JPH0358783A - Stabilization of enzyme - Google Patents

Abstract

PURPOSE:To keep the activity of glycerol kinase in an aqueous solution over a long period and obtain a stabilized enzyme useful for the quantitative determination of glycerol (derivative) by bonding glycerol kinase to a water-soluble polysaccharide through covalent bond. CONSTITUTION:(A) Glycerol kinase is stabilized by bonding to (B) a water-soluble polysaccharide through covalent bond. Preferably, the component A is an enzyme originated from genus Cellulomonas, etc., and the component B is a compound containing at least a pair of adjacent OH groups in the constituent unit, oxidizable with an oxidizing agent and capable of forming an aldehyde group reactive with amino group in the enzyme, especially preferably dextran, etc. The formation of the covalent bond is preferably carried out e.g. by oxidizing the component B to form an aldehyde of the component B, reacting the aldehyde with the component A in a buffer solution at pH5-10 and 15-40 deg.C, adding a reducing agent to the system and reacting at 5-40 deg.C.

Description

[Detailed Description of the Invention] [Field of Application of the Invention] The present invention provides a stabilization method for glycerol kinase that can stably maintain enzymatic activity for a long period of time in an aqueous solution state, a stabilized glycerol kinase, and the stabilized glycerol kinase. The present invention relates to a method for quantifying glycerin or/and glycerin derivatives using glycerol kinase.

[Background of the invention] Glycerol kinase (hereinafter abbreviated as GK).

) is an enzyme that catalyzes the reaction shown in the following formula.

Glycerin + ATP ≧→ Glycerol-3-phosphate + ADP ATP: Adenosine 5' monotriphosphate.

ADP: Adenosine 5'monodiphosphate.

Various measurement methods using this catalytic reaction are widely practiced in fields such as clinical chemistry, biochemistry, food chemistry, and food industry.

For example, there is a method in which GK is used as a conjugate enzyme when quantifying triglycerides in biological samples. This method is widely used because triglycerides can be determined with high accuracy under mild conditions. However, the stability of this enzyme in aqueous solution is low, and the usable period of the prepared enzyme solution is short, so in order to use it efficiently, it is necessary to prepare the required amount at the time of use. there were.

Therefore, in order to stabilize GK in an aqueous solution, methods of adding ammonium sulfate, ethylene glycol, glycerol, ATP, etc. as stabilizing agents have been attempted, but they have not been able to achieve a sufficient stabilizing effect. It cannot be said that this has been achieved, and further improvements are desired.

In addition, we screened persimmon microorganisms for GK that has good stability in aqueous solutions and has properties suitable for quantifying 1-liglyceride in biological samples.
■－． Methods are also being considered to try to solve the problems described above. However, this method not only requires a great deal of cost and time, but also provides a
There is no guarantee that you will be able to find this, so it is not a very practical method.

On the other hand, in general, methods for stabilizing enzymes include adding sugars such as sucrose and maltose, proteins such as anolepmin and skimminolec, and Ca' salts to the enzyme solution.
A method of adding salts such as g2+m, a reducing agent such as 2-merkab1 and ethanol, a method of nationalizing the enzyme to an appropriate I114 form, a method of modifying the amino acid sequence of the enzyme by illegal genetic engineering, etc. However, there is no general rule that says which of the above methods is best for a particular enzyme, and the method of stabilization for each enzyme is selected through trial and error. In reality, among these stabilization methods, the method of immobilization on a carrier is a method that is often used by Fanchika, and is known as a method with a relatively high stabilizing effect (Shimoto et al. Teuts Patent No. 2,
9] 9,622 shi;・Details j1～ etc.). however,
Even with this method, there are problems such as the enzymatic activity not being expressed if the amino acid residues involved in the expression of the enzymatic activity, such as the active center of the enzyme or the substrate binding site, are modified.
Even if we conduct various studies on binding force methods, binding agents, cross-linking agents, etc. in order to select the optimal conditions, it is not necessarily applicable to every enzyme, and in that respect it is exactly the same as other methods. It is.

[J-Object of the Invention] The present invention has been made in view of the above-mentioned circumstances, and provides a GK that can maintain the activity of GK at IIQ in an aqueous solution for a long period of time.
A method for stabilizing G K stabilized by this method
It is an object of the present invention to provide a method for quantifying glycerin or/and glycerin derivatives using the present invention.

[Configuration of the Invention] The present invention provides a method for stabilizing GK, which is characterized by covalently bonding GK to a water-soluble polysaccharide, and a method for stabilizing GK, which is characterized by covalently bonding GK to a water-soluble polysaccharide. In addition, in a method for quantifying glycerin or/and glycerin derivatives using the enzyme activity of GK, GK covalently bonded to a water-soluble polysaccharide is used as GK.
This invention is a method for quantifying glycerin or/and glycerin derivatives, characterized in that K is used.

That is, the present inventors have conducted extensive research to develop a method that can maintain the activity of GK in an aqueous solution for a long period of time.
By covalently bonding GK to a water-soluble polysaccharide, it can be stabilized for a long period of time while maintaining water solubility, and furthermore, GK stabilized in this way can be used as a conjugate enzyme in the measurement of glycerin or/and glycerin derivatives. The present inventors have found that the present invention can be effectively utilized and have completed the present invention.

The water-soluble polysaccharides used in the present invention include:
'1. If it has at least one adjacent hydroxyl group in it and is oxidized by an oxidizing agent such as periodate or metaperiodate to produce an aldehyde group that can react with the amino group in the enzyme molecule. may be a natural product or a synthesized product, and is not particularly limited, but preferably includes, for example, dex-1-lan, dextran sulfate, dextrin, pullulan, soluble starch, chondroitin sulfate, laminan, lichenan, methylcellulose, etc. Can be mentioned.

Furthermore, the molecular weight of these water-soluble polysaccharides is not particularly limited as long as they are water-soluble, but l. ,000 to 800,000 is preferably mentioned.

The GK used in the present invention is not particularly limited in its variety, but for example, Cellulomonas (Cel).u1 o
mon a s ) genus, Escherichia coli (E
．． CoLi) genus, Earthmouth Bacter (Art; hlob)
acl;er) genus, Strebtomyces (strepto
Preferred examples include those derived from the genus Myces.

As a method for producing a covalently bonded product of GK and a water-soluble polysaccharide according to the present invention, first, 1 part by weight of a water-soluble polysaccharide is added to
Add 1 part by weight of an oxidizing agent and react in a dark place at O'C to room temperature for several hours to several days to oxidize the water-soluble polysaccharide.

Next, the excess oxidizing agent is decomposed with a reducing agent, usually 1 to 2 parts by weight per 1 part by weight of the oxidizing agent used, and the reaction solution is further dialyzed in water or an appropriate buffer solution, or subjected to gel filtration. The water-soluble polysaccharide aldehyde (hereinafter referred to as PSA) is purified by
It is abbreviated as. ).

Next, this PS-A and GK are reacted in a buffer solution with a pH of 5 to 0 at a weight ratio of 30:1. Incidentally, the reaction temperature at this time is preferably in the range of 15 to 40°C.

In other words, if the reaction is carried out at a temperature lower than 15°C, the reaction may take a long time to complete or not proceed at all, and the stability of GK immobilized on the water-soluble polysaccharide may be affected. If anything, it gets worse, and 4
This is because, if the reaction is carried out at a temperature exceeding 0° C., problems such as a low recovery rate of GK after the reaction and an increase in production cost will occur.

Furthermore, a reducing agent (e.g., pyridine borane, sodium cyanoborate, etc.) is added to this so that the concentration in the reaction solution is 1 to 5.
It is added so that it becomes W/V%, and it is made to react at 5-40 degreeC for several hours thru|or several days.

It is preferable to coexist with a GK stabilizer such as glycerol, EDTA, protein, etc. during these reactions, since deactivation of GK activity after the reaction is less likely to occur.

The solution obtained by the above reaction is dialyzed against water or an appropriate buffer to form the desired covalently bound product of GK and water-soluble polysaccharide, that is, a covalently bound product of GK and PS-A ( GK/PS-A covalent bond) is obtained. The GK/PSA covalent bond thus obtained can be fully used as stabilized GK, but in order to further improve the stability of GK, it is desirable to inactivate the remaining aldehyde groups. That is, an appropriate reducing agent different from the one used previously is added to the reaction solution after reduction so that the concentration in the reaction solution is 1 to 5.
W/V%, and further reduce reaction at 0 to 40°C for 2 to 20 hours, or to the same reaction solution, add 0.1 to 0.5 M of a compound having an amino group. A reducing agent similar to that used previously was added so that the concentration in the reaction solution was 1 to 5 W/V%, and the mixture was heated again at 5 to 40°C for several hours to several hours. It is desirable to react for several days, and then purify by dialysis against water or an appropriate buffer to obtain a more stable GK/PS-A covalent bond.

Examples of the oxidizing agent used in producing PS-A include various types of periodate such as orthoperiodine 1g2 salt, nimesoperiodate, mesoperiodate, niorthoperiodate, and metaperiodate. Preferable examples include acid salts (alkali metal salts, etc.), and examples of reducing agents used to treat excess oxidizing agent include sodium bisulfite, sodium thiosulfate, hydrosulfite, and the like.

The buffer used for the buffer used when reacting PS-A and GK or when dialyzing the reaction solution at the end of each reaction may be one that does not deactivate GK and does not react with the reducing agent. Although they can be used without particular problems, preferred examples include phosphates and Good's buffers.

Further, as the reducing agent used when PS-A and GK are reacted, for example, pyridine borane, sodium cyanoborohydride, etc. are preferably mentioned.

A protein that is preferably added when reacting PS-A and GK may be included as an excipient in the lyophilized powder of GK that serves as a raw material. Preferred proteins used for this purpose include proteins such as albumin, globulin, casein, gelatin, etc.
The content of GK in the lyophilized powder is not particularly limited, but is preferably 10 to 90%, more preferably 30 to 50%.

Preferred examples of the reducing agent used to inactivate the aldehyde group remaining after covalently bonding GK and PS-A include sodium borohydride and dimethylamine borane.

In addition, compounds that destroy the amino group used to inactivate the aldehyde group remaining after covalent bonding with GK and PS-A are not particularly limited, but include glycine, ethanolamine, glycine ethyl ester, etc. Examples of the reducing agent added at this time include pyridine borane and sodium cyanoborohydride.

The GK. obtained in this way. The /PS-A covalent bond has excellent stability and can be stored for a long period of time in the form of an aqueous solution, but it is also naturally possible to freeze-dry it and store it as a powder.

The GK/PS-A covalent bond product according to the present invention is stable for a long period of time in an aqueous solution state, and moreover, its affinity with the substrate and specificity for the substrate are almost equivalent to those of the original GK. It can be widely used in fields such as clinical chemistry, biochemistry, food chemistry, and food industry. Especially for clinical chemistry! 1 in IIf
When used as a conjugate enzyme added to a reagent solution for measurement of GK, the reagent solution for measurement becomes stable for a long period of time, and has a significant advantage over conventional products. Since this is an enzyme that uses glycerin as a substrate, which has three types of glycerin, when it is combined with a polysaccharide containing many hydroxyl groups, there is a strong possibility that the reaction rate with its substrate specificity r1゛ and (quality) will change. However, GK covalently bonded to the water-soluble polysaccharide according to the present invention has almost the same substrate specificity and reaction rate with the A substance as that of GK before bonding to the water-soluble polysaccharide. It was completely unexpected that it would have the extremely beneficial properties of changing the chemical composition and significantly increasing its stability in aqueous solution.

The present invention will be described in more detail with reference to Examples below, but the present invention is not limited thereto.

[Example] Example 1-. 1 2 Dextran (hereinafter abbreviated as Dcx. Molecular weight: 100
,000-200,000. ) 20 ml of the IOW/Vl solution was cooled to 5° C., and 2 g of sodium metaperiodate was added in small portions with stirring. This was reacted in the dark at 5°C with stirring for 24 hours. After the reaction is complete, remove excess sodium metaperiodate at 40W/V%1I.
■Sodium hydrogen sulfate aqueous solution 7m. Add I and decompose,
This reaction solution was dialyzed against ion-exchanged water at room temperature, and D
45 m of an aldehyde derivative solution of ex] was obtained. 0.2 M N,N bis(2-hydroxyethyl)- in which 5 mg of GK (derived from Cellulomonas sp., 3411/mg) was dissolved in 0.6 ml of the obtained Dex aldehyde derivative solution.
2-aminoethanesulfonic acid (BES) buffer (pll
7.5)1. Add 0 ml of pyridine borane 2
0 μl was added and reacted at 30″C for 18 hours with stirring. Then, 0.3 μl of a 2M aqueous glycine solution was added to the reaction solution.
ml and 20μ of pyridine holane] were added, and the reaction was further stirred at 30"C for 8 hours. After the reaction was completed, the reaction solution was dialyzed against ]OmM phosphate buffer (pl 17.0), and G K / D cx covalent bond (GK and Ding) ex
3.5 ml of a solution containing a co-conjugate with the enzyme was obtained (enzyme activity recovery rate: 34.9%).

Example 2. In Example 1, GK derived from the genus Escherichiacoli was replaced with GK derived from the genus Cellulomonas ]. 5mg(]4
．． The reaction and post-treatment were carried out in exactly the same manner as in Example 1, except for the above, to obtain 3.7 ml of a solution containing the GK/D ex covalent bond (enzyme activity recovery rate =
7.0%), Example 3. In Example 1, GK derived from the genus Strebtomyces was replaced with GK derived from the genus Cellulomonas]. 5mg (]6
．． G K / D ex
4.0 ml of a solution containing a covalently bound substance was obtained (enzyme activity recovery rate = 74.7%).

Example 4. In Example 1, 1.5 mg (25.3 mg) of GK derived from the genus Arthrobacter was used instead of GK derived from the genus Cellulomonas.
G K / I) e x
4.9 ml of a solution containing a covalently bound substance was obtained (enzyme activity recovery rate: 27.6%).

Example 5. In Example 1, IO of soluble starch (hereinafter abbreviated as SS) was used instead of the IOW/V% aqueous solution of Dex.
Using a W/V% aqueous solution, the reaction and post-treatment were carried out in the same manner as in Example 1, except for the reaction and post-treatment, to obtain a GK/SS covalent bond (
5. A solution containing a covalent bond of GK and SS. ] mF was obtained (enzyme activity recovery rate: 58.3%).

Example 6. In Example 1, instead of the 1.0 W/V% aqueous solution of Dex, 10% of dextrin (hereinafter abbreviated as Din) was used.
Using a W/V% aqueous solution, the reaction and post-treatment were carried out in the same manner as in the example process, and G K / D j n
4.8 ml of a solution containing a covalent bond (a covalent bond between GK and Din) was obtained (enzyme activity recovery rate: 62.9%).

Example 7. In Example 1, instead of the 10 W/V% aqueous solution of Dex, I of pullulan (hereinafter abbreviated as Pu) was used.
Using an OW/V% aqueous solution, the reaction and post-treatment were carried out in the same manner as in Example 1 except for the reaction, and a solution containing a GK/Pul covalent bond (a covalent bond of GK and PuJ) was prepared. 5
．． 2 ml was obtained (enzyme activity recovery rate: 59.6%).

Example 8. Molecular weight distribution is 1.7,000, 60,000-90,0
00, 100,000-200,000, 200,00
0-300,000 Dex 2 g each for 25 m]
The solution dissolved in ion-exchanged water was cooled to 5° C., and 1.6 g of sodium metaperiodate was added little by little to each solution while stirring. These were stirred and reacted in the dark at 5°C for 19 hours. After the reaction, excess sodium metaperiodate was dissolved in 4.5 m of 40 W/V% sodium bisulfite aqueous solution.
These reaction solutions were dialyzed against ion-exchanged water at room temperature to obtain 1.50 m of Dex aldehyde derivative solutions with various molecular weight distributions. Each De obtained
20 mg of GK (derived from Cellulomonas sp., 300/mg) was dissolved in 1.5 ml of aldehyde derivative solution of x.
．． 2M BES buffer (containing 5mM EDTA. pl1
7.5) Add 3.5ml and then add 50μ of pyridine borane.
1 was added and reacted at 30° C. for 20 hours under pressure #1.

Then, add 1 ml of 2M chrysin ethyl ester solution to each reaction solution.
6 Add 0.5 ml of solution and 50 μl of pyridine borane, and add 2
The reaction was carried out at 8°C for 20 hours with stirring. After completion of the reaction, each reaction solution was diluted with 10mM phosphate buffer (p}!7.0, 2mM
Contains EDTA.

), and 8.4 ml of each solution containing the G K /D ex-containing conjugate was dialyzed. 8.4ml. 8.0ml
and 7.9 ml was obtained (recovery rate of each enzyme activity: 9.1%, 6
2.9%, 64.4% and 61.6%).

Example 9. Measurement of triglyceride (measurement sample solution) Solution A: 0.05M piperazine-N,N'-bis(2-
ethanesulfonic acid) (PIPES) buffer (pH 6.
5), N-ethyl-N-(2-hydroxy-3-sulfopyr)-3,5-dimethoxyaniline Na salt (D
AOS, manufactured by Dojindo Kagaku Kenkyusho C Co., Ltd.) 0.235 mg/m
l. ATP 1.7mg#nl-. The G obtained in Example 1 was dissolved in 82.5 U/ml of lipoprotein lipase (manufactured by Toyo Jozo Co., Ltd.) and 0.80 U/ml of ascorbic acid oxidase (manufactured by Boehringer Mannheim). GK/D ex covalent conjugate
It was added so that the activity was 3.5 U/ml to obtain a solution A.

Solution B: 0.05) IPIPEs buffer (pH 6.5)
0.55 mg/rrl],
Gly70phosphate oxidase (manufactured by Toyo Jozo Co., Ltd.) 22
．． 811/ml-. Peroxidase (manufactured by Toyobo Co., Ltd., type IT) was dissolved to a concentration of 5.40/ml and used as Solution B.

(Samples) 8 fresh serum samples and triglyceride 300mg/dl
The sample was a standard serum containing .

(Procedure) 5 ml of solution A was added to 20 μl of the sample and prewarmed at 37°C for 3 minutes. Next, 0.75 ml of solution B was added to this, mixed well, and heated at 37°C for 4.5 minutes. After 600n
The absorbance of m was measured.

Incidentally, the same operation was performed using ion-exchanged water instead of the sample, and the reagent blind value (EBB.) was measured.

The triglyceride values (TG) of 8 fresh serum samples were determined by substituting the measurement results into the following equation.

TG (mg/dl) = (Es-EBI) ÷ (Estd
- EBI) X 300Es: absorbance obtained with fresh serum.

Estd: Absorbance obtained with standard serum.

(result) The measurement results are shown in Table 1.

Example 1.0. 1-Liglyceride measurement example 9
In this case, the G K obtained in Example] was added to solution A of the measurement reagent solution
Example 3 instead of adding the right conjugate
The GK/Dex covalent bond obtained in Example 9 was added so that the GK activity was 3.5 U/ml, and the measurement was carried out in the same manner as in Example 9. The triglyceride values (G) of 8 freshly cleaned samples used were determined.

(result) The measurement results are also shown in Table 1.

Comparative example 1-. 8 of triglycerides! Each triglyceride sample of the 8 fresh serum samples used in Reconstitution Example 9 was
Using a commercially available triglyceride measurement reagent (Triglyceride E-Test Wako, manufactured by Wako Pure Chemical Industries, Ltd.),
It was determined using the standard operating method.

The measurement results are also shown in Table 1.

19 As is clear from the results in Table 1, the results obtained with the reagent for triglyceride measurement using GK stabilized by the method of the present invention as a conjugate enzyme are better than those obtained with the commercially available reagent for triglyceride measurement. The results were in good agreement with those obtained by.

2 0 Experimental example 1. Thermal stability test-1 (Sample) Each GK solution of Kumikai shown in Table 2 (2oIIl Asho liquid, p
I+7. 0, ill/m]. ) was used as a sample.

Table 2 phosphoric acid Untreated GK: derived from Cellulomonas.

(Procedure) After each sample was left at 55° C. for a predetermined period of time, the residual activity rate of GK was determined.

In addition, the activity measurement of GK is carried out using the Method of Enzyme Analysis.
aLie Ana. lysjs), Vol. II
, 2] on pages 6-217.

That is, using glycerin as a substrate at a reaction temperature of 5°C. 2
00mM G') SynIa buffer (pi{9.8, 24% hydrazine hydrate; 208mg/m] and MgC1 2.6
++20; 0.4 mg/m]. ) in 1 minute 111
1 + The amount of enzyme that releases 1 μmole of NAD I+ is 1 -. ! It was ranked Ji (U).

(Results) The obtained thermal stability curve is shown in FIG. In addition, the thermal stability curve is obtained by connecting the points obtained by plotting the logarithm of the residual activity rate of GK obtained for each time (minute) on the horizontal axis along the vertical axis. - indicates the results obtained with Sample 2, 1●1 indicates the results obtained with Sample 2, 1△- indicates the results obtained with Sample 3, and Sip- indicates the results obtained with Sample 4. show. Further, the logarithm value of the residual activity rate of GK was determined by the following formula.

Residual activity rate log value = 1. n (A t. / A.o
) Ao: GK activity value (U/ml.) immediately after sample preparation.

At: GK activity value (U/m
Ko).

As is clear from the results shown in Figure 1, the stabilized GK of the present invention is significantly more stable than untreated GK or untreated GK with 1% or 10% Dex added. I understand.

Experimental example 2. Thermal Stability Test-2 (Sample) The GK/Dex covalent bond obtained in Example 2 and the untreated GK derived from the genus Escherichiacoli were each tested at 20%
A sample was prepared by dissolving it in mM phosphate buffer (PII7.0) at a concentration of IU/ml.

(Operation method) Same as Experimental Example 1.

(result) The obtained thermal stability curve is shown in 2nd S-(1).

The thermal stability curve is a graph that connects the points obtained by plotting the logarithm of the residual activity of GK obtained for each time (minutes) on the horizontal axis along the vertical axis. is GK/D
The results obtained with the sample containing the ex covalent compound are expressed as -
●1 shows the results obtained with samples containing untreated GK.

Experimental example 3. Thermal Stability Test-3 (Sample) 2 3 The GK/D ex covalent bond obtained in Example 3 and the untreated GK derived from Streptomyces were each incubated at 20 m
The sample was dissolved in M phosphate buffer (p++7.0) to a concentration of 111/m].

(Operation method) Same as Experimental Example 1.

(result) The obtained thermal stability curve is shown in FIG. 2-(2).

The thermal stability curve is a graph that connects the points obtained by plotting the logarithm of the residual activity of GK obtained for each time (minutes) on the horizontal axis along the vertical axis. is GK/D
The results obtained with the sample containing the ex covalent bond are shown, and 1●- shows the results obtained with the sample containing untreated GK, respectively.

Experimental example 4. Thermal Stability Test-4 (Sample) The GK/Dex covalent bond obtained in Example 4 and the untreated GK derived from Earthmouth Bacter genus were each
The sample was prepared by dissolving it in mM phosphate buffer (pl+7.0) at a concentration of 1/ml.

2 4 (Operation method) Same as the experimental example.

(result) The obtained thermal stability curve is shown in FIG. 2-(3).

The thermal stability curve is a graph that connects the points obtained by plotting the logarithm of the residual activity of GK obtained for each time (minutes) on the horizontal axis along the vertical axis. is GK/D
The results obtained with the sample containing the e x covalent conjugate,
1●- indicates the results obtained with samples containing untreated GK, respectively.

As is clear from the results shown in FIG. 2, (1) to (3), the stabilized GK of the present invention is significantly more stable than untreated GK.

Experimental example 5. Thermal Stability Test-5 (Sample) Various GK/D ex covalent compounds obtained in Example 8 and untreated GK derived from the genus Cellulomonas were each treated with 20I
IU/ml in IIM phosphate buffer (p[+7.0)
The sample was prepared by dissolving it so that

(Operation method) Same as Experimental Example 1.

(Results) The obtained thermal stability curve is shown in FIG. The thermal stability curve is a graph that connects the points obtained by plotting the logarithm of the residual activity of GK obtained for each time (minutes) on the horizontal axis along the vertical axis. - has a molecular weight distribution of 1.7,000
The results obtained with the sample containing the GK/Dex covalent bond prepared using Dex of
G prepared using Dex of 0,000-90,000
The results obtained with the sample containing the K/D ex covalent conjugate were compared to those with a molecular weight distribution of 100,000-20
The results obtained with the sample containing the GK/Dex covalent compound prepared using Dex of 0,000 were compared with the results obtained with the sample containing the GK/D covalent compound prepared using Dex with a molecular weight distribution of 200,000-300,000. -111 indicates the results obtained with the sample containing the ex covalent bond, and -111 indicates the result obtained with the sample containing untreated GK.

As is clear from the results in Figure 3, the molecular weight distribution is 17,0
It can be seen that the stabilized GK of the present invention prepared using various Dex from 00 to 300,000 are significantly more stable than the untreated GK.

Experimental Example 6, Thermal Stability Test @-6 (Sample) Two volumes of each of the various GK/water-soluble polysaccharide covalently bonded products prepared in Examples 5 to 7 and the terminally treated GK derived from Cellulomonas sp.
0mM phosphate buffer (1) 117. 0) to 1-
A test sample was obtained by dissolving the solution at a ratio of U/m-1.

(Operation method) Experimental example] Same as -.

(Results) The obtained thermal stability operating curve is shown in Fig. 4. The thermal stability curve is obtained by connecting the points where the logarithm of the residual activity of GK obtained for each time (minute) on the horizontal axis is plotted along the vertical axis. , 1 - is GK/ prepared in Example 5
The results obtained with the sample containing the SS shared introduction 6 substances were
- indicates GK/D Jn manufactured by wM in Example 6.
The results obtained with the sample containing the conjugate, -◎1 the GK/P u prepared in Example 7] the results obtained with the sample containing the covalent conjugate, and the results obtained with the untreated G
The results obtained with 97 samples containing K are shown.

As is clear from the results in Figure 4, the stabilized GK of the present invention prepared using various water-soluble polysaccharides is significantly superior to untreated GK in terms of κ1, Experimental example 7. Storage stability test (trial) The GK/l)c x co-conjugate obtained in Example 1 and the untreated GK of Cellulomonas spp.
A sample was prepared by dissolving 1-U/ml in phosphate buffer (ptl 7.0).

(Procedure) Store the sample at 4°C or 30°C at a specified temperature,
The residual activity rate was measured. The GK activity was measured using the same procedure as in Experimental Example 1. In addition, the residual activity rate of GK was determined by the following formula.

Residual activity rate = (B ÷ Bo) × 100 13o: GK activity i (( (11/
ml).

Bt: Predetermined GK activity value after -1 number of points (] 1/ml
).

(Results) The obtained storage stability curve is shown in FIG. In addition, the storage stability curve is calculated by G
The residual activity rate of K is connected along the vertical axis with 1 point, and in the figure, 1 row is GK. The results obtained when a sample containing a /Dex covalent bond was stored at 4°C were
11- shows the results obtained when the sample containing the untreated GK was stored at 4℃, and 101 shows the result obtained when the sample containing the GK/Dcx covalent bond was stored at 30℃. The obtained results are also summarized as follows.
The results obtained when the files were stored are shown below.

As is clear from the results in Figure 5, even when stored at 4°C or 30''C, the stabilized G
Experimental example 8+p is found to be significantly more stable than K.
I-{Stability test-1- (Sample) Untreated GK of Cellulomonas sp. and GK/I'') ex covalent bond obtained in Example 1 were each tested at 211/m
It was dissolved in a buffer solution of a predetermined pu to give a test sample.

In addition, at pl14.0-6.0, 5 (lmMffI acid-forming sodium 1-lium buffer solution was used, and at pl16.0-9.0
Then, using 50mM phosphate buffer, pl+9.0
-]. 1. ．． 0, a 50 mM potassium carbonate-sodium bicarbonate buffer was used.

(Procedure) After each sample was left at 30° C. for 20 hours, the residual activity rate of GK was determined.

The GK activity was measured in the same manner as in Experimental Example 1. In addition, the residual activity rate of GK was determined by the following formula.

Residual activity rate = (C/Co) X]. OOco = G K activity value (IJ/ml-) immediately after sample preparation.

CL: G K activity value (IJ/m:L) after standing at 30°C for 20 hours in a predetermined pH solution.

(Results) The obtained pI{stability curve is shown in No. 61'J-(].).

In addition, the pH stability curve is a result of connecting the points obtained by plotting the residual activity rate obtained for each pH on the horizontal axis with the change in the vertical axis. In the figure, 1●- indicates G K / The results obtained with the sample containing the Dcx covalent conjugate, 10 is the untreated G
The results obtained from the assay containing K are shown below.

Experimental example 9. pH Stability Test-2 (Sample) Untreated GK derived from the genus E. coli and the GK/Dex covalent bond obtained in Example 2 were each dissolved in a predetermined po buffer solution to a concentration of 2 U/ml and used as a sample. .

In addition, at pH 4.0-6.0, 50mM sodium acetate monoacetate
pH 8. 0 to 9.0 using 50 mM phosphate buffer and pH 9.0 to 9.0. 0-1
1.0, a 50 mM potassium carbonate-sodium bicarbonate buffer was used.

(Operation method) Same as Experimental Example 8.

(result) The obtained pH stability curve is shown in No. 65D-(2).

In addition, the pH stability curve connects the points obtained by plotting the residual activity rate obtained for each pH on the horizontal axis along the vertical axis. The results obtained with the sample containing the conjugate are
The results obtained with samples containing .

31 Experimental example 10. p. H Stability Test-3 (Sample) 2 U/ml each of untreated GK derived from Streptococcus genus and the G K / D ax covalent compound obtained in Example 3.
It was dissolved in a buffer solution of a predetermined pH so as to be used as a sample.

In addition, for pH 4.0 to 6.0, use 50 mM sodium acetate monoacetate buffer, for pH 6.0 to 9.0, use 50 mM phosphate buffer, and for pH 9.0 to 1.1.0, use 50 mM sodium acetate buffer.
An mM potassium carbonate-sodium bicarbonate buffer was used.

(Operation method) Same as Experimental Example 8.

(result) The obtained pH stability curve is shown in FIG. 6 (3).

In addition, the pH stability curve connects the points obtained by plotting the residual activity rate obtained for each po on the horizontal axis along the vertical axis, and in the figure, 1●1 indicates G K / Dex sharing. 101 is the untreated GK
The results obtained with samples containing .

Experimental example 1]. ．． PH Stability Test-4 3 2 (Sample → Untreated GK derived from Earthmouth Bacterium and GK/Dex covalent bond obtained in Example 4 at 2 U/ml each
It was dissolved in a buffer solution of a predetermined pH so as to be used as a sample.

In addition, at pH 4.0-6.0, 50 mM sodium acetate monoacetate buffer was used, at pH 6.0-9.0, 50 mM phosphate buffer was used, and at pH 9. 0 to 11.0, a 50 mN potassium carbonate-sodium hydrogen carbonate buffer was used.

(Operation method) Same as Experimental Example 8.

(result) The obtained pH stability curve is shown in FIG. 6 (4).

In addition, the pH stability curve connects the points obtained by plotting the residual activity rate obtained for each pH on the horizontal axis along the vertical axis, and in the figure, 1 - indicates G K / Dex sharing 10 indicates the results obtained with the sample containing the conjugate, and 10 indicates the result obtained with the sample containing untreated GK.

As is clear from Figure 6 1 (1) to (4), the untreated G
p of the G K / De x covalent conjugate compared to K
It can be seen that the H stability region is clearly wide and stabilized.

Experimental example]. 2. Measurement of Km value (sample) Untreated GK derived from Cellulomonas genus and G obtained from the example plant
A K/D ex covalent bond was used as a sample.

(Operation method) Method of enzyme analysis (M
method of enzymatic analysis
is) r Vol. Using the reagent for measuring GK activity described in II +216-217, the Km value was determined by a conventional method (reaction temperature: 25°C).

(result) The results obtained are shown in Table 3.

Table 3 (margin below) As is clear from the results in Table 3, the Km value of the GK stabilized by the method of the present invention is almost the same as that of the untreated GK.

Experimental example 13. Study of optimal pH (sample) Untreated GK derived from Cellulomonas and G obtained in Example 1
K/I) cx co-right bound product was used as a sample.

(Operation method) Method of Enzymatic Analysis (M
et) iod of Enzymat. ic anal
ysis), Vol. Using the reagent for measuring GK activity described in J.R.L., pages 216-217, a 35 pT-{curve was determined by a conventional method (reaction temperature 25
``C) (Results) The obtained optimal p II-I curve is shown in the 71'% J.The optimal pH curve is calculated by dividing the phase activation rate obtained by cutting at each pl+ on the horizontal axis. In the figure, 101 indicates the results obtained with the sample containing the GK/Dex covalent bond, and -●1 indicates the results obtained with the untreated GK.
The results obtained with samples containing .

In addition, the relative activity rate is the converted Iα of the GK activity value obtained for each PI-I, when the maximum of the GK activity values obtained for each PH is taken as 100%.

As is clear from the results in FIG. 7, the optimal PI of the GK stabilized by the method of the present invention is almost the same as that of the untreated GK.

[Effects of the Invention] As described above, the present invention provides a method for stabilizing GK that can maintain the activity of GK in an aqueous solution for a long time, and stabilized GK, flf2, and the stabilized GK. G
This invention provides a method for quantifying glycerin and/or glycerin-36 serine derivatives using K, and is a great invention that contributes to this industry.

[Brief explanation of drawings]

FIG. 1 shows the thermal stability curve obtained in Experimental Example]. FIG. 2 shows the thermal stability curves obtained in Experimental Examples 2-4. FIG. 3 shows the thermal stability curve obtained in Experimental Example 5. FIG. 4 shows the thermal stability curve obtained in Experimental Example 6. FIG. 5 shows the storage stability curve obtained in Experimental Example 7. FIG. 6 shows the pH stability curves obtained in Experimental Examples 8-11. FIG. 7 shows the optimum pH curve obtained in Experimental Example 3.

Claims (3)

[Claims] (1) A method for stabilizing glycerol kinase, which comprises covalently bonding glycerol kinase to a water-soluble polysaccharide. (2) Glycerol kinase covalently bound to a water-soluble polysaccharide. (3) A method for quantifying glycerin or/and a glycerin derivative using the enzymatic activity of glycerol kinase, characterized in that glycerol kinase covalently bonded to a water-soluble polysaccharide is used as the glycerol kinase. and a method for quantifying glycerin derivatives.