JP2628310B2 - Enzyme stabilization method - Google Patents

Description

Description: FIELD OF THE INVENTION The present invention relates to a stabilization method in which α-glucosidase can maintain enzyme activity stably in an aqueous solution state for a long period of time.

BACKGROUND OF THE INVENTION α-Glucosidase (hereinafter abbreviated as AG) hydrolyzes the α1,4 glucosidic bond at the non-reducing terminal of a sugar chain to form α-glucosidase.
It is an enzyme that catalyzes the reaction that produces glucose. Utilizing this catalytic reaction, it is widely used in fields such as clinical chemistry, biochemistry, food chemistry, and the food industry. One example of its use is, for example, the use of a conjugated enzyme when measuring α-amylase activity in a biological sample.However, the stability of this enzyme in an aqueous solution is low, and Since the usable period is short, there is a limitation in use such that it is necessary to prepare a necessary amount at the time of use for efficient use.

Therefore, in order to stabilize AG in an aqueous solution, a method of adding glutathione, glycerose, sodium glutamate, albumin, and the like has been attempted so far.
It is hard to say that a sufficient stabilizing effect has been obtained, and further improvement has been desired.

On the other hand, generally, methods for stabilizing an enzyme include, for example, sugars such as sucrose and maltose, proteins such as albumin and skim milk, salts such as Ca ²⁺ salt and Mg ²⁺ salt, 2-mercaptoethanol and the like in an enzyme solution. A method of adding a reducing agent, etc., a method of immobilizing an enzyme on a suitable carrier, a method of modifying the amino acid sequence of the enzyme by genetic engineering techniques, and the like are known. There is no general rule that the method is good, and at present the stabilization method is selected by trial and error for each enzyme.

Among these stabilization methods, the method based on immobilization to a carrier is a method frequently used recently, and is known as a method having a relatively high stabilizing effect. However, even in this method, there is a problem that the enzyme activity is not expressed when an amino acid residue related to the expression of the enzyme activity such as the active center of the enzyme or the substrate binding site is modified. Even if various studies are conducted on the binding method, binder, cross-linking agent, etc. in order to select the conditions, it is not necessarily applicable to any enzyme, and in that respect it is exactly the same as the other methods.

[Object of the Invention] The present invention has been made in view of such circumstances, and an object of the present invention is to provide a method for stabilizing AG that can maintain the activity of AG in an aqueous solution for a long time.

[Configuration of the Invention] In order to achieve the object of the present invention, the present invention has the following configuration.

"A method for stabilizing α-glucosidase in an aqueous solution, wherein α-glucosidase is covalently bonded to a water-soluble polysaccharide (excluding chondroitin)." Has intensively studied to develop a method that can maintain the activity of AG in aqueous solution for a long period of time.As a result, by covalently bonding AG to a water-soluble polysaccharide, it was confirmed that AG could be stabilized for a long time while maintaining water solubility. The present invention has been completed.

As the water-soluble polysaccharide used in the present invention, periodate, oxidized by an oxidizing agent such as metaperiodate,
It generates an aldehyde group capable of reacting with an amino group in an enzyme molecule and has a molecular weight of 5,000 to 500,000, and is not particularly limited. Examples thereof include dextran, dextran sulfate, pullulan, and soluble starch. Preferably have a molecular weight of 5,000 to 500,000.

The origin of the AG used in the present invention is not particularly limited, but those derived from yeast are preferably used.

As a method for producing a covalent bond of AG and a water-soluble polysaccharide according to the present invention, first, 0.1 part by weight of the water-soluble polysaccharide is used.
An oxidizing agent is added to 05 to 1 part by weight, and the reaction is carried out in a dark place at 5 ° C. to room temperature for 1 to 24 hours with stirring to oxidize the water-soluble polysaccharide. Next, the excess oxidizing agent is decomposed with 1 to 2 parts by weight of a reducing agent to 1 part by weight of the oxidizing agent used, and the reaction solution is dialyzed against water or a suitable buffer to obtain a water-soluble polysaccharide. (Hereinafter, abbreviated as PS-A).

This PS-A and AG are mixed in a buffer of pH 6 to 8 by weight ratio of 1 to 3
0: 1, and further added with a reducing agent (eg, pyridine borane, sodium cyanoborohydride, etc.) and PS-A
0.1 to 1 part by weight per 1 part by weight, 0 to 40 ° C
For 6 to 24 hours. A freeze-dried product is usually used as AG, but it is more preferable to contain an appropriate protein as an excipient in a freeze-dried powder, since the inactivation of the enzyme activity after the reaction is completed is small.

This reaction solution is dialyzed against water or an appropriate buffer to form a covalent bond between the desired AG and the water-soluble polysaccharide,
That is, a covalent bond between AG and PS-A (AG / PS-A covalent bond) is obtained. The thus obtained AG / PS-A covalent bond can be sufficiently used as a stabilized AG, but it is desirable to inactivate the remaining aldehyde group in order to further improve the stability of AG. That is, a suitable reducing agent different from the previously used one is added to the reaction solution after the reduction by PS-A1.
0.1 to 0.5 parts by weight with respect to parts by weight,
For 20 hours, a reduction reaction is further performed, or a compound having an amino group is further added to the reaction solution to a concentration of 0.1 to 0.5 M / l, and a reducing agent similar to the previously used reducing agent is added to PS. -A
Add 0.1 to 1 part by weight to 1 part by weight, and again 0 to 40 ° C
For 2 to 20 hours under agitation, followed by dialysis against water or an appropriate buffer to obtain a more stable AG / PS-A covalent conjugate.

As the oxidizing agent used in producing PS-A, for example, alkali metal salts such as orthoperiodic acid, dimesioperiodic acid, mesoperiodic acid, diorthoperiodic acid, and metaperiodic acid are preferable. Preferred examples of the reducing agent used for treating the excess oxidizing agent include sodium bisulfite, sodium thiosulfate, and hydrosulfite.

When the PS-A and AG are reacted, or when the reaction solution is dialyzed at the end of each reaction, the buffer of the buffer used is one that does not deactivate AG and does not react with the reducing agent. Although it can be used without any particular problem, for example, phosphates, Good's buffer and the like are preferable.

Preferable examples of the reducing agent used for reacting PS-A with AG include pyridine borane, sodium cyanoborohydride and the like.

When PS-A is reacted with AG, preferable excipients to be contained in the lyophilized powder of AG include proteins such as albumin, globulin, casein, and gelatin. 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 the covalent bond between AG and PS-A include, for example, sodium borohydride, dimethylamine borane and the like.

Further, the compound having an amino group used to inactivate the aldehyde group remaining after the covalent bond between AG and PS-A is not particularly limited. For example, glycine,
Preferable examples include ethanolamine and glycine ethyl ester. As the reducing agent added at this time, for example, pyridine borane, sodium cyanoborohydride, and the like are preferable.

The AG / PS-A covalent conjugate thus obtained is
Because of its excellent stability, it can be stored for a long time in the state of an aqueous solution, but it is naturally also possible to freeze-dry and store it as a powder.

Since the AG / PS-A covalent conjugate according to the present invention is stable for a long time in the state of an aqueous solution, it can be widely used in the fields of clinical chemistry, biochemistry, food chemistry, food industry and the like. When used as a conjugated enzyme added to an α-amylase measurement test solution in the field of, the measurement test solution becomes stable for a long period of time, which is extremely advantageous as compared with the conventional product.

Hereinafter, the present invention will be described in more detail by way of Examples.
The present invention is not limited by these.

[Examples] Example 1. 25 g of dextran (molecular weight; 100,000 to 200,000, hereinafter abbreviated as Dex) is dissolved in 250 ml of distilled water, 11 g of sodium periodate is added and dissolved with stirring, and the mixture is dissolved in a dark place at 4 ° C. For 15 hours. After completion of the reaction, excess sodium periodate was decomposed by adding about 40 ml of a 40 W / V% aqueous sodium bisulfite solution, and the reaction solution was dialyzed against water at room temperature.
0.1 g of 5 g of AG (derived from yeast, containing 30% albumin, 100 U / mg) dissolved in the obtained aldehyde derivative solution of Dex
Phosphate buffer (pH7.0) 200ml, pyridineborane 10g
Was added and reacted at 20 ° C. for 24 hours with stirring. Thereafter, 250 ml of a 1 M glycine aqueous solution and 10 g of pyridineborane were added to the reaction solution, and the mixture was reacted at 37 ° C. for 5 hours with stirring. After completion of the reaction, the reaction solution was dialyzed against water and then freeze-dried to obtain 31.2 g (about 8 U / mg) of a freeze-dried powder of an AG / Dex covalent bond (a covalent bond of AG and Dex). (Enzyme activity recovery rate: 50%).

Example 2 AG (derived from yeast, containing no albumin, containing 70%) was added to the aldehyde derivative solution of Dex obtained in the same manner as in Example 1.
U / mg) was dissolved in 0.1 ml of a phosphate buffer (pH 7.0) (250 ml), and sodium cyanoborohydride (8 g) was added thereto. The mixture was reacted at 37 ° C for 20 hours with stirring. Next, 2 g of sodium borohydride was added to the reaction solution, and the mixture was further stirred and reacted at 30 ° C. for 3 hours. After completion of the reaction, the reaction solution was dialyzed against water to obtain an aqueous solution of an AG / Dex covalently bonded product (enzyme activity recovery rate: 35%).

Example 3. As AG, derived from yeast, containing 20% albumin, 110 U / mg
Using the same reagents as in Example 2 except that 7 g of AG was used, the same procedure was followed to obtain an AG / Dex covalently bound solution (enzyme activity recovery rate: 45%).

Example 4. As AG, yeast-derived, containing 40% albumin, 95 U / mg
Using the same reagent as in Example 2 except that 7 g of AG was used,
The same operation was performed to obtain an AG / Dex covalently bound solution (enzyme activity recovery rate: 50%).

Example 5 1 g of pullulan (molecular weight: 50,000 to 100,000, hereinafter abbreviated as Pul) was dissolved in 10 ml of distilled water, 0.5 g of sodium periodate was added and dissolved, and the mixture was stirred at 10 ° C. for 15 hours in a dark place. Was reacted. After completion of the reaction, the excess sodium periodate was decomposed by adding about 2 ml of a 30 W / V% aqueous sodium bisulfite solution, and the reaction solution was reacted at room temperature with 0.1 M BES [N, N-bis (2
-Hydroxyethyl) -2-aminoethanesulfonic acid]
-Dialyzed against NaOH buffer (pH 7.2). Pul obtained
AG (yeast-derived, albumin)
250 mg of 40% (95 U / mg) was dissolved, 0.5 g of pyridineborane was further added, and the mixture was reacted at 4 ° C. for 24 hours with stirring.
Then, add 1M glycine ethyl ester aqueous solution to the reaction solution.
10 ml and pyridine borane (0.3 g) were added and reacted at 37 ° C. for 5 hours with stirring. After completion of the reaction, the reaction solution was dialyzed against water to obtain an AG / Pul covalently bonded product (a covalently bonded product of AG and Pul) (enzyme activity recovery rate: 40%).

Comparative Example A solution of Dex aldehyde derivative obtained in the same manner as in Example 1 was added with AG (derived from yeast, containing 20% albumin, 110 U
(mg / mg) dissolved in 0.1 g phosphate buffer (pH 7.0) (250 ml) and reacted at 37 ° C. for 20 hours with stirring. After completion of the reaction, the reaction solution was dialyzed against water, and the conjugate of AG and Dex (A
(G / Dex conjugate) solution was obtained (enzyme activity recovery rate: 45)
%).

Experimental Example 1. Examination of thermal stability-1 (sample) Each AG solution having the composition shown in Table 1 (0.1 M BES-NaOH buffer,
pH 7.7, 20 U / ml) was used as a sample.

(Operation method) After each sample was allowed to stand at 37 ° C for 10 days, the residual activity ratio of AG was determined.

The activity of AG was measured using p-nitrophenyl-α-glucoside as a substrate and an enzyme that releases 1 μmole of p-nitrophenol per minute in a 100 mM phosphate buffer (pH 7.0) at a reaction temperature of 37 ° C. The amount was 1 unit (U).

(Results) The results are shown in Table 2.

As is clear from the results shown in Table 2, the stabilized AG of the present invention is significantly more stable than untreated AG or untreated AG added with 2% of Dex or Pul.

Experimental Example 2. Examination of thermal stability-2 (sample) Each AG solution having the composition shown in Table 3 (0.1 M BES-NaOH buffer,
(pH 7.7, 60 U / ml) was used as a sample.

(Operation method) The residual activity ratio of AG after heating each sample at 60 ° C for 10 minutes was determined.

 The activity of AG was measured in the same manner as in Experimental Example 1.

(Results) The results are shown in Table 4.

As is clear from the results in Table 4, the stabilized AG of the present invention was untreated AG, untreated AG with 2% of Dex added thereto, and
It can be seen that Dex is remarkably more stable than the one obtained by simply chemically bonding Dex (ie, one obtained by performing no reduction treatment after bonding).

Also, based on the residual activity rates of Sample Nos. 8, 9, and 10, when immobilizing AG, heat stability may be improved if albumin coexists, and the higher the concentration, the higher the concentration. I understand.

Experimental Example 3. Examination of pH Stability (Sample) Untreated AG and the AG / Dex covalent conjugate obtained in Example 1 were each dissolved in a buffer solution of a predetermined pH so as to be 20 U / ml to prepare a sample.

In the case of pH 4.0 to 6.0, a 50 mM acetic acid-sodium acetate buffer was used, and in the case of pH 5.5 to 8.5, a 50 mM phosphate buffer was used.

(Operation method) After each sample was allowed to stand at 37 ° C for 20 hours, the residual activity ratio of AG was determined.

 The activity of AG was measured in the same manner as in Experimental Example 1.

(Results) The results obtained with the AG / Dex covalent bond obtained in Example 1 are shown in FIG. 1, and the results obtained with the untreated AG are shown in FIG. These figures are obtained by connecting the points obtained by plotting the residual activity ratio obtained for each pH on the horizontal axis along the vertical axis, where-●-was obtained with a 50 mM acetic acid-sodium acetate buffer solution. In the results, -O- indicates the results obtained with the 50 mM phosphate buffer.

As is clear from FIGS. 1 and 2, the pH stable region of the AG / Dex covalently bonded product is clearly wider and stabilized as compared with untreated AG.

Experimental Example 4. The AG / Dex covalent conjugate of the present invention obtained in Example 1 was subjected to gel filtration chromatography under the following conditions.

Column; 2.5 φ × 100 cm.

Filler: Sephacryl S-300 (Pharmacia product name).

Eluent: 0.1 M BES-NaOH buffer (pH 7.7, containing 0.1 M NaCl) Fractionation volume: 10 ml / test tube.

The results are shown in FIG. Here, the horizontal axis indicates the fraction number (Fr. No.), and-●-indicates each Fr.N.
o. The absorbance (OD280nm) at o.
-○-shows the results obtained by linking the AG activity (U / ml) in each Fr. No. ↓ indicates the elution point when untreated AG was subjected to gel filtration chromatography under the same conditions.

 The activity of AG was measured in the same manner as in Experimental Example 1.

As is clear from these results, it is clear that the stabilized AG of the present invention is clearly polymerized as compared with the untreated AG.

[Effects of the Invention] As described above, the present invention is to provide a method for stabilizing AG that can maintain the activity of AG in an aqueous solution for a long period of time. is there.

[Brief description of the drawings]

FIG. 1 shows a pH stability curve of the α-glucosidase / dextran covalent conjugate according to the present invention obtained in Experimental Example 3, and the residual activity ratio obtained for each pH on the horizontal axis is shown on the vertical axis. The points plotted along are connected, and-●-is 50
The results obtained with the mM acetate-sodium acetate buffer were
-O- indicates the results obtained with the 50 mM phosphate buffer, respectively. FIG. 2 shows the pH stability curve of the untreated α-glucosidase obtained in Experimental Example 3, where the residual activity ratio obtained for each pH on the horizontal axis was plotted along the vertical axis. The results obtained with a 50 mM acetic acid-sodium acetate buffer, and the results obtained with a 50 mM phosphate buffer, are shown. FIG. 3 shows the elution results of gel filtration chromatography obtained in Experimental Example 4, where the horizontal axis represents the fraction number (F
r.No.), and-●-indicates the absorbance (O.
D.280 nm) and-○-is the result of tying the AG activity (U / ml) in each Fr. No. ↓ indicates the elution point when untreated AG was subjected to gel filtration chromatography under the same conditions.

 ──────────────────────────────────────────────────続 き Continuation of front page (56) References JP-A-62-255435 (JP, A) JP-A-61-249389 (JP, A) JP-A-59-22901 (JP, A) JP-A-59-22901 21626 (JP, A)

Claims (2)

(57) [Claims] [1] A method for stabilizing [alpha] -glucosidase in an aqueous solution, wherein [alpha] -glucosidase is covalently bonded to a water-soluble polysaccharide (excluding chondroitin). 2. The method according to claim 1, wherein the α-glucosidase is derived from yeast.