JPH06105693A - Dna fragment having base sequency coding esterase - Google Patents

Abstract

PURPOSE:To provide a new DNA fragment coding an esterase having high thermal stability and capable of forming an optically active carboxylic acid useful as a synthetic raw material for a physiologically active substance such as hypotensor. CONSTITUTION:A DNA fragment having a base sequence of formula II and coding an esterase catalyzing the asymmetric hydrolysis reaction of a carboxylic acid ester of formula I (R<1> is alkyl, aralkyl or aryl; R<2> and R<3> are independently alkyl; (n) is 1 or 2). It can be produced e.g. by cloning a chromosome of a microorganism capable of producing the desired esterase, e.g. Pseudomonas putida MR-2068 strain (FERM P-9677).

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cloned DNA fragment having a nucleotide sequence encoding an esterase having high thermostability which catalyzes asymmetric hydrolysis of carboxylic acid ester.

[0002]

PRIOR ART The following general formula (I):

[0003]

[Chemical 2] [In the formula (I), R ¹ is an alkyl group, an aralkyl group or an aryl group, R ² and R ³ are each independently an alkyl group, and n is 1 or 2] By asymmetric hydrolysis in the presence of esterase, the following general formula (II):

[0004]

[Chemical 3] [In the formula (II), R ¹ , R ² and n are defined in the same manner as in the above general formula (I)], and various physiologically active substances represented by the hypotensive agent captopril (trade name) An optically active carboxylic acid useful as a synthetic raw material can be obtained.

The alkyl group represented by R ^{1 in} the above general formulas (I) and (II) is, for example, a methyl group or an ethyl group, the aralkyl group is, for example, a benzyl group, and the aryl group is, for example, Examples thereof include a phenyl group and the like, and examples of the alkyl group as R ² and R ³ include a methyl group and an ethyl group.

Representative examples of the compound of the above-mentioned general formula (I) include, for example, methyl β-acetylthio-α-methylpropionate, methyl S-acetyl-β-mercaptoisobutyrate and S-acetyl-γ-mercapto-α. -Methyl-n-
Examples include methyl butyrate, methyl S-benzoyl-β-mercaptoisobutyrate and methyl S-phenylacetyl-β-mercaptoisobutyrate.

Utilization of the esterase activity in this asymmetric hydrolysis reaction is carried out, for example, by a method of allowing the reaction system to include the esterase-producing microorganism itself, or a method of allowing the reaction system to include the esterase separated and recovered from the esterase-producing microorganism. And the like (JP-A-60-12992, JP-A-60-
12993, etc.).

[0008] Further, in Japanese Patent Laid-Open No. 1-222798, Pseudomona ( Pseudomona ) newly isolated by the present inventors as a highly active esterase-producing bacterium.
s putida ) MR-2068 strain
No .: FERM P-9677) is disclosed.

On the other hand, various attempts have been made to mass-produce various useful enzymes and the like using recombinant DNA technology in response to the rapid progress of gene manipulation technology centering on recombinant DNA technology in recent years. As for the above esterase, Pseudomonas fluorescens IFO30 is disclosed in JP-A-1-67190.
Cloning of the esterase gene from 18 strains has been disclosed.

[0010]

The asymmetric hydrolysis reaction of the above-mentioned carboxylic acid ester can be carried out more efficiently by adopting higher temperature conditions, but an efficient reaction at high temperature is possible. In order to carry out the above, it is essential that the esterase has good thermal stability in addition to being capable of producing a carboxylic acid with high optical purity.

However, under the present circumstances, the cloning of the esterase gene necessary for mass production of the esterase satisfying these characteristics at the same time by the recombinant DNA technology has not been carried out.

For example, the esterase produced by recombinant DNA technology using the esterase gene from Pseudomonas fluorescens IFO 3018 strain described above has substantially the same properties as the esterase possessed by the gene donor, and is thermostable. However, it is inactivated in several hours under a temperature condition of 50 ° C. or higher, and cannot be used for an asymmetric hydrolysis reaction using a high temperature condition.

The object of the present invention is to provide a DNA encoding an esterase which has an activity to catalyze the asymmetric hydrolysis reaction of a carboxylic acid ester having a specific structure defined by the above general formula (I) and has a high thermostability. To provide a cloned DNA fragment containing the sequence.

[0014]

The DNA fragment of the present invention comprises:
It is characterized by having the following base sequence (SEQ ID NO: 1) encoding an esterase which catalyzes the asymmetric hydrolysis reaction of the carboxylic acid ester represented by the general formula (I). ATGAGCTATG TAACCACGAA GGACGGCGTA CAGATCTTCT ACAAGGACTG GGGCCCGCGC 60 GATGCGCCGG TCATCCACTT CCACCACGGC TGGCCGCTCA GTGCCGACGA CTGGGACGCG 120 CAGATGCTGT TCTTCCTCGC CCACGGTTAC CGCGTGGTCG CCCACGACCG CCGCGGCCAT 180 GGCCGCTCCA GCCAGGTATG GGACGGCCAC GACATGGACC ACTACGCCGA CGACGTAGCC 240 GCAGTGGTGG CCCACCTGGG CATTCAGGGC GCCGTGCATG TCGGCCACTC GACCGGTGGC 300 GGTGAGGTGG TGCGCTACAT GGCCCGACAC CCTGCAGACA AGGTGGCCAA GGCCGTGCTG 360 ATCGCCGCCG TACCGCCGTT GATGGTGCAG ACTCCCGATA ATCCCGGTGG CCTGCCCAAA 420 TCCGTTTTCG ACGGCTTCCA GGCCCAGGTC GCCAGCAACC GCGCGCAGTT CTACCGGGAT 480 GTGCCGGCAG GGCCGTTCTA CGGCTACAAC CGCCCCGGTG TCGACGCCAG CGAAGGCATC 540 ATCGGCAACT GGTGGCGCCA GGGCATGATC GGTAGCGCCA AGGCCCATTA CGATGGCATC 600 GTGGCGTTTT CCCAGACCGA CTTCACCGAA GACCTGAAGG GCATTACCCA GCCGGTGCTG 660 GTGATGCATG GCGACGACGA CCAGATCGTG CCGTATGAGA ACTCCGGGCT GCTGTCGGCC 720 AAGCTGCTGC CCAATGGCAC ACTGAAGACC TACCAGGGCT ACCCGCATGG CATGCCGACC 780 ACCCATGCCG ATGTGATCAA TGCGGATTTG CTGGCGTTTA TCCGTAGCTG A 831 ... ( Sequence number 1) Book Ming DNA fragments by cloning from chromosome of a microorganism producing the desired esterase, or D
It can be obtained by synthesis using an NA synthesizer, and its form may be either single-stranded or double-stranded.

Examples of chromosome-donating microorganisms obtained by cloning include Pseudomonas putida M.
The R-2068 strain (Ministry of Industrial Research, No. 9677) can be mentioned as a preferable example.

An example of a method for cloning a DNA fragment containing the entire base sequence (SEQ ID NO: 1) will be described below.

First, from a cultured bacterial cell of, for example, Pseudomonas putida MR-2068 strain as a chromosome donor,
The method of Marmur et al. [J. Marmur et al, J. Mol. Bio
l., 3 , 208 (1961)].
Partial digestion with coRI prepares a DNA fragment mixture.

On the other hand, the plasmid pUC19 was transformed with EcoRI.
It is opened at the EcoRI site at the multiple cloning site in lacZ within the lac operon (the lactose operon of E. coli) by digestion with and reacted with the DNA fragment mixture obtained above in the presence of T ₄ ligase. .

The reaction mixture obtained was treated with CaCl ₂ in advance to make it easier to receive foreign DNA. For example, E. coli (Esc)
It is introduced into JM109 strain, which is one of the K-12 strains of herichia coli.

The host E. coli strain into which the reaction mixture has been introduced,
Ampicillin, IPTG (isopropylthiogalactoside) and X-Gal (5-chloro-4-bromo-3-)
It is seeded on a plate made of LB agar medium containing indolyl-β-D-galactoside) and cultured.

In this culture, D at the EcoRI site
The transformant having pUC19 in which the NA fragment was not inserted acquired ampicillin resistance and IPTG.
Expresses β-galactosidase by the induction of
It grows on the plate of the above composition, and the colony is β
Cleavage of X-Gal by galactosidase activity produces a blue color.

On the other hand, DN was added to the EcoRI site of pUC19.
Since the expression of β-galactosidase is disabled when the A fragment is inserted, DNA is inserted at the EcoRI site of pUC19.
The transformant having the recombinant plasmid having the inserted fragment acquired ampicillin resistance, but did not cause blue coloration due to the expression of β-galactosidase activity.

Therefore, after the above culture, by selecting colorless colonies appearing on the plate, transformants having a recombinant plasmid having a DNA fragment inserted at the EcoRI site of pUC19 can be selected.

Next, a strain having an objective recombinant plasmid can be selected by screening a strain having an esterase activity from these colorless strains.

This screening can be carried out, for example, by the following operation.

The colonies on the culture plate were filtered with a filter paper [10 mM Tris-HCl (pH 7.5), 0.01%.
Bromocresol purple, which was impregnated with 100 ppm of methyl DL-β-acetylthio-α-methylpropionate as a carboxylic acid ester),
Leave to react at room temperature for several hours.

In this reaction, colonies having esterase activity produce carboxylic acid, and pH near the colonies
Decreases, so that the blue-purple color of bromocresol purple, which is a pH indicator, changes to yellow. Therefore, in the strain having the esterase structural gene, discoloration to yellow occurs around the colony, and its presence can be confirmed with the naked eye.

The position of the esterase structural gene in the EcoRI DNA fragment inserted from the plasmid into pUC19 can be determined, for example, by the following method.

First, a plasmid is isolated from the strain confirmed to have an esterase structural gene by the above-mentioned operation, and a chromosome-derived EcoRI DNA fragment is prepared.

Next, the EcoRI DNA fragment is treated with various restriction enzymes to prepare small DNA fragments corresponding to various portions of this EcoRI DNA fragment, which are individually incorporated into pUC19 to prepare a recombinant plasmid. .

Finally, the esterase activity of Escherichia coli transformed with the obtained recombinant plasmid is measured to identify the minimum DNA fragment region in the EcoRI DNA fragment showing the activity.

Further, the above-mentioned base sequence (SEQ ID NO: 1) can be obtained by cutting out the region containing the esterase structural gene thus determined and determining the base sequence of the region by various methods. .

The cloning of the DNA fragment of the present invention is not limited to the above-mentioned method, and various methods can be used. As a cloning vector, pUC19 or p
UC18, M13mp18, M13mp19 [all of which are commercially available from Takara Shuzo] can be selected and used according to the type of host.

D having the above base sequence (SEQ ID NO: 1)
By combining the NA fragment with a functional region such as a promoter capable of expressing the esterase encoded by the nucleotide sequence (SEQ ID NO: 1) and incorporating it into a vector having a replication origin in a host, recombinant DNA technology can be obtained. Recombinant DNA can be constructed to obtain a transformant having the esterase-producing ability used.

This transformant can be suitably used for mass production of esterase and for asymmetric hydrolysis of the carboxylic acid ester of the above general formula (I) using the transformant itself. Moreover, since the esterase produced by the transformant has thermostability, it is possible to perform an efficient asymmetric hydrolysis reaction under higher temperature conditions by using it.

[0036]

【Example】

Example 1 1-1) Preparation of chromosomal DNA The method of Marmur et al. [J. Marmur et al, J. Mol. Bio
l., 3 , 208 (1961)], the following operations were performed on Pseudomonas putida MR-2068 strain
No. 677) was isolated.

100 ml of LB medium (1% polypeptone, 0.5% yeast extract, 0.5% NaC
Pseudomonas putida MR-2068 strain (Ministry of Microbiology, Ltd. No. 9677) was inoculated in 1) and cultured overnight at 37 ° C.

After culturing, the cells were collected by centrifugation, and about 3
g (wet weight) was suspended in 24 ml of TEG buffer [25 mM Tris-HCl, 10 mM EDTA (ethylenediaminetetraacetic acid), 50 mM glucose, pH 8.0],
1 ml of a lysozyme solution (lysozyme dissolved in TEG buffer at a concentration of 10 mg / ml) was added, and this was stirred at 32 ° C. for 30 minutes.

After stirring, 2 ml of 12.5% SDS was added.
(Sodium dodecyl sulfate) solution is mixed, and further 5M
Sodium perchlorate solution (6.75 ml) was added and mixed well.

Next, 33.8 ml of a chloroform-isoamyl alcohol (24: 1, volume ratio) mixed solution was added to the obtained mixture and gently stirred for 30 minutes.

After completion of stirring, the mixture was centrifuged, and the supernatant was gently added to twice the amount of cold ethanol, and the precipitated DNA was attached to a glass rod and wound up, and taken out from the cold ethanol. Add 9 ml of this DNA to 0.1 x SS
C (1 × SSC: 0.15 M NaCl, 15 mM trisodium citrate), and the resulting solution was added to 10 ×.
1 ml of SSC was added to obtain a DNA solution.

Next, 5 parts of RNase A was added to this DNA solution.
The mixture was added to a concentration of 0 μg / ml and gently stirred for 30 minutes, then 10.2 ml of a chloroform-isoamyl alcohol (24: 1, volume ratio) mixed solution was added, and the mixture was gently stirred for another 30 minutes.

After completion of stirring, the reaction mixture was centrifuged, and the resulting supernatant was collected and added to 1 ml of acetic acid-EDTA.
(3M sodium acetate, 0.01M EDTA, pH
7.0) solution was added, and isopropyl alcohol was added dropwise to this with stirring with a glass rod (total amount 5.4 ml), and the precipitated DNA was attached to the glass rod and wound.

The DNA wound on a glass rod was further added to 1 × SS.
C (3 ml) and chromosomal DNA solution (2 μgD
NA / μl).

1-2) Preparation of recombinant plasmid 5 μl (10 μm) of the chromosomal DNA solution obtained in the above 1-1)
(corresponding to gDNA), a restriction enzyme EcoRI was added to 2 units / μg DNA, and the reaction was carried out according to a conventional method to obtain 3
The mixture was kept warm at 7 ° C for 30 minutes to obtain a DNA fragment mixture by partial digestion with EcoRI.

On the other hand, 1 μg of the plasmid pUC19 was digested with EcoRI by a conventional method to open the ring, and linear DN
A fragment was obtained.

Next, the mixture of DNA fragments from chromosomal DNA and the DNA fragment from plasmid pUC19 were treated with T ₄
Reaction was carried out in the presence of ligase according to a conventional method to obtain a reaction mixture.

1-3) Isolation of recombinant plasmid Escherichia coli JM109 strain (available from Takara Shuzo Co., Ltd.)
Was cultured in LB medium at 37 ° C. for 2 to 3 hours, and then the cultured bacterial cells were collected and suspended in an ice-cooled 50 mM CaCl ₂ solution. The cells were collected from this suspension by centrifugation again, suspended again in a fresh 50 mM CaCl ₂ solution, and allowed to stand for 30 minutes while cooling with ice.

A portion (about 100 to 200 μl) of this suspension was sampled in a test tube, the reaction mixture (the amount corresponding to 2 μg DNA) obtained in the above section 1-2) was added thereto, and the mixture was ice-cooled. While standing still for 30 minutes. Next, test tube 4
After heating at 2 ° C. for 2 minutes, 1 ml of LB medium was added thereto, and the mixture was shaken at 37 ° C. for 60 minutes.

After shaking, about 10 cell suspensions in a test tube were prepared.
0 μl was added to LB agar plates (100 μg / ml ampicillin, 0.5 mM IPTG, 0.2% X-G).
al.) and placed at 37 ° C. overnight.

Of the colonies appearing on the plate, the colorless colonies were assayed for esterase activity by the following method, and a strain having a recombinant plasmid into which the esterase structural gene had been inserted was selected. Measurement of esterase activity: 0.01% bromocresol purple and 100 ppm DL-β-acetylthio-
10 mM tris-containing methyl α-methylpropionate
A colorless colony was transferred onto a filter paper soaked with HCl (pH 7.5), and then left at room temperature for several hours.

[0052] A colony having a blue-purple to yellow discoloration which shows a pH decrease due to the production of carboxylic acid from a carboxylic acid ester by the expression of esterase is selected, and it is harbored with a recombinant plasmid into which an esterase structural gene has been inserted. It was used as a transformant.

One of the obtained transformants was isolated and cultured,
From the obtained cultured cells, the method of Birnboim et al. [Birnboim e
al., Nucleic Acid Res., 7, 1513-1523 (1979)] and prepared a restriction enzyme map. The result is shown in FIG. The recombinant plasmid having the structure shown in FIG. 2 was designated as pPE101.

1-4) Determination of Esterase Structural Gene Region pPE101 was digested with EcoRI according to a conventional method, and the resulting DNA fragment mixture was subjected to electrophoresis to separate and recover the EcoRI DNA fragment (large fragment). .

Next, this large DNA fragment was further treated with various restriction enzymes to obtain smaller DNA fragments. An example thereof is shown in FIG.

Next, each of the small DNA fragments was individually transformed into pUC19.
A recombinant plasmid was prepared by inserting the recombinant plasmid into the multi-cloning site in the lacZ region of E. coli and the esterase activity in JM103 transformed with each recombinant plasmid was measured.

The preparation of each recombinant plasmid, the transformation of the host and the measurement of esterase activity are carried out according to the above 1-2).
The method was carried out according to the method of items 1 to 3). Also, each small DN
When inserting the A fragment into pUC19, a linker was synthesized and used as necessary.

The presence or absence of esterase activity of JM103 transformed with each recombinant plasmid is as shown in FIG. 3. From the results, the esterase structure was found in the ClaI-SmaI DNA fragment (about 1.3 kb) in the EcoRI large DNA fragment. It turned out that the gene exists.

1-5) Determination of Nucleotide Sequence of Esterase Structural Gene The entire nucleotide sequence of the ClaI-SmaI DNA fragment obtained in the above 1-4) was used for dideoxy ch
ain terminater method [F. Sanger, Science, 214 , 1205 (198
1)]. In addition, S in the obtained nucleotide sequence
From the position of D sequence and the DNA region essential for enzyme activity,
The open reading frame and corresponding amino acids were identified. The results (SEQ ID NO: 2) are shown below. CCCGGGCCGT GAGCGATGCC ATCCTCGGTG ACGACGACCT GCTGGCGCTA TATCAAGGCA 60 TCGACAACGG CCGCTTCCCC GGTGGCGACC TGCTGGCCGC ACCGCTGGAA GCCGCCGCCA 120 AGGCCTGGTA CCGGATGCGC GACCGCGCCT GATCGCCTGG CACCGCTCCT ACACGGCGCC 180 GGGCAGGCCG GAAGCATGGT GCAAGCCCAC TGCAGTGCAG TCACCACAAA TTCCGGCGCC 240 AAGCAAAATT CCTCCTATTC TCAATAGCTC ACTTCGCTTC CTGCACACAG GAGACCCGAC 300 C ATG AGC TAT GTA ACC ACG AAG GAC GGC GTA CAG ATC TTC TAC AAG GAC 349 Met Ser Tyr Val Thr Thr Lys Asp Gly Val Gln Ile Phe Tyr Lys Asp 1 5 10 15 TGG GGC CCG CGC GAT GCG CCG GTC ATC CAC TTC CAC CAC GGC TGG CCG 397 Trp Gly Pro Arg Asp Ala Pro Val Ile His Phe His His Gly Trp Pro 20 25 30 CTC AGT GCC GAC GAC TGG GAC GCG CAG ATG CTG TTC TTC CTC GCC CAC 445 Leu Ser Ala Asp Asp Trp Asp Ala Gln Met Leu Phe Phe Leu Ala His 35 40 45 GGT TAC CGC GTG GTC GCC CAC GAC CGC CGC GGC CAT GGC CGC TCC AGC 493 Gly Tyr Arg Val Val Ala His Asp Arg Arg Gly His Gly Arg Ser Ser 50 55 60 CAG GTA TGG GAC GGC CAC GAC ATG GAC CAC TAC GCC GAC GAC GTA GCC 541 Gln Val Trp Asp Gly His Asp Met Asp His Tyr Ala Asp Asp Val Ala 65 70 75 80 GCA GTG GTG GCC CAC CTG GGC ATT CAG GGC GCC GTG CAT GTC GGC CAC 589 Ala Val Val Ala His Leu Gly Ile Gln Gly Ala Val His Val Gly His 85 90 95 TCG ACC GGT GGC GGT GAG GTG GTG CGC TAC ATG GCC CGA CAC CCT GCA 637 Ser Thr Gly Gly Gly Glu Val Val Arg Tyr Met Ala Arg His Pro Ala 100 105 110 GAC AAG GTG GCC AAG GCC GTG CTG ATC GCC GCC GTA CCG CCG TTG ATG 685 Asp Lys Val Ala Lys Ala Val Leu Ile Ala Ala Val Pro Pro Leu Met 115 120 125 GTG CAG ACT CCC GAT AAT CCC GGT GGC CTG CCC AAA TCC GTT TTC GAC 733 Val Gln Thr Pro Asp Asn Pro Gly Gly Leu Pro Lys Ser Val Phe Asp 130 135 140 GGC TTC CAG GCC CAG GTC GCC AGC AAC CGC GCG CAG TTC TAC CGG GAT 781 Gly Phe Gln Ala Gln Val Ala Ser Asn Arg Ala Gln Phe Tyr Arg Asp 145 150 155 160 GTG CCG GCA GGG CCG TTC TAC GGC TAC AAC CGC CCC GGT GTC GAC GCC 829 Val Pro Ala Gly Pro Phe Tyr Gly Tyr Asn Arg Pro Gly Val Asp Ala 165 170 175 AGC GAA GGC ATC ATC GGC AAC TGG TGG CGC CAG GGC ATG ATC GGT AGC 877 Ser Glu Gly Ile Ile Gly Asn Trp Trp Arg Gln Gly Met Ile Gly Ser 180 185 190 GCC AAG GCC CAT TAC GAT GGC ATC GTG GCG TTT TCC CAG ACC GAC TTC 925 Ala Lys Ala His Tyr Asp Gly Ile Val Ala Phe Ser Gln Thr Asp Phe 195 200 205 ACC GAA GAC CTG AAG GGC ATT ACC CAG CCG GTG CTG GTG ATG CAT GGC 973 Thr Glu Asp Leu Lys Gly Ile Thr Gln Pro Val Leu Val Met His Gly 210 215 220 GAC GAC GAC CAG ATC GTG CCG TAT GAG AAC TCC GGG CTG CTG TCG GCC 1021 Asp Asp Asp Gln Ile Val Pro Tyr Glu Asn Ser Gly Leu Leu Ser Ala 225 230 235 240 AAG CTG CTG CCC AAT GGC ACA CTG AAG ACC TAC CAG GGC TAC CCG CAT 1069 Lys Leu Leu Pro Asn Gly Thr Leu Lys Thr Tyr Gln Gly Tyr Pro His 245 250 255 GGC ATG CCG ACC ACC CAT GCC GAT GTG ATC AAT GCG GAT TTG CTG GCG 1117 Gly Met Pro Thr Thr His Ala Asp Val Ile Asn Ala Asp Leu Leu Ala 260 265 270 TTT ATC CGT AGC TGA TGTGATCGCC TGCACCGGCC TCTTCGCGGG CACTGGCAAC 1172 Phe Ile Arg Ser 275 276 ACACCTCCC CCAGGATTAC CATGTCACGC TTCTAGTGCG GCCCTTTGCC GCCCCTTGC 1230 CTCCCTGCC TGCCAAAACC CCATGCCCTT CGAACTCACC GTAGAACCCC TCACCCTGC 1298 TGATCCTGG CCCTGGTCGC CTTCGTCGCC GGTTTCATCG AT 1329 In this sequence, the base at the 5'end is numbered 1.

Example 2 Esterase thermostability test Pseudomonas fluorescens IFO 3018
Strain, Pseudomonas putida MR-2068 strain (Ministry of Industrial Science and Technology No. 9677), and Escherichia coli JM1 in the above 1-5).
The E. coli JM109 (pPE116) strain obtained by transforming the 09 strain with the recombinant plasmid pPE116 was cultured under the following conditions.

(A) Pseudomonas fluorescens IFO 3018 strain; medium: LB medium (500 ml), culture temperature: 30 ° C., culture time: overnight.

(B) Pseudomonas Petida MR-20
68 strains (Microtech Lab. No. 9677); Medium: LB medium (500 ml), culture temperature: 30 ° C., culture time: overnight.

(C) Escherichia coli JM109 (pPE116)
Strain; Medium: LB medium (500 ml) containing 50 μg / ml ampicillin, culture temperature: 37 ° C., culture time: overnight.

Next, the thermostability of esterase of the cultured bacterial cells obtained by the above-mentioned culture procedure was measured by the following method.

After culturing, the cultured bacterial cells were collected and 1.0 g
(Wet weight) is taken and this is DL-β-acetylthio-α
-0.05M containing 5.0 g of methyl methylpropionate
Suspend in 100 ml of phosphate buffer (pH 7.0),
This was kept warm at 30 ° C. to start an enzymatic reaction, and the pH of the suspension was lowered to 7.0 according to the reaction by dropping a 0.1 N NaOH solution. At this time, the total amount (Q ₁ ) of NaOH consumed for maintaining the pH was determined per hour at the beginning of the reaction.

Next, 1.0 g of the collected cultured bacterial cells
(Wet weight) and take this 0.05M phosphate buffer (p
H7.0) in 100 ml and this is 20-80
After incubating for 3 hours under the temperature condition selected from the range of ° C, 5.0 g of methyl DL-β-acetylthio-α-methylpropionate was added, and the mixture was kept at 30 ° C and maintained at the same pH as above. The total amount of NaOH consumed (Q ₂ ) was calculated, and the residual esterase activity was calculated from the following formula: residual esterase activity = (Q ₂ ÷ Q ₁ ) × 100.

The results are shown in Table 1.

[0068]

[Table 1] From the results of Table 1, Pseudomonas putida MR-206
It is understood that the esterase produced by the 8 strain (b) and the Escherichia coli JM109 (pPE116) strain (c) has higher thermostability than the esterase produced by Pseudomonas fluorescens IFO 3018 strain (a).

[0069]

INDUSTRIAL APPLICABILITY The present invention provides a cloned DNA fragment containing a structural gene of esterase having good thermostability, which can be suitably used when hydrolyzing the carboxylic acid ester represented by the above formula (I).

In the prior art, a DNA fragment corresponding to such an esterase having a high thermostability has not been cloned. The cloned DNA fragment according to the present invention is provided by a thermostable recombinant DNA technique. It is extremely important as the basis of mass production technology of esterase with good properties.

[0071]

[Sequence list]

SEQ ID NO: 1 Sequence length: 831 Sequence type: Nucleic acid Number of strands: Single-stranded or double-stranded Topology: Linear Sequence type: Genomic DNA Origin: Organism: Pseudomonas putid
a ) Share name: MR-2068 strain Ministry of International Trade and Industry, Institute of Industrial Technology Microbial Industrial Research Institute Deposit No:
MICRO LABORATORY NO. 9677 (FERM P-9677) Characteristic of sequence Symbol representing the characteristic: mat peptide Location: 1..828 Method of determining the characteristic: S sequence ATGAGCTATG TAACCACGAA GGACGGCGTA CAGATCTTCT ACAAGGACTG GGGCCCGCGCCTCCACCCGGCTCG GTGCCGACGA CTGGGACGCG 120 CAGATGCTGT TCTTCCTCGC CCACGGTTAC CGCGTGGTCG CCCACGACCG CCGCGGCCAT 180 GGCCGCTCCA GCCAGGTATG GGACGGCCAC GACATGGACC ACTACGCCGA CGACGTAGCC 240 GCAGTGGTGG CCCACCTGGG CATTCAGGGC GCCGTGCATG TCGGCCACTC GACCGGTGGC 300 GGTGAGGTGG TGCGCTACAT GGCCCGACAC CCTGCAGACA AGGTGGCCAA GGCCGTGCTG 360 ATCGCCGCCG TACCGCCGTT GATGGTGCAG ACTCCCGATA ATCCCGGTGG CCTGCCCAAA 420 TCCGTTTTCG ACGGCTTCCA GGCCCAGGTC GCCAGCAACC GCGCGCAGTT CTACCGGGAT 480 GTGCCGGCAG GGCCGTTCTA CGGCTACAAC CGCCCCGGTG TCGACGCCAG CGAAGGCATC 540 ATCGGCAACT GGTGGCGCCA GGGCATGATC GGTAGCGCCA AGGCCCATTA CGATGGCATC 600 GTGGCGTTTT CCCAGACCGA CTTCACCGAA GACCTGAAGG GCATTACCCA GCCGGTGCTG 660 GTGATGCATG GCGACGACGA CCAGATCGTG CCGTATGAGA ACTCCGGGCT GCTGTCGGCC 720 AAGCTGCTGC CCAATGGCAC ACTGAAGACC TACCAGGGCT ACCCGCATGG CATGCCGACC 780 ACCCATGCCG ATGTGATCAA TGCGGATTTG CTGGCGTTTA TCCGTAGCTG A 831 SEQ ID NO: 2 Sequence length: Number of two-chain sequences: Nucleic acid sequence: Number of two-chain sequences: Nucleic acid sequence: Nucleotide sequence: Nucleotide sequence number: Nucleotide sequence number: 1329 Genomic DNA Origin: Organism name: Pseudomonas putid
a ) Strain name: MR-2068 strain Ministry of International Trade and Industry, Institute of Industrial Technology, Microbial Industry Research Institute Deposit No: Microindustrial Research Institute of Microbiology No. 9677 (FERM P-9677) Sequence features Characteristic symbols: mat peptide Location: 302 ..1129 method to determine the characteristics: S sequence CCCGGGCCGT GAGCGATGCC ATCCTCGGTG ACGACGACCT GCTGGCGCTA TATCAAGGCA 60 TCGACAACGG CCGCTTCCCC GGTGGCGACC TGCTGGCCGC ACCGCTGGAA GCCGCCGCCA 120 AGGCCTGGTA CCGGATGCGC GACCGCGCCT GATCGCCTGG CACCGCTCCT ACACGGCGCC 180 GGGCAGGCCG GAAGCATGGT GCAAGCCCAC TGCAGTGCAG TCACCACAAA TTCCGGCGCC 240 AAGCAAAATT CCTCCTATTC TCAATAGCTC ACTTCGCTTC CTGCACACAG GAGACCCGAC 300 C ATG AGC TAT GTA ACC ACG AAG GAC GGC GTA CAG ATC TTC TAC AAG GAC 349 Met Ser Tyr Val Thr Thr Lys Asp Gly Val Gln Ile Phe Tyr Lys Asp 1 5 10 15 TGG GGC CCG CGC GAT GCG CCG GTC ATC CAC TTC CAC CAC GGC TGG CCG 397 Trp Gly Pro Arg Asp Ala Pro Val Ile His Phe His His Gly Trp Pro 20 25 30 CTC AGT GCC GAC GAC TGG GAC GCG CAG ATG CTG TTC TTC CTC GCC CAC 445 Leu Ser Ala Asp Asp Trp Asp Ala Gln Met Leu Phe Phe Leu Ala His 35 40 45 GGT TAC CGC GTG GTC GCC CAC GAC CGC CGC GGC CAT GGC CGC TCC AGC 493 Gly Tyr Arg Val Val Ala His Asp Arg Arg Gly His Gly Arg Ser Ser 50 55 60 CAG GTA TGG GAC GGC CAC GAC ATG GAC CAC TAC GCC GAC GAC GTA GCC 541 Gln Val Trp Asp Gly His Asp Met Asp His Tyr Ala Asp Asp Val Ala 65 70 75 80 GCA GTG GTG GCC CAC CTG GGC ATT CAG GGC GCC GTG CAT GTC GGC CAC 589 Ala Val Val Ala His Leu Gly Ile Gln Gly Ala Val His Val Gly His 85 90 95 TCG ACC GGT GGC GGT GAG GTG GTG CGC TAC ATG GCC CGA CAC CCT GCA 637 Ser Thr Gly Gly Gly Glu Val Val Arg Tyr Met Ala Arg His Pro Ala 100 105 110 GAC AAG GTG GCC AAG GCC GTG CTG ATC GCC GCC GTA CCG CCG TTG ATG 685 Asp Lys Val Ala Lys Ala Val Leu Ile Ala Ala Val Pro Pro Leu Met 115 120 125 GTG CAG ACT CCC GAT AAT CCC GGT GGC CTG CCC AAA TCC GTT TTC GAC 733 Val Gln Thr Pro Asp Asn Pro Gly Gly Leu Pro Lys Ser Val Phe Asp 130 135 140 GGC TTC CAG GCC CAG GTC GCC AGC AAC CGC GCG CAG TTC TAC CGG GAT 781 Gly Phe Gln Ala Gln Val Ala Ser Asn Arg Ala Gln Phe Tyr Arg Asp 145 150 155 160 GTG CCG GCA GGG CCG TTC TAC GGC TAC AAC CGC CCC GGT GTC GAC GCC 829 Val Pro Ala Gly Pro Phe Tyr Gly Tyr Asn Arg Pro Gly Val Asp Ala 165 170 175 AGC GAA GGC ATC ATC GGC AAC TGG TGG CGC CAG GGC ATG ATC GGT AGC 877 Ser Glu Gly Ile Ile Gly Asn Trp Trp Arg Gln Gly Met Ile Gly Ser 180 185 190 GCC AAG GCC CAT TAC GAT GGC ATC GTG GCG TTT TCC CAG ACC GAC TTC 925 Ala Lys Ala His Tyr Asp Gly Ile Val Ala Phe Ser Gln Thr Asp Phe 195 200 205 ACC GAA GAC CTG AAG GGC ATT ACC CAG CCG GTG CTG GTG ATG CAT GGC 973 Thr Glu Asp Leu Lys Gly Ile Thr Gln Pro Val Leu Val Met His Gly 210 215 220 GAC GAC GAC CAG ATC GTG CCG TAT GAG AAC TCC GGG CTG CTG TCG GCC 1021 Asp Asp Asp Gln Ile Val Pro Tyr Glu Asn Ser Gly Leu Leu Ser Ala 225 230 235 240 AAG CTG CTG CCC AAT GGC ACA CTG AAG ACC TAC CAG GGC TAC CCG CAT 1069 Lys Leu Leu Pro Asn Gly Thr Leu Lys Thr Tyr Gln Gly Tyr Pro His 245 250 255 GGC ATG CCG ACC ACC CAT GCC GA T GTG ATC AAT GCG GAT TTG CTG GCG 1117 Gly Met Pro Thr Thr His Ala Asp Val Ile Asn Ala Asp Leu Leu Ala 260 265 270 TT TAT ATC CGT AGC TGA TGTGATCGCC TGCAC
CGGCC TCTTCGCGGG CACTGGCAAC 1172 Phe Ile Arg Ser 275 276 ACACCTCCC CCAGGATTAC CATGTCACGC TTCTAGTGCG GCCCTTTGCC GCCCCTTGC 1230 CTCCCTGCC TGCCAAAACC CCATGCCTTT 12

[Brief description of drawings]

FIG. 1 is a diagram showing the construction of a cloning vector pUC19.

FIG. 2 DNA containing the esterase structural gene of the present invention
It is a figure which shows the restriction enzyme map of the recombinant plasmid pPE101 in which the fragment was integrated.

FIG. 3 EcoRI in recombinant plasmid pPE101
Positional relationship of small DNA fragments lacking various portions of large DNA fragment in EcoRI large DNA fragment, and each small DN
It is a figure which shows the presence or absence of esterase activity of the transformant transformed with the recombinant plasmid prepared by individually incorporating the A fragment into pUC19.

[Explanation of Codes] Amp ampicillin resistance coding region lacZ β-galactosidase structural gene region lai repressor structural gene region Part derived from chromosomal DNA Part derived from pU19 B BglII C ClaI EI EcoRI EV EcoRV P PstI PV SVuII Sm SmaI

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] June 13, 1991

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0019

[Correction method] Change

[Correction content]

The reaction mixture obtained is preliminarily treated with CaCl ₂
For example, E. coli (E
scherichia coli) is introduced into JM10 5 strain, which is one of the K-12 strain.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0033

[Correction method] Change

[Correction content]

The cloning of the DNA fragment of the present invention comprises
The method is not limited to the above-mentioned method, various methods can be used, and as the cloning vector, in addition to the above pUC19,
pUC18, M13mp18, M13mp19 [all commercially available from Takara Shuzo Co., Ltd. ] can be selected and used according to the type of host.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0041

[Correction method] Change

[Correction content]

After completion of stirring, the mixture was centrifuged, and the supernatant was gently added to twice the amount of cold ethanol, and the precipitated DNA was attached to a glass rod and wound up, and taken out from the cold ethanol. Add 9 ml of this DNA to 0.1 x S
Dissolve in SC (1 × SSC: 0.15M NaCl, 15 mM trisodium citrate) and add 1 ml to the resulting solution.
Was obtained in 10 × SSC was added the DNA solution.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0048

[Correction method] Change

[Correction content]

1-3) Isolation of recombinant plasmid E. coli JM105Stock (available from Takara Shuzo Co., Ltd.)
Were cultured in LB medium at 37 ° C. for 2 to 3 hours, and then cultured cells
Was collected and ice-cooled to 50 mM CaCl ₂ In solution
Suspended. The cells were collected from this suspension by centrifugation again and re-collected.
Fresh 50 mM CaCl₂ Suspend in solution and chill with ice
While standing still for 30 minutes.

[Procedure Amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0050

[Correction method] Change

[Correction content]

After shaking, about 1 of the bacterial cell suspension in the test tube was used.
00 μl was added to LB agar plate (100 μg / ml
Ampicillin, 0.5 mM IPTG, 0.2% X-
Dispersed on including gal), kept overnight at 37 ° C. this.

[Procedure correction 6]

[Document name to be amended] Statement

[Correction target item name] 0053

[Correction method] Change

[Correction content]

Isolation and culture of one of the obtained transformants
And, Birnboim et al's method [Birnboim et al, Nucleic Acid
Res., 7, 1513-1523 (1979)] and prepared a restriction enzyme map . The result is shown in FIG. The recombinant plasmid having the structure shown in FIG.
It was PE101.

[Procedure Amendment 7]

[Document name to be amended] Statement

[Correction target item name] 0056

[Correction method] Change

[Correction content]

Next, each small DNA fragment was individually treated with pUC1.
Recombinant plasmids were prepared by inserting into the multi-cloning site within the lacZ region of 9 and the esterase activity in the JM105 strain transformed with each recombinant plasmid was measured.

[Procedure Amendment 8]

[Document name to be amended] Statement

[Name of item to be corrected] 0058

[Correction method] Change

[Correction content]

The presence or absence of esterase activity of the JM105 strain transformed with each recombinant plasmid is as shown in FIG. 3, and the results show that the ClaI-SmaI DNA fragment (about 1.3 k) in the EcoRI large DNA fragment was obtained.
It was found that there is an esterase structural gene in b).

[Procedure Amendment 9]

[Document name to be amended] Statement

[Correction target item name] 0060

[Correction method] Change

[Correction content]

Example 2 Esterase thermostability test Pseudomonas fluorescens IFO 3018
Strain, Pseudomonas putida MR-2068 strain (Ministry of Industrial Science and Technology No. 9677), and Escherichia coli JM1 in the above 1-5).
0 E. coli JM10 which the 5 strains obtained by transformation with the recombinant plasmid pPE116 5 (pPE116) strains were each cultured under the following conditions.

[Procedure Amendment 10]

[Document name to be amended] Statement

[Correction target item name] 0063

[Correction method] Change

[Correction content]

(C) Escherichia coli JM10 5 (pPE116)
Strain; Medium: LB medium (500 ml) containing 50 μg / ml ampicillin, culture temperature: 37 ° C., culture time: overnight.

[Procedure Amendment 11]

[Document name to be amended] Statement

[Correction target item name] 0068

[Correction method] Change

[Correction content]

[0068]

[Table 1] From the results of Table 1, Pseudomonas putida MR-206
It is understood that the esterase produced by the 8 strain (b) and the Escherichia coli JM10 5 (pPE116) strain (c) has higher thermostability than the esterase produced by Pseudomonas fluorescens IFO 3018 strain (a).

[Procedure Amendment 12]

[Document name to be amended] Statement

[Correction target item name] Explanation of code

[Correction method] Change

[Correction content]

[Explanation of Codes] Amp ampicillin resistance coding region lacZ β-galactosidase structural gene region lai repressor structural gene region Partial derived from partial chromosomal DNA derived from pU19 B BglII C ClaI EI EcoRI EV EcoRV P PstI PV PvuII S S Sm SmaI

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location C12R 1:40) (C12N 9/18 C12R 1:19) (C12P 41/00 C12R 1:19)

Claims (1)

[Claims] 1. The following general formula (I): (Wherein R ¹ is an alkyl group, an aralkyl group or an aryl group, R ² and R ³ are each independently an alkyl group, and n is 1 or 2). A DNA fragment having the following base sequence (SEQ ID NO: 1) encoding an esterase that catalyzes a decomposition reaction. ATGAGCTATG TAACCACGAA GGACGGCGTA CAGATCTTCT ACAAGGACTG GGGCCCGCGC 60 GATGCGCCGG TCATCCACTT CCACCACGGC TGGCCGCTCA GTGCCGACGA CTGGGACGCG 120 CAGATGCTGT TCTTCCTCGC CCACGGTTAC CGCGTGGTCG CCCACGACCG CCGCGGCCAT 180 GGCCGCTCCA GCCAGGTATG GGACGGCCAC GACATGGACC ACTACGCCGA CGACGTAGCC 240 GCAGTGGTGG CCCACCTGGG CATTCAGGGC GCCGTGCATG TCGGCCACTC GACCGGTGGC 300 GGTGAGGTGG TGCGCTACAT GGCCCGACAC CCTGCAGACA AGGTGGCCAA GGCCGTGCTG 360 ATCGCCGCCG TACCGCCGTT GATGGTGCAG ACTCCCGATA ATCCCGGTGG CCTGCCCAAA 420 TCCGTTTTCG ACGGCTTCCA GGCCCAGGTC GCCAGCAACC GCGCGCAGTT CTACCGGGAT 480 GTGCCGGCAG GGCCGTTCTA CGGCTACAAC CGCCCCGGTG TCGACGCCAG CGAAGGCATC 540 ATCGGCAACT GGTGGCGCCA GGGCATGATC GGTAGCGCCA AGGCCCATTA CGATGGCATC 600 GTGGCGTTTT CCCAGACCGA CTTCACCGAA GACCTGAAGG GCATTACCCA GCCGGTGCTG 660 GTGATGCATG GCGACGACGA CCAGATCGTG CCGTATGAGA ACTCCGGGCT GCTGTCGGCC 720 AAGCTGCTGC CCAATGGCAC ACTGAAGACC TACCAGGGCT ACCCGCATGG CATGCCGACC 780 ACCCATGCCG ATGTGATCAA TGCGGATTTG CTGGCGTTTA TCCGTAGCTG A 831 ... ( SEQ ID NO: 1)