JPS63157986A - Production of l-phenylalanine by gene recombination - Google Patents

Abstract

PURPOSE:To obtain the titled compound at a low cost, by introducing an L- phenylalanine dehydrogenase gene into a proper vector to obtain a manifestation plasmid, transforming a microbial strain with said plasmid and using an L- phenylalanine dehydrogenase separated from the transformed strain as a reactant. CONSTITUTION:A DNA is extracted from a microbial strain belonging to Bacillus genus or Sporosarcina genus and capable of producing L-phenylalanine dehydrogenase [e.g. Bacillus sphaericus SCRC-R79a (FERM) BP-1013)] and the DNA is nicked with a restriction enzyme to obtain an L-phenylalanine dehydrogenase gene. Said gene is introduced into a proper vector (e.g. pUC9, pBR322, etc.) to obtain a manifestation plasmid. A host microorganism transformed with said plasmid is cultured to produce an L-phenylalanine dehydrogenase. The objective compound can be produced by reacting phenylpyruvic acid with ammonium ion in the presence of said enzyme and a reducing agent (e.g. reduced nicotinamide-adenine dinucleotide).

Description

Detailed Description of the Invention [Industrial Application Field] This invention relates to a plasmid containing an L-phenylalanine dehydrogenase gene, a microorganism transformed with the plasmid, and the production of L-phenylalanine dehydrogenase using the microorganism. The present invention relates to a method for producing L-phenylalanine using this enzyme.

[Conventional technology]

Attempts have been made to produce L-amino acids using α-ketocarboxylic acids as substrates through the action of microorganisms and enzymes. For example, a method of accumulating L-glutamic acid by adding α-ketogel tar acid and various amino acids to microbial cells [Katagiri et al., Amino Acid Nucleic Acids, (18(1960)
)) A method for obtaining L-phenylalanine by adding L-glutamic acid or L-aspartic acid to a reaction solution containing phenylpyruvic acid [Asai et al., Amino Acid Nucleic Acids, ↓, 114
(1960) ), L-glutamic acid and L-aspartic acid were added to the reaction solution containing indolepyruvic acid to produce L-
-) There are methods to synthesize liptophan [Aida et al.
Journal of General and Applied
Microbiology (Journal of Gen
eraland Applied Microbiol
ogy), ↓, 200 (195B)).

JP-A No. 60-164493 discloses that various microorganisms are cultured with phenylpyruvic acid and an amino group donor, or cells of the microorganism or a treated product thereof are allowed to act on phenylpyruvic acid and an amino group donor. L
- A method for producing phenylalanine is described.

JP-A-60-43391 discloses culturing a microorganism capable of converting α-keto acids into their corresponding L-amino acids, and in the process supplies α-keto acids to the culture to convert them into L-amino acids.
A method for producing L-amino acids by conversion to amino acids is described.

JP-A-59-198972 describes L-phenylalanine dehydrogenase and a method for producing L-α-aminocarboxylic acid using this enzyme.

JP-A-60-160890 discloses that various microorganisms are cultured with phenylpyruvic acid in the presence of an energy source, an inorganic ammonium compound or urea, and oxygen, or that a culture of microorganisms or a processed product thereof is cultured with energy. L-
A method for producing phenylalanine is described.

In JP-A-60-24192, Corynebacterium Co
It has been described that a microorganism of the genus Corynebacterium can be transformed with a plasmid incorporating a gene for L-phenylalanine synthase derived from an organism of the genus Corynebacterium and the transformant can be used to produce L-phenylalanine. There is.

JP-A-60-62992 describes the gene for an enzyme related to the synthesis of L-phenylalanine in E. coli (E. col.
It is described that L-phenylalanine was produced by cloning the gene according to i) and expressing the gene under the control of the trp promoter.

Also, JP-A-60-66984 and JP-A-60-210
993 contains the glutamate-producing bacterium Coryneform (Corineform).
Incorporating into a plasmid the gene for an enzyme related to L-phenylalanine synthesis derived from
This allows Brevibacterium (Brevibacterium
It is described that L-phenylalanine was synthesized using a production bacterium obtained by transforming a microorganism belonging to the genus Rium.

However, until now, L-phenylalanine dehydrogenase derived from microorganisms of the genus Bacillus and microorganisms of the genus Sporosarcina, and a method for producing L-phenylalanine using this enzyme, have not been described in known literature. There are no known L-phenylalanine dehydrogenase genes, plasmids containing the genes, microorganisms transformed with the plasmids, and methods for producing L-phenylalanine using such transformants.

In addition, microorganisms that produce L-phenylalanine dehydrogenase, methods for producing L-phenylalanine dehydrogenase using the microorganisms, and these phenylalanine dehydrogenases,
A method for producing α-amino acids (including L-phenylalanine) using these Shi-phenylalanine dehydrogenases is disclosed in Japanese Patent Application No. 60-80293 and Japanese Patent Application No. 60-12.
7118 and Japanese Patent Application No. 60-272494.

[Problem that the invention seeks to solve]

The production ability of wild strains that produce useful substances is generally low, and when trying to utilize the ability of these microorganisms to advantageous industrial purposes, it is necessary to improve the so-called microorganisms, that is, increase their production ability, by various methods. will be held. The present invention provides a more advantageous method for producing L-phenylalanine dehydrogenase by implanting L-phenylalanine dehydrogenase genes derived from microorganisms of the genus Bacillus and Sporosarcina into other microbial hosts by genetic recombination techniques. The present invention aims to establish a more advantageous method for producing L-phenylalanine.

[Failure to solve the problem]

The above object is an expression plasmid containing an L-phenylalanine dehydrogenase gene derived from a microorganism of the genus Bacillus or Sporosarcina; 1 a microorganism transformed with the plasmid; a method for producing L-phenylalanine dehydrogenase using this microorganism; This is achieved by providing a method for producing L-phenylalanine using this enzyme.

[Specific explanation]

In the present invention, DNA is extracted from a microorganism (gene source microorganism) that belongs to the genus Bacillus or Sporosarcina and has the ability to produce L-phenylalanine dehydrogenase, and this DNA is cut with a restriction enzyme to produce L-phenylalanine. The dehydrogenase gene is excised and introduced into an appropriate vector to create an expression plasmid, and this plasmid is used to transform a host microorganism to create an L-phenylalanine dehydrogenase producing strain. L-phenylalanine dehydrogenase can be produced by this microorganism. Furthermore, this L-
L-phenylalanine can be produced using phenylalanine dehydrogenase.

Soil EL Source Examples of microorganisms that can be used as gene sources in the present invention include bacteria belonging to the genus Sporosarcina or the genus Bacillus.

Examples of microorganisms belonging to the genus Sporosarcina include Sporosarcina ureae.

Specific strains include, for example, Sporosarcina ureae IFO12698 and Sporosarcina ureae IF.
O12699 (8TCC6473), as well as Sporosarcina ureae 5CRC-RO4. Each of the above-mentioned preserved bacteria has been deposited at IFO under the above-mentioned deposit number.
Alternatively, Sporosarcina ureae 5CRC-RO4 was sent to the Institute of Microbial Technology, Agency of Industrial Science and Technology, as part of the Microbiological Research Institute No. 1012 (FER).
M BP-1012). This 5C
The RC-ROd strain is aerobic, motile, and has spore-forming ability, and is a Durham-positive coccus with 2 to 4 series.
Bergey's Manual of Determinative Bacteriology
of Determi-native Bacter
It was identified according to the classification criteria of 8th edition (1974), and its detailed mycological properties are described in Japanese Patent Application No. 60-080293.

Examples of microorganisms belonging to Bacillus include Bacillus alvei IFO334.
3; Bacillus thiaminolyticus
thiaminoly-ticus) IAM 103
4.FeRM P-8528 No. 8528 (FERM P-8528)
) i Bacillus ba
dius) ■ M 11059 (8TCC14574)
FERMP-8529;
Bacillus sphaericus
ricus) IFO12622; Bacillus sphaericus IAM 1228
P-8527).

These are all IFO catalog, ATCC catalog,
Or, it is listed in the JFCC catalog and can be easily obtained, or it has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology. In addition, a new strain of Bacillus sp, 5CRC-R53b, which the present inventors recently isolated from soil,
Hasilus sp, 5CRC-R79a, Bacillus sp, 5
CRC-1018, and Bacillus sp, 5CRC414
D can be mentioned. The mycological properties of these strains are very similar, and Bacillus s is a representative strain of these strains.
p.

5CRC-R79a was awarded No. 1013 (FERM BP-101,
It has been deposited as 3). Also, Bacillus sp, 5
CRC-1140 is also published by FIKEN Jyoyori No. 1011 (F
ERM BP-1011). 5C
RC-R53b, 5CRC-R79a, 5CR
Strains C-1018 and 5CRC-1140 were both Durum-positive rods that formed endospores, and were identified as belonging to the genus Bacillus since they were found to produce catalase.

Detailed mycological properties of these strains can be found in patent application 1980-080.
It is described in the No. 293 specification.

In the present invention, as an example of the gene source, Pacillus sphaericus 5CRC-R79a (Feikoken Joyori No. 1013
), and Sporosarcina ureae 5CRC-RO4
(M Koken Joyori No. 1012) will be specifically explained. However, as mentioned above, various L
- It goes without saying that phenylalanine dehydrogenase-producing bacteria can be similarly used as a gene source. The gene cloning method will be explained in detail in Examples.

As the control region for the expression of the L-phenylalanine dehydrogenase of the present invention, it is possible to use the control regions originally associated with the genes of these enzymes, but it is also possible to use the control regions originally associated with the genes of these enzymes. In order to enable induction, it is preferable to provide a separate promoter/operator system. When using E. coli as a host, such promoter/operator systems include trp, t
ac.

1acUV5. Promoter/operator systems such as Pt + PR, IFF, etc. can be used, and SD sequences such as trp leader-peptide, 1acZ, metapyrocatechase or C■ gene can be used as the SD sequence. Furthermore, there is a transcription terminator downstream of the coding region.
1 For example, rrnBs T+ of E. coli glueposome gene
% T2 terminator etc. can be provided. Furthermore, in the expression of the above gene, Satucharomyces
Saccharom cescerev
isiae host/vector system can be used, and the promoters in this case include yeast alcohol dehydrogenase gene promoter, acid phosphatase gene promoter, glyceraldehyde-3-phosphate dehydrogenase gene promoter, and enolase gene promoter. A gene promoter, etc. can be used, in which case the plasmid contains a sequence for replication in yeast, and an auxotrophic marker, such as Leu, Trp, as a selection marker for selecting yeast containing the plasmid. .

Preferably, it contains a sequence such as His.

In general, the expression level of a specific protein in E. coli is influenced by gene copy number, transcription efficiency, mRNA stability, translation efficiency, protein stability, and the like. In order to modify these control regions such as the promoter and the SD region terminator using genetic engineering techniques, it is easier to handle the plasmid if it is small. Furthermore, the number of copies of a gene is related to the size of the plasmid encoding the gene, and the smaller the plasmid, the more the number of copies tends to increase.

Therefore, after inserting a DNA fragment containing the L-phenylalanine dehydrogenase gene into a plasmid, repeating subcloning of this plasmid to excise unnecessary portions of the inserted DNA fragment produces an even smaller expression type. Plasmids can be obtained.

As the host Escherichia coli of the present invention, any strain derived from the 112 strains of Escherichia coli can be used.

For example, JM83, JMIOI, JM103, JM105
, JM109, RR1, RB791, W3110
, C600, HBIOI, DH1, etc. can be used. In addition, host yeasts include AH22, DC5,
Examples include D-13-1a and YNN140.

When producing L-phenylalanine dehydrogenase according to the present invention, conventional methods for producing proteins using genetic engineering methods can be used. For example, a microorganism such as E. coli transformed with an expression plasmid containing the L-phenylalanine dehydrogenase gene is cultured in an appropriate medium, and when the bacterial cell concentration reaches a predetermined value, the control region in the plasmid By performing appropriate guidance operations, L
- Produces phenylalanine dehydrogenase.

Next, the L-phenylalanine dehydrogenase of the present invention is collected from the obtained culture, and a conventional enzyme purification method can be used as a purification method. The bacterial cells are collected by centrifugation or the like, and then disrupted by a mechanical method such as ultrasonication or Dynomill. For centrifugation of solid materials such as cell debris. 1 was removed to obtain a crude enzyme, which was further treated by adding protamine sulfate or streptomycin sulfate, followed by salting out, organic solvent precipitation, adsorption chromatography, ion exchange chromatography, gel filtration chromatography, etc. A homogeneous crystalline enzyme preparation can be isolated by known methods such as crystallization by adding a salt such as ammonium sulfate or polyethylene glycol.

In the present invention, the titer of L-phenylalanine dehydrogenase is measured as follows. Glycine-KCI
-KOH buffer (pH 10,5) 100 μmol,
NAD"2-5 t' mol -, L-phenylalanine l Q μmol.

and an appropriate amount of sample were mixed to 1rrl and reacted, and the increase in NADH at 25°C was reduced to 340n
The amount of enzyme that increases NADH by 1 micromole per minute is defined as one unit.

11. Method for producing L-phenylalanine In the method for producing L-phenylalanine of the present invention,
L-phenylalanine is produced by reacting phenylpyruvic acid, a reducing agent, and ammonium ions in the presence of L-phenylalanine dehydrogenase produced as described above, and the phenylalanine is collected.

The usage form of L-phenylalanine dehydrogenase used in this method is not particularly limited.

For example, it is a matter of course that the enzyme purified by this invention can be used in a culture solution containing cells, a cultured live bacterial cell, a dried bacterial cell dehydrated with acetone, etc., a crushed bacterial cell product, etc. It is possible to use enzyme-containing materials such as partially purified enzyme preparations that have been purified to the stage of. Furthermore, it is also possible to use these enzymes or enzyme-containing substances immobilized according to conventional methods. In industrial implementation, it is advantageous to use live microbial cells, immobilized microbial cells, etc.

Phenylpyruvic acid or a salt thereof, such as sodium salt, potassium salt, lithium salt, calcium salt, ammonium salt, etc., can be used as a substrate. In this case, this ammonium salt functions as a source of phenylpyruvic acid and at the same time as a source of ammonium ions, which will be described later. In a batch reaction, phenylpyruvic acid or a salt thereof can be added all at once at the start of the reaction, or can be added in multiple portions or continuously as the reaction progresses.

Conveniently, the source of ammonium ions is in the form of ammonium salts, such as ammonium chloride or ammonium sulfate. It is also possible to continuously introduce ammonia gas or ammonium hydroxide aqueous solution as the reaction progresses while maintaining the pH of the reaction solution at a predetermined value. When ammonium phenylpyruvate is used as described above, this material also serves as a source of ammonium salt. The amount of ammonium salt used is the same molar amount as the amount of phenylpyruvic acid or more. It is generally convenient that this amount is 1 to 2 times the amount of phenylpyruvic acid. By increasing the molar amount of ammonium salt, the equilibrium of the enzyme reaction can be tilted toward L-phenylalanine, and the yield of L-phenylalanine based on phenylpyruvic acid can be increased.

As the reducing agent, for example, reduced nicotinamide adenine dinucleotide (NADH) can be used. In this case, NADH may be added in the same molar amount as phenylpyruvic acid, but since NADH is very expensive,
From an industrial standpoint, in addition to the above reaction system, there is also an NADH regeneration system, that is, a system that converts NAD+ produced by the above reaction into NADH.
It is preferable to coexist with a system that reduces to . Such a system includes a combination of an enzyme that converts "NAD" to NADH and its substrate, such as formate dehydrogenase (EC1, 2, 1,
2) and formic acid, L-glutamate dehydrogenase (EC1,4
, 1, 2) and glutamic acid, alcohol dehydrogenase (E
C1,1,1,1) and ethanol, aldehyde dehydrogenase (Ec 1.2.1.3) and acetaldehyde, glucose-6-phosphate dehydrogenase (EC1,1,1,49
) and glucose-6-phosphate, etc. 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 dihydroriboamide undergo diaphorase (EC 1, 6
, 4, 3) of NAD+ due to oxidation by
A reduction reaction to can also be used. When using formic acid dehydrogenase and formic acid, NAD' is reduced and NAD
At the same time as H, formic acid is oxidized to generate carbon dioxide, which is easily removed from the reaction system and the reaction always proceeds in the desired direction, which is particularly preferred.

Formate dehydrogenase is commercially available and can be easily obtained. Also, for example, Candida boidini (Candida boidini)
da boidinii) Th2201 (8KU 47
05) and Hansenula polymorph y (Hansen
ula polymorpha) (ATCC2601
2) known method [Katu et al., Agricultural
and Biological Chemistry (Agricu)
Ltural and Btological Che
mistry) 3B +111~116 (1974
)) It can also be used after being purified. The enzyme concentration of the NADH regeneration system varies depending on the concentration of L-phenylalanine dehydrogenase, etc., and generally reduces NAD+ to NADH at a rate comparable to the rate of reductive amination of the substrate phenylpyruvate (therefore, the rate of NAD production). This is the amount necessary to do so.

As a substrate for formate dehydrogenase, it is convenient to use salts of formic acid, such as sodium formate, potassium formate, ammonium formate, and the like. The amount of formate used is preferably 1 to 2 times the molar amount of phenylpyruvic acid or its salt. When using an NADH regeneration system, NAD+ or NAD
H may be added at a normal physiological concentration of 0.1 to 10 mM.

For the production of L-phenylalanine, a growing culture of transformed bacteria is used, such as a culture solution containing bacterial cells, isolated live bacterial cells, or bacterial cells that have been treated to the extent that the enzyme system is not destroyed. NADH contained in these cases
If a regenerative system can be used, NAD+ and NAD+ (there is no need to artificially add a regenerative system; for example, sugars, alcohols, or organic acids can be used as an energy source in a bacterial culture solution or bacterial reaction solution. Sugars include arabinose, ribose, ribulose, xylose, fucose, rhamnose, fructose, galactose, gluconic acid, trehalose, glucose, mannitol, mannose, sorbitol, sorbose, inositol, lactose, maltose, sucrose, raffinose. , glycerol, starch, inulin, glycogen, carboxymethyl cellulose, etc. Examples of the alcohol include ethanol, methanol, etc.

Organic acids include propionic acid, acetic acid, formic acid, citric acid,
Examples include pyruvic acid, succinic acid, malic acid, α-ketogeltaric acid, and the like.

Water or an aqueous liquid, for example an aqueous buffer, can be used as the reaction medium. As a buffer solution, for example, Tris-H
Cl buffer, glycine-NaOH buffer, etc. can be used.

As the pH of the reaction solution, if the NADH regeneration system described above is not used, a pH suitable for reductive amination by L-phenylalanine dehydrogenase can be used, and a pH in the range of 8 to 11 is preferable, for example. When regenerating an NADH regeneration system together with a phenylpyruvic acid reductive amination system, it is necessary to select a pH range in which both reactions proceed well, for example, a pH range of 8.5 to 9.5 (pi)
A range is preferred.

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

The reaction time is not particularly critical; the substrate temperature of the reaction mixture,
Depending on the enzyme titer, etc., the reaction is maintained until the substrate phenylpyruvate is converted to L-phenylalanine in sufficient yield.

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

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

For the determination of phenylalanine, for example, Leuconostoc 0
Mesenteroides (Leuconostoc mese)
(nte-roides) 8 TCC8042 can be carried out by a bioassay using TCC8042.

Next, the present invention will be explained in more detail with reference to Examples.

Bacillus sphaericus 5CRC-R79a (FER
M BP-1013) in 31 medium <0.2g/dl
L-phenylalanine, 0.5g/yeast extract, 1
．． 0 g/m peptone, 0.2 g/dIK2HPO4
,0,1g/dINaC1,0,05g/dIM
gSOn・7 HzO, pH 7,0) and (z5
) Cultured with shaking at 30°C for about 10 hours. 00616, which was confirmed in advance to be in the logarithmic growth phase under these conditions =
Bacteria were collected in step 1.1.

Chromosomal DNA was extracted from bacterial cells weighing about 10 g wet by the method of Doi (Reference 1). The bacterial cells were soaked in 4QmA TEN buffer (10mM Tris-■C1 pH 7, 6, 1m
After suspension in M'EDTA, 10 mM NaCl), the mixture was centrifuged. The precipitate was added to 2Qm# SET buffer (20% sucrose, 50 mM Tris-HCI pH 7,6).
, 50mM EDTA), IQmg of lysozyme was added, and the mixture was incubated at 37°C for 30 minutes. After confirming the formation of spheroplasts using a microscope, 1 Qrrl of TEN buffer and 0.25 g of SDS were added to perform bacteriolysis. Conventional phenol/chloroform treatment was followed by ethanol precipitation and the DNA was plated onto glass rods. 1Q
Dissolve the DNA in TEN buffer at 0.5 m
g of RNase was added, and the mixture was kept at 37°C for 2 hours. Next, 1 mg of pronase was added and the mixture was kept warm for an additional hour.

Phenol/chloroform treatment was followed by ethanol precipitation and drying under reduced pressure. Finally, it was dissolved in TEN buffer.

Either pUC9 or pBR322 was used as a vector for vectoring DNA L.

10 gg of either pUC9 or pBR322 was digested with the restriction endonuclease old ndI[I for 2 hours at 37°C, followed by treatment with calf thymus phosphatase for 1 hour at 37°C and with phenol.

This solution was subjected to 1% agarose gel electrophoresis, and the 2.6 kb DNA fragment of pUc9 or the 4.6 kb DNA fragment of pBR322 was isolated.
A 4 kb DNA cleavage fragment was isolated by electroelution.

This solution was treated with phenol/chloroform, precipitated with ethanol, and then dissolved in 20 μβ sterile water. 50 gg of chromosomal DNA obtained in Example 1 was digested with 50 units of old nd■ at 37°C for 16 hours, and then phenol/
50μ after chloroborum treatment and ethanol precipitation
1 of sterile water.

0.5 μg of vector DNA obtained by the above method
and 2 μg of chromosomal DNA were mixed, and the DNA strands were ligated at 12.5° C. for 16 hours using T4 phage-derived DNA ligase in the presence of ATP and dithiothreitol.

E. coli was transformed using these reaction mixtures according to a conventional method. As for Escherichia coli, 12 strains/JM 103 were used when pUc9 was used as a vector, and 112 strains/RRI were used when pBR322 was used as a vector.

The transformant solution obtained in Example 2 was applied onto a nitrocellulose filter placed on an LB agar medium containing 50 μg/ml of ampicillin so that 500 to 1000 plants were grown. After incubating at 37°C for 16 hours, it was replicated onto another nitrocellulose filter and further incubated at 37°C for 3
Cultured for hours.

This nitrocellulose was mixed with Q, 5mA Norizozyme solution (
5mg/mff lysozyme, 10mM Tris-H
CI pH 7.5, 1mM EDTA) and left at room temperature for several tens of minutes to lyse the bacteria. This was frozen at -70°C and then thawed at room temperature. This was mixed with 0.5rrl of tetrazolium solution (0.2M Tris-■C1 pH8,
5,1,5mM NAD", 0.8mM 2-p-phenyl iodide-3-p-nitrophenyl-5-phenyltetrazolium chloride (INT), 0.32mM phenazine meso sulfate (PMS)] and concentrated. A transformed strain exhibiting a reddish-purple color was selected.

In this way, a recombinant using pUC9 as a vector and pBR
As a result of searching for about 2,000 ampicillin-resistant strains of each of the recombinants using No. 322 as a vector, one strain of each strain positive for L-phenylalanine dehydrogenase activity was obtained.

3 (4 Awashishio [Ayu Taun's Plasmid'' (1
) A plasmid was extracted from the obtained L-phenylalanine dehydrogenase activity positive strain. The recombinant plasmid obtained using pUC9 as a vector was named pBPDI, and pBR
The recombinant plasmid obtained using 322 as a vector was pB
The plasmid was named PDI (3). Each plasmid was cut with various restriction enzymes and restriction enzyme maps were prepared. These restriction maps are shown in FIG. 1 (pBPDI3).

In addition, the base sequence and the corresponding amino acid sequence of the DNA fragment containing the region encoding L-phenylalanine dehydrogenase in plasmid pBPD)+1 are shown in Figures 3-1 and 3.
- Shown in Figure 2. This sequence is pBPDHl-
Also present in A and pBPDHl-BS, Figure 3-1~
The coding region in Figure 3-2 is present in a series of plasmids of the invention shown in Figures 1 and 2.

Subcloning of Sousei 1BPDH1 (1nu) 10μ
The plasmid pBPDI of g was digested with old ndlll, electrophoresed on a 0.7% agarose gel, and a DNA fragment of approximately 6 kb was isolated by electroelution.

2 μg of pUC9 was digested with old ndI, treated with calf thymus phosphatase, and treated with phenol/chloroform. Both reactants were mixed and Elutip-d (Sc
After purification with a column (manufactured by Hlei-Cher & 5Chuel1), it was precipitated with ethanol.

This was mixed with 10μl of T4 ligase buffer (50mM Tr
is-HCI pH 7, 4, 10mM MgC1z, 1
The mixture was dissolved in 0mM DTT, 1mM ATP), and a ligation reaction was performed using T4 ligase.

This reaction solution was used to transform Escherichia coli JM103 strain.

50. ampicillin at czg/m4, 0.3mM isopropylthio-galactoside, 0.03% 5-bromo-
A strain exhibiting no β-galactosidase activity and exhibiting a white color on an LB agar medium containing 4-chloro-3-indolyl-β-D-galactoside was selected. Plasmids were extracted from these strains, and pUC was determined by the restriction enzyme digestion pattern.
A recombinant plasmid in which approximately 6 kb of the old ndlI cut fragment was inserted at the old ndlI site of 9 was selected, and pBPD
It was named H1-A.

Practical visit Plasmid BPDH3 'I (1) 2μ
pBR322 of g was digested with old ndIII, treated with calf thymus phosphatase, and then treated with phenol/chloroform. Approximately 6 kb isolated in Example 5
The DNA fragments were added and mixed, purified using an Elutip-d column, and then precipitated with ethanol. 10μ of this
1 of T4 ligase buffer (50mM Tris-HC
I pH7,4,10mM MgC1z, 10mM
DTT, 1mM ATP), and a ligation reaction was performed using T4 ligase. Using this reaction solution, E. coli R
The RI strain was transformed. The transformed bacteria were screened by conventional means to obtain plasmid pBPDH3.

Subcloning to Jitseijo1BPDH1- (1nu10
After digesting μg of pBPDHl-A with BamHI and 5ail, it was electrophoresed on a 0.7% agarose gel, and a DNA fragment of approximately 3 kb was isolated by electroelution.

pUC9 was digested with BamHI and 5alI, then treated with calf thymus phosphatase and phenol/chloroform. The reaction mixture was mixed in the same manner as in Example 5, purified using an Elutip-d column, precipitated with ethanol, ligated with T4 ligase, and used to transform E. coli strain JM103. From the obtained ampicillin-resistant transformants, a white coloring strain that did not show β-galactosidase activity was selected in the same manner as in Example 5. Plasmids were extracted from these strains, and BamHI-3 of pUC9 was determined by the restriction enzyme digestion pattern.
A recombinant plasmid with an approximately 3.2 kb BamHI-Sail cleavage fragment inserted into the ail site was selected, and pBP
It was named DHL-BS. In addition, plasmid pBPDI
The base sequence and the corresponding amino acid sequence of the DNA fragment containing the region encoding L-phenylalanine dehydrogenase in +1-BS are shown in Figure 3-1.3-2. Base sequencing was performed according to the dideoxy method (Reference 4).

W5 Subcloning of BPDHI-BS (10/jg of pBPDH1-BS was digested with DraI and then precipitated with ethanol. 0.010D26° units of BamHI linker phosphorylated at the 5' end and aI The digested DNA was ligated with 350 units of T4 ligase in 20 μl of T4 ligase buffer at 22°C for 5 hours, and then precipitated with ethanol.
After digestion with HI, electrophoresis was performed on a 0.7% agarose gel, and a DNA fragment of approximately 2 kb was isolated by electroelution.

2 μg of pUC9 was digested with BamH, treated with calf thymus phosphatase, and then treated with phenol/chloroform. Similar to Example 5, the re-reactants were mixed and El
After purification with a utip-d column, ethanol precipitation was performed,
ligated with T4 ligase and used this to isolate E. coli JM1.
09 strain was transformed. From the obtained transformed strains, strains negative for β-galactosidase activity were selected in the same manner as in Example 5.

Plasmids were extracted from these strains, and approximately 1.8 kb was extracted from the BamHI site of pUC9 using the restriction enzyme digestion pattern.
A recombinant plasmid into which the DNA fragment was inserted was selected and named pBPDHl-Dr.

Example: Subcloning of pBPDH1-Dr (10 μg of pBPDH1-Dr was digested with Ba1I and precipitated with ethanol. 0.010D260 units of Bam old linker Ba1I with 5' end phosphorylated)
10 μ of digested DNA! 3 in glue gauze buffer
After a ligation reaction using 50 units of T4 ligase at 16° C. for 6 hours, precipitation was performed with ethanol.

This was digested with BamHI, electrophoresed on a 0.7% agarose gel, and DNA fragments were isolated by electrophoretic elution, treated with phenol/chloroform three times, and then precipitated with ethanol.

2 μg of pUC8 was digested with BamHI, treated with calf thymus phosphatase, treated with phenol/chloroform, and precipitated with ethanol.

The re-reactant was dissolved in T4 ligase buffer and mixed,
4 ligase, and transformed into E. coli strain JM109. From the obtained transformed strains, strains exhibiting no β-galactosidase activity were selected in the same manner as in Example 5. Plasmids were extracted from these strains, and approximately 1.3 k was added to the BamH1 site of pUC8 using the restriction enzyme digestion pattern.
Recombinants with the DNA fragment b inserted were selected, and the plasmids were transformed into pBPDHl-DBL and pBPDHl-DBR.
It was named. These differ in the direction of the inserted fragment on the restriction enzyme map.

Each of the E. coli bacteria containing the obtained recombinant plasmids has L-phenylalanine dehydrogenase activity.

Escherichia coli containing the above plasmid pBPDH1-DBL
i) JM109/pBPDHl-DBL has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as FERM P-8873. Also, plasmid pBPD! (Escherichia coli containing 1-DBR)
JM109/pBPDH1-DBR is available from Microtechnical Laboratory No. 8
794 (FE, RM P-8794).

E. coli JM103 strain containing pBPD old and pBPDH
The L-phenylalanine production activity of the E. coli RRI strain containing No. 3 was investigated.

E. coli JM103 strain containing pBPDHl was added to 140 rr.
50 μg/m I! The cells were cultured in LB medium containing thiancillin at 37°C for 6 hours. As a control, Bacillus sphaericus 5CRC-R79a was added to 140 ml of 0.
1% Phe medium (0.1% L-phenylalanine, 1%
Peptone, 0.5% yeast extract, 0.2% 2HPOA
, 0.1% NaCl, 0.02% MgSO4・7H20
, pH 7,0) at 30°C overnight.

After collecting the bacteria by centrifugation, the bacteria were transferred to 70ml of 0.
．． Washed with 85% NaCl solution. 10mA 0.1M
After suspension in potassium phosphate (pl! 7.0) buffer 2
Ultrasonic disruption was carried out at 200 W for 20 minutes at ℃. Centrifuge this and remove the supernatant as 101 0.01M potassium phosphate (p)17
．． 0) Dialyzed against buffer overnight at 4°C.

Using this sample, L-phenylalanine dehydrogenase activity was measured by the method described above. The results are shown in the table below.

E. coli JM103 /pBPDH11500 E. coli
RR1/pBPDH32600 E. coli JM103/
E. coli containing the pBPDH1-DBL 3000 recombinant plasmid is
~50 times more enzyme activity was observed.

m day, A of L-phenylalanine (1) pBPDH3
L-phenylalanine was synthesized using E. coli RRI strain containing the following. E. coli glycolytic enzyme was used as the NADH regeneration system. LB medium containing 50 μg/mj2 ampicillin was inoculated with 1% of the E. coli and cultured at 37°C for 6 hours.

After centrifuging the culture solution of 5mff1, it was washed with 0.85% NaCl solution. This bacterial cell was added to 3 ml of reaction solution [200 u
mol NH4Cl-NH2OH (pF! 9.0) buffer, 109 pmol sodium phenylpyruvate, 267 μmol lactose] at 30°C for 24 hours.
Let it stand for a while. As a result, '1. Om g / m 1
L-phenylalanine of 2 was accumulated and the yield was 33%. L-phenylalanine was quantified by the method described above.

n, addition of phenylalanine (2) Next, L-phenylalanine was synthesized using Candida boidini No. 2201 containing formate dehydrogenase as an NAD)I regeneration system. Candida boidini 1Vh2201 cultured according to the method of Tani et al. (Reference 2) and Escherichia coli RRI strain containing ρBPDH3 cultured according to the method described above were collected and then treated with acetone (Reference 3).

15 mg of Escherichia coli acetone-treated cells, which corresponds to 6.3 mβ culture solution and has 6.3 units of L-phenylalanine dehydrogenase activity, and 23 m (corresponds to 1 culture solution, 1
．． The reaction was carried out using 30 mg of acetone-treated Candida boidini cells having 4 units of formate dehydrogenase activity. For the reaction, the bacterial cells were mixed with 3mj2 reaction solution (250, t+
mol Tris-14C1 (ph8.5) buffer,
728 μmol (0,136 g) phenylpyruvic acid, 2 mmol (0,122 g) ammonium formate,
After suspending in 2.5 μmol (1.8 g) NAD''
The test was carried out by standing at 30°C for 53 hours. As a result, the yield was 10
0.126 g (42 mg/mA) of L-phenylalanine, corresponding to 0%, was obtained.

Physiolitis U, panchromatic DNA nore'+ (2) According to the method of Doi et al. (Reference 1), Sporosarcina
Urea SCRC-RO4 (FERM BP-1012
) chromosomal DNA was prepared.

L-phenylalanine 1%, yeast extract 0.5%, peptone 1.0%, KIT2PO4,0,2%, NaCl0
．． A medium containing 1% MgSO4.7 H2O and 0.02% and adjusted to pH 7.0 was sterilized in an autoclave at 1°C, then inoculated with the above bacterial strain and cultured overnight at 30°C.

The bacterial cells collected after centrifugation were suspended in 20 ml of TEN buffer,
After further centrifugation to make the SET buffer solution 19 constant A, 1 mj
Add 5 mg/m j2 of 2 lysozyme to 37
Shake at °C for 30 minutes. After further centrifugation, the precipitate
Suspended in TEN buffer of rrl, 25% of Q, 5mj2
SDS, 1 mj2 of 5 M NaCl, and lQmj
! After extracting the DNA by adding saturated phenol, the supernatant obtained by centrifugation was mixed with chloroform/iso-amyl alcohol (2
4:1 volume) was added for washing. The supernatant obtained by centrifugation was centrifuged at 50 m
The DNA was then gently placed in ethanol (1) to precipitate and recover. After the DNA was dried, it was dissolved in lQm# TEN buffer.

111(2) of the 7-color DNA library
50 μg of the chromosomal DNA obtained in Example 13 was treated with restriction enzyme B.
am old, old ndIII, or EcoRI 20 each
After digestion in units at 37°C for 12 hours, the mixture was extracted with phenol/chloroform (1:1 volume) and precipitated by adding 2.5 volumes of ethanol. On the other hand, vector plasmid pUC9
5 μg of the same enzyme as above was added to each of the 10 cells at 37°C.
After 2 hours of reaction, DNA was collected in the same manner.

A recircularization reaction was performed using T4 DNA ligase using a restriction enzyme digest of vector plasmid pUC9 and 0.5 μg and 4 μg of inserter 1-DNA, respectively, and this reaction mixture was used to transform Escherichia coli with 112/JM103 to generate chromosomal DNA live cells. It was Larry.

Each chromosomal DNA library has 20,000 ~
It consisted of 30,000 transformants.

A nitrocellulose filter (0,45 μm
XTYPE TM-2 (Toyo Roshi) was placed thereon, and the transformed bacteria (library) was plated and cultured thereon to form 1,000 to 2,000 colonies.

This was further replicated onto two nitrocellulose filters, and the culture was continued. Rear ff nitrocellulose filter, 20 m of 0.5 mj+
g/m/! It was left standing on a lysozyme aqueous solution at room temperature for 3 minutes and dried at 45°C for 1 hour. These filters were treated with 50mM L-phenylalanine, 50mM
Tris-11cI (pH 8), 0.625 mM
Colonies colored reddish-brown were placed on 0.5 m# of active staining solution containing NAD, 0.064 mM phenazine methosulfate (PMS), and 0.24 mM nitroblue tetrazolium (INT) for 1 to 5 minutes at room temperature. selected (Reference 5). Several colonies selected by this activity staining method were further incubated at 50°C g/m j! The cells were cultured overnight in an LB liquid medium containing thiancillin, and the cells were sonicated. The above-mentioned activity staining solution was added to the cell-free extract obtained by centrifugation, and the enzyme activity was confirmed. In this way, a transformed strain JM103/pSPD old containing one L-phenylalanine dehydrogenase gene was isolated.

and subcloning (2) of transformed bacteria JM103/pSPD old at 50℃g/mj
2. Cultured at 37°C overnight in 200 mA of LB liquid medium containing ampicillin, Birnboin+, Doly
Prepare plasmid DNA according to the method of (Reference 6),
15cIg was obtained. pSPD) 11 with various restriction enzymes (B
amHI, EcoRI.

BgllI, PvuI[, NaeI, Hin
clI, etc.), and a restriction enzyme map was prepared (Fig. 4A).

The old insert DNA of pSPD was fragmented to approximately 5.6 kb, and the old ndl of vector plasmid pUc19 was fragmented.
ll -5ma I site of pSPD) II insert DNA Nael-Hindll I fragment approx. 1.4
112/J into E. coli using a plasmid inserted with kb.
Transform M103 to obtain a transformed strain JM103/psPDH that has the ability to produce L-phenylalanine dehydrogenase.
I got 2. The restriction enzyme map of pSPDH2 is shown in Figure 4B.

In addition, the DN containing the region encoding L-phenylalanine dehydrogenase in plasmids pSPD old and pSPDH2
The base sequence and the corresponding amino acid sequence of the A fragment are shown in No. 5-1.
and Fig. 5-2.

The base sequence was determined using the dideoxy method (Reference 4) and MB1.
The 3-dideoxy chain termination method (Reference 7) was followed.

The transformed bacterium Escherichia coli
-hia colt) JM103/psPDH2 has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as FERM P-8793.

Implementation ± u, ■ Awato Ikusu (2) Escherichia coli JM103/pSPDH2 and JM103/
In order to examine the enzyme production amount of psPDHl, these E. coli were cultured in LB medium containing 50 μg/mρ ampicillin at 37° C. for 6 hours. On the other hand, as a control, Sporosarcina ureae 5CRC-RO4 was cultured in the medium shown in Example 1 at 30°C overnight (16 hours).

After collecting the bacterial cells by centrifugation, 0.1 m'M ED
It was suspended in 0.1M potassium phosphate buffer (pH 7,0) containing TA and 5mM 2-mercaptoethanol and disrupted by ultrasonication. These were mixed with 0.1 mM EDTA.
and 1 containing 5 m M 2-mercaptoethanol.
4 to 0mM potassium phosphate buffer (pH 7,0)
Dialysis was performed overnight at °C. The activity of L-phenylalanine dehydrogenase in these samples was measured by the method described above, and the results shown in Table 2 were obtained.

E. coli JM103/psPDH1175 E. coli JM10
In the recombinant E. coli strain 3/pSPDH2340, the amount of enzyme activity expressed per culture solution was approximately 6 times that of the wild strain.

W, L-phenylalanine (3) Escherichia coli JM10
3/pSPDH2 was cultured as described above. On the other hand,
Candida boidini & 220 used for NADH regeneration system
1 was cultured according to the method of Tani et al. (Reference 2). Bacterial cells were collected from each of these two cultures and treated with acetone (
Reference 3) and 0.48 g/I!, respectively! and 1.
A processed product of 3 g/β was obtained.

Sodium phenylpyruvate 0.136 g (7
28μmol), ammonium formate 0.122g (2
mmol), NAD” 1.8 mg (2.5 μmol
), Tris-HCI 250 pmol (pH
8,5), 15 mg of acetone-treated E. coli cells (30
rrl culture solution, 6 units), Candida boidini acetone-treated bacterial cells 3" g (2.3 ml culture solution, 0.1
3 mj including 4 units)! When the reaction solution was reacted at 30°C for 53 hours, 0.120 g (yield 100%)
Noshi-phenylalanine was obtained.

The words of the DNA. '+(3) Bacillus badius IAM1 according to the method of Doi et al. (Reference 1)
Chromosomal DNA of 1059 (FERM P-8529)
was prepared.

L-phenylalanine 1%, yeast extract 0.5%, peptone 1.0%, K2HPO40.2% and Mg5O,
・After autoclaving 1.2 L of a medium containing 0.02% H2O and adjusted to pH 7.0, the above strain was inoculated and cultured at 30"C for 6 hours. Chromosomal DNA was the same as shown in Example 1. obtained by the method.

(3) 50 μg of the chromosomal DNA obtained in Example 19 was treated with the restriction enzyme Eco.
After overnight digestion at 37° C. using 300 units of RI, extraction was performed with phenol/chloroform (1:1 volume) and precipitation was performed by adding 2 volumes of ethanol. On the other hand, 1910 μg of vector plasmid pUc1 was digested overnight at 37° C. using 60 units of restriction enzyme ECORI, and DNA was recovered in the same manner.

A recircularization reaction was performed with DNA ligase using 0.5 μg and 1 μg of the restriction enzyme EcoRI digest of vector plasmid pUc19 and the restriction enzyme EcoRI digest of chromosomal DNA, respectively, and this mixture was used to infect Escherichia coli with 1 μg.
2/JM103 was transformed into a chromosomal DNA library. The chromosomal DNA library consisted of 23,000 transformants.

A nitrocellulose filter was placed on an LB agar medium containing 50 μg/rrl of ampicillin, and the transformed bacteria was plated and cultured on the filter to obtain 1,000 to 2,000 colonies. This was replicated on a nitrocellulose filter and culture was further continued. The latter nitrocellulose filter, ], Qmj? After leaving it for 10 minutes at 30°C on a 20 mg 7 ml aqueous solution of lysozyme, 10 m! ! 0.
The mixture was incubated at 50° C. for 10 minutes over IMTris-HCI (pH 8,0). These nitrocellulose filters were treated with 50 mM Tris-HCI (pH 8).
, 0.625 m M NAD”, 0.06
5mM PMS.

Colonies that colored reddish-purple were selected by standing on active staining solution 1.0 containing 0.24 mM INT at room temperature for 1 to 5 minutes (Reference 5). Approximately 50 active strains were obtained from approximately 23,000 transformed strains. 50μg of 12 of them
37° in LB liquid medium IL containing /ml ampicillin.
C - overnight culture, Birn-boim.

When plasmid DNA was prepared according to Doly's method (Reference 6) and the gene size was analyzed by agarose gel, one clone had a gene fragment of about 7 Kbp inserted and one clone had a gene fragment of about 3.8 Kbp inserted. There were 11 clones. All of these transformed bacteria had L-phenylalanine dehydrogenase activity.

One of the clones into which a 3.8 Kbp gene fragment was inserted was named JM103/pBBPDH19.

[Fruit] l Elementary School, Torso 11j Pull I Figure-q Harukai Transformed Bacteria JMIOI / pBBPDl119 50
3 in LB liquid medium IL containing μg/mA ampicillin.
After culturing at 7°C overnight, plasmid DNA was prepared according to the method of Birnboim and Doly (Reference 6).
50 μg was obtained. pBBPDH19 was digested with various restriction enzymes (
Sma I, Xma I + BamHI, EcoR
I, Hind I, Pst I, Sal I, A
cc I etc.) and created a restriction enzyme map (6th
figure).

12 strains of JM103/pBBPDH were added to the transformed bacteria.
pBBPDH19 was isolated according to a conventional method. Using this plasmid, 12 strains of RRI were transformed into E. coli. The transformed bacterium Escherichia coli (E
scherichiacoli) RRI/pBBPD
H19 is FERMP-889
0) has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology.

Next jl coarse, Byorin! In order to examine the enzyme production amount of E. coli RRI/pBBPDH19 under L1, these E. coli were cultured in LB medium containing 50 μg/mβ ampicillin at 37° C. for 12 hours. On the other hand, as a control, Bacillus badius IAM 1105
9 was cultured in the medium shown in Example 1 at 30°C for 20 hours. After collecting the bacterial cells by centrifugation, 0.1 m
The cells were suspended in 0.1M potassium phosphate buffer containing MEDTA and 5mM 2-mercaptoethanol and disrupted by ultrasonication. Remove the crushed bacterial cells by centrifugation and reduce the supernatant to 0.1
Dialyzed overnight against 10 mM potassium phosphate buffer containing mM EDTA and 5 mM 2-mercaptoethanol. The L-phenylalanine dehydrogenase activity in these samples was measured by the method described above, and the results shown in Table 3 were obtained.

To Bacillus Badius■ M 11059 1,090
E. coli containing the E. coli RRI (pBBPDH19) 6,900 recombinant plasmid was found to have about 6 to 7 times more enzyme activity per culture solution than the wild strain.

E. coli RRI/pBBPDH19 at 50μg/rrl
1.8 L of LB medium containing ampicillin at 37°C for 12
Cultured for hours. After washing the bacterial cells with physiological saline, 0,
The cells were suspended in 1 liter of 0.1M phosphate buffer (pH 7,0) containing 1mM EDTA and 5mM 2-mercaptoethanol, and sonicated at 9 KHz for 1 hour to disrupt the bacterial cells. Crushed bacterial cells are 14,000 x g
was removed by centrifugation for 20 minutes to obtain a raw extract containing L-phenylalanine dehydrogenase. After this cell-free extract was heat-treated at 50°C for 110 minutes, solid ammonium sulfate was added to make it 30% ammonium sulfate saturated. The precipitate formed after stirring for 30 minutes was removed by centrifugation at 14.0 OOX g for 20 minutes, and solid ammonium sulfate was further added to the supernatant to achieve 60% ammonium sulfate saturation. The precipitate containing enzyme activity was centrifuged at 14.0 OOX g for 20 minutes, and then mixed with a small amount of 0.01 M phosphate buffer (pH (7,0)
further dissolved in 0.1mM EDT8 and 5mM 2
- Dialyzed against 0.01M phosphate buffer (pH 7.0) containing mercabutethanol. Add this enzyme solution to zero in advance.
．． Passed through a column of DEAE-1-Yopal 650M equilibrated with 0.01M phosphate buffer (pH 7.0) containing 1mM EDTA and 5mM 2-mercaptoethanol, 0.1mM EDTA, 5mM 2-mercaptoethanol. 0.1M containing mercaptoethanol and 0.1M NaCl
Elution was performed with phosphate buffer (pH 7,0).

The active fraction was collected, concentrated after dialysis, and diluted with 0.1 mM EDTA.
, 5mM 2-mercaptoethanol and 0,1 MN
Gel filtration chromatography was performed using Sephadex G-200 equilibrated with 0.05M phosphate buffer (pH 7.0) containing aCl. In this way, L-phenylalanine dehydrogenase was purified approximately 15 times with a yield of approximately 54%. Table 4 shows the specific activity and recovery rate in this purification process. This enzyme was demonstrated to be homogeneous in polyacrylamide gel electrophoresis and 5DS-polyacrylamide gel electrophoresis.

Table 1 Process Summary Total protein Specific activity Recovery (unit) (■) (unit/mg) (%) 1, cell-free extract 12,400 1,760 7.05
1002, heat treatment 11,900 1,3
10 8.78 963, Ammonium sulfate fraction 7
, 680 611 12.6 624, DE
AE-) Yopal 7,100 1
88 37.8 57 Escherichia coli R
RI/pBBPDH19 was cultured as described above. On the other hand, Candida boidini No. 2201 used in the NADH regeneration system was cultured according to the method of Tani et al. (Reference 2). Bacterial cells were collected from each of these two cultures, treated with acetone (Reference 3), and incubated at 0 and 5 g/i, respectively.
1! And 1.3g/A of treated product was obtained.

Sodium phenylpyruvate 0.136g (728
μmol), ammonium formate 0.122 g
(2 mmol), NAD′″1.8 mg (2,5
μmol), Tris-HCI 250 μmol+(p
H8,5), 5 mg (10 m
j! When a 3 mA reaction solution containing 3 mg of Candida boidini acetone-treated bacterial cells (2,3 culture solution, 0.14 units) was reacted at 30°C for 48 hours, 0.120 g (Yield: 100%) Noshi-phenylalanine was obtained.

References 1) R, L, Rodriguez et al., Recom
binant DNA Teqhnique +, An
Introduction, Addison-We
sleyPublishing Company (1
983) 162 pages.

2) Y, Tan1 et al., Agric, Biol,
Chem, 36+68+ (1972).

3) Y., Izumi et al., J., Perment, T.
echnol, +6L 135 (1983).

4) H. Hattori et al., Nucl, 8cids Res, 13 7813 (1985
).

5) L. Mollering et al., Metho
ds of enzymatic eight analysis
Academic Press, 1 (1974H
pp. 36-144.

6) H.C. Birnboim et al., Nucl, Ac.
1ds Res, +7.

1513 (1979).

7) F. Sanger et al., Proc. Natl.
, Acad, Sci, USA5463 (19
77).

[Brief explanation of the drawing]

Figures 1 and 2 show plasmids pBPDH3, pBPDH1-DBL, and pB
It is a systematic diagram showing PDI+1-DBR production. Figures 3-1 and 3-2 show the base sequence (upper row) and the corresponding amino acid sequence (lower row) of a DNA fragment containing the region encoding the L-phenylalanine dehydrogenase of the present invention in plasmid pBPDH1. Figure 4 shows the restriction map of plasmids pspoold and pSPDH2. Figures 5-1 and 5-2 show plasmid psPDHL
The base sequence (upper row) and the corresponding amino acid sequence (lower row) of the DNA fragment containing the region encoding the L-phenylalanine dehydrogenase of the present invention are shown in FIG. Figure 6 shows the restriction map of plasmid pBBPDH19. Procedural amendment (voluntary) February 1985 Director General of the Japan Patent Office Kunio Ogawa 1, Indication of the case 1980 Patent Application No. 280654 2, Name of the invention Process for producing L-phenylalanine by genetic recombination 3. Relationship with the case of the person making the amendment Patent applicant name: Sumo Central Chemical Research Institute name (220) Central Glass Co., Ltd. 4, Agent address: 8-10-5 Toranomon-chome, Minato-ku, Tokyo 105
The following statement is added in column 6 of "Detailed Description of the Invention" of the specification to be amended, between lines 5 and 6 on page 53 of the specification of contents of the amendment. “The deposit of the following microorganisms has been transferred to the International Deposit under the Budapest Treaty.
li) JM109/pBPDH1-DBL Former accession number FERNP-8
873) New accession number FERMBl'
-1433) Escherichia coli
li) RRI/pBBPDH19 Old accession number: FEBMP-8
890) New accession number FERMBP-
1436) Escherichia co
li) JM103/pSPDH2 Former accession number FERMP-8
793) New accession number FERMBP-
1435) Escherichia coli
li) JM109/ pBPDHl -DBR Old accession number: FERMP-8794 (FERMP-8794) New accession number: FERMP-8794 (FERMBP-8794) New accession number: FERMP-8794 (FERMBP-8794)
1434)

Claims (1)

[Scope of Claims] 1. L derived from a microorganism of the genus Bacillus or Sporosarcina
-Phenylalanine dehydrogenase gene. 2. The Bacillus microorganism is Bacillus sphaericus (
The gene according to claim 1, which is Bacillus sphaericus. 3. The Bacillus microorganism is Bacillus badius (B
acillus badius). 4. The gene according to claim 1, wherein the microorganism of the genus Sporosarcina is Sporosarcina ureae. 5. L derived from microorganisms of the genus Bacillus or Sporosarcina
- An expression plasmid containing the phenylalanine dehydrogenase gene. 6. The Bacillus microorganism is Bacillus sphaericus (
The plasmid according to claim 5, which is Bacillus sphaericus. 7. The Bacillus microorganism is Bacillus badius (B
5. The plasmid according to claim 5, which is a plasmid of A. acillus badius. 8. The plasmid according to claim 5, wherein the microorganism of the genus Sporosarcina is Sporosarcina ureae. 9. A microorganism transformed with an expression plasmid containing an L-phenylalanine dehydrogenase gene derived from a microorganism of the genus Bacillus or Sporosarcina. 10. The microorganism according to claim 9, wherein the transformed microorganism is Escherichia coli. 11. Cultivating a microorganism transformed with an expression plasmid containing an L-phenylalanine dehydrogenase gene derived from a microorganism of the genus Bacillus or Sporosarcina,
A method for producing L-phenylalanine dehydrogenase, which comprises collecting L-phenylalanine dehydrogenase from the culture. 12. L-phenylalanine dehydrogenase produced by a microorganism transformed with an expression plasmid containing an L-phenylalanine dehydrogenase gene derived from a microorganism of the genus Bacillus or Sporosarcina, and phenyl in the presence of a reducing agent. A method for producing L-phenylalanine, which comprises producing L-phenylalanine by reacting pyruvic acid and ammonium ions, and collecting the product. 13. The method according to claim 8, wherein the reducing agent is reduced nicotinamide adenine dinucleotide. 14. The method according to claim 8, further comprising a system for regenerating oxidized nicotinamide adenine dinucleotide produced by the reaction into reduced nicotinamide adenine dinucleotide.