months, whereas the lyophilized product containing only trehalose was added. The dried product shows a residual activity rate of 25%, trehalose and glutathione (or N-acetylcysteine)
It can be seen that the freeze-dried product added in combination with the above exhibits a residual activity of 89 to 95%. From these facts, by using trehalose in combination with glutathione (or N-acetylcysteine), the stability of L-methionine γ-lyase at the time of lyophilization can be improved as compared with the case of trehalose alone. I understand.

[0024]

As is apparent from the above description, the present invention provides a method for stabilizing L-methionine γ-lyase useful for detection and quantification of amino acids such as L-methionine and L-cysteine. By utilizing the present invention, L-methionine γ-lyase, which was conventionally difficult to store, can be stored easily and stably for a long period of time. is there.

Continuation of front page (56) References JP-A-62-221697 (JP, A) JP-A-57-118790 (JP, A) JP-A-2-23866 (JP, A) JP-A-62-130687 (JP) , A) Analytical Biochemistry, Vol. 138, (1984), p. 421-424 (58) Field surveyed (Int. Cl. ⁷ , DB name) C12N 9/00-9/99 BIOSIS (DIALOG) MEDLINE (STN) WPI (DIALOG)

Claims (13)

(57) [Claims] 1. L-methionine γ-lyase, (1) oligosaccharide, (2) water-soluble polysaccharide, (3) sugar alcohol,
Lyophilizing a solution in which one or more substances selected from the group consisting of (4) protein, (5) acidic amino acid, (6) glutathione, and (7) N-acetylcysteine are present. Characteristic method for stabilizing L-methionine γ-lyase. 2. The method according to claim 1, wherein the oligosaccharide is saccharose, maltose,
2. The method according to claim 1, wherein the method is lactose, trehalose, raffinose or maltotriose. 3. The method according to claim 1, wherein the oligosaccharide is trehalose or raffinose. 4. The method according to claim 1, wherein the water-soluble polysaccharide is dextran. 5. The method according to claim 1, wherein the sugar alcohol is sorbitol, mannitol or xylitol. 6. The method according to claim 1, wherein the protein is a hydrolyzate of gelatin. 7. The method according to claim 1, wherein the acidic amino acid is glutamic acid, aspartic acid or an alkali metal salt thereof. 8. One or two selected from the group consisting of L-methionine γ-lyase, oligosaccharide and (1) protein, (2) amino acid, (3) glutathione and (4) N-acetylcysteine. A method for stabilizing L-methionine γ-lyase, comprising freeze-drying a solution in which the above-mentioned substance is coexisted. 9. An oligosaccharide comprising saccharose, maltose,
9. The method according to claim 8, wherein the method is lactose, trehalose, raffinose or maltotriose. 10. The method according to claim 8, wherein the protein is a hydrolyzate of gelatin. 11. The method according to claim 8, wherein the amino acid is glutamine, glutamic acid or a salt of glutamic acid. 12. L-methionine γ-lyase, and (1)
Oligosaccharide, (2) water-soluble polysaccharide, (3) sugar alcohol,
A solution obtained by freeze-drying a solution containing one or more substances selected from the group consisting of (4) a protein, (5) an acidic amino acid, (6) glutathione, and (7) N-acetylcysteine. , Stabilized L-methionine γ-lyase. 13. An L-methionine γ-lyase, an oligosaccharide, and one or more selected from the group consisting of (1) protein, (2) amino acid, (3) glutathione, and (4) N-acetylcysteine. A stabilized L-methionine γ-lyase, which is obtained by freeze-drying a solution in which a substance and a substance are coexisted.