JPH04271784A - 5-substituted hydantoin racemase and gene capable of coding the same - Google Patents

Abstract

PURPOSE:To obtain the subject gene capable of mass-producing a 5-substituted hydantoin racemase converting DL-5-substituted hydantoin into the corresponding optically active amino acid by coding the aforementioned enzyme, integrating the coded enzyme in a vector and making a host hold the vector. CONSTITUTION:A plasmid of a microorganism NS671 (FERM P-9543) belonging to the genus Pseudomonas is treated with a restriction enzyme, linked to a vector and transduced into Escherichia coli to carry out transformation. Screening is then performed to select a clone containing a DNA capable of coding a 5-substituted hydantoin racemase, and a plasmid is collected from the resultant clone and treated with a restriction enzyme to provide a 5-substituted hydantoin racemase gene having a sequence from the 202nd base to the 951st base expressed by the formula. Furthermore, the resultant gene is linked to a vector and transduced into Escherichia coli to preform transformation. The obtained transformant is cultured in a culture medium to afford the objective 5-substituted hydantoin racemase that is a protein, composed of 249 amino acids and having 27090 molecular weight.

Description

[Detailed description of the invention]

0001

FIELD OF THE INVENTION This invention relates to a process for racemizing 5-substituted hydantoins.

0002

[Prior Art] Microorganisms that convert DL-5-substituted hydantoins into the corresponding optically active amino acids include Flavobacterium (Japanese Patent Publication No. 61-12296) and Bacillus (Japanese Patent Publication No. 61-12296).
(Japanese Patent Publication No. 63-24895), Pseudomonas (Special Publication No. 64-71476), Arthrobacter (Japanese Patent Publication No. 60-Sho.
-214889) are known. DL-
Racemization of 5-substituted hydantoin is necessary to efficiently convert 5-substituted hydantoin into the corresponding optically active amino acid, and racemization of 5-substituted hydantoin occurs chemically or enzymatically in these bacteria. It is believed that there are.

0003

[Problem to be solved by the invention] At a pH near neutrality,
Chemical racemization of 5-substituted hydantoins hardly proceeds. Furthermore, conventional bacteria did not produce enough racemization enzymes.

0004

[Means for Solving the Problems] In order to solve the above-mentioned problems, the present invention clones a gene from a bacterium that has a racemization enzyme, and increases gene amplification, transcription, and translation activities to achieve the objective. The aim is to increase enzyme production. That is, the present invention provides a gene for a 5-substituted hydantoin racemase, a DNA fragment containing the gene, a vector DNA incorporating the DNA fragment, a microorganism transformed with the vector DNA, and a 5-substituted hydantoin produced using the microorganism. 5 using a racemization method, a racemization enzyme prepared from the microorganism, and the present racemization enzyme.
- A method for racemizing substituted hydantoins.

The present invention will be explained in more detail.

The 5-substituted hydantoin to be racemized in the present invention is represented by general formula (I). (However, in the formula, R represents a 5-substituent group.) Specific examples of R include those corresponding to 5-substituted hydantoins corresponding to various amino acids; for example, as a side chain of β-lactam antibiotics. Important p-hydroxy-D-phenylglycine, D-phenylglycine, D-cysteine, D-aspartic acid, D-homophenylalanine as a synthetic raw material for angiotensin converting enzyme inhibitor, or L-2-amino as a herbicide. There is an amino acid corresponding to -4-methylphosphinobutyric acid, and there is also an amino acid corresponding to 5-substituted hydantoin, such as alanine/CH3CH(N
H2) COOH, valine (CH3)2 CHCH(N
H2) COOH, leucine (CH3)2 CHCH2
CH(NH2)COOH, isoleucine/CH3C
H2 CH(CH3)CH(NH2)COOH, serine・HOCH2 CH(NH2)COOH, threonine・
CH3 CH(OH)CH(NH2)COOH, Methionine CH3 SCH2(NH2)COOH, Phenylalanine C6H5CH2 CH(NH2)COOH,
Tyrosine・HOC6 H4 CH2 CH (NH2)
COOH is important, and examples of alkyl groups include methyl, isopropyl, isobutyl, 1-methylpropyl, hydroxyl,
As an alkyl group substituted with an alkylthio group, hydroxyphenyl group, or indolyl group, hydroxymethyl group, 1-hydroxyethyl group, 2-methylthioethyl group, benzyl group, p-hydroxybenzyl group, or indolylmethyl group There is. (1) Gene cloning Pseudomonas NS671 (FERM P-95
43) converted the DL-5-substituted hydantoin to the corresponding L-
A bacterium that has the ability to convert amino acids into amino acids. We obtained genes involved in 5-substituted hydantoin conversion from this bacterium, namely DL-specific hydantoinase and L-specific N-
The gene for carbamyl amino acid hydrolase was cloned (Japanese Patent Application No. 2-137120). It was revealed that the gene cloned at the same time was that of a 5-substituted hydantoin racemase.

[0006] Pseudomonas NS671 (FERM
Plasmid vector pUC obtained by partially digesting plasmid pHN671 of P-9543) with restriction enzyme MboI, digesting with restriction enzyme BamHI, and treating with phosphatase.
It was ligated to 18. E. coli JM103 was transformed using this recombinant DNA, and the resulting transformant was spread on an agar medium containing DL-5-(2-methylthioethyl)hydantoin as the sole nitrogen source. The transformed strain containing the recombinant DNA containing the gene of interest is DL-5-(
It uses ammonium ions produced when converting 2-methylthioethyl)hydantoin into L-amino acids as a nitrogen source, grows on the above-mentioned agar medium, and forms colonies that can be easily separated. As a result of culturing transformants from colonies formed on this selective medium and preparing plasmid DNA, two types of plasmids with sizes of approximately 10.2 kilobases and approximately 15.5 kilobases were obtained. . The former plasmid is pHPB12, the latter plasmid is pHPB12, and the latter plasmid is pHPB12.
It was named PB14. Plasmid pHPB12 is DL
Specific hydantoinase genes (Fig. 1, ORF2 and O
RF3) and L-specific N-carbamyl amino acid hydrolase gene (Fig. 1, ORF4) (Japanese Patent Application No. 137120/1999). Analysis of DNA fragments obtained by restriction enzyme digestion revealed that plasmid pHPB14 has a structure in which the downstream part of the inserted DNA of plasmid pHPB12 is extended by about 5.3 kilobases.

(2) Transformability encoded by plasmid pHPB14 Escherichia coli JM103 was transformed with plasmid pHPB14, and a transformed strain was obtained using ampicillin resistance as an indicator. This transformed strain is D-5-(2-methylthioethyl)
It had the ability to convert all hydantoin into L-methionine. On the other hand, the transformed strain (FERM P-11176) using plasmid pHPB12 was D-
5-(2-methylthioethyl)hydantoin can only be converted to N-carbamyl-D-methionine.

(3) Reduction of plasmid pHPB14 The restriction enzyme map of plasmid pHPB14 is shown in FIG. The solid line portion represents vector DNA pUC18, and the bar line portion represents Pseudomonas NS671 (FERMP-954).
3) shows the inserted DNA derived from plasmid pHN671. The inserted DNA upstream of the restriction enzyme BamHI site at the 7.5 kilobase position is identical to that of plasmid pHPB12. The number appended to the restriction enzyme name indicates the distance (kilobases) from the starting point of the inserted DNA. Open reading frame (ORF) discovered as a result of base sequencing
) area and direction are indicated by arrows.

Plasmid p digested with restriction enzyme SalI
HPB14 was treated with restriction enzymes AflII, SnaBI, Sp
This was double digested with lI, SphI, or XhoI, and if necessary, the ends were blunted by Klenow enzyme reaction, followed by self-ligation to obtain a deletion plasmid. E. coli JM103 was transformed with these deletion plasmids, and the conversion ability of the resulting transformants was examined. As a result, even if the region downstream of the restriction enzyme SnaBI site at the 10.2 kilobase position of plasmid pHPB14 was deleted, D
-5-(2-methylthioethyl)hydantoin is all L
- Retains the ability to convert to methionine, but 9.
When the region downstream of the restriction enzyme SplI site at the 1 kilobase position is deleted, the above-mentioned conversion ability is lost (D-5-(
2-methylthioethyl)hydantoin to N-carbamyl-D-methionine).

(4) Determination of base sequence 11 from 7.0 kilobase position of plasmid pHPB14
．． The restriction enzyme EcoRI digested fragment up to 1 kilobase position was subcloned into M13mp18 phage. This subcloned inserted DNA portion was shortened using various restriction enzymes or a kilosequencing dilation kit (Takara Shuzo), and the base sequence was determined by the dideoxy method. The nucleotide sequence upstream of the restriction enzyme BamHI site at the 7.5 kilobase position of plasmid pHPB14 is as follows:
The base sequence was the same as the inserted DNA of plasmid pHPB12. As a result, two new open reading frames, ORF5 and ORF6, were discovered (Figure 1
).

(5) Preparation of deletion plasmid Plasmid p
HPB14 was double digested with restriction enzymes SalI and SnaBI, treated with Klenow enzyme, self-ligated, and plasmid pSNA101 (ORF2, ORF3,
(including ORF4, ORF5, and ORF6) were created. Plasmid pSNA101 was digested with the restriction enzyme KpnI and self-ligated to create plasmid pKPN1.
7 (containing ORF6) was produced. Plasmid pKPN
17 was digested with the restriction enzyme EcoRI, treated with phosphatase, and a 6.4-kilobase restriction enzyme EcoRI-digested fragment of plasmid pHPB12 (ORF2, ORF3, and
RF4). Select a DNA fragment into which the open reading frame is oriented in the same direction, and add the plasmid pSNAdEE (
(including ORF2, ORF3, ORF4, and ORF6)
It was named. E. coli JM1 with plasmid pSNAdEE
The transformed strain obtained by transforming D-5-(
It had the ability to convert all 2-methylthioethyl)hydantoin into L-methionine. That is, ORF5
It turns out that is not necessary for this conversion. Therefore, O
It was thought that RF6 plays an important role in this conversion.

(6) E. coli JM1 transformed with ORF6 functional plasmid pKPN17
When D-5-(2-methylthioethyl)hydantoin was added to a mixed culture of Escherichia coli JM103 (FERM P-11176) transformed with 03 and plasmid pHPB12, all of the D-5-(2-methylthioethyl)hydantoin was converted to L-methionine. This indicates that ORF6 functions normally even when expressed in cells different from ORF2, ORF3, and ORF4. D-5-(2-methylthioethyl)hydantoin was added to the culture of E. coli JM103 transformed with plasmid pKPN17, and after reacting for a certain period of time, the cells were removed by filtration, and E. coli JM103 transformed with plasmid pHPB12 was cultured. (FERM P-11176
) instead of L-
The production ratio of methionine increased and the production ratio of N-carbamyl-D-methionine decreased, reaching an equilibrium of 1:1 (molar ratio). This indicates that ORF6 is a gene for 5-substituted hydantoin racemase.

(7) Escherichia coli JM103 was transformed with the molecular weight plasmid pKPN17 of the ORF6 gene product (5-substituted hydantoin racemase) to obtain a transformed strain. This transformed strain was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology (FERM BP-3188) as FERM BP-3188. The cells obtained by culturing this transformed strain were disrupted, the soluble fraction was fractionated by ammonium sulfate salting out, hydrophobic chromatography,
The 5-substituted hydantoin racemization enzyme was purified by ion exchange chromatography and gel filtration chromatography. The apparent molecular weight of the purified enzyme on SDS-polyacrylamide gel electrophoresis was 31,900. The molecular weight determined from gel filtration chromatography was approximately 130,000. From this, it is presumed that this racemizing enzyme has a tetrameric structure. The partial base sequence of the inserted DNA of plasmid pKPN17 containing ORF6 is shown in the sequence listing. ORF6 is A at the 196th base
It may begin with two start codons: TG and ATG at base 202. As a result of analyzing the N-terminal amino acid sequence of the purified enzyme, MetLysIleLysValIl
It was revealed that the sequence was eAsnProAsnThr. Therefore, this gene has AT at the 202nd base.
It can be seen that G is used as the start codon. The molecular weight of the 5-substituted hydantoin racemase determined from the base sequence is 27,090.

(8) Method of using the 5-substituted hydantoin racemase gene A microorganism such as Escherichia coli into which the recombinant DNA of the DNA fragment containing the gene of the present invention and the vector DNA has been introduced is cultured in a medium, and D-5 If a -substituted hydantoin or an L-5-substituted hydantoin is added, a racemic 5-substituted hydantoin can be obtained. The same effect can also be obtained by preparing a 5-substituted hydantoin racemizing enzyme from the microorganism and carrying out the enzymatic reaction. The method of the present invention includes a method for converting a 5-substituted hydantoin into a corresponding N-carbamyl amino acid by a hydantoinase reaction, and a method for converting an optically active N-carbamyl amino acid into a corresponding optically active amino acid by an N-carbamyl amino acid hydrolase reaction. When used in combination with a conversion method, the entire amount of DL-5-substituted hydantoin as a raw material can be efficiently converted into an optically active amino acid.

[0015]

[Example] Example 1. E. coli JM1 transformed with racemized plasmid pKPN17 of various 5-substituted hydantoins
03 (FERM BP-3188) in 50ml LB
The cells were inoculated into a medium and cultured with shaking at 30°C until the OD at 600 nm reached approximately 0.2. 250μl of 200mM I
PTG was added and cultured with shaking at 30°C for 2 hours. A portion of 20 OD (600 nm) of this culture was centrifuged to obtain bacterial cells. This was suspended in 10 ml of 20 mM Tris-HCl buffer (pH 7.5) and 0.1 M sodium chloride containing 0.2% of D- or L-form various 5-substituted hydantoins, and shaken at 30°C. After a certain period of time, 1ml
The cells were removed by centrifugation and filtration to obtain a supernatant.

[0016] The ratio of D-form and L-form of 5-substituted hydantoin present in this supernatant was measured using CHIR, a column for optical isomer separation.
HP using ALPAK WH (Daicel Chemical Industries)
It was analyzed by LC and the results are shown in Table 1. As a control, E. coli JM1 transformed with plasmid pUC18
As a result of performing the same operation as above using 03, the production of L-form or D-form 5-substituted hydantoin from D-form or L-form 5-substituted hydantoin, respectively, could not be detected.

[0017]

Example 2. Purification of racemization enzyme E. coli JM103 (FE) transformed with plasmid pKPN17
RM BP-3188) was inoculated into 50 ml of LB medium and precultured at 30°C overnight. Transfer this entire volume to 3L of medium (
6g/L disodium phosphate, 3g/L monopotassium phosphate, 0.5g/L sodium chloride, 1g/L
L ammonium chloride, 5mM magnesium sulfate, 0.5mM calcium chloride, 1% glycerol, 2% yeast extract) and cultured at 30°C in a jar fermenter until the OD at 550nm reached about 10. After adding 15 ml of 200 mM IPTG to this and further culturing for about two and a half hours, the cells were collected by centrifugation to obtain about 100 g of wet bacterial cells.

The wet bacterial cells were suspended in 500 ml of 20 mM Tris-HCl buffer (pH 7.5) and 0.1 M sodium chloride, and the cells were disrupted using a Gorlin homogenizer. This was centrifuged to remove insoluble matter to obtain a supernatant. The 60-90% saturated ammonium sulfate salting out fraction of this supernatant was added to 100 ml of 20 mM Tris-HCl buffer (pH
7.5), 0.1M sodium chloride, 0.5M
It was subjected to hydrophobic chromatography on butyl-Toyopearl (TOSOH) dissolved in ammonium sulfate and equilibrated with the same buffer.

The activity of racemization enzyme was detected as follows. That is, plasmid pHP was added to 1 ml of LB medium.
E. coli JM103 transformed with B12 was inoculated with 100 μl of 2% D-5-(2-methylthioethyl)hydantoin and 5 μl of 200 mM IPTG.
Then, 1 μl of the test solution was added, and cultured with shaking at 30° C. overnight. The culture solution was analyzed by TLC to examine the production of methionine (L-form) or N-carbamylmethionine (D-form). The production of methionine (L-form) indicates the presence of racemization enzyme activity. Moreover, when there is no racemization enzyme activity, only N-carbamylmethionine (D form) is produced.

D
It was subjected to ion exchange chromatography on EAE-Sephacel (Pharmacia). Racemization enzyme activity is 100-
It was eluted at about 150mM sodium chloride.

The racemization enzyme active fraction obtained from DEAE-Sephacel chromatography was concentrated using a PM10 membrane (Amicon), and Sephadic was equilibrated with 20mM Tris-HCl buffer (pH 7.5) and 0.1M sodium chloride. It was subjected to gel filtration chromatography using G-200 Superfine. As a result, racemization enzyme activity was eluted at a molecular weight of approximately 130,000. This racemization enzyme active fraction was collected using SDS-
As a result of analysis by polyacrylamide gel electrophoresis, a band appeared at a molecular weight of 31,900. Therefore, this racemizing enzyme is presumed to have a tetrameric structure.

[0023]

According to the present invention, it has become possible to stably and highly express the gene for 5-substituted hydantoin racemase in microorganisms such as Escherichia coli. As a result, the present enzyme can now be easily prepared from such microorganisms.

[0024]

[Sequence list] Sequence length: 1282 Sequence type: Number of nucleic acid strands: Double stranded Topology: Linear Sequence type: Other nucleic acids Plasmid DNA source organism name: Pseudomonas
sp. ) Strain name: NS671 Sequence characteristics Symbol representing characteristics: CDS Location: 202. ．． 951 How the characteristics were determined: E Sequence GAATTCGAGC TCGGTACCTG AAA
TTGATGG TGTAGTAGTG ATTGAT
AGTT TATAGAAACA 60ATTGT
TGACC CTTGGAAAAAAA
AC TAAAAACTTA TAAATCTCCT
GAAAAGAAAG 120AATATTGCGG
GAAATTAAGA AATTTGGGTC CG
TGGAAAAAAGCTATAAAATAAATT
CACTA 180AATTCTAGGA GGAA
A ATG GTT ATG AAA ATT AAA
GTT ATC AAC CCG AAT ACA
231
Met Lys Ile Lys Val
Ile Asn Pro Asn Thr
1
5
10ACT TTG GCA ATG A
CA AAG GGA ATT GAA CAT GC
T GCT AAG TCA GCT GCA
279Thr Leu Ala Met Thr Ly
s Gly Ile Glu His Ala Ala
Lys Ser Ala Ala
15
20 2
5 AGA TCT GAT ACT CAA ATT
GTT GCA GTG AGT CCT AAA
ATG GGT CCG GCT 327Arg
Ser Asp Thr Gln Ile Val
Ala Val Ser Pro Lys Met G
ly Pro Ala
30 35
40 TCA AT
T GAA TCC TAC TAT GAT GAA
TAT TTA AGC ATT CCA GGA
GTA ATT 375Ser Ile Glu
Ser Tyr Tyr Asp Glu Tyr
Leu Ser Ile Pro Gly Val I
le 45
50
55 GAG GAA
ATT AAA AAG GGA GAA GAA G
AA GGA GTA GAT GCA TTT GT
T ATA 423 Glu Glu Ile L
ys Lys Gly Glu Glu Glu Gl
y Val Asp Ala Phe Val Ile
60
65 70
GCA TGC TGG GG
A GAC CCT GGA TTG CAT GCT
GCG AGA GAA GTA ACA GAT
471Ala Cys Trp Gly Asp
Pro Gly Leu His Ala Ala
Arg Glu Val Thr Asp 75
80
85
90 AAA CCA GTT GTA
GGC ATT GCG GAA TCC TCC G
TT TAT TTA GCA TCA ATG
519Lys Pro Val Val Gly I
le Ala Glu Ser Ser Val Ty
r Leu Ala Ser Met
95
100 1
05 CTT GCA GCC AGA TTT TC
C GTG GTC ACA GTT TTA CCT
AGA ATT AAA ACA 567Le
u Ala Ala Arg Phe Ser Val
Val Thr Val Leu Pro Arg
Ile Lys Thr 1
10 115
120A
TG TTA GAA GAC CTG GTT GA
T TCA TAC GGT ATG CAA AAA
CGT GTA CTA 615Met Le
u Glu Asp Leu Val Asp Ser
Tyr Gly Met Gln Lys Arg
Val Leu 125
130
135 AAC
ATC CGT ACG ACA CCG ATG
GGC GTA TTA GAT TTT GAG A
GA GAT CCA 663 Asn Ile
Arg Thr Thr Pro Met Gly V
al Leu Asp Phe Glu Arg As
p Pro 140
145
150 GAA GCG G
GA ATT GAA ATG TTA AGG CA
G GAA GGG AAA AGA GCG GTA
GAG 711Glu Ala Gly Il
e Glu Met Leu Arg Gln Glu
Gly Lys Arg Ala Val Glu
155 160
165
170 GAA GAT AAT
GCA GAA GCT ATT TTA CTT
GGA TGT GCT GGT ATG GCA G
AA 759Glu Asp Asn Ala
Glu Ala Ile Leu Leu Gly C
ys Ala Gly Met Ala Glu
175
180
185 TTT GCG GAT AGC CTT GAA A
AA GAA TTA GGA GTT CCC GT
T ATC GAT GGA 807Phe A
la Asp Ser Leu Glu Lys Gl
u Leu Gly Val Pro Val Ile
Asp Gly 190
195
200 GTT GTA GC
G GGT GTG AAA TTC GCC GAA
ACA ATT GTT GAT CTA GGA
AAG 855 Val Val Ala Gly
Val Lys Phe Ala Glu Thr
Ile Val Asp Leu Gly Lys
205
210 215
AAA ACA AGT
AAA CTA AAA ACT TAT AAA T
AT CCA GAG AAA AAA GAA TA
T 903Lys Thr Ser Lys L
eu Lys Thr Tyr Lys Tyr Pr
o Glu Lys Lys Glu Tyr
220 225
230
GTT GGG GCA TTG GA
G AAC TTT GGA CGG AAT CAA
ACA ACA ACA AAA TAA 9
51Val Gly Ala Leu Glu Asn
Phe Gly Arg Asn Gln Thr
Thr Thr Lys 235
240
245 AAAATTA
AAG AGTTTTTCACA CTTGTTAAAT
CATAGATAGT TTATATCAAT TT
TATTTAAT 1011TTTGGGCGAA T
TTCAAATTAC TAGTGAAATT GCCC
ATTTTT TAAAAGCATA ACAGGGA
TTT 1071CAGTGATATT GACTGT
TAGG CAGAACAGAA ATTTTATTA
T ACATCCCTTG AATGAATTAT 1
131TGCATAGCAA ATGGGTGAAC
TATAAAAGTT CTTACATCAA TAT
GTATCAA AAAGGAGGTGC 1191AG
TTATTCAT GGATTTAAAA TCTAA
CAGCG TCATCAATCC AGATCCAA
AT AGGATTCAAA 1251CAAATAA
TTT TAACATCATA AAAACAAGCT
T
1282

[Brief explanation of the drawing]

FIG. 1 is a restriction enzyme map of plasmid pHPB14.

Claims (2)

[Claims] 1. A 5-substituted hydantoin racemase gene, which has a sequence from the 202nd base to the 951st base described in the sequence listing. 2. A 5-substituted hydantoin racemase having a sequence from the 1st amino acid to the 249th amino acid listed in the sequence listing.