JP5234452B2 - Alkali resistant glutaminase - Google Patents

Description

  The present invention relates to an alkali-resistant glutaminase.

  Glutaminase is known to play an important role in the food industry, particularly in the production of seasoned foods by enzymatically degrading proteins. This glutaminase has attracted particular attention in recent years also in the biochemical and medical fields, and the production of a stable enzyme agent is expected. For example, L-theanine can be obtained in high yield by allowing glutaminase to act on a mixture of glutamine and an ethylamine derivative. If the protein can be enzymatically degraded under alkaline conditions, application to various fields such as the food field is expected. Although development of technology for imparting alkali resistance to glutaminase has been carried out, it is difficult to say that such technology is sufficiently developed.

On the other hand, porous silica having regular pores having a pore diameter of about 2 to 20 nm is particularly called mesoporous silica, and since its synthesis was reported in the 1990s, it has been used as an adsorbent, catalyst, and various carriers. Have been proposed (Non-Patent Document 1). Since the pore diameter of mesoporous silica corresponds to the sizes of various enzymes, it is expected to be used as an enzyme carrier for immobilizing enzymes inside the pores. Up to now, a technology has been developed to impart an effect such as acid resistance, heat resistance, or substrate specificity improvement of isomerization reaction to the enzyme by supporting the enzyme in the pores of the porous silica ( Patent Documents 1 and 2). In general, porous silica has a drawback that silica as a constituent component is easily dissolved in a solution under an alkaline condition of pH 8 or higher and the pore structure cannot be maintained. Further, under alkaline conditions, the enzyme is dissolved in the solution, and there is a disadvantage that a stable reaction cannot be obtained.
Therefore, it has been difficult to use porous silica as an enzyme carrier that catalyzes a reaction under alkaline conditions.
JP 2001-128672 A JP 2002-95471 A Yoshiaki Fukushima, Ceramics, 34, pp722-725, 1999

  This invention is made | formed in view of the said situation, The objective is to obtain the glutaminase which has the outstanding alkali resistance which is not before by making glutaminase adsorb | suck in the pore of a silica porous body.

The present invention for achieving the above object is as follows.
(1) An alkali-resistant glutaminase comprising a porous silica having an average pore size of more than 3 nm and a metal content in the porous silica of 1 to 20% by mass, and glutaminase.
(2) The alkali-resistant glutaminase according to (1) having at least one peak at a position where the d interval in the X-ray diffraction of the porous silica material is larger than 2 nm.
(3) The alkali-resistant glutaminase according to (1) or (2), wherein the metal is zirconium.
(4) The alkali-resistant glutaminase according to any one of (1) to (3), wherein the content of glutaminase in the alkali-resistant glutaminase is 2 to 20% by mass.

  ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to improve the alkali resistance of the conventional glutaminase remarkably, and can make glutaminase act very efficiently on the objective of manufacturing a seasoning food etc.

Next, embodiments of the present invention will be described with reference to the drawings. However, the technical scope of the present invention is not limited by these embodiments, and various forms are possible without changing the gist of the invention. Can be implemented. Further, the technical scope of the present invention extends to an equivalent range.
The porous silica in the present invention means a substance mainly composed of a silicon oxide having a porous structure.
If the average pore diameter of the pores in the porous silica material is less than 3 nm, it is not preferable because the glutaminase is not sufficiently adsorbed inside the porous silica material. In addition, those having an average pore diameter exceeding 20 nm are substantially difficult to produce. Therefore, from the above viewpoint, the average pore diameter of the pores in the present invention is 3 to 20 nm, preferably 3 to 14 nm.
The average pore diameter of the present invention was calculated by known nitrogen adsorption / desorption. That is, the average pore diameter was calculated by a known BJH method.
As for content of the metal in the silica porous body of the silica porous body in this invention, 1-20 mass% is preferable. The metal content of the porous silica material of the present invention was calculated using an ICP emission spectrometer.
Moreover, it is preferable that the porous silica in the present invention contains zirconium as a metal. The zirconium content in the porous silica is preferably 1 to 20% by mass, more preferably 3 to 15% by mass, and most preferably 5 to 10% by mass from the viewpoint of alkali resistance.

The method of introducing zirconium into the porous silica material is not particularly limited. For example, the porous silica material is immersed in 50-fold 0.2 M zirconia nitrate solution, stirred, filtered, dried, and fired at 580 ° C. The porous silica in the present invention that can be performed preferably has at least one peak at a position where the d interval of X-ray diffraction is larger than 2 nm. The X-ray diffraction pattern of the present invention was measured by RINT ULTIMA II, which is an existing X-ray diffractometer, manufactured by Rigaku Corporation.
When the specific surface area of the porous silica material in the present invention is less than 100 m ² / g, adsorption of glutaminase supported on the porous silica material may not be sufficient. Moreover, it is substantially difficult to manufacture a specific surface area larger than 2000 m < ² > / g. Therefore, from the above viewpoint, the specific surface area of the porous silica material in the present invention is preferably 100m ² / g~1500m ² / g, more preferably 300m ² / g~1500m ² / g, most preferably 500 meters ² / g to 1500 m ² / g. The specific surface area of the porous body of the present invention can be calculated by known nitrogen desorption.

Although it does not specifically limit as a manufacturing method of the silica porous body in this invention, For example, it can manufacture as follows. First, an inorganic raw material and an organic raw material are mixed and reacted to form an organic matter-inorganic matter composite in which an inorganic matter skeleton is formed around the organic matter as a template. Next, a porous silica material is produced by removing organic substances from the obtained composite.
As the inorganic material, for example, a silicon-containing inorganic material can be used. Examples of such raw materials include substances containing silicates such as layered silicates and non-layered silicates, and substances containing silicon other than silicates. Examples of layered silicates include kanemite (NaHSi ₂ O ₅ · 3H ₂ O), sodium disilicate crystal (Na ₂ Si ₂ O ₅ ), macatite (NaHSi ₄ O ₉ · 5H ₂ O), and Iraite (NaHSi ₈ O ₁₇ · XH ₂ O), magadiite (Na ₂ HSi ₁₄ O ₂₉ · XH ₂ O), Kenyaite (Na ₂ HSi ₂₀ O ₄₁ · XH ₂ O) and the like. Non-layered silicates include water glass (sodium silicate). ), Glass, amorphous sodium silicate, tetraethoxysilane (TEOS), tetramethoxysilane (TMOS), tetramethylammonium (TMA) silicate, silicon alkoxide such as tetraethylorthosilicate, and the like. Examples of the substance containing silicon other than silicate include silica, silica oxide, and silica-metal composite oxide. These can be used alone or in admixture of two or more.

  Although it does not specifically limit as an organic raw material used as a casting_mold | template, For example, surfactant is mentioned. The surfactant is not particularly limited, but a nonionic surfactant is preferable. The nonionic surfactant is not particularly limited. For example, polyoxyethylene alkyl ether, polyoxyethylene secondary alcohol ether, polyoxyethylene alkylphenyl ether, polyoxyethylene sterol ether, polyoxyethylene Lanolinic acid derivatives, polyoxyethylene polyoxypropylene alkyl ethers, ether type compounds such as polypropylene glycol and polyethylene glycol, and nitrogen-containing compounds such as polyoxyethylene alkylamine can be used. Polyglycerin fatty acid ester obtained by esterifying can be preferably used. You may use these individually or in mixture of 2 or more types.

The polyglycerol fatty acid ester preferably has an HLB of 14.0 to 18.0 from the viewpoint of glutaminase adsorption. Here, HLB represents the balance between the hydrophilic group and the lipophilic group in the molecule, and is equally divided into 0 when the hydrophilic group in the molecule is 0% and 20 when the hydrophilic group is 100%.
When mixing an inorganic raw material and an organic raw material, a suitable solvent can be used. Such a solvent is not particularly limited, and examples thereof include water and alcohol.
The mixing method of the inorganic raw material and the organic raw material is not particularly limited, but after dissolving the surfactant in the acidic solution, the basic substance and the inorganic raw material are added to the solution, and the temperature is from 20 ° C to 60 ° C. It is preferable to mix for 3 hours to 24 hours. The mixing ratio (weight ratio) between the inorganic raw material and the surfactant is not particularly limited, but inorganic raw material: surfactant = 1: 0.5 to 1: 2 is preferable. The mixing ratio (mass ratio) of the inorganic raw material and the basic substance is not particularly limited, but is preferably inorganic raw material: basic substance = 100: 0.1 to 100: 10.

The acidic substance for preparing the acidic solution is not particularly limited, and an inorganic acid or an organic acid can be used. For example, hydrochloric acid, hydrogen bromide, hydrogen iodide, formic acid, acetic acid, nitric acid, sulfuric acid, phosphoric acid and the like can be exemplified.
The pH condition when the inorganic raw material and the organic raw material are stirred and reacted is not particularly limited as long as it is acidic, but is preferably pH 3 or less.
Examples of the method for removing the organic substance from the complex of the organic substance and the inorganic substance include a method in which the complex is filtered, washed with water and dried, then baked at 400 ° C. to 600 ° C., and extracted with an organic solvent. Can be mentioned.

The alkali-resistant glutaminase of the present invention is composed of a porous silica and glutaminase. The glutaminase in the porous silica is preferably 2% by mass to 20% by mass, more preferably 3% by mass to 20% by mass, and most preferably 5% by mass to 10% by mass.
Adsorption of glutaminase to the porous silica can be carried out by adding 4 ml of glutaminase solution (1 mg / ml) to 20 mg of porous silica and mixing and stirring at 4 ° C. for 1 day.
The prepared porous silica = glutaminase complex is separated from unadsorbed glutaminase by centrifugation.
The glutaminase adsorption rate can be calculated from the amount of added glutaminase and the glutaminase adsorbed on the porous silica as shown in the following formula 1.

  The glutaminase activity of the alkali-resistant glutaminase of the present invention can be measured with an existing kit (for example, Yamasa L-glutamic acid measurement kit (Yamasa Shoyu Co., Ltd.)). That is, after leaving the alkali-resistant glutaminase in a constant temperature water bath at 37 ± 0.2 ° C. for 5 minutes, 10 ml of 2% glutamic acid previously maintained at 37 ± 0.2 ° C. is added and vigorously stirred with a test tube mixer. After leaving in a constant temperature water bath at 37 ± 0.2 ° C. for exactly 10 minutes, add 10 ml of 0.75 mol / L perchloric acid, vigorously stir with a test tube mixer, and immediately put on ice water. After leaving it for 5 minutes, it is taken out from the ice water, 10 ml of 0.75 mol / L sodium hydroxide is added, and vigorously stirred with a test tube mixer (test solution on the reaction side).

Separately, 10 ml of 0.75 mol / L perchloric acid is added to a 50 ml plastic tube (Falcon tube), and vigorously stirred with a test tube mixer. After leaving in a constant temperature water bath at 37 ± 0.2 ° C. for 5 minutes, add 10 ml of 2% glutamine solution, vigorously stir with a test tube mixer, and then put on ice water. After leaving it for 5 minutes, it is taken out from ice water, added with 10 ml of 0.75 mol / L sodium hydroxide, and vigorously stirred with a test tube mixer (blank side test solution).
A test tube (reaction side), a test solution (blank side), a glutamic acid standard solution (100 μg / ml), and water (200 μL) separately added with 3 ml of the coloring solution of Yamasa L-glutamic acid measurement kit (Yamasa Shoyu Co., Ltd.) were added separately. The mixture is allowed to stand for 25 to 30 minutes, and then the absorbance at a wavelength of 600 nm is measured.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further more concretely, this invention is not limited to a following example.
Production Example 1
After mixing HLB 15.0 polyglycerol fatty acid ester and 200 ml of 1N hydrochloric acid to completely dissolve the polyglycerol fatty acid ester, 9 g of TEOS (tetraethoxysilane) and 20 ml of decane were added. This solution (pH 3 or lower) was stirred at 25 ° C. for 12 hours in a sealed system, and the resulting precipitate was collected by filtration. Thereafter, washing and filtering with ion-exchanged water were repeated three times, and after washing and filtering with ethanol, the solid was dried at 50 ° C. for 2 hours, and further calcined at 550 ° C. for 6 hours. 0.8 g was obtained.
1 g of this porous silica was immersed in 0.05 L of a 0.2 M zirconia nitrate solution in an amount of 50 times for 1 hour, stirred at room temperature for 2 hours and filtered. The obtained solid was dried and then calcined at 580 ° C. for 6 hours to obtain 0.9 g of porous silica A containing zirconium.

When the pore diameter distribution of the obtained porous silica A was measured with a nitrogen adsorption device (BELsorb II) manufactured by BEL and the average pore diameter was determined by the BJH method, the average pore diameter was 10.6 nm. Moreover, when the zirconium content of the obtained porous silica A was measured with an ICP emission spectrometer, the content with zirconium metal was 3.0%.
The obtained porous silica A had one peak at a position where the d interval of X-ray diffraction was larger than 2 nm.
It was 523 m < ² > / g when the specific surface area of the obtained silica porous body A was computed by nitrogen desorption.

Comparative product production example 1
0.5 g of the porous silica A obtained in Production Example 1 was immersed in 0.025 L of a 50-fold amount of 0.2 M aluminum nitrate solution for 1 hour, stirred at room temperature for 2 hours, and then filtered. The obtained solid was dried and then calcined at 580 ° C. for 6 hours to obtain 0.4 g of silica porous body B containing aluminum.
The pore diameter distribution of the obtained porous silica B was measured with a nitrogen adsorption device (BELsorb II) manufactured by BEL, and the average pore diameter was determined by the BJH method. The average pore diameter was 10.6 nm.

When the aluminum content of the obtained porous silica B was measured with an ICP emission spectrometer, the content of aluminum metal was 0.3%. Further, when the X-ray diffraction pattern of the obtained porous silica B was measured, it had one peak at a position where the d interval was larger than 2 nm.
It was 508 m < ² > / g when the specific surface area of the obtained silica porous body was computed by nitrogen desorption.

Example 1
5 ml of 1.0 mg / ml glutaminase and 0.01 M ethylamine solution are added to 50 mg of the porous silica A obtained in Production Example 1, and stirred at 4 ° C. for 24 hours to obtain the alkali-resistant glutaminase A1 of the present invention. It was. For the glutaminase solution used at this time, the protein concentration of the supernatant after the reaction was measured by the CBB method using a protein quantification kit manufactured by Dojindo Chemical. The glutaminase content in the obtained alkali-resistant glutaminase A1 was determined from Formula 1 and found to be 7.4% by mass.

Comparative Example 1
Instead of the porous silica A, a glutaminase porous material mixture B1 was obtained in the same manner as in Example 1, except that the porous silica B obtained in Comparative Production Example 1 was used. For the glutaminase solution used at this time, the protein concentration of the supernatant after the reaction was measured by the CBB method using a protein quantification kit manufactured by Dojindo Chemical. The glutaminase content in the obtained glutaminase B1 was calculated from Formula 1 and found to be 8.2% by mass.

Test example 1
For the alkali-resistant glutaminase A1 obtained in Example 1 and the glutaminase B1 obtained in Comparative Example 1, 10 ml of 0.02M L-glutamine / 0.12M ethylamine hydrochloride solution having a pH of 10 was added to 50 mg each. For 20 hours. After centrifugation, the solid content was collected, and the enzyme activity was calculated as the enzyme activity when the enzyme activity of the original glutaminase was taken as 100. The results are shown in Table 1.

Test example 2
With respect to the alkali-resistant glutaminase A1 obtained in Example 1 and the glutaminase B1 obtained in Comparative Example 1, 10 ml of 0.02M L-glutamine / 0.12M ethylamine hydrochloride solution having a pH of 10 was added to 0.05 g. The mixture was stirred and reacted at 20 ° C. for 20 hours. Thereafter, the alkali-resistant glutaminase A1 and glutaminase B1 were centrifuged to remove the upper reaction solution, and the precipitated alkali-resistant glutaminase A1 and glutaminase B1 were collected (single synthesis). To the recovered alkali-resistant glutaminase A1 and glutaminase B1, 10 ml of 0.02M L-glutamine / 0.12M ethylamine hydrochloride solution each having a pH of 10 was added, stirred and reacted at 4 ° C. for 20 hours, and then precipitated by centrifugation. The same operation for recovering alkaline glutaminase A1 and glutaminase B1 was repeated a total of 6 times. In the collected alkali-resistant glutaminase A1 and glutaminase B1, the enzyme activity after synthesis once, three times and six times was calculated as the enzyme activity when the original enzyme activity was taken as 100. The results are shown in Table 2.

  As described above, according to the present embodiment, it was possible to provide an alkali-resistant glutaminase in which the alkali resistance of the conventional glutaminase is remarkably improved. By using this alkali-resistant glutaminase, glutaminase can be made to act very efficiently for the purpose of producing seasoned foods and the like.

Claims (3)

It is composed of a porous silica material having an average pore size of 3 nm to 14 nm and a metal content in the porous silica material of 1 to 20% by mass, and glutaminase, and the metal is zirconium. together with the porous silica alkali resistance glutaminase the glutaminase that is adsorbed in the pores of the. The alkali-resistant glutaminase according to claim 1, wherein the porous silica has at least one peak at a position where the d interval in X-ray diffraction is larger than 2 nm. The alkali-resistant glutaminase according to claim 1 or 2 , wherein the content of glutaminase in the alkali-resistant glutaminase is 2 to 20% by mass.