JPH0829079B2 - L-phenylalanine dehydrogenase and method for producing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to an L-phenylalanine dehydrogenase useful for producing L-phenylalanine, quantifying L-phenylalanine and phenylpyruvic acid, and a method for producing the same.

(Prior Art and Problems Thereof) L-phenylalanine dehydrogenase having an action similar to that of L-phenylalanine dehydrogenase of the present invention and a method for producing an L-amino acid using this enzyme are disclosed in JP-A-59-198972 and JP-A-61721. -146183, JP-A-61-
239887 and JP-A-61-239888. However, L- as described in this published specification
Phenylalanine dehydrogenase is a bacterium belonging to the genus Brevibacterium (Rhodococcus), which is a normal-temperature bacterium, and Sporosarcina.
It was produced by a genus and a bacterium of the genus Bacillus, both of which were active at room temperature but inactivated at 50 ° C or higher. However, from the viewpoint of industrial use of enzymes, it is desired to use enzymes that have a high reaction rate and can be used under conditions in which the reaction solution is unlikely to be contaminated by various bacteria.

To meet such conditions, it is preferable to use a thermostable enzyme, and various thermostable enzymes have been searched.

However, so far, a thermostable L-phenylalanine dehydrogenase has not been found.

(Means for Solving the Problem) The present inventors eagerly searched for a strain having the enzyme-producing ability for the purpose of searching for L-phenylalanine dehydrogenase excellent in heat resistance, and diligently including known preserved strains. As a result of continued research, they have found that Thermoactinomyces intermedius ATCC33205 produces highly thermostable L-phenylalanine dehydrogenase, which is completely different from the hitherto known ones, and arrived at the present invention.

That is, the present invention provides (1) L-phenylalanine dehydrogenase having the following physicochemical properties (a) Action: 1 mol of L-phenylalanine, 1 mol of NAD ⁺ and 1
From 1 mol of water, 1 mol of phenylpyruvic acid, 1 mol of
It catalyzes the reaction that produces NADH and 1 mol of ammonium ion (oxidative deamination reaction) and its reverse reaction (reductive amination reaction).

(B) Substrate specificity: In the oxidative deamination reaction, it specifically acts on L-phenylalanine and very little or no action on other amino acids.

In the reductive amination reaction, it acts specifically on phenylpyruvic acid and has very little or no effect on other α-keto acids. (Table-1) (c) Optimum pH: pH around 10.8 in oxidative deamination reaction and around pH9.2 in reductive amination reaction (Fig. 1) (d) Stable pH range: 50 ° C for 10 minutes No decrease in activity was observed in the treatment range of pH 5.5 to 10.8.

No decrease in activity was observed in the pH range of 5.5 to 9.8 after treatment at 70 ° C for 10 minutes. (Fig. 2) (e) Thermal stability: No decrease in activity is observed after treatment at 70 ° C for 60 minutes.

After treatment at 75 ° C for 30 minutes, about 25% of the activity remains. (Figure −
3) (f) Molecular weight: Total molecular weight-about 270,000-290,000 (gel filtration method) Molecular weight of subunit-47,000 (hexameric structure of the same subunit) and (2) Thermoactinomyces (Thermoactinomyces)
A method for producing L-phenylalanine dehydrogenase, which comprises culturing an L-phenylalanine dehydrogenase-producing bacterium belonging to the genus and collecting the above L-phenylalanine dehydrogenase from the culture.

The physicochemical properties of the L-phenylalanine dehydrogenase of the present invention will be described below.

(A) Action: 1 mol of L-phenylalanine, 1 mol of NAD ⁺ and 1
From 1 mol of water, 1 mol of phenylpyruvic acid, 1 mol of
It catalyzes the reaction that produces NADH and 1 mol of ammonium ion (oxidative deamination reaction) and its reverse reaction (reductive amination reaction).

(B) Substrate specificity: Enzymatic activity was measured using L-amino acid as a substrate in the oxidative deamination reaction and α-keto acid as a substrate in the reductive amination reaction. The results are shown in Table 1-1 and 1-2. The enzyme activity was expressed as a relative activity with the activity for L-phenylalanine and phenylpyruvic acid as 100. Table 1-1 (Oxidative deamination reaction) Substrate Relative activity L-Phenylalanine 100 Lp-Aminophenylalanine 7.2 L-Leucine 3.9 In addition, D-phenylalanine, DL-phenylglycine, L-tyrosine, DL-tert- Leucine, L-tryptophan, L-methionine, L-arginine, L-alanine, L-valine, glycylglycine, L-histidine,
It has no effect on L-proline, L-glutamic acid and L-isoleucine. Table 1-2 (Reductive amination reaction) Substrate Relative activity Phenylpyruvic acid 100 Ketomethionine 13.9 α-Ketoisocaproic acid 5.5 α-Ketoisovaleric acid 5.0 α-Ketoisoleucine 3.3 α-Ketobutyric acid 1.3 Pyruvic acid 0.7 Oxaloacetic acid 0.9 α-Ketoglutarate 0 p-Hydroxyphenylpyruvate 0 As can be seen from Table-1, the L-phenylalanine dehydrogenase of the present invention is L-phenylalanine dehydrogenase in the oxidative deamination reaction.
It acts specifically on phenylalanine and has very little or no effect on other amino acids.

In the reductive amination reaction, it acts specifically on phenylpyruvic acid and has very little or no effect on other α-keto acids.

The L-phenylalanine dehydrogenase of the present invention is
It can be said that the enzyme has extremely high substrate specificity for L-phenylalanine and phenylpyruvic acid as compared with the conventional enzyme.

(C) Optimum pH: In the oxidative deamination reaction, 0.2 M Gly-KCl-KOH buffer (pH 9 to 11), 50 mM K ₂ HPO ₄ -KOH buffer (pH 11 to 1)
2) was used for the examination. 200 mM for reductive amination reaction
NH ₄ Cl-NH ₄ OH buffer _{(pH8~10), 100mM Na 2 CO} 3 -Na
It was examined using HCO ₃ buffer instead of 2M NH ₄ Cl.

As a result, as shown in Figure 1, the optimum pH was around pH 10.8 in the oxidative deamination reaction and in the reductive amination reaction.
It is around 9.2.

(D) Stable pH range: 0.1 ml of this enzyme solution with 0.4 buffer solution of each pH value described later.
ml was added, treated at 50 ° C. and 70 ° C. for 10 minutes, and cooled on ice to obtain a sample. The specific activity of this was determined. The buffer solution has a pH of 4 to
50 mM CH ₃ COOH-CH ₃ COONa for 6, 50 mM KH ₂ PO ₄ -K ₂ H PO ₄ for pH 6-8, 50 mM Tris for pH 8-8.6
-50 mM Gly-KCl-KOH, pH 11-12 for HCl, pH 9-11.
For 5, 50 mM K ₂ HPO ₄ -K ₃ PO ₄ was used, respectively.

As a result, as shown in Fig. 2, this enzyme is stable up to pH 5.5 to 10.8 when treated at 50 ° C for 10 minutes. 70 ℃ 1
With 0 minute treatment, it is stable up to pH 5.5-9.8.

From this fact, it can be said that the phenylalanine dehydrogenase of the present invention is easy to handle since it is stable in a wide range as compared with the conventional enzyme.

(E) Thermostability: 10 mM potassium phosphate buffer solution (pH7.) Containing 0.01% 2-mercaptoethanol and 1 mM EDTA per 0.1 ml of this enzyme solution.
2) Prepare 10 test tubes with 0.4 ml added, 20 ℃, 37 ℃, 45
Each was treated for 5 minutes at a temperature of 50 ° C, 50 ° C, 55 ° C, 60 ° C, 65 ° C, 70 ° C, 75 ° C, and 80 ° C, and ice-cooled to obtain a sample.
The specific activity of this was determined. Furthermore, 70 ℃ and 75 ℃
Was treated for 0 to 60 minutes and the specific activity was similarly determined. as a result,
As shown in Fig.-3, this enzyme was treated at 70 ℃ for 5 minutes.
It is stable until about 50%, and no decrease in activity is observed, and about 50% of the activity remains at 75 ° C. Also, at 70 ° C, even after 60 minutes of treatment, the activity was stable and no decrease in activity was observed, and at 75 ° C, about 25% of the activity remained after 30 minutes of treatment.

From this, the phenylalanine dehydrogenase of the present invention shows no decrease in activity at 70 ° C., which is 20 ° C. higher than that of the enzyme derived from a normal temperature bacterium that is inactivated at about 50 ° C. It can be said that the enzyme has excellent thermostability.

(F) Molecular weight: Toyopearl HW55F column (diameter 1.5)
X63.5 cm) (manufactured by Toyo Soda Co., Ltd.) was used for gel filtration. The standard protein is ferritin (molecular weight 460,00
0), Bacillus sphaericus
Derived alanine dehydrogenase (molecular weight 235,000),
Lactate dehydrogenase derived from horse heart (molecular weight 140,000), bovine serum albumin (molecular weight 68,000), and ovalbumin (molecular weight 43,000) were used.

The molecular weight of the subunit was measured by SDS-slab electrophoresis. Bovine serum albumin (molecular weight 68,000), α-chymotrypsinogen A (molecular weight 25,700), catalase (molecular weight 60,000), ovalbumin (molecular weight 43,000), and cytochrome (molecular weight 12,384) were used as standard proteins.

As a result, the total molecular weight is 270,000 to 290,000. In addition, the molecular weight of the subunit was 47,000, and there was only one SDS protein band, indicating that this enzyme has a hexameric structure of the same subunit.

(G) Effect of Metal Ions Various metal ions were mixed as chlorides in the reaction solution for enzyme activity measurement described below, and the activity was measured. As a result, as shown in Table 2, this enzyme is inhibited by Fe ³⁺ and Hg ²⁺ .

Regarding the conventionally known enzyme, it is often reported that the enzyme activity is increased by Fe ³⁺ , but the enzyme according to the present invention is also a novel enzyme because it is inhibited by Fe ^3+. .

The L-phenylalanine dehydrogenase of the present invention is
For example, a phenylalanine dehydrogenase-producing bacterium belonging to the genus Thermoactinomyces is cultured, and L-
It can be produced by collecting phenylalanine dehydrogenase.

Microorganisms used in the method for producing L-phenylalanine dehydrogenase of the present invention include all strains, mutants and variants belonging to the genus Thermoactinomyces capable of producing L-phenylalanine dehydrogenase. A preferred specific example thereof is Thermoactinomyces intermedius, and a conserved bacterium belonging to this species is Thermoactinomyces intermedius.
The ATCC 33205 can be mentioned.

Cultivation of the L-phenylalanine dehydrogenase-producing bacterium for obtaining the L-phenylalanine dehydrogenase of the present invention can be carried out in an ordinary basal nutrient medium. For example, a medium containing peptone, yeast extract, meat extract, inorganic salts and the like is used. Moreover, when a small amount of L-phenylalanine is added as an inducer to this basal nutrient medium, L-phenylalanine dehydrogenase is induced. The amount of L-phenylalanine added varies depending on the composition of the medium and the nature of the strain, but is preferably about 0.1-1%.

The culture may be carried out using either a solid medium or a liquid medium, but in order to obtain a large amount of the target enzyme, the liquid medium is used, and the culture is carried out under aerobic conditions by shaking culture, aeration stirring culture or the like. It is preferable. At the culturing temperature, the bacteria grow and L
It may be within the temperature range where phenylalanine dehydrogenase is produced, but it is preferably 25 to 60 ° C. The culture time may be selected such that the enzyme activity is expressed, but it is preferably 6 to 110 hours. The pH of the culture is preferably 7 to 7.5.

By this culture, most of the enzyme is accumulated in the cells.

After completion of the culture, the culture may be used as it is as an enzyme source, but it is usually separated and purified. As a purification method,
Conventional enzyme purification methods can be used. For example, the cells are collected by centrifugation and disrupted by a mechanical method such as ultrasonic treatment or Dynomill. Then, solid matters such as cell debris are removed by centrifugation or the like to obtain a crude enzyme solution. Then, this crude enzyme solution is fractionated by the ammonium sulfate precipitation method, and a uniform crystalline enzyme preparation can be isolated by dialysis by addition of mercaptoethanol or EDTA, affinity chromatography or the like.

Next, the method for measuring the activity of L-phenylalanine dehydrogenase of the present invention will be described.

The method for measuring the activity of Nase will be described.

In the oxidative deamination reaction, the reaction solution containing coenzyme NAD ⁺ and the substrate as shown in Table 3-1 is used, while in the reductive amination reaction, the reaction solution containing coenzyme NADH and the substrate 2 is used.
The reaction solution as shown in (3) is incubated at 37 ° C. for 2 to 3 minutes, and the reaction rate is determined by quantifying the increase / decrease amount of NADH accompanying each reaction. However, the reaction is started with coenzyme. Since NADH has a spectrum having an absorption maximum at 340 nm, the amount of change with time at 340 nm is measured with a spectrophotometer. In the measurement, the amount of enzyme is adjusted so that the absorption increases and decreases linearly.

The enzyme activity unit of this enzyme is 1 μmol / min NADH.
The amount of enzyme that produces is defined as 1 unit. Specific activity is expressed as the number of units per mg of enzyme protein.

Next, the method for producing L-phenylalanine dehydrogenase of the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto.

(Example) First stage Thermoactinomyces intermedius ATCC33
205 was inoculated into the medium shown in Table 4 with a platinum loop and cultured at 50 ° C. for 7 hours with shaking. Centrifuge the medium containing the cells (8000G, 1
(0 minutes) to collect the cells, wash with 0.85% saline,
The cells were suspended in a 10 mM potassium phosphate buffer (pH 7.2) containing 0.01% 2-mercaptoethanol and 1 mM EDTA, and disrupted with an ultrasonic disruptor while cooling with ice. In addition, 20000
G was sedimented by a centrifuge for 20 minutes, and the supernatant was separated as a crude enzyme solution.

Table 4 peptone 5g glycerol _{5g KH 2 PO 4 1g K 2} HPO 4 2g MgSO 4 · 7H 2 O 0.1g CaCl 2 0.05g Yeast Extract 2g meat extract 2g biotin 9 [mu] g L-Tyrosine _{0.4g NH 4 Cl 5g H 2 O} 1 pH7.2 Second step Stir the crude enzyme solution obtained in the first step while cooling with ice,
Ammonium sulfate was gradually added to the enzyme solution so that the final concentration was 25% saturation. When the pH of the enzyme solution was lowered, 14% aqueous ammonia was added to adjust the pH to 7-8. Next, this solution was centrifuged (10000 G, 10 minutes) to remove the precipitate. In the supernatant,
Ammonium sulfate was added so that the final concentration was 60% saturation, and the precipitate and the supernatant were separated in the same manner as above. This precipitate should be added as little as possible to 0.01% 2-mercaptoethanol, 1 mM EDTA.
Dissolved in 10 mM potassium phosphate buffer (pH 7.2) containing
The buffer solution (pH 6.7) 1 having the same composition was dialyzed twice for about 20 hours in a low temperature room (5 to 10 ° C) while stirring with a stirrer to obtain a dialyzed enzyme solution.

Third step The dialyzed enzyme solution obtained in the second step was preliminarily adjusted to 0.
Adsorbed on a Red Sepharose 4B column (diameter 3 x 22 cm) equilibrated with 10 mM potassium phosphate buffer (pH 6.7) containing 01% 2-mercaptoethanol and 1 mM EDTA, and 0.01% 2
Non-adsorbed proteins were washed with 500 ml of 10 mM potassium phosphate buffer (pH 6.7) containing mercaptoethanol and 1 mM EDTA. Then 0.1M NaCl and 0.3M NaCl were added respectively.
0.01% 2-mercaptoethanol, 10 mM containing 1 mM EDTA
Phenylalanine dehydrate genase was eluted by gradually flowing 500 ml of potassium phosphate buffer (pH 6.7).

4th step The oxygen solution obtained in the 3rd step was dialyzed twice with a 10 mM potassium phosphate buffer (pH 6.7) containing 0.01% 2-mercaptoethanol and 1 mM EDTA for 2 times for about 20 hours and then subjected to ultrafiltration. It concentrated with the container (filter paper UHP43, UP20).

The obtained concentrated liquid is slab gel (thickness 1 × 10 × 10 cm: 7.5
Electrophoresis was performed for about 6 hours using a polyacrylamide gel (pH 8.9), and one end was immediately cut out in a width of 5 mm for activity staining. The fragment was returned to the original state and the phenylalanine dehydrogenase portion was cut off using the active band as an index.
The obtained gel was ground with a Potter type Teflon homogenizer while cooling with ice, and the enzyme was extracted using a 10 mM potassium phosphate buffer (pH 7.2) containing 1 mM EDTA. Next, this extract was centrifuged (10000 G, 10 minutes) to obtain a supernatant as an enzyme solution.

Next, the total protein amount, the total activity and the specific activity of the enzyme solution obtained in each of the first to fourth purification steps were measured. The results are shown in Table-5. The total amount of protein was measured by the Kalb and Bernlohr method for the first to third steps, and the Folin-Lowry method for the fourth step. The total activity was measured according to the activity measuring method described above.

Next, regarding the enzyme solution obtained in the fourth step, Davis an
Purity was assayed by the d Ornstein method. That is, a 7.5% (W / V) polyacrylamide gel (pH 9.4) was prepared in a glass tube (diameter 0.5 × 7.5 cm), and a current of 2 mA was applied to each of the gels, and electrophoresis was performed for about 1 hour.

Protein staining is Coomasie brilliant blue R-250
The activity staining was carried out by the PMS (phenazine methosulfate) -INT (iodonitrotetrazolium) method at 37 ° C using the reaction solution shown in Table-6. After that, decolorization solution (10% (V / V) acetic acid + 10% (V / V) ethyl alcohol)
Decolorization was carried out and the purity was checked.

As a result, 1 band was obtained for both protein staining and activity staining.
It became a book, and the Rf values agreed. From this, it was found that the present enzyme was successfully purified.

(Effect) The L-phenylalanine dehydrogenase of the present invention is
It has excellent heat resistance compared to conventional products.

Substrate specificity is very high for L-phenylalanine and phenylpyruvic acid.

 Wide stable pH range.

It has the advantage of. The L-phenylalanine dehydrogenase of the present invention having these properties is extremely useful for production of L-phenylalanine, quantification of L-phenylalanine and phenylpyruvic acid, and the like.

Phenylketonuria is a hereditary amino acid metabolism disorder that results in an increase in blood phenylalanine levels and urinary phenylketone body concentrations such as phenylpyruvic acid due to the lack of phenylalanine hydroxylase in the liver, leading to a mental retardation zone. This phenylketonuria can be diagnosed by measuring the blood phenylalanine concentration or the urine phenylpyruvic acid concentration, but the L-phenylalanine dehydrogenase of the present invention has the extremely excellent properties described above. Therefore, it is also very useful for the microquantification of phenylalanine and phenylpyruvic acid as a diagnosis of phenylketonuria.

[Brief description of drawings]

FIG. 1 is a diagram showing the optimum pH of phenylalanine dehydrogenase of the present invention. FIG. 2 is a diagram showing a stable pH range of the phenylalanine dehydrogenase of the present invention. FIG. 3 is a diagram showing the thermostability of phenylalanine dehydrogenase of the present invention.

Claims (3)

[Claims] 1. L-Phenylalanine dehydrogenase having the following physicochemical properties (a) Action: From 1 mol L-phenylalanine, 1 mol NAD ⁺ and 1 mol water to 1 mol phenylpyruvic acid, 1 mol NA
It catalyzes the reaction that produces DH and 1 mol of ammonium ion (oxidative deamination reaction) and its reverse reaction (reductive amination reaction). (B) Substrate specificity: In the oxidative deamination reaction, it specifically acts on L-phenylalanine and very little or no action on other amino acids. In the reductive amination reaction, it acts specifically on phenylpyruvic acid and has very little or no effect on other α-keto acids. (C) Optimum pH: pH around 10.8 in the oxidative deamination reaction and around pH 9.2 in the reductive amination reaction. (D) Stable pH range: No decrease in activity is observed in the pH range of 5.5 to 10.8 after treatment at 50 ° C for 10 minutes. No decrease in activity was observed in the pH range of 5.5 to 9.8 after treatment at 70 ° C for 10 minutes. (E) Thermal stability: No decrease in activity is observed after treatment at 70 ° C. for 60 minutes. After treatment at 75 ° C for 30 minutes, about 25% of the activity remains. (F) Molecular weight: The total molecular weight is about 270,000 to 290,000 (gel filtration method). The molecular weight of the subunit is 47,000 (6 for the same subunit).
Quantum structure). 2. A thermoactinomycete.
ces) L-phenylalanine dehydrogenase-producing bacterium is cultivated, and L-phenylalanine dehydrogenase having the following physicochemical properties is obtained from the culture (a) Action: 1 mol L-phenylalanine, 1 mol NAD ⁺ and 1 mol From water, 1 mol phenylpyruvic acid, 1 mol NA
It catalyzes the reaction that produces DH and 1 mol of ammonium ion (oxidative deamination reaction) and its reverse reaction (reductive amination reaction). (B) Substrate specificity: In the oxidative deamination reaction, it specifically acts on L-phenylalanine and very little or no action on other amino acids. In the reductive amination reaction, it acts specifically on phenylpyruvic acid and has very little or no effect on other α-keto acids. (C) Optimum pH: pH around 10.8 in the oxidative deamination reaction and around pH 9.2 in the reductive amination reaction. (D) Stable pH range: No decrease in activity is observed in the pH range of 5.5 to 10.8 after treatment at 50 ° C for 10 minutes. No decrease in activity was observed in the pH range of 5.5 to 9.8 after treatment at 70 ° C for 10 minutes. (E) Thermal stability: No decrease in activity is observed after treatment at 70 ° C. for 60 minutes. After treatment at 75 ° C for 30 minutes, about 25% of the activity remains. (F) Molecular weight: The total molecular weight is about 270,000 to 290,000 (gel filtration method). The molecular weight of the subunit is 47,000 (6 for the same subunit).
Quantum structure). A method for producing L-phenylalanine dehydrogenase, which comprises: 3. An L-phenylalanine dehydrogenase-producing bacterium belonging to the genus Thermoactinomyces belongs to Termoactinomyces i.
ntermedius) ATCC 33205, the manufacturing method according to claim 2.