JP2006025688A - New thermostable amp deaminase and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new thermostable AMP deaminase capable of being simultaneously used with a nuclease, enabling the contamination with various germs to be prevented by shortening the production process and carrying out a treating process of the enzyme at a high temperature, and having excellent thermal stability allowing the utilization at 65°C; and to provide a method for producing the AMP deaminase. <P>SOLUTION: The new thermostable AMP deaminase has the following physicochemical properties: (1) substrate specificity: catalyzing the reaction of 5'-adenylic acid+H<SB>2</SB>O→5'-inosinic acid+NH<SB>3</SB>; (2) stable temperature: stable up to 65°C; (3) optimum pH: about 6.0; (4) molecular weight: 85,000±3,000 (by SDS-PAGE), and 88,000±3,000 (by gel filtration). The method for producing the thermostable AMP deaminase comprises culturing microorganisms having thermostable AMP deaminase-producing ability in a nutrient medium to produce the thermostable AMP deaminase, and collecting the produced AMP deaminase. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a novel heat-resistant AMP deaminase and a method for producing the same, and more particularly to a novel AMP deaminase stable up to 65 ° C. and a method for producing the same.

AMP deaminase is also called adenyl deaminase, AMP aminohydrolase, etc., and catalyzes a reaction that hydrolyzes 5′-adenylic acid to produce 5′-inosinic acid and ammonia. AMP deaminase is widely present in animal biological tissues and has been separated from various tissues of various species so far (see Non-Patent Document 1 and Patent Document 1). On the other hand, mainly from the viewpoint of industrial use, search for AMP-derived deaminase derived from microorganisms has been energetically performed. In particular, there are many studies on filamentous fungus-derived AMP deaminase, and some AMP deaminases such as Aspergillus mereus AMP deaminase have been industrially used for the purpose of enhancing umami in the production of yeast extract. Currently, in the production of yeast extract, AMP deaminase and nuclease are used in combination to enhance umami.
Tetsuro Fujishima and Hiroshi Yoshino, Amino Acid / Nucleic Acid, No. 16, pp 45-55 (1967) Japanese Patent Laid-Open No. 55-120788

  However, in general, the optimum temperature of nuclease is about 65 ° C., whereas the optimum temperature of AMP deaminase derived from Aspergillus mereus currently used is about 50 ° C., and the activity is remarkably reduced at around 65 ° C. Therefore, in production, it is impossible to cause two enzymes to act simultaneously at a high temperature, and nuclease treatment and AMP deaminase treatment must be performed as separate steps, and the production process is complicated. In addition, since a process for lowering the treatment temperature to about 50 ° C., which is the reaction temperature of AMP deaminase, is required in the production process, there is a problem that contamination with various bacteria cannot be effectively prevented.

  The present invention has been made in view of the above circumstances, and can be used at the same time as nuclease. It can be used at 65 ° C., which can effectively prevent contamination by shortening the production process and treating the enzyme at a high temperature. It is an object of the present invention to provide an AMP deaminase having excellent heat resistance and a method for producing the same.

In light of the above circumstances, the present inventors conducted screening for microorganisms for AMP deaminase with excellent heat resistance, and as a result, microorganisms newly collected from soil produced AMP deaminase with high thermal stability. As a result, the present invention has been completed.
That is, the gist of the present invention is a thermostable AMP deaminase having the following physicochemical properties.
(1) Substrate specificity: Catalyze the reaction of 5′-adenylic acid + H ₂ O → 5′-inosinic acid + NH ₃ .
(2) Stable temperature: Stable up to 65 ° C.
(3) Optimal pH: around 6.0.
(4) Molecular weight: 85,000 ± 3,000 (by SDS-PAGE), 88,000 ± 3,000 (by gel filtration).

  Summary of the method for producing thermostable AMP deaminase characterized in that the above-mentioned microorganisms capable of producing thermostable AMP deaminase are cultured in a nutrient medium, and thermostable AMP deaminase is produced in the culture medium and collected. .

  In the production method of the above-mentioned thermostable AMP deaminase and thermostable AMP deaminase, a microorganism belonging to the genus Aspergillus, Aspergillus fumigatus, or Aspergillus fumigatus N0.4 (FERM P-20075) may be used. .

  The gist is Aspergillus fumigatus No. 4 (FERM P-20075) having the above-mentioned physicochemical properties and the ability to produce thermostable AMP deaminase.

  Since the thermostable AMP deaminase of the present invention has high activity even at 65 ° C., it is suitable for applications where a reaction at such a high temperature is desired. For example, it can be operated simultaneously with other enzymes that act at high temperatures, such as nucleases used in the production process of yeast extract, and the production process can be shortened and the enzyme reaction can be carried out in a situation where there is little risk of contamination. it can.

The thermostable AMP deaminase of the present invention catalyzes the reaction of (1) substrate specificity: 5′-adenylic acid + H ₂ O → 5′-inosinic acid + NH ₃ , (2) stable temperature: stable to 65 ° C. (3) Optimal pH: around 6.0, (4) Molecular weight: 85,000 ± 3,000 (according to SDS-PAGE), 88,000 ± 3,000 (according to gel filtration), especially in heat resistance Excellent. Here, "stable up to 65 ° C" means that the enzyme activity adjusted to pH 6.0 with phosphate buffer at 65 ° C for 30 minutes is the standard (100% ) Means that 80% or more of the activity remains.

  As long as the thermostable AMP deaminase having the above physicochemical properties is produced, the microorganism to be produced is not limited, but a microorganism belonging to the genus Aspergillus is preferable, and Aspergillus fumigatus is more preferable. Further, a microorganism having the following mycological properties newly collected from soil is most preferable.

Growth in various media (1) Potato dextrose agar slope
Grows at 40 ° C and 45 ° C, powder to velvet, surface is dark green.
(2) Malt extract agar plate The settlement surface is velvety to wooly. Initially white, dark green when many conidia are formed, the village surface is pale yellow.
(3) Czapek Docs agar plate Dark green, deep green, grayish green, good growth at 25 ° C.
Form (1) Mycelium: White (colorless)
(2) Conidial head: actuated or cylindrical, diameter 16-40μm
(3) Conidial pattern: 160-320 μm long, 4-10 μm in diameter
(4) Apical sac: 12 to 28 μm in diameter, flask-type, spatula-type, light green, usually forming phialide from about the upper half.
(5) Fear ride: 4-6 × 2 μm arranged in parallel in a single row.
(6) Conidia: 2 to 3 μm in diameter, spherical to subspherical, rough surface but no protrusions.
(7) Metre: Not formed.
(8) Closure capsular shell: not formed.

A dark green slant surface is formed with potato dextrose agar and malt extract agar. The conidia head is radial or cylindrical and not a club. In addition, the phialides are strictly arranged in parallel in a single row. A closed crust is not formed. As a result of referring to the above characteristics and the following references, this strain was found to have Aspergillus fumigatus (Aspergillu
fumigatus) and named Aspergillu fumigatus No.4.

References (1) Mold Inspection Manual Color Chart 2002 298-299 pages (issued March 29, 2002) Publisher: Technosystem Co., Ltd. (2) Fungal Encyclopedia Volume 1018-1021 (issued February 20, 1980) Publisher: Kodansha (3) Identification of Common Aspergillus Species Maren A. Klich 2002 50-51 CBS UTRECHT

This strain is deposited as follows.
Depositary institution name: Tsukuba City, Ibaraki Prefecture 1-1-1 Central 6th
National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center Date of deposit: June 3, 2004
Deposit No. 20075 (FERM P-20075)

  The thermostable AMP deaminase of the present invention can be produced by culturing a microorganism having the above-mentioned physicochemical properties with the ability to produce AMP deaminase in a nutrient medium, producing the thermostable AMP deaminase in the culture solution, and collecting it. .

  The culture of each microorganism belonging to the genus Aspergillus that produces the thermostable AMP deaminase of the present invention can be carried out using a conventional method. Medium is glucose, sucrose, gentiobiose, soluble starch, glycerin, dextrin, molasses, organic acid and other carbon sources, ammonium sulfate, ammonium carbonate, ammonium phosphate, ammonium acetate, or polypeptone, yeast extract, corn steep liquor, casein Nitrogen sources such as hydrolysates, bran and meat extracts, and those containing inorganic salts (inorganic ions) such as potassium, magnesium, sodium, phosphate, manganese, iron, and zinc salts as required Can be used. In order to promote the growth of microorganisms belonging to the genus Aspergillus, a medium supplemented with vitamins, amino acids and the like can also be used. The pH of the medium is adjusted to, for example, 5.0 to 8.0, preferably 5.5 to 7.5. The culture temperature is, for example, in the range of 15 ° C to 65 ° C, preferably in the range of 30 ° C to 60 ° C, and more preferably in the range of 40 ° C to 55 ° C. The culture time is not particularly limited, but is, for example, 3 days or longer. As the culture method, for example, a shaking culture method or an aerobic deep culture method using a jar fermenter can be used. The various culture conditions described above are appropriately changed depending on the subject to be cultured, and the conditions are not particularly limited as long as the thermostable AMP deaminase of the present invention is produced.

  The thermostable AMP deaminase of the present invention can be separated from a culture solution or cells obtained after culturing a microorganism belonging to the genus Aspergillus for a desired time. For example, after filtering and centrifuging the culture supernatant to remove insolubles from the culture solution, heat-stable AMP deaminase purified using a known purification method such as a combination of protamine sulfate treatment, dialysis, various chromatographies, etc. Although it can be obtained, it is preferable to carry out using hydrophobic chromatography and gel filtration after the protamine sulfate treatment. On the other hand, when the cells are separated from the cells, for example, after the cells are crushed by pressure treatment, ultrasonic treatment, etc., purified AMP deaminase can be obtained by performing purification in the same manner as described above. In addition, after collect | recovering a microbial cell from a culture solution previously by filtration, a centrifugation process, etc., you may perform said series of processes (crushing, isolation | separation, refinement | purification of a microbial cell). In each purification step, in principle, fractionation can be performed using AMP deaminase activity as an index.

    EXAMPLES Next, although an Example is given and this invention is demonstrated in detail, this invention is not limited to a following example.

[Reference Example] (Method for measuring thermostable AMP deaminase activity)
The activity of thermostable AMP deaminase (hereinafter referred to as “the present enzyme”) was measured as follows.
(1) Enzymatic reaction
Add this enzyme solution (0.1ml) to a solution (0.9ml) of 1mM 5'AMP-2Na and 100mM potassium phosphate buffer (pH 6.0) to make 1ml reaction solution, and react at 30 ° C for 30 minutes I let you. The reaction was stopped by adding a 20% perchloric acid solution to 100 μl of the enzyme reaction solution, and then centrifuged at 4 ° C. and 13,000 rpm for 10 minutes. To 5 μl of the supernatant, 995 μl of 20 mM potassium phosphate buffer (pH 7.0) was added, and 100 μl was subjected to HPLC.
(2) Determination of IMP produced by HPLC Reagent A: 5 mM tetra-n-butylammonium phosphate, 20 mM potassium phosphate buffer (pH 7.0)
Reagent B: Methanol Reagent C: [Reagent A: Reagent B = 4: 1]
[Analysis conditions] Column type: 5C ₁₈ -AR-II Size: 4.6x150mm
The measurement was performed at a flow rate of 1.0 ml / min (using reagent C). A blank that was measured in the same manner with a reaction time of 0 minutes was used. One unit was defined as 1 μmol of IMP produced per minute under the above conditions.
(3) Determination of produced ammonia by GIDH method
The enzyme solution (0.1 ml) was added to a solution (0.9 ml) in which 1 mM AMP and 100 mM potassium phosphate buffer (pH 6.0) were mixed to obtain a 1 ml reaction solution, which was reacted at 30 ° C. for 60 minutes. The enzyme reaction solution was boiled (3 minutes) to stop the reaction, and the amount of ammonia produced was quantified by the GIDH method.
Ammonia quantitative reaction composition (in 1 ml)
Tris-HCl buffer (pH 8.0) 50 mM
2-Oxoglutaric acid 5 mM
β-NADH 0.12mM
ADP 2.5mM
Enzyme reaction solution 12.5U / ml

[Example 1] (Separation and purification of thermostable AMP deaminase)
1% glucose, 0.5% of polypeptone, 0.05% yeast extract, KH ₂ PO ₄ 0.1% was sterilized 30 minutes adjusted medium at 121 ° C. to pH6.0 by addition of MgSO ₄ 0.01%. Aspergillus fumigatus No. 4 (FERM P-20075) was inoculated into the medium, and the culture solution obtained by culturing at 50 ° C. for 9 days was filtered with ADVATEC FILTER PAPER 5C, and 10 mg per 100 mg of protein was obtained. Of protamine sulfate was added slowly. Thereafter, the enzyme solution obtained by centrifugation at 8000 rpm for 40 minutes was passed through DEAE-Toyopearl (diameter 3 cm volume 60 ml; product name of Tosoh Corporation) to adsorb the enzyme, and then 20 mM potassium phosphate buffer containing 50 mM NaCl. After washing with (pH 6.0), the enzyme was eluted with 20 mM potassium phosphate buffer (pH 6.0) containing 120 mM NaCl. Subsequently, the enzyme solution was passed through butyl-Toyopearl (diameter 1.5 cm volume 12 ml; product name of Tosoh Corporation), the enzyme was adsorbed, washed with 20 mM potassium phosphate buffer (pH 6.0) containing 30% saturated ammonium sulfate, The enzyme was eluted with 20 mM potassium phosphate buffer (pH 6.0) containing 20% saturated ammonium sulfate. Furthermore, the eluted enzyme solution was passed through octyl-cellulofine (diameter 1.5 cm volume 12 ml; product name of Chisso Corporation) to adsorb the enzyme and washed with 20 mM potassium phosphate buffer (pH 6.0) containing 20% saturated ammonium sulfate. did. Then, the enzyme is eluted with 20 mM potassium phosphate buffer (pH 6.0) containing 10% saturated ammonium sulfate, and the enzyme solution is passed through Mono Q (1 ml, FPLC; product name of Pharmacia) to adsorb the enzyme and 205 mM NaCl. After washing with 20 mM potassium phosphate buffer (pH 6.0) containing 20 ml, the enzyme was eluted with 20 mM potassium phosphate buffer (pH 6.0) containing 210 ml NaCl to obtain a purified enzyme. The purified enzyme thus obtained was subjected to the following physicochemical properties.
The results of purification at each stage are shown in FIG. The specific activity at the final stage was 120 times that of the crude enzyme. When the purified enzyme was subjected to SDS-PAGE (CBB staining), a single band was shown.

[Example 2] (Physicochemical properties of thermostable AMP deaminase)
Substrate specificity: This enzyme is added to a substrate solution in which each substrate concentration 1 mM and 100 mM potassium phosphate buffer (pH 6.0) are mixed, reacted at 50 ° C. for 20 minutes, and enzyme activity is measured by the GIDH method. Substrate specificity was examined. The results are shown in FIG. Enzyme activity was expressed as relative activity when the enzyme activity for AMP (5′-adenylic acid or adenosine monophosphate) was taken as 100%. As is clear from FIG. 2, for adenosine, ATP (adenosine triphosphate), ADP (adenosine diphosphate), adenine, NAD (nicotinamide adenine dinucleotide), NADP (nicotinamide adenine dinucleotide phosphate) However, it did not act at all on 3′-AMP, GMP, and 3′-CMP. From the above results, it was found that the thermostable AMP deaminase may be used for reactions using adenosine, ADP, NAD or the like as a substrate in addition to AMP.

  Working temperature: The relationship between the reaction temperature and activity of this enzyme was examined. After adjusting the enzyme (0.1 ml) to pH 6.0 using a potassium phosphate buffer solution of pH 6.0, the reaction temperature during the activity measurement reaction was changed from 10 ° C. to 80 ° C. to 1 mM AMP and The reaction was conducted and the activity was examined. The results are shown in FIG. FIG. 3 shows the relative activity (%) when the maximum activity is 100%. As is apparent from FIG. 3, the optimum temperature is around 55 ° C., and a high activity of about 70% or more is observed in a wide range of 30 ° C. to 70 ° C., and the enzyme works well under a wide range of temperature conditions. I understood that.

  Thermostability: The thermostability of this enzyme was examined. After adjusting the enzyme (0.1 ml) to pH 6.0 with potassium phosphate buffer solution at pH 6.0, each temperature (30 ° C, 40 ° C, 50 ° C, 60 ° C, 65 ° C, 70 ° C, 80 ° C) Then, the mixture was reacted with 1 mM AMP and 100 mM potassium phosphate buffer (pH 6.0) at 50 ° C. for 20 minutes, and then the activity was measured. The results are shown in FIG. From FIG. 4, when the enzyme activity in the untreated case was used as a reference (100%), the activity of about 80% or more was maintained up to 65 ° C. Further, even when treated at 70 ° C., the activity of about 17% was maintained. From this, it was found that the enzyme was excellent in thermal stability.

  Action pH: The relationship between pH and activity of the enzyme was examined. In the presence of 1 mM AMP and 100 mM buffer (pH 3 to 6 is citrate phosphate buffer, pH 6 to 8 is potassium phosphate buffer (KPB), pH 8 to 9 is Tris-HCl buffer, pH 9 to 11 was used with a carbonate buffer and pH 11-12 was used with a sodium phosphate buffer, respectively) at 30 ° C. for 30 minutes, and the relationship between pH and activity was examined. The results are shown in FIG. FIG. 5 shows the relative activity (%) when the maximum activity is 100%. From FIG. 5, the optimum pH was around 6.0.

  pH stability: The pH stability of thermostable AMP deaminase was examined. This enzyme solution (0.1 ml) was added to 100 mM of each buffer solution (pH 3 to 6 is citrate phosphate buffer, pH 6 to 8 is potassium phosphate buffer (KPB), pH 8 to 9 is Tris-HCl buffer and pH 9 to 11 is treated with carbonate buffer solution at 50 ° C for 30 minutes (final amount 1ml), then reacted with 1mM AMP, 100mM potassium phosphate buffer solution (pH6.0) at 50 ° C for 20 minutes, Activity was measured. The results are shown in FIG. From FIG. 6, when the enzyme activity in the case of untreated is used as a reference (100%), a relatively high activity is maintained in the pH range of about 4.0 to about 9.0, and about 80% in the range of about 6.0 to about 8.0. It was found that the above activity was maintained.

  Kinetic parameters: Kinetic parameters for thermostable AMP deaminase were determined under the conditions of reaction temperature of 50 ° C. and reaction for 20 minutes. The Km value was 1.1 mM, and the Vmax was 10.63 U / mg.

  Molecular weight: The molecular weight by SDS-PAGE was 85,000 ± 3,000, and the molecular weight by gel filtration was 88,000 ± 3,000.

  Effect of metal ions: 1 mM and 10 mM metal ions were added to the enzyme in 100 mM potassium phosphate buffer (pH 6.0), treated at 50 ° C for 10 minutes, and then added with 1 mM AMP at 50 ° C. The reaction was performed for 20 minutes. The relative activity was examined with 100 as the case where no metal ion was added. The results are shown in FIG. From FIG. 7, inhibition of this enzyme was recognized by Cu ions and Fe ions.

  Effect of modifying reagents and inhibitors: Add 1 mM of various modifying reagents or inhibitors to this enzyme in 100 mM potassium phosphate buffer (pH 6.0), treat at 50 ° C for 10 minutes, then add 1 mM AMP. The reaction was performed at 50 ° C. for 20 minutes. The relative activity was examined with 100 as the case where no modifier or inhibitor was added. The results are shown in FIG. In FIG. 8, PCMB is parachloromer benzoate, PMSF is phenylmethanesulfonyl fluoride, EDC is 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, DEPC is diethyl pyrocarbonate, and TNBS is triphenyl. Nitrobenzenesulfonic acid, DTT is dithiothreitol, EDTA is an abbreviation for ethylenediaminetetraacetic acid, and EGTA is an abbreviation for ethylene glycol bis (β-aminoethyl ester) tetraacetic acid. This enzyme was found to be strongly inhibited by PCMB and monoiodoacetic acid, but not by 4- (2-aminoethyl) benzenesulfohydrochloride, EDTA and EGTA.

It is a table | surface which shows the total enzyme activity amount, specific activity, total protein amount, total activity, activity multiplication factor, and yield in each purification process of thermostable AMP deaminase. It is a table | surface which shows the substrate specificity of thermostable AMP deaminase. It is a graph which shows the working temperature of thermostable AMP deaminase. It is a graph which shows the thermostability of thermostable AMP deaminase. It is a graph which shows the action | operation pH of thermostable AMP deaminase. It is a graph which shows the pH stability of thermostable AMP deaminase. It is a table | surface which shows the influence of the metal ion of thermostable AMP deaminase. It is a table | surface which shows the influence of the modification reagent or inhibitor of thermostable AMP deaminase.

Claims (9)

A thermostable AMP deaminase having the following physicochemical properties:
(1) Substrate specificity: Catalyze the reaction of 5′-adenylic acid + H ₂ O → 5′-inosinic acid + NH ₃ .
(2) Stable temperature: Stable up to 65 ° C.
(3) Optimal pH: around 6.0.
(4) Molecular weight: 85,000 ± 3,000 (by SDS-PAGE), 88,000 ± 3,000 (by gel filtration).   The thermostable AMP deaminase according to claim 1, which is produced by a microorganism belonging to the genus Aspergillus. The microorganism belonging to the genus Aspergillus is Aspergillus fumigatus (Aspergillus
The thermostable AMP deaminase according to claim 2, which is fumigatus).   The thermostable AMP deaminase according to claim 3, wherein the Aspergillus fumigatus is Aspergillus fumigatus N0.4 (FERM P-20075).   A method for producing a thermostable AMP deaminase, comprising culturing the microorganism having the ability to produce thermostable AMP deaminase according to claim 1 in a nutrient medium, producing the thermostable AMP deaminase in a culture solution, and collecting the same.   The method for producing a thermostable AMP deaminase according to claim 5, wherein the microorganism belongs to the genus Aspergillus. The microorganism belonging to the genus Aspergillus is Aspergillus fumigatus (Aspergillus
fumigatus). The method for producing a thermostable AMP deaminase according to claim 6.   The method for producing a thermostable AMP deaminase according to claim 7, wherein the Aspergillus fumigatus is Aspergillus fumigatus No. 4 (FERM P-20075).   Aspergillus fumigatus No. 4 (FERM P-20075) having the ability to produce the thermostable AMP deaminase according to claim 1.

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