JPH07163364A - Esterase gene - Google Patents

Abstract

PURPOSE:To obtain the subject gene capable of efficiently producing an esterase by preparing a plasmid integrated into a vector DNA and transferring it to a microorganism so as to remarkably improve the productivity of an esterase in the resultant transformant microorganism. CONSTITUTION:An esterase gene coding at least region from the first amino acid to the 362th amino acid of the amino acid sequence represented by the formula, a recombinant plasmid containing the gene, a microorganism containing the plasmid and a method for producing an esterase by using the microorganism containing the plasmid.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a gene encoding the amino acid sequence of esterase used in ester hydrolysis reaction, ester synthesis reaction, transesterification reaction, etc., a plasmid containing the gene, a microorganism containing the plasmid, Further, the present invention relates to a method for producing esterase, which comprises culturing the microorganism and producing esterase by the microorganism.

[0002]

2. Description of the Related Art In recent years, attempts have been actively made to utilize enzymes such as esterase in organic synthetic reactions such as hydrolysis. Examples of the microorganism-derived esterase used for such purpose include, for example, Arthrobacter.
globifomis IFO 12958 (JP-A-1-181788), Bacillus stearo
thermophilus (Archiv. Bioch
em. Biophys. 160, 504-513 (19
74)), Geotrichum candidum
(Agric. Biol. Chem. 37 (6), 14
57-1464 (1973)), Pseudomona
s aeruginosa (J. Biochem. 8
6, 643-656 (1979)), Pseudomo
NAS Fluorescens (J. Bioche
m. 95, 1047-1054 (1984)) and the like are known.

[0003]

However, the productivity of the enzymes in the microorganisms that produce them has not always been sufficient.

[0004]

Under these circumstances, the inventors of the present invention have conducted extensive studies and as a result, found a DNA fragment containing an esterase gene derived from a certain kind of microorganism, and analyzed the DNA fragment. By constructing a plasmid incorporated into vector DNA and introducing it into a microorganism, the productivity of esterase in a transformant microorganism was dramatically increased, and the present invention was completed. That is, the present invention provides an esterase gene (hereinafter referred to as the gene of the present invention) encoding an amino acid sequence represented by at least the 1st to 362nd amino acids of the amino acid sequence represented by SEQ ID NO: 1 and a plasmid containing the gene ( Hereinafter, referred to as the plasmid of the present invention), a microorganism containing the plasmid (hereinafter referred to as the microorganism of the present invention) and a method for producing esterase, which comprises culturing the microorganism and producing esterase by the microorganism (hereinafter, referred to as The present invention provides a method).

The present invention will be described in more detail below. The gene of the present invention is a chromobacterium (Chromobacteriu).
m) It is derived from a microorganism belonging to the genus, and the esterase encoded by it has the following properties. Molecular weight: 3
9348, pI: 3.9, optimum reaction pH: 9.0, optimum reaction temperature: 40 ° C. Further, this enzyme is dimethyl glutarate, dimethyl adipate, dimethyl pimelate, cis-
It shows activity against compounds such as carboxylic acid esters such as imidazolicin dicarboxylic acid methyl ester, triglyceride esters such as tripropionin, and amino acid ester compounds such as diethyl glutamic acid. The microorganisms belonging to the genus Chromobacterium are Ch
romobacterium SC-YM-1 (FERM P-14009)
Has been deposited with the Institute of Biotechnology, Industrial Technology Institute.

The gene of the present invention is, for example, J. Sambrook, EF
Fritsch, T. Maniatis; Molecular Cloning 2nd edition, Cold Spring Harbour.
Bor Laboratory), 1989, etc., and can be obtained according to the usual genetic engineering techniques. That is, in order to determine the N-terminal amino acid sequence of esterase, a microorganism is cultured by a usual culture method and the esterase produced by the microorganism is purified. In addition, for the culture, for example, a carbon source such as glucose, starch hydrolysate, molasses, etc., a nitrogen source such as yeast extract, meat extract, polypeptone, tryptone, inorganic ions and, if necessary, sulfate, phosphate, etc. as a medium are used. Using the usual ones containing, under aerobic conditions,
This can be performed by adjusting the pH and temperature of the medium as appropriate. The culturing time is preferably performed until the esterase activity in the medium is maximized, but it is not always necessary. The obtained cultured bacterial cells are ultrasonically disrupted and fractionated with ammonium sulfate, and then esterase is purified by a usual isolation / purification method using various chromatography such as ion exchange, hydrophobicity, gel filtration and the like. The obtained esterase is subjected to an amino acid sequence analyzer to determine the N-terminal amino acid sequence, and a synthetic DNA probe is prepared from the amino acid sequence information. Separately cultivated microbial cells are disrupted by an ordinary method such as ultrasonic disruption, treated with protease and the like, and then genomic DNA is extracted. The obtained genomic DNA is cleaved with an appropriate restriction enzyme and inserted into, for example, a phage vector λgt11 or a plasmid vector pUC19 using a ligase to prepare a genomic DNA library. For example, an immunological method using an antibody against esterase,
Synthetic DNA corresponding to partial amino acid sequence of esterase
The gene of the present invention can be obtained by a screening method such as a hybridization method using a probe or a method for measuring the activity of esterase. By using the gene of the present invention thus obtained, a large amount of esterase can be produced and obtained according to a usual genetic engineering method. More specifically, a plasmid that allows the gene of the present invention to be expressed in a host microbial cell is prepared, introduced into a host microbial cell for transformation, and the transformant microorganism is cultured. The above-mentioned plasmid contains a genetic information that can be replicated in a host microbial cell, is capable of autonomous growth, is easy to isolate and purify from the host microbial cell, and has an expression with a detectable marker. A vector into which the gene of the present invention is introduced can be preferably mentioned. Various kinds of expression vectors are already on the market, and the restriction enzyme for cleaving the expression vector used for introducing the gene of the present invention is also on the market. For example, for expression in E. coli, lac, tr
Expression vectors containing promoters such as p and tac (these are commercially available as expression vectors or promoter cartridges from Pharmacia PL) can be used. Furthermore, by connecting a ribosome binding region upstream of the gene of the present invention, higher expression can be achieved. Guarent as a ribosome binding region
Report by eL et al. (Cell 20 p543 (1980)) and Taniguchi et al.
The report by etics of Industrial Microorganisms p202 (1982) Kodansha) is known, but SD-1 and SD-2 described later can be preferably mentioned. Thus, a ribosome binding region suitable for expressing the gene of the present invention may be designed and synthesized. In order to introduce the above-mentioned plasmid into host microbial cells, ordinary genetic engineering methods are used. As the host microbial cell, either a eukaryote or a prokaryote can be used, and preferably E. coli or the like can be mentioned. The host microbial cell into which the above-mentioned plasmid has been introduced can be cultivated by a culturing method usually used for the microorganism. For example, in the case of Escherichia coli, it is cultivated in an ordinary medium containing appropriate carbon sources, nitrogen sources and trace nutrients such as vitamins. The culturing method may be either solid culturing or liquid culturing, but preferably the liquid culturing method with aeration and stirring can be mentioned. Collection of esterase from the cultured bacterial cells of the microorganism of the present invention thus obtained may be carried out by appropriately combining ordinary isolation / purification methods. For example, after completion of the culture, the bacterial cells are centrifuged or the like. It may be purified by combining the steps of using various chromatographies such as ion exchange, hydrophobicity, gel filtration, etc. The recombinant bacterial cell producing esterase thus produced can be used as a bioreactor for ester hydrolysis to produce useful compounds such as intermediates for pharmaceuticals and agricultural chemicals. Further, by culturing the microorganism of the present invention, it is also possible to obtain a cultured microbial cell in which a large amount of esterase is accumulated and use this as it is as an enzyme reactor.

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

Example 1 (Separation of esterase and N
Determination of terminal amino acid sequence) Chromobacter which is a strain belonging to the genus Chromobacterium
ium SC-YM-1 was added to 5 ml of preculture medium (glucose 1
% (W / V), yeast extract 1% (W / V), K₂HPO _Four0.1
% (W / V), MgSO_Four0.02% (W / V), pH 7.3)
After shaking culture at 30 ° C for 24 hours, the culture solution obtained
1000 ml of medium for main culture (glucose 1% (W / V)
 , Yeast extract 1% (W / V), K₂HPO_Four0.1% (W / V)
 , MgSO_FourInoculate to 0.02% (W / V), pH 7.3)
And incubate at 30 ° C until the OD 660 reaches 10.
Cultivate and centrifuge (8000g, 10 minutes, 4 ℃)
I recovered my body. Recovered cells were treated with 0.1M phosphate buffer
(1 mM EDTA, 5 mM MgCl₂, PH 7.5)
Suspend in 800 ml, French press treatment (2000p
It was crushed by si). Centrifuge (8
000g, 10 minutes, 4 ° C) to obtain the supernatant. further
This was ultracentrifuged (110,000 g, 60 minutes, 4 ° C)
To obtain the supernatant. Ammonium sulfate (25% (W / V) was added to this supernatant.
Saturation) was added and the mixture was allowed to stand at 4 ° C. for 2 hours.
Removed by centrifugation (10000g, 15 minutes, 4 ° C)
did. Subsequently, ammonium sulfate (55% (W / V) was added to the obtained supernatant.
Saturation) was added and the mixture was allowed to stand at 4 ° C. for 2 hours.
Centrifuge (10000 g, 15 minutes, 4 ° C) to remove the precipitate.
Recovered. This precipitate was added to 0.01M phosphate buffer (pH
After suspending it in 7.5), add 150 ml of this to anion exchange.
Replacement resin (DEAE-Sepharose fastfl
ow, manufactured by Pharmacia) 200 ml packed column
The target enzyme was adsorbed on the carrier. 0.15M N
Color with aCl + 0.01M phosphate buffer (pH 7.5)
After thoroughly washing the membrane and removing the non-adsorbed components, 0.15-
Dissolve the target enzyme by the 0.35M NaCl linear gradient method.
I put it out. Collect active fractions of the target enzyme esterase
Therefore, hydrophobic resin (Butyl-Toyopearl 65
Column filled with 200 ml of 0S, manufactured by Toyo Soda Kogyo Co., Ltd.
Passed through. 10% (W / V) (NH_Four)₂SO_Four+0.01
Thoroughly wash the column with M phosphate buffer (pH 7.5)
Then, after removing the non-adsorbed portion, 10-0% (W / V) saturated sulfuric acid
The target enzyme was eluted by the linear ammonium gradient method.
Collect the active fractions of the target enzyme esterase and collect by gel filtration.
Over (Sephacryl S-200, Pharmacia
Column chromatography, 0.01M phosphorus
It was eluted with an acid buffer (pH 7.5). Is the target enzyme
Collect the active fractions of esterase to obtain purified esterase.
It was The esterase activity is p, which is a general substrate.
-Activity against nitrophenyl acetate (pNPA)
Was measured. The activity was measured by dissolving in 2% (W / V) acetone.
0.1M phosphate buffer containing 40 μM substrate (pH 7.
2) Add enzyme solution and incubate at 37 ℃ to release
The amount of p-nitrophenol to increase the absorbance at 405 nm.
It was measured by adding. The activity is 1 μmol p / min
-The amount of enzyme that releases nitrophenol is 1 unit
did. Dissolve 1 ng of purified esterase in 30 μl of distilled water
Gas phase sequencer (Applied Biosystems
N-terminal amino acid sequence was determined with a model 373A)
The amino acid sequence as shown below (shown in SEQ ID NO: 1)
The amino acid sequence shown from the 2nd to the 32nd
It was). Thr Leu Phe Asp Gly Ile Thr Se
r Arg Ile Val Asp Thr Asp Arg Leu Thr Val Asn Ile
Leu Glu Arg Ala Ala Asp Asp Pro Gln Thr Pro

Example 2 (single clone of the gene clone of the present invention)
Separation: Preparation of genomic DNA) Chromobacter which is a strain belonging to the genus Chromobacter
ium SC-YM-1 was added to 5 ml of preculture medium (glucose 1
% (W / V), yeast extract 1% (W / V), K₂HPO _Four0.1
% (W / V), MgSO_Four0.02% (W / V), pH 7.3)
After shaking culture at 30 ° C. for 24 hours, the culture solution obtained
1000 ml of medium for main culture (glucose 1% (W / V)
 , Yeast extract 1% (W / V), K₂HPO_Four0.1% (W / V)
 , MgSO_FourInoculate to 0.02% (W / V), pH 7.3)
And cultured at 30 ° C. At that time, OD 660 was 3.4
When reached, the penicillin G is used as the final concentration for 2 units.
OD 660 of 10
The culture was continued until the temperature reached. Centrifuge (8000g,
10 minutes, 4 ℃) to collect the cells, 80ml of 10m
M Tris-HCl buffer (pH 8.0), 25% (W / V)
Final concentration of egg white lysozyme (Seikagaku Corporation) in sugar solution
To 5 mg / ml, and then at 37 ℃ for 30 minutes
Incubated for a period of time. Next, 10 ml of 10% (W / V) S
DS was added, and protease K (Boehringer
Product) to a final concentration of 200 μg / ml,
Incubated at 37 ° C for 3 hours. Then an equal amount
0.1M Tris saturated phenol 3 times, ether 2 times
After extraction, add 2 volumes of ethanol to the aqueous layer,
Precipitate the nucleic acid and centrifuge (12000 g, 30 minutes, 4
℃). After drying the obtained nucleic acid, 20 ml of Tris E
DTA buffer (10 mM Tris-HCl, 1 mM EDT
A, pH 8.3) and equilibrated with CsCl-EtBr.
Gradient ultracentrifugation (275000g, 18 hours, 25
℃), collect the DNA band, and use it for Tris-EDT
A buffer (10 mM Tris-HCl, 1 mM EDTA, p
Dialysis against H8.3) and about 5.4 mg of genomic DN
I got A.

Example 3 (Isolation of gene clone of the present invention: preparation of genomic DNA library) 100 μg of the genomic DNA obtained in Example 2 was digested with XhoI (restriction enzyme manufactured by Takara Shuzo) to obtain a genomic DNA fragment. On the other hand, 1 μg of λ phage λZAPII (manufactured by Stratagene) was also digested with XhoI, and
Mix with A fragment, add ligase (Takara Shuzo),
C reacted overnight. Next, this reaction solution was packaged in Escherichia coli XL-1blue strain (attached to the kit) using an in vitro packaging kit (manufactured by Stratagene) to prepare a genomic DNA library.

Example 4 (Isolation of gene clone of the present invention: screening of genomic DNA library) Preparation of synthetic DNA probe and isotope labeling Based on the N-terminal amino acid sequence of esterase determined in Example 1, the following is shown. A 44 mer oligonucleotide (mixed probe) was synthesized. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Thr Leu Phe Asp Gly Ile Thr Ser Arg Ile Val Asp Thr Asp Arg 5'ACI CTI TTC GAC GGI ATC ACI TGI CGI ATC GTI GAC ACI GAC CG 3 'C Oligonucleotides were synthesized using an automatic DNA synthesizer (Applied Biosystems model 380A). 50 μmol of this probe DNA contained 3 μl of 0.5 M Tris-HCl (pH 7.6) and 0.1 M MgC.
l ₂ , 0.05M DTT, 0.001M EDTA,
10 units T4 polynucleotide kinase (manufactured by Takara Shuzo) and 10 μl [γ ³² P] ATP (manufactured by Amersham) were mixed and reacted at 37 ° C. for 60 minutes, and then gel filtration using Sephadex G-50 (manufactured by Pharmacia). Was carried out to prepare a DNA probe labeled with an isotope. Screening of genomic DNA library As shown in Example 3, phage-infected E. coli was plated using an in vitro packaging kit (Stratagene), the plate was cooled to 4 ° C., and then surface-coated. A nitrocellulose filter was brought into close contact with and phages were transferred onto the filter. Soak the filter in 1.5M NaCl-0.5M NaOH solution,
Denature the attached DNA, then add 1.5M NaCl
-Neutralized with 0.5 M Tris-HCl (pH 8.0) solution.
Then 0.36M NaCl-20 mM NaH ₂ PO
_The filter was washed with ₄ (pH 7.5) -2 mM EDTA (pH 7.5) and then dried. Next, using the isotope-labeled DNA probe corresponding to the N-terminal amino acid sequence of esterase prepared as described above, the genomic DNA library prepared in Example 3 was subjected to plaque hybridization according to the following method. . That is, filter 1) 4xSSC, 1% (W
/ V) SDS, 10x Denhardt (0.2% (W / V) Ficoll, 0.2% (W / V) Polyvinylpyrrolidone, 0.2%
(W / V) Bovine serum albumin) in solution containing 60 ℃, 30
After incubation for 2 minutes, 2) a solution containing 5xSSC, 5xDenhardt, 100 μg / ml salmon testis DNA,
The reaction was carried out by incubating at 60 ° C for 5 hours. Hybridization was carried out by adding a filter and the solution of 2) above to a plastic bag, and isotopically labeled D
About 5x10 ⁵ cp of NA probe per filter
m and added and the mixture was heated at 60 ° C. overnight. The hybridized filter was 1) 2xSSC, 0.5.
% (W / V) SDS solution at 60 ℃, 15 minutes 2) 2x
SSC, 0.5% (W / V) SDS solution at 25 ℃, 3
0 minutes 3) After washing with a solution containing 2xSSC and 0.5% SDS (W / V) at 60 ° C for 15 minutes in that order, air-drying, and applying X-ray film (FUJI RX) and intensifying screen to -80 ℃,
I took an autoradiogram overnight. As a result, a pCC3 strain giving a positive signal was obtained. As a result of determining the nucleotide sequence by the dideoxy method, the obtained clone strain did not encode the entire length of the esterase gene. Therefore, a part of the sequence was again used as a DNA probe for screening by plaque hybridization. At that time, a library prepared by partially digesting genomic DNA with Sau3AI and similarly ligating to λ phage λZAPII (manufactured by Stratagene) was used. As a result, 9 strains of the gene clone of the present invention giving a positive signal were obtained.

Example 5 (Determination of restriction enzyme map and total nucleotide sequence of gene of the present invention) A restriction enzyme map of insert DNA was prepared for the gene clone of the present invention obtained in Example 4. λZAPII
The insert DNA cloned in J. Sambrook, E.
F. Fritsch, T. Maniatis; Molecular Cloning 2nd edition, Cold Spring Harbor Laboratory.
arbor Laboratory), 1989, etc. and infecting the helper phage R408 with the usual method described in
It was excised in vivo and subcloned. The thus obtained plasmid pCC6 (see FIG. 4) was digested with various restriction enzymes, and the DNA fragment was analyzed by 1% agarose gel and 5% polyacrylamide gel electrophoresis.
A restriction enzyme map was prepared by comparing the chain lengths of various DNA fragments. DN of about 1.8 kbp containing a region having an affinity for the probe DNA present in the vector pCC6
The base sequence of the A fragment was determined using a DEAZA sequence kit (Takara Shuzo). The determined nucleotide sequences are shown in FIGS. 1 to 3 and SEQ ID NO: 2. As a result of searching the open reading frame from the base sequence, as shown in FIGS. 1 to 3 and SEQ ID NO: 1, it is found that the ATG at the base number 343th to the GAC at the 1452th position is the only open reading frame. found. In addition, since the N-terminal amino acid sequence of esterase determined in Example 1 and the amino acid sequence below the start codon of the above open reading frame matched, this open reading frame encodes esterase. It became clear.
As a result, it was revealed that esterase is a protein having a molecular weight of 39348 consisting of 370 amino acid residues.

Example 6 (Expression plasmid containing the gene of the present invention) Expression vector pKK223-3 (Boyer et al., Prot. Natl.
Acad.Sci.USA, 80,21-25 (1983), made by Pharmacia)
By partial digestion with BamHI, blunting with T4 DNA polymerase, and ligation
PKK223-4 having one BamHI site deleted was prepared. On the other hand, around the start codon of the gene of the present invention and its 5 ′
In order to convert the upstream nucleotide sequence into a sequence suitable for gene expression in Escherichia coli, a DNA fragment having the following nucleotide sequence was synthesized using Applied DNA Synthesizer Model 380A. LP-1 (shown in SEQ ID NO: 3) LP-2 (shown in SEQ ID NO: 4) ES-1 (shown in SEQ ID NO: 5) ES-2 (shown in SEQ ID NO: 6) ES-3 (shown in SEQ ID NO: 7) ES-4 (shown in SEQ ID NO: 8) ES-5 (shown in SEQ ID NO: 9) ES-6 (shown in SEQ ID NO: 10) ES-7 (shown in SEQ ID NO: 11) Synthesized LP-2, ES-1, ES-2, ES-3, E
The 5'ends of the S-5, ES-6 and ES-7 fragments were phosphorylated, and each untreated fragment was ligated and annealed to give the following two types of double-stranded DNA fragments (SD-1, SD-2) was prepared. Prepared double-stranded D
The NA fragments (SD-1, SD-2) were phosphorylated at both ends. SD-1 <LP-1><ES-1><AATTCTTTTT TAATAAAATC AGGAGGAAAA ACATATGACT CTGTTCGATG GTATCACTTC GAAAAA ATTATTTTAG TCCTCCTTTT TGTATACTGA GACAAGCTAC CATAGTGAAG <LP-2><ES-2 EcoRI ES-3><ES-5> GCGAATCGTA GTGACTGATCCATC CGCTTAGCAT CTATGACTAG CAGACTGACA ATTGTAGGAC CTTGCACGCC GG><ES-4> Eco52I SD-2 <LP-1><ES-7><AATTCTTTTT TAATAAAATC AGGAGGAAAA AACATATGAC TCTGTTCGAT GGTATCACTT GAAAAAA ATTATTTTAG TCCTCCTTTT TGCTATACATA <TGATA LPTG -3><ES-5> CGCGAATCGT AGATACTGAT CGTCTGACTG TTAACATCCT GGAACGTGC GCGCTTAGCA TCTATGACTA GCAGACTGAC AATTGTAGGA CCTTGCACGC CGG><ES-4> Eco52I On the other hand, the SacI fragment in pCC6 (about 3.5 kbp) was cloned into a sub company, pUC118.
CC30 was produced. This pCC30 is Eco52I,
The translation region of esterase (about 1.2 Kbp) was excised by digestion with SacI. On the other hand, pUC118 having a lac promoter was transformed with EcoRI and Sac.
It was digested with I and treated with alkaline phosphatase.
Then, between the EcoRI and SacI sites of pUC118, the DNA fragment (SD-1 or SD-2) and the Eco52I-Sa containing the gene translation region of the present invention are contained.
cI fragment is DNA ligation kit (Takara Shuzo)
Ligation with pCC100 and pC
C101 was produced (see FIG. 5). Then this pCC1
00 and pCC101 to EcoRI and HindIII
Digested with, translation region of esterase (about 1.3 Kbp)
Cut out. On the other hand, pK having the tac promoter
K223-4 was digested with EcoRI and HindIII,
Alkaline phosphatase treatment was performed. Then this p
EcoRI-Hind containing an esterase translation region between the EcoRI and HindIII sites of KK223-4
The III fragment (1.3 Kbp) was ligated using a DNA ligation kit (Takara Shuzo) to obtain pC.
C200 and pCC201 were produced (see FIG. 5).
In this way, four types of expression plasmids pCC100 (SD-1, pUC11 for Escherichia coli having modified 5'terminal base sequences downstream of the lac and tac promoters were used.
8), pCC101 (SD-2, pUC118), pC
C200 (SD-1, pKK223-4) and pCC
201 (SD-2, pKK223-4) was obtained. ─────────────────────────────────── The constructed expression plasmid name Modified 5'terminal nucleotide sequence vector ─── ─────────────────────────────────────── pCC100 SD-1 pUC118 pCC101 SD-2 pUC118 pCC200 SD-1 pKK233-4 pCC201 SD-2 pKK223-4 ────────────────────────────────────

Example 7 (Production of Esterase by Transformant Microorganism Introduced with Gene of the Present Invention) Escherichia coli JM109 was transformed with each of the four expression plasmids obtained in Example 6 according to a conventional genetic engineering method. Then, a recombinant microorganism was prepared. LB medium (tryptone 1% (W / V), yeast extract 0.
5% (W / V), NaCl 0.5% (W / V))
Cultivated at ℃, IPTG (isopropyl-β
-D-thiogalactopyranoside) was added to a final concentration of 1 mM to induce the expression of esterase. After the completion of the culture, the cells were collected by centrifugation (8000 g, 10 minutes, 4 ° C.), and part of them was subjected to SDS-PAGE. As a result, esterase was recognized as a main band at a position of molecular weight of about 40,000, It was found that all of the enzymes were highly expressed in the microorganism. Therefore, p-nitrophenylacetate (pNP), which is a general substrate for esterase, is used.
The esterase production of these transformant microorganisms was measured relative to A). The amount of p-nitrophenol released was measured by adding the enzyme solution to 0.1 M phosphate buffer (pH 7.2) containing 40 μM of substrate dissolved in 2% (W / V) acetone and incubating at 37 ° C. Was measured from the increase in absorbance at 405 nm. Activity is 1μ per minute
The amount of enzyme that releases mol of p-nitrophenol is 1
It was a unit. The results are shown in Table 1.

[Table 1] ─────────────────────────────── Bacterial strain production (units / OD 660) ───── ────────────────────────── pCC100 / JM109 27.0 pCC101 / JM109 63.5 pCC200 / JM109 45.4 pCC201 / JM109 35.7 ─────────────────────────────── Furthermore, E. coli JM105 and JM103 were respectively transformed with the above four expression plasmids, Similarly, after inoculating into LB medium, it was cultured at 37 ° C., and IPTG (isopropyl-β-D-thiogalactoside) was grown in the logarithmic growth phase.
Was added to a final concentration of 1 mM to induce the expression of esterase. The esterase production of these transformant microorganisms was measured by the same method as above. The results are shown in Table 2.

[Table 2] ─────────────────────────────── Bacterial strain production (units / OD ₆₆₀ ) ──── ─────────────────────────── pCC100 / JM105 25.4 pCC101 / JM105 142.0 pCC200 / JM105 42.0 pCC201 / JM105 40. 4 pCC100 / JM103 55.4 pCC101 / JM103 55.2 pCC200 / JM103 41.1 pCC201 / JM103 56.1 ────────────────────────── ──────

Example 8 (Purification and Characterization of Esterase Produced by Transformant Microorganism Introduced with Gene of the Present Invention) The recombinant microorganism (pCC101 / JM105) cultured by the method shown in Example 7 was ultrasonically disrupted. (20KH
z, 15 minutes, 4 ° C.) and then centrifugation (12000 g, 30)
Minutes, 4 ° C.) to obtain the supernatant. The obtained supernatant 15
0 ml of anion exchange resin (DEAE-Sepharo
se fastflow made by Pharmacia) 200m
The target enzyme was adsorbed on the carrier by passing it through a column filled with l. The column was thoroughly washed with 0.15 M NaCl + 10 mM Tris-HCl buffer (pH 7.5) to remove the non-adsorbed portion, and then the target enzyme was eluted by the 0.15-0.35 M NaCl linear concentration gradient method. The active fractions of esterase, the target enzyme, were collected and collected by a hydrophobic resin (Butyl-Toy
pearl 650S, manufactured by Toyo Soda Kogyo Co., Ltd.) 200 ml
Was passed through a column packed with. 10% (W / V) of (NH ₄ )
_The column was thoroughly washed with ₂ SO ₄ +10 mM Tris-HCl buffer (pH 7.5) to remove non-adsorbed components, and then 10-
The target enzyme was eluted by a 0% (W / V) saturated ammonium sulfate linear concentration gradient method. The active fraction of esterase, which is the target enzyme, was collected to obtain a purified esterase. The activity was measured according to the method described in Example 1. When the obtained purified esterase was subjected to mass spectrometry by ESI (electrospry ionization) mass spectrum and C-terminal amino acid analysis by an enzymatic method, a full-length esterase having a molecular weight of 39348 (hereinafter referred to as L-form) and a C-terminal amino acid having a molecular weight of 38508 were identified. Esterase degradation products (hereinafter referred to as S-form) with 8 deletions were detected. The obtained full-length esterase (L
Substrate specificity of various fatty acid esters, triglycerides and amino acid esters for the compounds was examined by the following activity measuring method. The hydrolysis activity was measured by adding the enzyme solution to 0.2 M phosphate buffer (pH 8.0) containing 0.5 M of each substrate and reacting at 35 ° C.
The produced fatty acid and amino acid were titrated and quantified with a 0.05N NaOH solution using phenolphthalein as an indicator. The activity was defined as 1 unit of the amount of enzyme that liberates 1 μmol of fatty acid / amino acid in 1 minute. The results are shown in Table 3. The enzyme activity in Table 3 is shown as a relative value (%) when the activity against dimethyl glutarate is 100. Further, the obtained esterase degradation product (S form) also had almost the same effect.

[Table 3] ────────────────────────── Substrate Relative activity (%) ────────────── ───────────── Dimethyl malonate 50 Dimethyl succinate 16 Dimethyl glutarate 100 Dimethyl adipate 32 Dimethyl pimelate 15 Dimethyl suberate 8 Diethyl glutarate 82 Methyl glutarate 45 Methyl acetate 11 Propionate Methyl 14 Methyl butyrate 12 Methyl valerate 7 Methyl caproate 3 Methyl heptanoate 6 Tributyrin 3 Tripropionine 4 Glycine methyl ester 2 Glutamic acid diethyl ester 58 Arginine methyl ester 6 Phenylalanine methyl ester 4 ───────────── ─────────────

[0015]

By constructing a plasmid in which a DNA fragment containing the gene of the present invention is inserted into a vector and introducing it into a microorganism, the productivity of esterase in a transformant microorganism can be dramatically increased, Esterase can be produced efficiently.

[0016]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 370 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence 5 10 15 Met Thr Leu Phe Asp Gly Ile Thr Ser Arg Ile Val Asp Thr Asp Arg 20 25 30 Leu Thr Val Asn Ile Leu Glu Arg Ala Ala Asp Asp Pro Gln Thr Pro 35 40 45 Pro Asp Arg Thr Val Val Phe Val His Gly Asn Val Ser Ser Ala Leu 50 55 60 Phe Trp Gln Glu Ile Met Gln Asp Leu Pro Ser Asp Leu Arg Ala Ile 65 70 75 80 Ala Val Asp Leu Arg Gly Phe Gly Gly ser Glu His Ala Pro Val Asp 85 90 95 Ala Thr Arg Gly Val Arg Asp Phe Ser Asp Asp Leu His Ala Thr Leu 100 105 110 Glu Ala Leu Asp Ile Pro Val Ala His Leu Val Gly Trp Ser Met Gly 115 120 125 Gly Gly Val Val Met Gln Tyr Ala Leu Asp His Pro Val Leu Ser Leu 130 135 140 Thr Leu Gln Ser Pro Val Ser Pro Tyr Gly Phe Gly Gly Thr Arg Arg 145 150 155 160 Asp Gly Ser Arg Leu Thr Asp Asp Asp Ala Gly Cys Gly Gly Gly Gly 165 170 175 Ala Asn Pro Asp Phe Ile Gln Arg Leu Ile Asp His Asp Thr Ser Asp 180 185 190 Asp Ala Gln Thr Se r Pro Arg Ser Val Phe Arg Ala Gly Tyr Val Ala 195 200 205 Ser Asp Tyr Thr Thr Asp His Glu Asp Val Trp Val Glu Ser Met Leu 210 215 220 Thr Thr Ser Thr Ala Asp Gly Asn Tyr Pro Gly Asp Ala Val Pro Ser 225 230 235 240 Asp Asn Trp Pro Gly Phe Ala Ala Gly Arg His Gly Val Leu Asn Thr 245 250 255 Met Ala Pro Gln Tyr Phe Asp Val Ser Gly Ile Val Asp Leu Ala Glu 260 265 270 Lys Pro Pro Ile Leu Trp Ile His Gly Thr Ala Asp Ala Ile Val Ser 275 280 285 Asp Ala Ser Phe Tyr Asp Leu Asn Tyr Leu Gly Gln Leu Gly Ile Val 290 295 300 Pro Gly Trp Pro Gly Glu Asp Val Ala Pro Ala Gln Glu Met Val Ser 305 310 315 320 Gln Thr Arg Asp Val Leu Gly Arg Tyr Ala Ala Gly Gly Gly Thr Val 325 330 335 Thr Glu Val Ala Val Glu Gly Ala Gly His Ser Ala His Leu Glu Arg 340 345 350 Pro Ala Val Phe Arg His Ala Leu Leu Glu Ile Ile Gly Tyr Val Gly 355 360 365 Ala Ala Ala Asp Pro Ala Pro Pro Thr Glu Ala Ile Ile Ile Arg Ser 370 Ala Asp

SEQ ID NO: 2 Sequence length: 1110 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: genomic DNA Origin organism name: Chromobact
erium) strain name; SC-YM-1 Sequence features: Characteristic symbol: CDS Location: 1. ． ． 1110 method to determine the characteristics: E SEQ ATGACCCTGT TCGACGGCAT CACGTCTCGC ATCGTCGACA CCGACCGCCT GACCGTGAAC 60 ATCCTGGAGC GCGCGGCCGA CGACCCGCAG ACCCCGCCCG ACCGCACGGT CGTGTTCGTC 120 CACGGGAATG TGTCCTCCGC GCTGTTCTGG CAGGAGATCA TGCAGGACCT GCCGAGCGAC 180 CTGCGCGCCA TCGCGGTCGA CCTGCGCGGC TTCGGCGGCT CGGAGCACGC GCCGGTCGAC 240 GCCACCCGCG GCGTCCGCGA CTTCAGCGAC GATCTGCACG CGACCCTCGA GGCGCTCGAC 300 ATCCCGGTCG CGCATCTGGT CGGCTGGTCG ATGGGCGGCG GCGTCGTCAT GCAGTATGCC 360 CTCGACCACC CGGTGCTGAG CCTGACCCTG CAGTCGCCGG TGTCGCCCTA CGGCTTCGGC 420 GGCACCCGCC GTGACGGCTC ACGCCTCACC GACGACGATG CCGGCTGCGG TGGCGGCGGT 480 GCGAACCCCG ACTTCATCCA GCGCCTCATC GACCACGACA CCTCCGACGA TGCGCAGACC 540 TCGCCCCGGA GCGTCTTCCG CGCCGGCTAC GTCGCCTCGG ACTACACCAC CGACCACGAG 600 GACGTGTGGG TCGAATCGAT GCTCACCACG TCCACCGCCG ACGGAAACTA CCCCGGCGAT 660 GCGGTGCCGA GCGACAACTG GCCGGGCTTC GCCGCCGGCC GCCACGGCGT GCTGAACACC 720 ATGGCCCCGC AGTACTTCGA TGTGTCGGGG ATTGTCGACC TGGCCGAGAA GCCTCCGATC 780 CTGTGGATCC ACGGCACCGC GGACGCGATC GTCT CCGACG CGTCGTTCTA CGACCTCAAC 840 TACCTCGGCC AGCTGGGCAT CGTCCCCGGC TGGCCCGGCG AAGACGTCGC GCCCGCGCAG 900 GAGATGGTGT CGCAGACCCG CGATGTCCTC GGCCGCTACG CTGCGGGCGG CGGAACGGTC 960 ACCGAGGTCG CCGTCGAGGG CGCGGGCCAC TCCGCGCACC TGGAGCGTCC CGCGGTGTTC 1020 CGCCACGCGC TGCTCGAGAT CATCGGCTAC GTCGGCGCGG CGGCCGACCC CGCCCCGCCG 1080 ACCGAGGCGA TCATCATCCG CTCCGCCGAC 1110

SEQ ID NO: 3 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence AATTCTTTTT TAATAAAATC 20

SEQ ID NO: 4 Sequence length: 26 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TTTTCCTCCT GATTTTATTA AAAAAG 26

SEQ ID NO: 5 Sequence length: 29 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence AGGAGGAAAA ACATATGACT CTGTTCGAT 29

SEQ ID NO: 6 Sequence length: 36 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GATTCGCGAA GTGATACCAT CGAACAGAGT CATATG 36

SEQ ID NO: 7 Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GGTATCACTT CGCGAATCGT AGATACTGAT 30

SEQ ID NO: 8 Sequence length: 45 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GGCCGCACGT TCCAGGATGT TAACAGTCAG ACGATCAGTA TCTAC 45

SEQ ID NO: 9 Sequence length: 29 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CGTCTGACTG TTAACATCCT GGAACGTGC 29

SEQ ID NO: 10 Sequence length: 37 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GATTCGCGAA GTGATACCAT CGAACAGAGT CATATGT 37

SEQ ID NO: 11 Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence AGGAGGAAAA AACATATGAC TCTGTTCGAT 30

[Brief description of drawings]

FIG. 1 is a diagram showing the 1st to 642nd base sequences of the insert DNA of vector pCC6 and the amino acid sequence of esterase deduced therefrom. The probe DNA used for the screening was base numbers 346 to 39.
Corresponds to the 0th.

FIG. 2: 643 of insert DNA of vector pCC6
It is a figure which shows the amino acid sequence of the esterase inferred from the nucleotide sequence from the 10th to the 1074th nucleotide.

FIG. 3: 107 of insert DNA of vector pCC6
It is a figure which shows the amino acid sequence of the esterase inferred from the 5th to 1639th base sequence.

FIG. 4 is a diagram showing a restriction enzyme map of vector pCC6 obtained by plaque hybridization. In the figure, the white outline indicates the gene of the present invention derived from a microorganism belonging to the genus Chromobacterium, and the hatched portion indicates the translation region of esterase.

FIG. 5: Expression plasmid pCC containing the gene of the present invention
100, pCC101, pCC200 and pCC20
2 is a diagram showing a construction strategy of No. 1; FIG. In the figure, the arrow is l
Indicates the ac or tac promoter.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location C12R 1:19)

Claims (6)

[Claims] 1. An esterase gene encoding the amino acid sequence represented by at least the 1st to 362nd amino acids of the amino acid sequence represented by SEQ ID NO: 1. 2. The esterase gene according to claim 1, which has at least the 1st to 1086th nucleotide sequences among the nucleotide sequences shown in SEQ ID NO: 2. 3. A plasmid having the esterase gene according to claim 1 or 2. 4. pCC100, pCC101, pCC20
The plasmid according to claim 3, designated 0 or pCC201. 5. A microorganism containing the plasmid according to claim 3. 6. A method for producing esterase, which comprises culturing the microorganism according to claim 4 and producing esterase by the microorganism.