JPH0630573B2 - L-phenylalanine dehydrogenase and method for producing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to an L-phenylalanine dehydrogenase and a method for producing sesame.

[Conventional technology]

JP-A-59-198972 describes L-phenylalanine dehydrogenase having an action similar to that of L-phenylalanine dehydrogenase of the present invention and a method for producing L-α-aminocarboxylic acid using this enzyme. However, the L-phenylalanine dehydrogenase described in this published specification is a Brevibacterium.
It was produced by a bacterium belonging to the genus cterium, and there is no suggestion in this specification that the bacterium belonging to the genus Sporosarcina or the bacterium belonging to the genus Bacillus produces a similar enzyme. In addition, this L-phenylalanine dehydrogenase has a molecular weight of 130,000 ± 10,000 and is composed of subunits with a molecular weight of 66,000 ± 5,000, and for a wide range of substrates such as p-hydroxyphenylpyruvic acid and indolepyruvic acid as well as phenylpinvic acid. And has a high specificity, and is completely different from the L-phenylalanine dehydrogenase of the present invention.

[Problems to be solved by the invention]

Therefore, the present invention aims to provide a novel L-phenylalanine dehydrogenase derived from a microorganism which has not been known to produce L-phenylalanine dehydrogenase, and a novel method for producing the enzyme. Is.

[Means for solving problems]

The purpose of the above is the following properties: (1) 1 mol of L-phenylalanine, 1 mol of NAD ⁺ and 1 mol of water to 1 mol of phenylpyruvic acid, 1 mol of
A reaction that produces NADH and 1 mole of ammonium ion,
And catalyze this reverse reaction; (2) about 2 in high performance liquid chromatography gel filtration
It has a molecular weight of 90,000 and is about 340,000 in sedimentation equilibrium
And a subunit having a molecular weight of about 38,000 to 39,000 in SDS-polyacrylamide gel disc electrophoresis; (3) L-tryptophan and L-tryptophan and L-tryptophan which act specifically on L-phenylalanine and L-tyrosine. L-phenylalanine dehydrogenase having a very low specificity for methionine; and the production of said enzyme characterized by culturing these bacteria and collecting said enzyme from this culture. Method).

[Specific explanation]

(1) Microorganisms The microorganisms used in the present invention may be those belonging to the genus Sporosarcina or the genus Bacillus and capable of producing L-phenylalanine dehydrogenase, and such microorganisms are selected from the preservation bacteria. In some cases, it can be newly separated from the natural world.

Examples of the microorganism belonging to the genus Sporosarcina include sporosarcina ureae. Examples of conserved bacteria belonging to this species include Sporosarcina ureae IFO126
98, and Sporosarcina ureae IFO12699 (ATCC6473)
Examples of the new strain include sporosarcina ureae SCRC-R04 isolated by the present inventors. Each of the above-mentioned preserved bacteria can be freely obtained from IFO or ATCC under the above-mentioned deposit number, and a new strain Sporosarcina ureae SCRC-R04 Institute of Microbial Science and Technology, Institute of Microbiology Research, Microtechnology Research Institute Issue (FERM P-8178)
, And was transferred to the International Deposit under the Budapest Treaty as NIRS No. 1012 (FERM BP-1012).

Examples of the microorganism belonging to Bacillus include a new strain Bacillus sp. SCRC-R53b and Bacillus isolated by the present inventors.
sp.SCRC-R79a, Bacillus sp.SCRC-101A, and Bacillus s
p.SCRC-114D can be mentioned. The mycological properties of these strains are very close to each other, and Bacillus sp.SCRC-R79a is a representative strain of these strains at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology, Microindustry Research Institute No. 8179 (FERM P-8179). ), And was transferred to the International Deposit under the Budapest Treaty as NIRS 1013 (FERM BP-1013). See also Bacillus sp.S
CRC-114D has been deposited internationally under the Budapest Treaty as NIKKOSHI No. 1011.

The new strain was isolated as follows. A medium having the composition shown in Table 1 below was prepared.

Dispense 5 ml of this medium into a test tube (18 mm), and
Sterilized for 15 minutes at ° C. A small amount of a soil sample collected from various places was added to this medium and shake-cultured at 30 ° C. for 3 days.
Take one platinum loop of this culture and inoculate the same medium.
The culture was carried out at 0 ° C. for 3 days with shaking. A part of the culture solution was streaked on a plate medium prepared by adding 2% agar to the medium shown in the table with a platinum loop and kept at 30 ° C. for several days. The colonies that appeared were picked up on a slant medium having the same medium composition.

In this way, many strains were isolated from the soil samples collected from various places. Next, 200 ml of the medium in the table was dispensed into a 500-ml Erlenmeyer flask and sterilized in the same manner. Each strain was cultivated in this medium at 30 ° C. for 24 hours with rotary shaking, and the obtained bacterial cells were washed and then disrupted by ultrasonication. The supernatant obtained after stretching was dialyzed against 0.01 M phosphate buffer (pH 7.0) containing 0.1 mM EDTA and 5 mM 2-mercaptoethanol. The L-phenylalanine dehydrogenase activity contained in this supernatant was measured by the method described below.

In this way, the following 5 strains that remarkably produce L-phenylalanine dehydrogenase were obtained. The sources of isolation of these strains were as follows.

The above-mentioned novel 5 strains have the following mycological properties.

Based on the above mycological properties
Of Determinative Bacteriology (Berge
y's Manual of Determinative Bacteriology) 8th edition,
According to the 1974 classification criteria, the 5 strains were identified as follows.

(I) The SCRC-R04 strain belongs to the genus Sporosarcina because it is aerobic, has motility and sporulation ability, and is a Gram-positive 2 to 4 consecutive cocci. Since Sporosarcina ureae is known as the only species in the genus Sporosarcina, and the above-mentioned properties are almost the same as those described in the literature, SCRC-R04
The strain is identified as Sporosarcina ureae.

(ii) SCRC-R53b, SCRC-R79a, SCRC-101A, and SCRC-114D
It is clear that all the strains belong to the genus Bacillus, because they form endospores in Gram-positive bacilli and produce catalase.

In addition, strains SCRC-R04, SCRC-R53b, SCRC-R79a, SCRC-101
Electron micrographs of A and SCRC-114D are shown in Fig. 1 ~.
It is shown in FIG.

Although the strains isolated from nature have been described above in detail, it is also possible to mutate these strains to obtain strains with higher productivity. In addition, the gene involved in the production of L-phenylalanine dehydrogenase existing in the cells of these strains is excised and inserted into an appropriate vector such as a plasmid, and this vector is used to produce an appropriate host such as Escherichia coli. (Eshcerichia c
It is also possible to artificially create the L-phenylalanine dehydrogenase-producing strain of the present invention by transforming a heterologous host such as oli) or yeast or a homologous host such as Bacillus strain or Sporosarcina strain.

The strain of the present invention can be stored according to a conventional method,
For example, it can be stored on an agar slant medium or by a freeze-drying method. As the agar slant medium, a medium commonly used for the preservation of sporosarcin or Bacillus bacteria, for example, the medium described above for the isolation of bacteria can be used. In addition, freeze-drying can be preserved according to a conventional method.

(2) Method for producing enzyme When culturing the above-mentioned microorganism to produce the L-phenylalanine dehydrogenase of the present invention, any basal nutrient medium can be used as long as the microorganism of the present invention can grow. You may. This medium contains one or a plurality of types such as yeast extract, peptone, and meat extract as a nitrogen source. If necessary, glucose, starch, glycerin, etc. can be added to this medium as a carbon source. It is preferable to add inorganic salts such as dipotassium phosphate, sodium chloride and magnesium sulfate to this medium.

In producing L-phenylalanine dehydrogenase, it is preferable to add a small amount of L-phenylalanine as an inducer to the basal medium. The amount of L-phenylalanine added is about 0.01 to 10 w / v%, preferably 0.1 to 1 w / v%, although it varies depending on the composition of the basal medium, the properties of the strain to be cultured, and the like.

The culture may be performed using either a solid medium or a liquid medium, but in order to obtain a large amount of the target enzyme, a liquid medium is used and cultured under aerobic conditions by shaking culture, aeration / agitation culture, etc. Is preferably performed.

The culture temperature may be any temperature within the temperature range in which the bacterium grows and L-phenylalanine dehydrogenase is produced, but it is preferably 25 to 45 ° C. The pH is in the range of 6-11, preferably 7-10. The culture time may be selected such that the enzyme activity is expressed, but it is preferably 6 to 48 hours.

Next, the L-phenylalanine dehydrogenase of the present invention is collected from the obtained culture, and a usual enzyme purification method can be used as a purification method. The cells are collected by centrifugation or the like, and the cells are disrupted by a mechanical method such as ultrasonic treatment or Dynomill. Solid substances such as cell debris are removed by centrifugation, etc. to obtain a crude enzyme, which is further treated with protamine sulfate or streptomycin sulfate, salting out, organic solvent precipitation, adsorption chromatography, ion exchange chromatography, gel A uniform crystalline enzyme preparation can be isolated by filtration chromatography or the like, and by a known method such as crystallization by adding a salt such as ammonium sulfate or polyethylene glycol. In addition,
A specific example of the method for producing the enzyme of the present invention will be described in Examples.

(3) Method for measuring titer In the present invention, titer was measured by the following method.

Oxidative deamination reaction: Glycine-KCl-KOH buffer (pH
0.5) 100 μmol, NAD ⁺ 2.5 μmol, L-phenylalanine 10
μmol and an appropriate amount of sample were mixed to make 1 ml and reacted, and the increase in NADH at 25 ° C was measured as an increase in absorbance at 340 nm, and the amount of enzyme that increased 1 μmol NADH per minute was 1 The unit was used.

Reductive amination reaction: Various buffers 100 μmol, NADH 0.1 μ
mol, NH ₄ Cl 200 μmol, sodium phenylpyruvate 1
React by mixing 0 μmol and an appropriate amount of sample to 1 ml to react, and measure the decrease of NADH at 25 ° C as the decrease of absorbance at 340 nm, and measure 1 unit of the enzyme amount to decrease 1 μmol of NADH per minute. And

The rate of the reductive amination reaction of phenylpyruvic acid is approximately the same as the above oxidative deamination rate at the optimum pH.
5.5 times faster. Therefore, when the reductive amination reaction rate was measured as described in JP-A-59-198972 and the titer was defined as described above, the same amount of enzyme showed about 5.5 times the number of units.

(4) Properties of enzyme The L-phenylalanine dehydrogenase of the present invention has the following properties.

A. Enzyme produced by Sporosarcina ureae SCRC-R04 (1) Action: Catalyze the reaction shown in the following formula.

L-Phenylalanine + NAD ⁺ + H ₂ O Phenylpyruvate + NADH + NH ₃ + H ⁺ (2) Substrate specificity: As shown in Table 3, this enzyme has very little effect on L-amino acids other than L-phenylalanine. No reaction or no reaction.

The above table shows the results measured for the oxidative deamination reaction. The substrate concentration was 10 mM except that L-tyrosine was 1.4 mM.

D-phenylalanine, L-alanine, L-histidine, L-arginine, L-lysine, L-ornithine, L
-Asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline, L-serine, L
-Threonine, L-cysteine and DL-phenylglycine do not serve as substrates.

NAD ⁺ is required as a coenzyme, and NADP ⁺ shows only about 3.9% activity against NAD ⁺ .

(3) Optimal pH: A pH of around 10.5 is optimal for the oxidative deamination reaction, and a pH of around 9.0 is optimal for the reductive amination reaction (Fig. 6).

(4) pH stability: When the residual activity after incubation at 30 ° C for 1 hour in buffer solution (0.05M) of each pH was measured for oxidative deamination, it is shown in Fig. 7 (activity before treatment). Is stable around pH 9 as shown in FIG.

(5) Optimum temperature: maximum activity around 40 ° C (Fig. 8).

(6) Temperature stability: 0.1 M glycine-NaOH buffer (pH 9.0)
When the residual activity after 10 minutes of treatment at each temperature was measured by oxidative deamination reaction, half of the activity was lost at 42 ° C. as shown in FIG. 9 (assume the activity before treatment is 100%). .

(7) Absorption spectrum: It has a maximum absorption at 278 nm and a shoulder near 283 nm. No visible absorption. This is shown in FIG.

(8) Effect of metal ions and inhibitors: Metal ions such as silver and mercury, and SH inhibitors such as PCBM, N-ethylmaleimide, and 5,5′-dithio-bis (2-nitrobenzoic acid) can be used for activity. It is inhibited (Table 4).

The concentration of metal ion and inhibitor is 1 m unless otherwise specified.
It is M.

(9) Isoelectric point: 5.3 to 5.4 when measured by focal electrophoresis using an ampholine.

(10) Molecular weight: High performance liquid chromatography (TSK 3000 S
It was calculated to be about 290,000 by W) and about 30 by sedimentation equilibrium method.
Calculated as 0,000.

(11) Molecular weight of subunit: Calculated to be about 38,000 to 39,000 by SDS-polyacrylamide gel disk electrophoresis.

(12) Uniformity: polyacrylamide gel electrophoresis (7.5%, pH
8.4) gives a single band as shown in Figure 11A. In addition, SDS-polyacrylamide gel electrophoresis (10.0%,
pH 7.2) gives a single band as shown in Figure 11B.

(13) Crystal form: It has a plate shape as shown in an enlarged scale in FIG.

As described above, the L-phenylalanine dehydrogenase of the present invention is clearly different from those of other microbial origin,
It is a new enzyme.

B. Enzyme produced by Bacillus sp. SCRC-R79a (1) Action: Catalyze the reaction shown in the following formula.

L-Phenylalanine + NAD ⁺ + H ₂ O Phenylpyruvate + NADH + MH ₃ + H ⁺ (2) Substrate specificity: As shown in Table 5, this enzyme has L-amino acids other than L-phenylalanine and L-tyrosine. Very little or no reaction.

The above table shows the results measured for the oxidative deamination reaction. The substrate concentration was 10 mM except that L-tyrosine was 1.4 mM.

D-phenylalanine, L-alanine, L-histidine, L-arginine, L-lysine, L-ornithine, L
-Asparagine, L-aspartic acid, L-glutamine, L-flutamic acid, L-proline, L-serine, L
-Threonine, L-cysteine, L-valine, L-leucine, L-isoleucine, and L-α-amino-n-
Butyric acid does not serve as a substrate.

In the reductive amination reaction, phenylpyruvic acid was added to L
The relative rate of converting p-hydroxyphenylpyruvic acid to L-tyrosine is 176% at pH 9.0, given that the rate of converting to -phenylalanine is 100%.

NADP in oxidative deamination of L-phenylalanine
⁺ Has only 2.9% coenzyme activity of NAD ⁺ .

(3) Optimum pH: pH around 10.6 to 11.3 is optimal for oxidative deamination reaction, and pH 9.8 to 10.8 for reductive amination reaction.
The vicinity is optimal (Fig. 13).

(4) pH stability: When the residual activity after incubation at 30 ° C. for 1 hour in buffer solution (0.05 M) of each pH was measured for oxidative deamination, it is shown in FIG. 14 (before treatment). 100% activity
Is stable in the range of pH 4 to 11.3,
It was stable in the pH range of 9-11.

(5) Optimum temperature: maximum activity around 50 ° C (Fig. 15).

(6) Temperature stability: 0.1 M glycine-NaOH buffer (pH 9.0;
Fig. 16A), and 0.1 M glycine-KCl-KOH buffer (p
H11.0; Fig. 16B), when the residual activity after treatment for 10 minutes at each temperature was measured for the oxidative deamination reaction, the activity was halved at 57 ° C at pH 9.0.
At 1.0, the activity halves at 48 ° C.

(7) Absorption spectrum: It has a maximum absorption at 278 nm and a shoulder near 283 nm. No visible absorption. This state is shown in FIG.

(8) Effects of metal ions and inhibitors: The activity is inhibited by metal ions such as silver and mercury and PCBM (Table 6).

The concentration of metal ion and inhibitor is 1 m unless otherwise specified.
It is M.

(9) Isoelectric point: 4.3 to 4.4 when measured by focal electrophoresis using an ampholine.

(10) Molecular weight: High performance liquid chromatography (TSK 3000 S
Calculated as about 290,000 by W) and about 34 by sedimentation equilibrium method.
Calculated as 0,000.

(11) Molecular weight of subunit: Calculated to be about 38,000 to 39,000 by SDS-polyacrylamide gel disk electrophoresis.

(12) Uniformity: polyacrylamide gel electrophoresis (7.5%, pH
8.4) gives a single band as shown in Figure 18A. In addition, SDS-polyacrylamide gel electrophoresis (10.0%,
pH 7.2) gives a single band as shown in Figure 18B.

(13) Crystal form: Needle-like as shown in an enlarged view in FIG.

(5) Method for producing L-phenylalanine In the method for producing L-phenylalanine of the present invention,
L-phenylalanine is produced by reacting phenylpyruvic acid, NADH and ammonium ions in the presence of L-phenylalanine dehydrogenase produced by Sporosarcina or Bacillus bacteria,
Collect the phenylalanine.

The usage form of L-phenylalanine dehydrogenase used in this method is not particularly limited. For example, it is needless to say that the enzyme purified by the present invention can be used, a cell-containing culture solution, live cultured cells, dried cells dehydrated by acetone or the like, crushed cells,
Enzyme-containing substances such as partially purified enzyme preparations purified to various stages can be used. Further, those obtained by immobilizing these enzymes or enzyme-containing substances according to a conventional method can also be used. In industrial practice, it is advantageous to use live cells, immobilized cells and the like. The amount of L-phenylalanine dehydrogenase in the reaction solution varies depending on the concentration of phenylpyruvic acid or its salt as a substrate and is not particularly limited, but usually 10 to 10,000 units / convenient is convenient.

As the substrate, phenylpyruvic acid or a salt thereof such as sodium salt, potassium salt, lithium salt, calcium salt and the like can be used. The addition amount of phenylpyruvic acid or its salt varies depending on the concentration of the enzyme in the reaction solution and the like, and is not particularly limited, but it is convenient to be 1 to 500 g /. When used at a low concentration, it can be used in the form of a free acid, but when used at a relatively high concentration, it is preferably used in the form of a salt from the viewpoint of pH adjustment. For example, sodium phenylpyruvate is not completely dissolved at a high concentration, but there is no problem even if undissolved sodium salt is present in the reaction solution. It is also possible to use ammonium phenylpyruvate or ammonium phenylpyruvate neutralized, in which case this ammonium salt functions as a source of phenylpyruvate and at the same time as a source of ammonium ions described later. Phenylpyruvic acid or a salt thereof may be added all at once at the start of the reaction in a batch reaction, or may be added in a plurality of divided portions as the reaction progresses, or continuously.

It is convenient to use the ammonium salt in the form of an ammonium salt, for example ammonium chloride or ammonium sulphate, as a source of ammonium ions. It is also possible to introduce ammonia gas or an ammonium hydroxide aqueous solution continuously with the progress of the reaction while maintaining the pH of the reaction solution at a predetermined value. When ammonium phenylpyruvate is used as described above, this substance also functions as a source of ammonium salt. The amount of ammonium salt used is the same as or more than the amount of phenylpyruvic acid. It is generally convenient to use this amount in an amount of 1 to 2 times the molar amount of phenylpyruvic acid. By increasing the molar amount of the ammonium salt, the equilibrium of the enzymatic reaction can be shifted to the L-phenylalanine side, and the yield of L-phenylalanine with respect to phenylpyruvic acid can be increased.

NADH may be added in an equimolar amount to phenylpyruvic acid, but since NADH is very expensive, from an industrial standpoint, in addition to the above reaction system, NADH regeneration system, that is, NAD ⁺ produced by the above reaction. It is preferable to share a system for reducing NADH. As such a system, a combination of an enzyme that converts NAD ⁺ into NADH and its substrate, such as formate dehydrogenase (EC1.
2.1.2) and formic acid, L-glutamate dehydrogenase (EC 1.4.1.
2) with glutamate and alcohol dehydrogenase (EC 1.1.1.1)
And ethanol, aldehyde dehydrogenase (EC 1.2.1.3) and acetaldehyde, glucose-6-phosphate dehydrogenase (E
C1.1.1.49) and glucose-6-phosphate and the like can be used. In addition, hydrogenase (EC1.18.3.1) reduces NAD ⁺ to NADH using molecular hydrogen as an electron donor, and electrochemically reduced methyl viologen and dihydrolipoamide diaphorase (EC 1.6.4. The reduction reaction of NAD ⁺ to NADH that accompanies oxidation by 3) can also be used. When using formic acid dehydratase and formic acid, NAD ⁺ is reduced to NADH and at the same time formic acid is oxidized to produce carbon dioxide, which is easily removed from the reaction system and the reaction always proceeds in the desired direction. Therefore, it is particularly preferable. Formate dehydrogenase is commercially available and can be easily obtained. or,
For example, Candida boidinii No. 220
1 (AKU4705) and Hansenula Bolimorpha (Hansenula p
olymorpha) (known from the ATCC26012) [Katoura, Agricultural And Biological Chemistry (Agricultural and Biological Chemistry) 38, 111
~ 116 (1974)]. N
Enzyme concentration of ADH reproducing system, L- phenylalanine vary depending on the dehydrogenase concentration, etc., generally at a rate comparable to the reductive amination rate of substrate phenylpyruvic acid (hence NAD ⁺ generating rate) of NAD ⁺ to NADH This is the amount necessary for reduction. For example, as described above, 10 to 10,000 units / L-
When phenylalanine dehydrogenase is used and formate dehydrogenase is used as the NADH regenerating enzyme, the amount of this enzyme used is preferably 10 to 10,000 units / about. It is convenient to use a formate salt such as sodium formate, potassium formate, or ammonium formate as a substrate for formate dehydrogenase. The amount of formate used is preferably 1 to 2 times the molar amount of phenylpyruvic acid or its salt. NA
When using a DH regeneration system, NAD ⁺ or NADH may be added at a normal physiological concentration of 0.1 to 10 mM.

Water or an aqueous liquid such as an aqueous buffer can be used as the reaction medium. Examples of buffer solutions include Tris-HC
1 buffer, glycine-NaOH buffer, etc. can be used.

As the pH of the reaction solution, a pH suitable for reductive amination with L-phenylalanine dehydrogenase can be used when the NADH regeneration system is not used. For example, when an enzyme derived from Sporosarcina is used. Is pH 8-10,
The pH is preferably about 9, and when an enzyme derived from Bacillus bacterium is used, the pH is about 9 to 11, preferably about 10.
When the NADH regeneration system is used together with the reductive amination system of phenylpyruvic acid, it is necessary to select a pH range in which both of these reactions favorably proceed. Such a pH is
For example, when using L-phenylalanine dehydrogenase derived from Sporosarcina spp. And formate dehydrogenase derived from Candida voidini, pH is usually 7.5 to 9.5, preferably
pH is 8.0 to 9.0. When L-phenylalanine dehydrogenase derived from Bacillus bacterium and formate dehydrogenase derived from Candida voidini are used, the pH is usually 8 to 10, preferably pH 8.5 to 9.5.

The reaction temperature can be considered in the same manner as in the case of the reaction pH, but is usually 20 ° C to 50 ° C in any combination of enzymes.
C, preferably 25 to 40 ° C.

The reaction time is not particularly critical, the substrate concentration of the reaction mixture,
The reaction is maintained until the substrate phenylpyruvic acid is converted to L-phenylalanine in sufficient yield depending on the enzyme titer and the like.

The reaction system may be a batch system or a continuous system, and the reaction time varies depending on which system is used.

The produced L-phenylalanine can be purified and collected according to any conventional method. For example, after completion of the reaction, trichloroacetic acid is added to precipitate the protein, the protein is filtered off together with the bacterial cells (if present), and the filtrate is purified by ion exchange resin or the like and crystallized.

For example, leuconostoc
Leuconostoc mesentroides ATCC 804
Bioassay using 2 can be performed.

Next, the present invention will be described more specifically by way of examples.

Example 1. (Reference Example) Sporosarcina-Ureae SCRC-R04 L- phenylalanine dehydrogenase purified L- phenylalanine 0.2% of the enzyme from 0.5% yeast extract, 1.0% _{peptone, K 2 HPO 4 0.2%,} 0.1% NaCl and MgSO ₄ · 7H ₂ O 0.
A medium 30 containing 02% and adjusted to pH 7.0 was stored at 120 ° C for 1
After heat sterilization for 5 minutes, Sporosarcina ureae SCRC
-R04 (Micromachine Lab. No. 8178 (Micromachine Lab. No. 1012) was inoculated and cultivated aerobically at 30 ° C for 24 hours. After culturing, the cells were collected by a centrifuge and wet. A microbial cell having a weight of about 380 g was obtained, which was washed once with 0.85% saline and then suspended in a phosphate buffer (pH 7.0) 1 containing 0.1 mM EDTA and 5 mM 2-mercaptoethanol, The cells were sonicated at 9 KHz for about 10 hours to crush the cells, which were 14,000x.
It was removed by centrifugation for 20 minutes at g to obtain a crude extract containing L-phenylalanine dehydrogenase. A 5% aqueous solution of protamine sulfate was added to this cell-free extract at 0.1 g per 1 g of protein, and the mixture was stirred for 30 minutes. Generated precipitate 14,0
Centrifuge for 20 minutes at 00xg, and get the crude enzyme solution 0.1m.
It was dialyzed against 0.01 M phosphate buffer (pH 7.0) containing M EDTA and 5 mM 2-mercaptoethanol. Solid ammonium sulfate (412 g) in the enzyme solution (1990 ml) after dialysis
Was added to 30% ammonium sulfate saturation. After stirring for 30 minutes, centrifuge at 14,000 xg for 20 minutes to obtain the supernatant (210
Solid ammonium sulfate (416 g) was further added to 0 ml) and 60%
It was saturated with ammonium sulfate. The precipitate having the enzyme activity obtained by centrifugation at 14,000 xg for 20 minutes was dissolved in a small amount of 0.01 M phosphate buffer (pH 7.0), and 0.01 M phosphorus containing 0.1 mM EDTA and 5 mM 2-mercaptoethanol was added. It dialyzed with the acid buffer solution (pH 7.0). This enzyme solution was equilibrated with 0.01 M phosphate buffer (pH 7.0) containing 0.1 mM EDTA and 5 mM 2-mercaptoethanol in advance.
EAE-Toyopearl 650M column, 0.1 mM E
0.1 with DTA and 5 mM 2-mercaptoethanol
Elution was performed with M phosphate buffer (pH 7.0).

The active fractions were collected, dialyzed against 0.01 M phosphate buffer containing 0.1 mM EDTA and 5 mM 2-mercaptoethanol, and then passed through a hydroxyapatite column equilibrated with the same buffer in advance to obtain 0.1 mM EDTA and 5m
0.01M to 0.15M containing M 2-mercaptoethanol
The enzyme was eluted with a linear concentration gradient of phosphate buffer solution (pH 7.0). This active fraction was collected and 0.1mM EDTA, 5m
Contains M 2-mercaptoethanol and 0.1M NaCl
Gel filtration chromatography was performed on Sephadex G-200 equilibrated with 0.05 M phosphate buffer (pH 7.0). The enzyme solution thus obtained was concentrated by ultrafiltration, and ammonium sulfate was added for crystallization. Thus, plate-like crystal L-phenylalanine dehydrogenase was obtained with a yield of 31%. An enlarged view of this crystal is shown in FIG. In addition, Table 7 shows the specific activity and the recovery rate in the purification process from the cell extract to the crystallization.

Example 2. Bacillus sp.SCRC-R79a of L- phenylalanine dehydrogenase from the purified (1) L- phenylalanine 0.2%, 0.5% yeast extract, 1.0% _{peptone, K 2 HPO 4 0.2%,} 0.1% NaCl and MgSO ₄ · 7H ₂ O 0.
The medium 10 containing 02% and adjusted to pH 7.0 was heated at 120 ° C for 1
After heat sterilization for 5 minutes, inoculate Bacillus sp. SCRC-R79a (Microtechnological Research Institute No. 8179 (Microtechnical Research Article No. 1013) at 30 ° C.
The cells were cultured aerobically for 24 hours. After culturing, cells were collected from the 10 cultures by a centrifuge to obtain cells having a wet weight of about 108 g. After washing the cells once with 0.85% saline, 0.1 mME
The cells were suspended in about 0.4 phosphate buffer (pH 7.0) containing DTA and 5 mM 2-mercaptoethanol, and sonicated at 9 KHz for about 6 hours to disrupt the cells. The crushed cells were removed by centrifugation at 14,000 xg for 20 minutes to obtain a crude extract containing L-phenylalanine dehydrogenase. To this cell-free extract, a 5% aqueous solution of protamine sulfate was added at 0.1 g per 1 g of protein.
It was added to 1 g and stirred for 30 minutes. The resulting precipitate was centrifuged at 14,000 xg for 20 minutes, and the resulting crude enzyme solution was dialyzed against 0.01 M phosphate buffer (pH 7.0) containing 0.1 mM EDTA and 5 mM 2-mercaptoethanol. Solid ammonium sulfate (70.4 g) was added to the enzyme solution (400 ml) after dialysis to make 30% ammonium sulfate saturation. After stirring for 30 minutes, the supernatant (430 ml) obtained by centrifugation at 14,000 xg for 20 minutes was added with solid ammonium sulfate (85.6
g) was added to make 60% ammonium sulfate saturation. 14,000xg
The precipitate having enzyme activity obtained by centrifugation at 20 ° C. for 20 minutes was dissolved in a small amount of 0.01 M phosphate buffer (pH 7.0), and
It was dialyzed against 0.01 M phosphate buffer (pH 7.0) containing 0.1 mM EDTA and 5 mM 2-mercaptoethanol. This enzyme solution was previously added with 0.1 mM EDTA and 5 mM 2
-0.01M phosphate buffer containing mercaptoethanol (pH
It was passed through a DEAE-Toyopearl 650M column equilibrated with 7.0) and eluted with 0.1M phosphate buffer (pH 7.0) containing 0.1 mM EDTA and 5 mM 2-mercaptoethanol.

The active fractions were collected, dialyzed against 0.01 M phosphate buffer containing 0.1 mM EDTA and 5 mM 2-mercaptoethanol, and then passed through a hydroxyapatite column equilibrated with the same buffer in advance to obtain 0.1 mM EDTA and 5m
0.01M to 0.4M containing M 2-mercaptoethanol
The enzyme was eluted with a linear concentration gradient of phosphate buffer solution (pH 7.0). The active fractions were collected and added to 0.1 mM EDTA, 5
Gel filtration chromatography was performed on Sephadex G-200 equilibrated with 0.05 M phosphate buffer (pH 7.0) containing mM 2-mercaptoethanol and 0.1 M NaCl. Thus, L-phenylalanine dehydrogenase was purified about 1800 times with a yield of about 60%. Table 8 shows the specific activity and recovery rate in this purification process. This enzyme proved to be homogeneous on polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis.

Example 3. Bacillus sp.SCRC-79a from L- phenylalanine purification of dehydrogenases (2) L- phenylalanine 0.2%, 0.5% yeast extract, 1.0% _{peptone, K 2 HPO 4 0.2%,} 0.1% NaCl and MgSO ₄ · 7H ₂ O 0.0
Medium 100 containing 2% and adjusted to pH 8.0, 120 ℃,
After heat sterilization for 15 minutes, inoculate Bacillus sp. SCRC-R79a (Fermentation Research Institute for Microbiology No. 8179 (FERMP-8179) (No. 1013) to aerobically at 30 ° C for 22 hours. After culturing, after culturing
Cells were collected from 100 cultures by a centrifuge to obtain cells having a wet weight of about 0.7 kg. Bacteria are 0.1 mM EDTA and 5 mM
0.1M phosphate buffer containing 2-mercaptoethanol
(pH 7.9) Suspended in about 4 and sonicated at 9 KHz for about 41 hours to crush the cells. 14,000x crushed cells
It was removed by centrifugation for 20 minutes at g to obtain a crude extract containing L-phenylalanine dehydrogenase. This cell-free extract was heat-treated at 50 ° C. for 10 minutes and immediately cooled with ice. Solid ammonium sulfate (678 g) in the treated enzyme solution (3,850 ml)
Was added to 30% ammonium sulfate saturation. After stirring for 30 minutes, the formed precipitate was removed by centrifugation at 14,000 xg for 20 minutes. Solid ammonium sulfate (673 g) was added to this supernatant (3,400 ml) to make 60% ammonium sulfate saturation. 1
The precipitate having enzyme activity obtained by centrifugation at 4,000 xg for 20 minutes was dissolved in a small amount of 0.01 M phosphate buffer (pH 7.9), and 0.01 M phosphate buffer containing 0.1 mM EDTA and 5 mM 2-mercaptoethanol was added. It dialyzed with (pH7.9). This enzyme solution was previously prepared with 0.1 mM EDTA and 5 m
DEAE-Toyopearl 650M column equilibrated with 0.01 mM phosphate buffer (pH 7.9) containing M2-mercaptoethanol is passed through a column of 0.1 mM EDTA, 5 mM 2-mercaptoethanol and 0.1 M NaCl, and 0.1 M phosphorus is added. Elution was performed with an acid buffer (pH 7.9).

The active fractions were collected, dialyzed against 0.01 M phosphate buffer containing 0.1 mM EDTA and 5 mM 2-mercaptoethanol, and then passed through a second DEAE-Toyopearl 650 M column that had been equilibrated with the same buffer in advance. Elute with buffer. This active fraction was collected and 0.1 mM EDTA 5 mM2-
After dialyzing with 0.01 M phosphate buffer containing mercaptoethanol, it is passed through a hydroxyapatite column previously equilibrated with the same buffer, and 0.1 mM EDTA and 5 mM 2
The enzyme was eluted with a linear gradient of 0.01M to 0.4M phosphate buffer (pH 7.9) containing mercaptoethanol. Sephadex G-2 was collected by collecting active fractions and concentrating and equilibrating with 0.05 M phosphate buffer (pH 7.9) containing 0.1 mM EDTA 5 mM 2-mercaptoethanol and 0.1 mM NaCl.
Gel filtration chromatography on 00 was performed. Thus, L-phenylalanine dehydrogenase was purified about 1400 times with a yield of about 54%. Table 9 shows specific activity and recovery rate in this purification process. The enzyme solution thus obtained was concentrated, and ammonium sulfate was added for crystallization. Needle-like crystals as shown in FIG. 19 were obtained.

Example 4. (Reference Example) Production of L-phenylalanine using a crude enzyme derived from Sporosarcina ureae SCRC-R04 Sodium phenylpyruvate 5 g (22 mmol), ammonium formate 3 g (49 mmol), NAD ⁺ 0.21 g (0.29 mmol), Tris-HCl buffer Liquid (pH 8.5) 18 mmol, crude L-phenylalanine dehydrogenase 43.2 units (corresponding to the crude enzyme fraction partially purified to the ammonium sulfate fraction of Example 1, Step 3 in Table 7) and crude formate dehydrogenase 49.0 300 ml containing the unit ((pH8.5), partially purified from Candida Boydini No.2201)
The reaction solution of was reacted at 30 ° C. for 24 hours. When the amount of L-phenylalanine produced in the reaction solution was quantified by a microbial quantification method using Leuconostoc mesenteroides, 1.91 g (11.6 mmol, 52.7% conversion) of L-phenylalanine was produced. To this reaction solution, 30 ml of 20% trichloroacetic acid was added to remove the protein, which was then adsorbed on a cation exchange resin Amberlite IR-120 (H ⁺ ) column and eluted with 1M aqueous ammonia. Fractions containing L- phenylalanine, concentrated after the anion exchange resin Amberlite (Amberlite) IRA-400 (OH -) adsorbed on a column,
Elution with 1M formic acid. The fraction containing L-phenylalanine was concentrated to dryness. It was dissolved in a small amount of warm water, ethanol was added to 50% and refrigerated to precipitate crystals.
This crystal was recrystallized by the same operation to obtain 0.458 g of a colorless solid. The elemental analysis value of this standard product was as follows.

Melting point: Decomposed at 270 ° C.

It was an optically pure L-form with a specific rotation ▲ [α] ²⁰ _D ▼ -35.5 ° (c = 0.48, H ₂ O). The analysis results by mass spectrum, nuclear magnetic resonance absorption spectrum, and infrared absorption spectrum all showed that the product was L-phenylalanine.

Example 5. (Reference example) High-concentration synthesis of L-phenylalanine using a partially purified enzyme derived from Sporosarcina ureae SCRC-R04 Sodium phenylpyruvate, (3.57 mmol), NAD ⁺ 100
μmmol, NH ₄ Cl 5 mmol, Tris-HCl buffer (pH 8.5) 272
μmol, sodium formate (7.84 mmol), 35 units of L-phenylalanine dehydrogenase (Example 1, Step 4 of Table 7)
30 reaction solutions containing DEAE-toyopearl column fraction) and formate dehydrogenase 10.2 units ((pH8.5), partially purified from Candida voidini No. 2201) in 5 ml
It was kept at 24 ° C for 24 hours. When quantified by the microorganism quantification method, 580 mg (3.51 mmol, conversion rate of 98.5%) of L-phenylalanine was formed.

Example 6. (Reference example) Synthesis of L-phenylalanine using crude enzyme from Sporosarcina ureae SCRC-R04 200 μmol sodium phenylpyruvate, NAD ⁺ 20 μmo
l, sodium formate 200 μmol, Tris-HCl buffer (pH 8.
5) 600 μmol, formate dehydrogenase 11.9 units ((pH8.5), Candida voidini No. 2201 cell-free extract) and L
-Phenylalanine dehydrogenase 13.2 units (crude enzyme fraction obtained by precipitating a cell-free extract of cultured cells of the SCRC-R04 strain as 30 to 60% saturation with ammonium sulfate; corresponding to step 3 in Table 1 of Example 1) 13.0 ml of the reaction liquid containing it was reacted at 30 ° C. for 15 hours. When the amount of L-phenylalanine produced in the reaction solution was measured by a microorganism quantification method, it was 32.8 mg (198.8 μmo
L-phenylalanine with a conversion rate of 1,98.4%) was produced.

Example 7. Synthesis of L-phenylalanine using partially purified enzyme from Bacillus sp. SCRC-R79a Sodium phenylpyruvate 400 µmol, ammonium formate 800 µmol, NAD ⁺ 5 µmol, Tris-HCl buffer (pH 8.
5) 260 μmol, 0.5 units of crude formate dehydrogenase ((pH8.5),
And L-phenylalanine dehydrogenase 0.25 unit (SCRC
-Protamine treatment from cell-free extract of R79a, ammonium sulfate fractionation, D
A 5.0 ml reaction solution containing an enzyme preparation purified about 180 times by column chromatography of EAE-Toyopearl and hydroxyapatite was reacted at 30 ° C for 24 hours. 64.4 mg (390.0
μmol, conversion rate of 97.5%) L-phenylalanine was produced.

From this reaction solution, L-phenylalanine was obtained according to the method of Example 3.

Example 8. Synthesis of L-phenylalanine using partially purified enzyme derived from Bacillus sp. SCRC-R79a Sodium phenylpyruvate 0.91 g (4 mmol), ammonium formate 0.75 g (11.9 mmol), NAD ⁺ 36 mg (50 μmol), Tris-HCl buffer ( pH8.5) 20 units of L-phenylalanine dehydrogenase (fraction passed through DEAE-Toyopearl column) and 15 units of crude formate dehydrogenase (pH 8. 5, partially purified from Candida voidini No. 2201) and reacted at 30 ° C. for 24 hours. After the reaction, the tube containing the enzyme was taken out, immersed in a new reaction solution, and reacted in the same manner. This was repeated and 28 times of reaction was performed. When the amount of L-phenylalanine produced in each reaction solution was measured by a microbial quantification method using Leuconostoc mesenteroides, a total of 10.74 g (65 mmol, 58% conversion) of L-phenylalanine was produced. A part of this reaction liquid (containing 450 ml and 3.58 g of L-phenylalanine) was used as a cation exchange resin Amberlite I.
R-120 (H ⁺ column was adsorbed and eluted with IM ammonia water. Fractions containing L-phenylalanine were collected and concentrated, and after concentration, anion exchange resin Amberlite IRA-400 (O
H ^-) adsorbed on a column and eluted with IM formic acid. The fraction containing L-phenylalanine was concentrated to dryness. When dissolved in a small amount of warm water and refrigerated, crystals were precipitated. This crystal was recrystallized by the same operation to obtain 1.235 g of a colorless solid. The elemental analysis values of this preparation were as follows.

Melting point: Decomposed at 252-254 ° C.

It has a specific optical rotation [α] ²⁰ _D = -34.1 ° (c = 1.72, H ₂ O) and is an L-form, and its optical purity is 98% ee. The analysis results by mass spectrum, nuclear magnetic resonance absorption spectrum, and infrared absorption spectrum all showed that the product was L-phenylalanine.

Example 9. High-concentration synthesis of L-phenylalanine using partially purified enzyme from Bacillus sp. SCRC-R79a Sodium phenylpyruvate 1.8 g (8 mmol), ammonium formate 0.98 g (15.54 mmol), NAD ⁺ 72 mg (100 μmol), Tris-HCl buffer Liquid (pH 8.5) 5 mmol, L-phenylalanine dehydrogenase 40 units (DEAE-fraction passed through Toyopearl column) and formate dehydrogenase 20 units pH 8.5, partially purified from Candida voidini No. 2201) The reaction solution containing 10 ml of the above was kept at 30 ° C. for 24 hours. When quantified by the microorganism quantification method, 1.12 g (6.78 mmol, conversion rate of 86%) of L-phenylalanine was formed.

Example 10. Synthesis of L-phenylalanine using crude enzyme from Bacillus sp. SCRC-R79a Sodium phenylpyruvate 400 μmol, ammonium chloride 1.2 mmol, NADH 480 μmol, Tris-HCl buffer (pH 8.
5) 250 μmol and crude L-phenylalanine dehydrogenase 2.
0 unit (50% in cell-free extract of cultured cells of SCRC-R79a strain)
After heat treatment at ℃ for 10 minutes, ammonium sulfate
5 ml of a reaction solution containing a crude enzyme fraction precipitated as 30 to 60% saturation (corresponding to step 2 of Example 3) was reacted at 30 ° C. for 24 hours. When the amount of L-phenylalanine produced in the reaction solution was measured by a microorganism quantification method, it was 63.5 mg (384 μmo
L, 96.0% conversion rate) of L-phenylalanine was produced.

Example 11. Synthesis of L-phenylalanine using crude enzyme from Bacillus sp. SCRC-R79a 400 μmol sodium phenylpyruvate, 1.2 mmol ammonium formate, NAD ⁺ 2.5 μmol, Tris-HCl buffer (pH 8.
5) 250 μmol, formate dehydrogenase 1.5 units (pH 8.5, partially purified from Candida voidini No. 2201) and crude L-phenylalanine dehydrogenase 2.0 units (in a cell-free extract of cultured bacterial cells of SCRC-R79a strain) After heat treatment at 50 ° C. for 10 minutes, 5 ml of a reaction solution containing a crude enzyme fraction precipitated as 30 to 60% saturation of ammonium sulfate (corresponding to step 2 of Example 3) was reacted at 30 ° C. for 24 hours. . When the amount of L-phenylalanine produced in the reaction solution was measured by a microorganism quantification method, 62.8 mg (380 μmol, conversion rate of 95.0%) of L-phenylalanine was produced.

Example 12. Synthesis of L-phenylalanine using crude enzyme from Bacillus sp. SCRC-R53b Sodium phenylpyruvate 400 μmol, ammonium formate 1.2 mmol, NAD ⁺ 2.5 μl Tris-HCl buffer (pH 8.5)
250 μmol, formate dehydrogenase 1.5 units (pH 8.5, Candida
Partially purified from Boydini No.2201) and 2.0 units of crude L-phenylalanine dehydrogenase (cell-free extract of cultured bacterial cells of SCRC-R53b strain was subjected to heat treatment at 50 ° C for 10 minutes, and then ammonium sulfate 30 to 60%. 5 ml of the reaction solution containing the crude enzyme fraction precipitated as saturated) was reacted at 30 ° C. for 20 hours. When the amount of L-phenylalanine produced in the reaction solution was measured by a microorganism quantification method, it was 57.3 mg (347 μmol, 8
L-phenylalanine with a conversion of 6.8%) was produced.

Example 13. Synthesis of L-phenylalanine using crude enzyme from Bacillus sp. SCRC-101A Sodium phenylpyruvate 400 μmol, ammonium formate 1.2 mmol, NAD ⁺ 2.5 μmol, Tris-HCl buffer (pH 8.
5) 250 μmol, formate dehydrogenase 1.5 units (pH 8.5, partially purified from Candida vodini No.2201) and crude L-phenylalanine dehydrogenase 2.0 units (in a cell-free extract of cultured bacterial cells of SCRC-101A strain) After heat treatment at 50 ° C. for 10 minutes, 5 ml of a reaction solution containing a crude enzyme fraction (precipitated with ammonium sulfate of 30 to 60% saturation) was reacted at 30 ° C. for 20 hours. When the amount of L-phenylalanine produced in the reaction solution was measured by a microorganism quantification method, it was 58.3 mg (353 μmol,
(88.3% conversion rate) of L-phenylalanine was produced.

Example 14. Synthesis of L-phenylalanine using crude enzyme from Bacillus sp. SCRC-114D Sodium phenylpyruvate 400 μmol, ammonium formate 1.2 mmol, NAD ⁺ 2.5 μmol, Tris-HCl buffer (pH 8.
5) 250 μmol, formate dehydrogenase 1.5 units (pH 8.5, partially purified from Candida vodini No. 2201) and crude L-phenylalanine dehydrogenase 2.0 units (cell-free extract of cultured bacterial cells of SCRC-114D strain) 5 ml of a reaction solution containing a crude enzyme fraction (precipitated with ammonium sulfate of 30 to 60% saturation) was reacted at 30 ° C. for 20 hours. When the amount of L-phenylalanine produced in the reaction solution was measured by the microorganism quantification method, it was 52.5.
mg (318 μmol, 79.5% conversion) of L-phenylalanine had formed.

Reference Example The Bacillus-derived L-phenylalanine dehydrogenase of the present invention strongly acts on L-tyrosine as well as on L-phenylalanine, and 1 mol of L-tyrosine, 1 mol.
It catalyzes a reaction that produces 1 mol of p-hydroxyphenylpyruvic acid, 1 mol of NADH and 1 mol of ammonium ion from 1 mol of NAD ⁺ and 1 mol of water, and vice versa.

Therefore, L-tyrosine can be produced using a method similar to that described for the method for producing L-phenylalanine, but using p-hydroxyphenylpyruvic acid as the substrate instead of phenylpyruvic acid. Next, a production example of L-tyrosine will be described for reference.

L-using partially purified enzyme from Bacillus sp. SCRC-R79a
Synthesis of tyrosine 1.1 g (6 mmol) of p-hydroxyphenylpyruvic acid, 4.5 g (71.3 mmol) of ammonium formate, NAD ⁺ 216 mg (300 μmol),
Tris-HCl buffer (pH 8.5) 15 mmol, L-phenylalanine dehydrogenase 24 units (DEAE-fraction passed through Toyopearl column) and crude formate dehydrogenase 30 units (pH
When 300 ml of a reaction solution containing 8.5, partially purified from Candida boindini No. 2201) was reacted at 30 ° C. for 45 hours, crystals were precipitated. The crystals were collected by filtration and recrystallized to obtain 0.648 g of colorless solid. The elemental analysis values of this preparation were as follows.

It has a specific optical rotation of [α] ²⁵ _D ▼ = −7.33 ° (c = 4, 6N HCl) and is in the L-form with an optical purity of 100% ee. Analysis results by mass spectrum, nuclear magnetic resonance absorption spectrum, and infrared absorption spectrum all showed that the product was L-tyrosine.

[Brief description of drawings]

Fig. 1 is an electron micrograph of Sporosarcina ureae SCRC-R04, which is a photograph instead of a drawing showing the morphology of organisms; Fig. 2 is an electron micrograph of Bacillus sp. SCRC-R53b 3 is an electron micrograph of Bacillus sp. SCRC-R79a and is a photograph replacing a drawing showing the morphology of living organisms; and FIG. Fig. 5 is an electron micrograph of SCRC-101A, which is a photograph instead of a drawing showing the morphology of living organisms; Fig. 5 is an electron micrograph of Bacillus sp. SCRC-114D, which is a drawing showing the morphology of living things. Fig. 6 is a graph showing the relationship between pH and reaction rate of L-phenylalanine dehydrogenase produced by Sporosarcina ureae SCRC-R04, where A is for oxidative deamination reaction and B is for Shows the results for the reductive amination reaction; Fig. 7 is a graph showing the pH stability of L-phenylalanine dehydrogenase produced by Sporosarcina ureae SCRC-R04; Fig. 8 is the temperature of L-phenylalanine dehydrogenase produced by Sporosarcina ureae SCRC-R04. FIG. 9 is a graph showing the relationship with the reaction rate, showing the results of the oxidative deamination reaction; FIG. 9 is a graph showing the temperature stability of L-phenylalanine dehydrogenase produced by Sporosarcina ureae SCRC-R04. Fig. 10 is an ultraviolet absorption spectrum of L-phenylalanine dehydrogenase produced by Sporosarcina ureae SCRC-R04; Fig. 11 is a spectrum of L-phenylalanine dehydrogenase produced by Sporosarcina ureae SCRC-R04. In the electropherogram showing homogeneity, A is polyacrylamide gel electrophoresis (7.
5% gel, pH 8.4) and B shows SDS-polyacrylamide gel electrophoresis (10.0% gel, pH 7.2); Figure 12 shows L-phenylalanine dehydrogenation produced by Sporosarcina ureae SCRC-R04. Fig. 13 is a microscopic enlarged sketch of the enzyme; Fig. 13 is a graph showing the relationship between pH and reaction rate of L-phenylalanine dehydrogenase produced by Bacillus sp. SCRC-R79a, where A represents oxidative deamination reaction. , B
Shows the results of the reductive amination reaction; Fig. 14 is a graph showing the pH stability of L-phenylalanine dehydrogenase produced by Bacillus sp. SCRC-R79a; Fig. 15 is Bacillus sp. SCRC-. FIG. 16 is a graph showing the relationship between temperature and reaction rate of L-phenylalanine dehydrogenase produced by R79a, showing the results of oxidative deamination reaction; FIG. 16 shows that produced by Bacillus sp. SCRC-R79a. 1 is a graph showing the temperature stability of L-phenylalanine dehydrogenase, A showing the results in 0.1 M glycine-NaOH buffer (pH 9.0), and B showing 0.1 M glycine-KCl-KOH buffer ( Fig. 17 shows the results in pH 11.0); Fig. 17 is an ultraviolet absorption spectrum of L-phenylalanine dehydrogenase produced by Bacillus sp. SCRC-R79a; Fig. 18 is produced by Bacillus sp. SCRC-R79a. L-phenylalanine dehydration It is an electropherogram showing the homogeneity of elementary enzymes, where A is polyacrylamide electrophoresis (7.5% gel, pH
8.4), and B shows the result of SDS-polyacrylamide gel electrophoresis (10.0% gel, pH7.2); and FIG. 19 is a microscope of L-phenylalanine dehydrogenase produced by Bacillus sp. SCRC-R79a. It is an enlarged sketch.

Claims (4)

[Claims] 1. The following properties: (1) Action: 1 mol of L-amino acid, 1 mol of NAD ⁺ and 1 mol of water to 1 mol of phenylpyruvic acid, 1 mol of
A reaction that produces NADH and 1 mole of ammonium ion,
And catalyze this reverse reaction; (2) Molecular weight: a high-performance liquid chromatography gel filtration method with a molecular weight of 290,000 and a precipitation equilibrium method with 3
It shows a molecular weight of 40,000 and a subunit having a molecular weight of 38,000 to 39,000 in SDS-polyacrylamide gel disc electrophoresis; (3) Substrate specificity: other than L-phenylalanine and L-tyrosine as shown in the table below. Reacts very weakly or not at all to the L-amino acids of: (The above table shows the results of measurement of oxidative deamination reaction); (4) Optimum pH: pH of oxidative deamination reaction is 10.6-11.3.
The optimum range is around pH 9.8-10 in the reductive amination reaction.
Around 8 is optimal; (5) pH stability: pH 4 to 4 when the residual activity after incubation at 30 ° C for 1 hour in buffer solution (0.05M) of each pH is measured for oxidative deamination. L-phenylalanine dehydrogenase, which is stable in the range of 11.3; and (6) Optimum temperature: maximum activity at around 50 ° C. 2. The enzyme according to claim 1, which is produced by a bacterium belonging to the genus Bacillus. 3. The following properties: (1) Action: 1 mol of L-amino acid, 1 mol of NAD ⁺ and 1 mol of water to 1 mol of phenylpyruvic acid, 1 mol of
A reaction that produces NADH and 1 mole of ammonium ion,
And catalyze this reverse reaction; (2) Molecular weight: a high-performance liquid chromatography gel filtration method with a molecular weight of 290,000 and a precipitation equilibrium method with 3
It shows a molecular weight of 40,000 and a subunit having a molecular weight of 38,000 to 39,000 in SDS-polyacrylamide gel disc electrophoresis; (3) Substrate specificity: other than L-phenylalanine and L-tyrosine as shown in the table below. Reacts very weakly or not at all to the L-amino acids of: (The above table shows the results of measurement of oxidative deamination reaction); (4) Optimum pH: pH of oxidative deamination reaction is 10.6-11.3.
The optimum range is around pH 9.8-10 in the reductive amination reaction.
Around 8 is optimal; (5) pH stability: pH 4 to 4 when the residual activity after incubation at 30 ° C for 1 hour in buffer solution (0.05M) of each pH is measured for oxidative deamination. In the method for producing L-phenylalanine dehydrogenase, which is stable in the range of 11.3;
a method of culturing a bacterium of the genus lus), and collecting the enzyme from the culture. 4. The bacterium is Bacillus sp. SCR.
C-R53b, SCRC-R79a (Microtechnical Lab. No. 8179; Microtechnical Lab. No. 8
No. 1013), SCRC-101A, or SCRC-114D strain
No. 1011), the method according to claim 3.