JP3400809B2 - Substantially pure microorganism producing acyl Co-A synthetase - Google Patents

Description

Detailed Description of the Invention

[0001]

The present invention relates to a substantially pure microorganism producing acyl Co-A synthetase, substantially pure DNA expressing acyl Co-A synthetase and acyl C.
The present invention relates to a method for producing o-A synthetase.

[0002]

2. Description of the Related Art Conventionally, acyl Co-A synthetase has been
Reaction formula in the presence of adenosine triphosphate (ATP)

[0003]

[Chemical 1]

(In the formula, RCOOH means a fatty acid having 10 to 24 carbon atoms, AMP means adenosine monophosphate, and PPi means pyrophosphate.) As shown in the formula, the free fatty acid (eg palmitic acid) is acylated. It has a catalytic action to convert to Co-A (for example, palmitoyl Co-A) and has an enzyme number E.I. C. It is known as 6.2.1.3 (Enzyme Handbook, pp. 770-771, 1982, published by Asakura Shoten). Free fatty acids in serum are said to be generated by the decomposition of triglyceride in adipose tissue, or by the action of lipoprotein lipase when serum triglyceride is absorbed into tissues. Most of free fatty acids are usually present in blood in a state of being bound to albumin and circulate in the body. Its concentration is significantly lower than other serum lipids. However, free fatty acids are the most metabolically active lipids, and accurate understanding of the dynamics of free fatty acids in the blood minutely reflects the energy state based on the metabolism of carbohydrates and lipids in the body. [Vitamins, 54
Vol. 489-501 (1980)]. Free fatty acids increase during diseases such as diabetes, hyperthyroidism, myocardial infarction,
It is known that free fatty acids decrease in diseases such as hypothyroidism and Addison's disease, and it is clinically extremely significant [Clinical Examination Guide, Bunkodo Publishing, 277-278 ( 1987)].

This acyl Co-A synthetase is produced by rat liver [J. Biol. Chem. , 265,8681-
8685 (1990)], and bacteria, fungi, yeasts, and carrier bacteria [vitamins, 54 volumes, 489-501 (1980)], and the like, are widely known to exist.
Among various acyl Co-A synthetases, Pseudomonas (Pseudomonas)
aeruginosa) IFO3919 produces an enzyme whose physicochemical properties are well known [vitamins,
54, 489-501 (1980)].

Free fatty acids exist in the body as fatty acids having a plurality of chain lengths. Therefore, the acyl Co-A synthetase used for measuring free fatty acids is required to have an enzyme with wide specificity. Further, the color development reaction when measuring the free fatty acid is in the alkaline region (pH 7.5 to 8.
0) is often done. However, Pseudomonas aerugin
osa) Acyl Co-A synthetase from IFO3919 is only stable between pH 4.5 and pH 7.0. Furthermore, Pseudomonas aerugino-sa (P
seudomonas aeruginosa) IFO
The acyl-Co-A synthetase derived from 3919 has many problems such that it hardly acts on arachidic acid (Arachidic acid).

[0007] When we made an earnest effort and searched for an acyl-Co-A synthetase which also acts on arachidic acid and has excellent substrate specificity, stability, etc., Pseudomonas fragii B-0771
Acyl C of strain (Ministry of Engineering deposit No. FERMP-5701)
We have found that o-A synthetase is suitable for this purpose. That is, the present enzyme sufficiently reacted with arachidic acid as a substrate and had an optimum pH of 6.0 to 8.0. However, Pseudomonas frazi
The production of acyl Co-A synthetase produced by the B. onas fragi) B-0771 strain is extremely low, and the development of a mass production method using a method such as genetic engineering has been awaited for a long time.

For the rat liver-derived acyl Co-A synthetase, the gene has already been isolated, the nucleotide sequence of the gene has been determined, and the amino acid sequence has been deduced [J. Bi
ol. Chem. , 265, 8681-8685 (19
90)]. We prepared various probes with reference to this nucleotide sequence and amino acid sequence, and analyzed Pseudomonas fragii B-0771.
An attempt was made to obtain the acyl Co-A synthetase gene of the strain, but it could not be obtained. That is, as will be described later, the amino acid sequence of the acyl-Co-A synthetase of the present invention and the nucleotide sequence of DNA encoding the amino acid sequence of the enzyme have extremely low homology of 10% or less with those derived from rat liver, It was impossible to predict what kind of amino acid sequence the acyl-Co-A synthetase had depending on the producing bacterium.

Under such circumstances, Pseudomonas vulgaris, which has high reactivity and stability and is effective for measuring free fatty acids,
Since the productivity of the Acyl-Co-A synthetase derived from the Frazi B-0771 strain is low, it has been desired to develop a microorganism that efficiently produces this enzyme using genetic engineering and the like. The primary structure of the polypeptide constituting the enzyme and the nucleotide sequence of the DNA encoding the amino acid sequence of the enzyme have not yet been published.

Therefore, it has been desired to determine the primary structure of the polypeptide constituting the acyl-Co-A synthetase, the nucleotide sequence of the DNA encoding the amino acid sequence of the enzyme, and the production of the enzyme by genetic engineering.

[0011]

Under the circumstances described above, the present invention provides acyl Co- which is useful for quantifying free fatty acids.
It was made for the purpose of developing a microorganism that efficiently produces A synthetase and providing a method for mass-producing the enzyme using this microorganism.

[0012]

The inventors of the present invention have conducted extensive studies to achieve the above-mentioned object, and as a result, acyl Co-A
A chromosomal DNA library derived from a microorganism that produces a synthetase is screened for a gene DNA that expresses the enzyme, and then an expression vector is constructed using this DNA. For example, Escherichia coli (E
scherichia coli; hereinafter E. It was found that the acyl Co-A synthetase can be efficiently mass-produced by introducing it into a microorganism belonging to (A. coli) to produce a transformed microorganism, and culturing the transformed microorganism in a medium. The invention was completed.

That is, the present invention relates to E. coli transformed with a recombinant plasmid having a DNA expressing an acyl Co-A synthetase. A substantially pure microorganism that produces an acyl Co-A synthetase, which is a microorganism belonging to Escherichia coli, and an acyl Co-A synthetase having a base sequence encoding the amino acid sequence shown in FIG. -Substantially pure DNA expressing
And said transformed microorganism, acyl Co-A
Substantially pure microorganisms producing synthetase are cultivated in a medium, and the acyl Co-A synthetase is then extracted from the culture.
The present invention provides a method for producing an acyl Co-A synthetase, which comprises collecting the enzyme.

The present invention will be described in detail below. In the present invention, acyl Co- used to produce a transformed microorganism that produces acyl Co-A synthetase.
The gene DNA expressing A synthetase can be obtained by screening, for example, from a chromosomal DNA library derived from a microorganism that produces the enzyme.

In the present invention, the aforementioned acyl Co-A is used.
As a microorganism producing synthetase, Pseudomonas fradi B-0771 strain is preferably used. A specific example of the method for screening a gene DNA expressing the enzyme from the chromosomal DNA library of Pseudomonas fradizi B-0771 strain will be described.
About 100 to 2000 μg of chromosomal DNA of the microorganism is extracted by a commonly used method, and then 1 to
Partially cleave about 10 μg with the restriction enzyme Sau3AI,
For example, one kind of lambda phage vector, ligated to the BamHI site of lambda emble 3 and then the recombinant DNA
Recombinant recombinant phage particles having as a genome are reconstituted in a test tube to prepare a chromosomal DNA library consisting of about 10 ⁷ clones. The host microorganism used at this time is E. E. coli is used.

Lambda phage was transformed into E. coli. As a method for introducing into a microorganism belonging to E. coli, for example, competent cell method, in vitro packaging method, spheroplast method, etc. may be used, or lambda phage may be introduced in the presence of calcium ion. . Regarding the selection of the presence or absence of lambda phage infection in the host microorganism,
Confirmed lambda phage infection by plaque formation,
The infected host microorganism may be selected.

On the other hand, the N-terminal side amino acid sequence, the lysyl endopeptidase-treated fragment amino acid sequence, and the asparaginyl endopeptidase-treated fragment amino acid sequence of the purified preparation of acyl-Co-A synthetase were determined, and various oligonucleotides were determined based on the determined amino acid sequences. Is synthesized and then labeled with, for example, an isotope to prepare a radioactive oligonucleotide probe.

Then, using these radioactive oligonucleotide probes, according to the method conventionally used conventionally,
From the above chromosomal DNA library, acyl Co
Although recombinant phage having a gene expressing -A synthetase is screened, this step is a very difficult part in the present invention, and acyl Co-A is used.
Various probes were prepared from a partial amino acid sequence of synthetase, but it was difficult to find a clone carrying the acyl-Co-A synthetase gene. Specifically, when designing a probe, generally, a sequence in which the deduced combination of DNA sequences is as small as possible is selected to synthesize the probe. However, in the case of the acyl Co-A synthetase of the present invention, a sequence with few combinations is left over. It does not exist, and it is possible to create a probe that narrows the combination from many combinations with reference to the codon usage of the Pseudomonas gene, or to create a probe in which the bases of multiple putative moieties are inosine. Of the N-terminal amino acid sequence, which will be described in the examples below, as a result of various examinations on the length of the DNA, and various examinations on the conditions of hybridization and subsequent washing (composition of the solution, temperature, time). -Based radioactive oligonucleotide probe is used for hybridization and washing under specific conditions. Under obtained recombinant phage it was found to be those with the gene of interest DNA.

Next, from the recombinant phage containing the target gene DNA, for example, Maniat
is) et al. ["Molecular Cloning: Cold Spring Harbor (Molecular Clo
Ning: Cold Spring Harbor) "
(1982)] and the like, and a recombinant plasmid (pA containing a DNA expressing an acyl Co-A synthetase).
(Named CS1) can be prepared. A schematic diagram showing the constitution of this plasmid is shown in FIG. In addition, Pseudomonas fradi B-0771 strain chromosome D in the plasmid
The restriction map of the NA-derived site is shown in FIG. The plasmid thus constructed was transformed into E. col
As a method for introducing into a microorganism belonging to i, the competent cell method may be used, or the recombinant DNA may be introduced in the presence of calcium ions. Regarding selection of whether or not the desired recombinant DNA is introduced into the host microorganism, the host microorganism may be cultured in a selection medium based on the drug resistance marker of the vector constituting the recombinant DNA, and the growing host microorganism may be selected. .

Next, an expression vector incorporating the gene DNA for expressing the acyl Co-A synthetase is constructed. As the expression vector, a vector constructed for gene recombination from a phage or a plasmid capable of autonomously growing in a host microorganism is suitable. Examples of the former phage include E. coli. When E. coli is used as the host microorganism, λgt · λC, λgt · λB and the like are used. As the plasmid, E. When E. coli is used as the host microorganism, for example, pBR322, pBR32
5, pACYC184, pUC12, pUC13, pU
C18, pUC19, pUC118, pUC119, Blue Script SK +, Blue Script KS +, Blue Script SK-, Blue Script KS-, Blue Script SKII +, Blue Script KSII +,
Blue Script SKII-, Blue Script KSI
I- or the like is used. Furthermore, when Bacillus is a host microorganism, for example, pHY300PLK or the like may be used, and when Saccharomyces is a host microorganism, for example, pYAC5 or the like may be used.

The method for incorporating the acyl-Co-A synthetase gene DNA into these vectors is not particularly limited, and a conventionally used method can be used. For example, using the appropriate restriction enzyme, the acyl C
The recombinant plasmid DNA containing the o-A synthetase gene DNA and the expression vector were treated to obtain a DNA fragment and a vector fragment containing the acyl Co-A synthetase gene, respectively. An expression vector is obtained by ligation using DNA ligase.

The expression vector in the examples described below was obtained from the above-mentioned recombinant plasmid pACS1 and vector plasmid Bluescript SK-, and was named pACS12 (the construction flow of the vector is shown in FIGS. 9 and 10). FIG. 6 shows a schematic diagram of the structure. Thus constructed plasmid E. For the introduction into E. coli and the selection of the presence or absence of introduction of the desired recombinant DNA into the host microorganism, the method described above may be used.

In the present invention, E. coli transformed with the recombinant plasmid pACS12 is used. The microorganism belonging to E. coli is Escherichia coli DH1-pACS.
12 (FERM P-13130). Cultivation of the transformed microorganism thus obtained can be carried out in a medium containing a nutrient source such as a carbon source and a nitrogen source necessary for the growth of the microorganism and an inorganic component.

The carbon source is, for example, glucose, starch, sucrose, molasses, dextrin, etc., and the nitrogen source is, for example, peptone, meat extract, casein hydrolyzate, corn steep liquor, nitrate, ammonium salt, etc. As the inorganic component, for example, sodium, potassium, calcium, magnesium, cobalt, zinc,
Examples thereof include salts containing cations such as manganese and iron and anions such as chlorine, sulfuric acid and phosphoric acid.

The culturing method is not particularly limited and may be a known method such as aeration and agitation culture, shaking culture, rotary culture, stationary culture, etc., usually at 20 to 50 ° C., preferably 25 to 42 ° C., more preferably 12 ~ at around 37 ℃
A method of culturing for about 48 hours is used. After culturing in this way, the cells are collected by means such as centrifugation, and then extraction containing the target enzyme by destroying the cells by enzyme treatment, autolysis, French press, ultrasonic treatment, etc. Get the liquid. To isolate and purify the enzyme from the extract, for example, salting out, desalting, adsorption / desorption treatment with an ion exchange resin, and the like, and further purification by adsorption chromatography, gel filtration, electrophoresis, etc. do it.

By examining the enzymatic activity and physicochemical properties of the acyl-Co-A synthetase in this purified preparation, it was confirmed that the transformed microorganism had the ability to produce acyl-Co-A synthetase. Therefore,
It is clear that the gene DNA expressing the acyl Co-A synthetase used in the present invention has a base sequence encoding the amino acid sequence shown in FIG. 1, and the base sequence is the sequence shown in FIG. is there.

The acyl-Co-A synthetase thus obtained has a catalytic action for effectively converting a free fatty acid (eg palmitic acid) into an acyl-Co-A (eg palmitoyl-Co-A) in the presence of ATP. Therefore, it is useful as a clinical enzyme for quantifying free fatty acid in serum, for example. The symbols for base sequences and symbols for amino acid sequences described in the specification of the present invention are based on conventional abbreviations in the art, and examples thereof are listed below. In addition, all amino acids are L-form.

DNA: Deoxyribonucleic acid A: Adenine T: thymine G: Guanine C: cytosine H: adenine, thymine or cytosine N: Adenine, thymine, guanine or cytosine R: adenine or guanine Y: thymine or cytosine Ala: Alanine Arg: Arginine Asn: Asparagine Asp: Aspartic acid Cys: Cysteine Gln: Glutamine Glu: Glutamic acid His: Histidine Ile: Isoleucine Leu: Leucine Lys: Lysine Met: methionine Phe: Phenylalanine Pro: Proline Ser: Serine Thr: Threonine Trp: Tryptophan Tyr: Tyrosine Val: Valine

[0029]

EXAMPLES Next, the present invention will be explained in more detail by reference examples and examples, but the present invention is not limited to these examples.

Reference Example 1 Isolation of chromosomal DNA Pseudomonas fradi B-0771 strain was used as a medium [10 g
/ Liter, Bactopeptone (manufactured by Difco), 5 g /
Liter, yeast extract (manufactured by Difco), 2 g / liter, sodium chloride, 2 g / liter, potassium chloride,
4 g of 1 g / liter, 1 potassium hydrogen phosphate, 1 g / liter, magnesium sulfate heptahydrate, pH 7.5] were shake-cultured at 37 ° C. for a whole day and night, and then this culture solution was subjected to a high-speed cooling centrifuge (Tomy CX-250). Type), 3500r
The cells were collected by centrifugation at pm (2000G) for 5 minutes.

Then, the cells were treated with 100 mM Tris-hydrochloric acid (pH 8.0) and 10 mM EDTA (pH 8.0).
After being washed with 1 ml of a solution consisting of, the suspension was suspended in 500 μl of the same solution, lysozyme (manufactured by Seikagaku Corporation) was added to give a final concentration of 0.5 mg / ml, and the cell wall of the strain was treated at 37 ° C. for 5 minutes. Destroyed. Next, 50 μl of a 10% sodium lauryl sulfate (manufactured by Nakarai Co., Ltd.) aqueous solution was added thereto, treated at 37 ° C. for 5 minutes, and then sodium chloride was added to a final concentration of 250 mM. Stir on ice for 30 minutes, mix equal amounts of phenol, then 12000 rpm (1
The aqueous phase was recovered by centrifugation at 8000 G for 5 minutes. Then add an equal volume of phenol / chloroform (1
/ 1) The mixed solution was added and treated in the same manner as above to separate the aqueous phase. This aqueous phase was gently mixed with an equal amount of isopropyl alcohol, left standing at -20 ° C for 2 hours, and then the DNA was sprinkled on a glass rod to separate it.
H8.0) and 1 mM EDTA solution 200μ
Dissolved in 1. RNase A (manufactured by Sigma) was added to this so that the final concentration was 100 μg / ml, and the mixture was incubated at 37 ° C. for 30 minutes.
After allowing to stand for minutes, proteinase K (manufactured by Boehringer) was added to a final concentration of 2.5 mg / ml, and the mixture was heated at 68 ° C for 30
I left it for a minute. This was mixed with an equal amount of phenol, a phenol / chloroform (1/1) mixed solution, and chloroform in this order, and treated in the same manner as above to separate the aqueous phases.

Next, twice the amount of ethanol was added to this aqueous phase and the DNA was separated again by the above-mentioned method.
g of DNA was obtained and it was added to 10 mM Tris-HCl (pH
200 μl of a solution consisting of 8.0) and 1 mM EDTA
Dissolved in.

Reference Example 2 Preparation of Pseudomonas fragilis gene library Pseudomonas fragii chromosome 5μ obtained in Reference Example 1
g using 1 unit of the restriction enzyme Sau3AI (Takara Shuzo), 50 mM Tris-acetic acid (pH 7.5), 100 mM
NaCl, 10 mM MgCl ₂ and 1 mM DTT
In a solution consisting of
-Centrifuge using a 40% sucrose density gradient to give approximately 13
A -16 kb DNA fragment was recovered.

In addition, 1 μg of lambda phage vector, Lambda Emble 3 (manufactured by Stratagene), was placed in a separate container.
Using 10 units of the restriction enzyme BamHI (Takara Shuzo), 5
0 mM Tris-acetic acid (pH 7.5), 100 mM Na
Cl, 10 mM MgCl ₂ and 1 mM DTT were cleaved at 37 ° C. for 2 hours, and then 1 unit of alkaline phosphatase (Boehringer) was added to the reaction solution to dephosphorylate the 5 ′ end. 1 at 37 ° C
Time processed. An equal amount of phenol was added to the reaction solution for treatment, and the aqueous phase was separated by centrifugation. Then, an equal volume of phenol / chloroform (1
/ 1) After adding and treating the mixed solution, the aqueous phase was separated by centrifugation. Furthermore, 1/10 amount of 3
DNA was recovered by adding M sodium acetate solution and 2 times amount of ethanol and centrifuging, drying under reduced pressure, 10 mM Tris-hydrochloric acid (pH 8.0) and 1 m.
It was dissolved in a solution consisting of M EDTA solution.

S of Pseudomonas fragilis chromosomal DNA
After mixing the au3AI cleavage product and the vector DNA digested with the restriction enzyme BamHI, the DNA was recovered by adding 1/10 volume of 3M sodium acetate solution and 2 volumes of ethanol and centrifuging, and drying under reduced pressure. The DNA was treated with 10 mM Tris-hydrochloric acid (pH 8.0).
And 66 mM Tris-hydrochloric acid (pH 7.6), 6.6 mM after dissolution in a solution consisting of 1 mM EDTA solution
MgCl ₂ , 10 mM DTT and 660 μM AT
T4DN in the presence of P (Boehringer Mannheim)
Ligation was performed for 16 hours at 4 ° C. using 100 units of A ligase (Takara Shuzo).

Next, the recombinant DNA thus prepared
Recombinant phage particles having as a genome were reconstituted in vitro using the Lambda Phage In Vitro Packaging Kit (Stratagene).
It was used as a fragrance gene DNA library.

Reference Example 3 Preparation of Radioactive Oligonucleotide Probe The amino acid sequences of the N-terminal side amino acid sequence, the lysyl endopeptidase-treated fragment, and the asparaginyl endopeptidase-treated fragment of the acyl Co-A synthetase purified preparation were examined. The sequence shown in 7 was determined. Based on this information, the base sequence was predicted from the 5'end side of the gene. Since various combinations are conceivable for this predicted nucleotide sequence, an experiment was conducted by designing an oligonucleotide of a part having a small number of combinations. This oligonucleotide was prepared by the method of R. Lessinger et al. ["Journal of American Chemical Society (JA
m. Chem. Soc. ) ”, Vol. 98, page 3655], using a DNA synthesizer [Cyclone (manufactured by Biosearch)].

That is, the DNA combination corresponding to Lys from the amino acid sequence Met in the underlined portion of FIG. 7 is 20 m.
Considering er's one, there are 48 types. Therefore, a 20-mer oligonucleotide shown in FIG. 8 which is complementary to the mRNA deduced from the amino acid sequence was prepared.
5 pmol of the oligonucleotide thus obtained
T4 polynucleotide kinase buffer [50 mM
Tris-hydrochloric acid (pH 8.0), 10 mM MgCl ₂ ,
4 mM DTT, 0.1 mM EDTA, 0.1 mM spermidine] and 3 megabecquerel [γ- ³² P] AT
In the presence of P (manufactured by Amersham), 8.5 units of T4 polynucleotide kinase (manufactured by Toyobo Co., Ltd.) were used to react at 37 ° C. for 30 minutes to incorporate isotope 32P.
A radioactive oligonucleotide probe was made.

Example 1 Screening for DNA Containing Acyl Co-A Synthetase Gene The Pseudomonas fradi gene DNA library obtained in Reference Example 2 was used as a host E. E. coli was allowed to form and about 10 ⁴ plaques were formed on the plate agar medium. in addition,
Nitrocellulose Membrane Filter (S & S)
After transferring a part of the phage particles in the plaque on the filter, the filter is immersed in an alkaline denaturing solution (0.5 M NaCl-containing 0.5 N-NaOH solution) for 5 minutes to further neutralize the solution. It was immersed in [0.5 M Tris-hydrochloric acid (pH 7.0) and 3 M NaCl mixed solution] for 5 minutes and then dried.

Next, this filter was heated at 80 ° C. for 2 hours to immobilize the recombinant phage DNA on the filter. In addition, this filter is
0.18M sodium citrate, 0.05% sodium diphosphate, 0.1% sodium lauryl sulfate, 0.1%
Ficoll, 0.1% polyvinylpyrrolidone, 0.1%
It was immersed in a prehybridization solution containing BSA and 0.01% salmon sperm DNA, and prehybridized at 37 ° C. for 2 hours. The filter was then filtered with 1.8 M NaCl, 0.18 M sodium citrate, 0.05% sodium diphosphate, 0.1% sodium lauryl sulfate, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1%. After immersing in a hybridization solution containing 1% BSA and 0.002% Escherichia coli-derived transfer RNA, the radioactive oligonucleotide probe obtained in Reference Example 3 was added, and hybridization was carried out at 37 ° C. for 16 hours.

After hybridization, 1.8 M N
The filter was washed three times with a washing solution containing aCl, 0.18 M sodium citrate, and 0.05% sodium diphosphate, and then immersed in a washing solution at 42 ° C. for 10 minutes to wash off excess probe. Then, after air-drying the filter, it was layered on an X-ray film (XAR manufactured by Kodak Co., Ltd.) and protected from light -8
Autoradiography was performed at 0 ° C for 24 hours.

Then, when the film was developed, plaques showing a positive signal were confirmed. A restriction enzyme digestion map of the inserted DNA of the recombinant phage forming the plaque is shown in FIG. The recombinant phage forming the plaque was designated as φ2.

Example 2 Extraction and Structural Analysis of Recombinant Phage DNA The recombinant phage φ2 obtained in Example 1 above was transformed into E. coli contained in the Stratagene Lambda Emble 3 kit. coliP2392 share ^[F -, hsdR514
(Rk ⁻ , mk ⁻ ), supE44, supF58, l
acY1, galK2, galT22, metB1, t
rpR55, λ ⁻ , mcrA ⁻ , mcrB ⁺ , P2] and cultured overnight in LB medium at 37 ° C., followed by the method of T. Maniatis et al. [“Molecular Cloning: Cold Spring Harbor (Molecu
lar Cloning: Cold Spring H
Arbor) ”, pp. 76-85 (1982)], and the φ2 genomic DNA was extracted. φ2 genome DN
A schematic diagram showing the configuration of A is shown in FIG. Next, a DNA fragment of about 11 kb obtained by cleaving this recombinant phage DNA with a restriction enzyme BamHI is recombined into a plasmid vector Bluescript SK ⁻ to construct a plasmid pACS1, and to construct a plasmid pACS1 of K. Shigesada. Method ["Cell Engineering" Volume 2, 616-626 (1
983)] and made E. co
^{liDH1 (ATCC33849) [F -,} recA
1, endA1, gyrA96, thi-1, hsdR
17 (rk-, mk +), SupE44, relA1,
λ ^-] [ "Morekyuraru Cloning: Cold Spring Harbor (Molecular Clonin
g: Cold Spring Harbor) "504
.About.506 pages (1982)].

E. coli transformed with the plasmid pACS1.
From E. coli DH1 the method of T. Maniatis et al. ["Molecular Cloning: Cold Spring Harbor:
Cold Spring Harbor) "No. 86-9
P. 4 (1982)], pACS1DNA
Was extracted. A schematic diagram showing the construction of this plasmid is shown in FIG.
Shown in. The site derived from the Pseudomonas frazi chromosome in the plasmid was subjected to the dideoxy method ["Science (Scie
No.), 214, 1205-1210 (1981)].
It was confirmed that the total DNA encoding the o-A synthetase was contained, the total base sequence thereof was determined, and it was confirmed that at least the sequence shown in FIG. 2 was contained.

The amino acid sequence of the N-terminal side of the acyl-Co-A synthetase analyzed this time, the amino acid sequence of the lysyl endopeptidase-treated fragment, and the amino acid sequence of the asparaginyl endopeptidase-treated fragment completely matched in the same frame.

Example 3 Activity expression of acyl Co-A synthetase in Escherichia coli 0.5 μg of the plasmid pACS1 obtained in Example 2
Was cleaved with restriction enzyme SacI (Takara Shuzo) to give acyl C
DN of about 10.2 kb containing o-A synthetase gene
The A fragment was separated and collected by 0.5% agarose gel electrophoresis.

The recovered DNA solution was ligated in the same manner as in Reference Example 2, and E. BH containing 50 μg / ml ampicillin transformed with E. coli DH1
It was spread on I agar medium and cultured at 37 ° C. overnight. In this way, the vector plasmid Bluescript SK ^- to obtain a transformant microorganism which holds the SacI and plasmid pACS12 the DNA fragment of approximately 7.3kb which contain the acyl Co-A synthetase gene was inserted between the BamHI site, performed Recombinant plasmid p
ACS12 was obtained.

The transformed microorganism carrying the plasmid pACS12 was cultured overnight at 37 ° C. in a BHI medium containing 50 μg / ml of ampicillin, and then the culture broth was 15,000 rp.
The precipitate was recovered by centrifugation at m for 1 minute. To this precipitate, the same amount of 10 M Tris-hydrochloric acid (pH 8.
0) was added and ultrasonic disruption was performed. After appropriately diluting the disrupted solution, 50 μl was taken, and 200 μl of 0.2 M phosphate buffer (pH 7.5) and 10 mM ATP (p
H7.5) 100 μl, 10 mM MgCl ₂ 100 μl
1, 10 mM Co-A (pH 6.5) 50 μl, 5%
200 mM of 1 mM palmitic acid containing Triton-X100
l, 1000 μl of reaction solution consisting of 350 μl of water was added,
The reaction was carried out at 37 ° C for 10 minutes. After that, 0.2M
Phosphate buffer (pH 7.5) 500 μl, 20 mM
N-ethylmaleimide 100 μl, 15 mM 4-aminoantipyrine 300 μl, 0.3% 3-methyl-
N-ethyl-N- (β-hydroxymethyl) aniline 2
50 μl, 100 units / ml of peroxidase 1
00 μl, 0.5% sodium azide 100 μl, 12
0 unit / ml of acyl Co-A oxidase 100
2000 μl of a reaction solution consisting of μl and 550 μl of water was added, and the mixture was reacted at 37 ° C. for 5 minutes. 550n of this reaction solution
Acyl Co by measuring the absorbance at m
-A synthetase activity was quantified. For comparison, E. coli obtained by transforming Blue Script SK ⁻ was used. The same treatment as described above was performed on the disrupted liquid of E. coli, and the acyl Co-A synthetase activity was measured.

As a result, the activity in the transformed microorganism carrying the plasmid pACS12 was 500 mU / ml, but the activity of Bluescript SK- was 0 U.
/ Ml. From this, it was confirmed that a transformant having an acyl Co-A synthetase activity was obtained. This transformant was designated as Escherichia coli DH1.
-PACS12 (Escherichia coli
DH1-pACS12) (FERM P-13130)
I named it. Further, the obtained acyl Co-A synthetase was isolated and purified to have a molecular weight of 60,000 ± 15,000.
The physicochemical properties of the expressed protein were confirmed to be 0 (gel filtration method using TSK gel G3000SW) and have an isoelectric point of pI5.2 ± 0.5. In addition, when the relative activity when using palmitic acid as the substrate was 100%, the relative activity when using arachidic acid as the substrate was 152%, confirming that it is an expressed protein that also acts well on arachidic acid as originally intended. did. As a result, it was confirmed that the expressed protein was identical to Pseudomonas fragii acyl Co-A synthetase.

[0050]

INDUSTRIAL APPLICABILITY According to the present invention, from a chromosomal DNA library derived from Pseudomonas fradi B-0771 strain,
A gene plasmid that expresses acyl Co-A synthetase was screened, and a recombinant plasmid of an expression vector constructed using this was screened, for example, E. coli. By introducing into a microorganism belonging to E. coli, the obtained transformed microorganism can efficiently produce acyl Co-A synthetase. In addition, the present invention has made it possible to determine the entire amino acid sequence of acyl Co-A synthetase and the base sequence of the gene DNA encoding this amino acid. Therefore, the substrate for the enzyme and the protein such as coenzyme specificity conversion and heat resistance improvement can be determined. Engineering became possible.

[Sequence list]

SEQ ID NO: 1 Sequence length: 1724 base pairs Sequence type: Nucleic acid chain number: Double-stranded topology: Linear sequence type: Genomic DNA Origin organism name: Pseudomonas fradi strain name: B- 0771 Sequence features: 30-1698 E Acyl Co-A Synthase gene sequence GAATTCGCGA CGCTGAGGAG TGGGCTTCC ATG ATT GAA GGC TTT TGG AAG GAT 53 Met Ile Glu Gly Phe Trp Lys Asp 15 AAG TAC CCA GCT GGG ATT GCT GCT GAG AAT CCT GAT GAA TAC CCA 101 Lys Tyr Pro Ala Gly Ile Ala Ala Glu Ile Asn Pro Asp Glu Tyr Pro 10 15 20 AAT ATT CAG ACG GTA CTG CGG GAA TCT TGT CAG CGG TTC GCC GAC AAA 149 Asn Ile Gln Thr Val Leu Arg Glu Ser Cys Gln Arg Phe Ala Asp Lys 25 30 35 40 CCG GCG TTC AGT AAC CTC GGC AAA ACC ATC ACC TAC GGT GAG CTT TAT 197 Pro Ala Phe Ser Asn Leu Gly Lys Thr Ile Thr Tyr Gly Glu Leu Tyr 45 50 55 GAG CAG TCC GGC GCG TTT GCA GCC TGG TTG CAA CAG CAC ACC GAC CTG 145 Glu Gln Ser Gly Ala Phe Ala Al a Trp Leu Gln Gln His Thr Asp Leu 60 65 70 CAG CCG GGC GAT CGC ATC GCC GTG CAG TTG CCC AAC CTG CTG CAA TAC 293 Gln Pro Gly Asp Arg Ile Ala Val Gln Leu Pro Asn Leu Leu Gln Tyr 75 80 85 CCG ATC GCT GTG TTC GGA GCC ATC CGC GCC GGC TTG ATC GTG GTC AAC 341 Pro Ile Ala Val Phe Gly Ala Ile Arg Ala Gly Leu Ile Val Val Asn 90 95 100 ACC AAC CCG CTG TAC ACC GCG CGG GAA ATG GAA CAC CAA TTC AAG GAT 389 Thr Asn Pro Leu Tyr Thr Ala Arg Glu Met Glu His Gln Phe Lys Asp 105 110 115 120 TCC GGC GCC AAG GCC CTG GTG TGC CTG GCG AAC ATG GCG CAC CTG GCC 437 Ser Gly Ala Lys Ala Leu Val Cys Leu Ala Asn Met Ala His Leu Ala 125 130 135 GAA AAG GTC GTG CCG CAG ACT TCG GTC AAG TAT GTG ATC GTC ACT GAA 485 Glu Lys Val Val Pro Gln Thr Ser Val Lys Tyr Val Ile Val Thr Glu 140 145 150 GTG GCC GAC ATG CTG TCG CCG TTC AAG CGC GTA CTG ATC AAC AGC GTC 533 Val Ala Asp Met Leu Ser Pro Phe Lys Arg Val Leu Ile Asn Ser Val 155 160 165 ATC AAG TAT GTG AAG AAG ATG GTC CCG GCT TAT CAC TTG CCG CAG GCC 581 Ile Lys Tyr Val Lys Lys Me t Val Pro Ala Tyr His Leu Pro Gln Ala 170 175 180 ATC AAG TTC AAC GAT GTG CTG AGC AAG GGG CAG GGC CAG CCG GTC AAG 629 Ile Lys Phe Asn Asp Val Leu Ser Lys Gly Gln Gly Gln Pro Val Lys 185 190 195 200 GAA GCC AGC CCG GTA AGC AGT GAT GTT GCC GTG CTG CAG TAC ACC GGC 677 Glu Ala Ser Pro Val Ser Ser Asp Val Ala Val Leu Gln Tyr Thr Gly 205 210 215 GGC ACC ACC GGT GTG GCC AAG GGC GCG ATG CTG AGC CAC CGC AAC CTG 725 Gly Thr Thr Gly Val Ala Lys Gly Ala Met Leu Ser His Arg Asn Leu 220 225 230 ATT GCC AAC ATG TTG CAG TGC AAG GCG CTG ATG GCT TCC AAC CTC GAT 773 Ile Ala Asn Met Leu Gln Cys Lys Ala Leu Met Ala Ser Asn Leu Asp 235 240 245 GAA GGT TGT GAA ATC CTG ATC ACG CCA CTG CCG CTG TAC CAC ATC TAT 821 Glu Gly Cys Glu Ile Leu Ile Thr Pro Leu Pro Leu Tyr His Ile Tyr 250 255 260 GCT TTT ACC TTT CAC TGC ATG GCG ATG ATG TTC CTG GGT AAC CAC AAC 869 Ala Phe Thr Phe His Cys Met Ala Met Met Leu Leu Gly Asn His Asn 265 270 275 280 ATA CTG ATC AGC AAC CCC CGG GAT TTG CCG GCG ATG GTT AAA GAG CTG 917 Ile Leu Il e Ser Asn Pro Arg Asp Leu Pro Ala Met Val Lys Glu Leu 285 290 295 TCC AAG TGG AAG TTC ACC GGC TTT GTG GGC CTT AAC ACC CTG TTC GTG 965 Ser Lys Trp Lys Phe Thr Gly Phe Val Gly Leu Asn Thr Leu Phe Val 300 305 310 GCG TTG TGC AAT AAC GAA GCG TTC CGC AAA CTG GAC TTT TCG GCC CTC 1013 Ala Leu Cys Asn Asn Glu Ala Phe Arg Lys Leu Asp Phe Ser Ala Leu 315 320 325 AAG GTG ACG TTG TCG GGC GGC ATG GCC TTG CAG CTG TCG GTG GCT GAA 1061 Lys Val Thr Leu Ser Gly Gly Met Ala Leu Gln Leu Ser Val Ala Glu 330 335 340 CGC TGG AAA ACC GTG ACC GGT TGC CCG ATC TGC GAA GGT TAC GGC ATG 1109 Arg Trp Lys Thr Val Thr Gly Cys Pro Ile Cys Glu Gly Tyr Gly Met 345 350 355 360 ACC GAA ACC AGC CCG GTG GCC ACG GTC AAC CCG GCC CAG AGC ATC CAG 1157 Thr Glu Thr Ser Pro Val Ala Thr Val Asn Pro Ala Gln Ser Ile Gln 365 370 375 GTT GGC ACC ATT GGC ATT CCA GTG CCT TCG ACC TTG TGC AAA GTG ATC 1205 Val Gly Thr Ile Gly Ile Pro Val Pro Ser Thr Leu Cys Lys Val Ile 380 385 390 AAC GAT GCC GGT GAA GAG CTG CCG CTG GGT GAA GTC GGC GAG CTG T GC 1253 Asn Asp Ala Gly Glu Glu Leu Pro Leu Gly Glu Val Gly Glu Leu Cys 395 400 405 ATC AAG GGC CCG CAA GTC ATG AAA GGC TAC TGG GAG CGC CCT GAC GCT 1301 Ile Lys Gly Pro Gln Val Met Lys Gly Tyr Trp Glu Arg Pro Asp Ala 410 415 420 ACC GCC GAA ATC ATG GAC AGT GAA GGC TGG TTG AAG TCC GGT GAT ATC 1349 Thr Ala Glu Ile Met Asp Ser Glu Gly Trp Leu Lys Ser Gly Asp Ile 425 430 435 440 GCC GTG ATT CAG CCC GAT GGT TAT ATC CGG ATT GTC GAC CGC AAA AAA 1397 Ala Val Ile Gln Pro Asp Gly Tyr Ile Arg Ile Val Asp Arg Lys Lys 445 450 455 GAC ATG ATT CTG GTG TCG GGG TTC AAT GTG TAC CCC AAT GAG CTG GAA 1445 Asp Met Ile Leu Val Ser Gly Phe Asn Val Tyr Pro Asn Glu Leu Glu 460 465 470 GAT GTC ATG GCT TCC TTG CCT GGC GTG CTG CAA TCT GCG GCA ATC GGT 1493 Asp Val Met Ala Ser Leu Pro Gly Val Leu Gln Ser Ala Ala Ile Gly 475 480 485 ATT CCG GAT GAG AAA ACC GGT GAG CTG ATC AAG GTG TTT ATC GTG GTC 1541 Ile Pro Asp Glu Lys Thr Gly Glu Leu Ile Lys Val Phe Ile Val Val 490 495 500 AAG CCG GGC ATG ACC TTG ACC AAG GAT GAG GT C ATG AAG CAT ATG CGC 1589 Lys Pro Gly Met Thr Leu Thr Lys Asp Glu Val Met Lys His Met Arg 505 510 515 520 GCC AAT CTG ACG GCT TAT AAA GCG CCC AAA TTC GTC GAG TTC CGC GAC 1637 Ala Asn Leu Thr Ala Tyr Lys Ala Pro Lys Phe Val Glu Phe Arg Asp 525 530 535 AGC TTG CCG ACT ACC AAT GTA GGC AAG ATC TTG CGT CGT GAG CTG CGC 1685 Ser Leu Pro Thr Thr Asn Val Gly Lys Ile Leu Arg Arg Glu Leu Arg 540 545 550 GAT GAA GAA CTG AAA AAG CTG GGT CTC AAG AAG TAACCGGCCT GGATACGAAA 1718 Asp Glu Glu Leu Lys Lys Leu Gly Leu Lys Lys 555 560 563 AAGCCC 1724

SEQ ID NO: 2 Sequence length: 20 base pairs Number of strands: Single-stranded topology: Linear sequence type: Other nucleic acid Synthetic DNA Sequence name: Acyl Co-A Synthase gene probe ACS1 Array ATGATHGARG GNTTYTGGAA 20

[Brief description of drawings]

FIG. 1 shows the amino acid sequence of acyl Co-A synthetase.

FIG. 2 is a diagram showing the base sequence of the acyl Co-A synthetase gene DNA encoding the amino acid of FIG.

FIG. 3 is a restriction enzyme map of chromosomal DNA derived from Pseudomonas fradi in plasmid pACS1.

FIG. 4 is a schematic diagram showing the structure of recombinant phage φ2.

FIG. 5 is a schematic diagram showing the structure of plasmid pACS1.

FIG. 6 is a schematic diagram showing the structure of plasmid pACS12.

FIG. 7: Analysis of the N-terminal side amino acid sequence, lysyl endopeptidase-treated fragment amino acid sequence, and asparaginyl endopeptidase-treated fragment amino acid sequence of a purified preparation of acyl Co-A synthetase, and the joined partial acyl Co- It is a figure which shows the amino acid sequence of A synthetase. In addition, X shows an unknown amino acid.

FIG. 8 is a diagram showing a nucleotide sequence of an oligonucleotide used for producing an oligonucleotide probe.

FIG. 9 is a schematic diagram showing the flow of construction of the acyl Co-A synthetase gene expression vector used in the present invention.

FIG. 10 is a schematic diagram showing a part of the construction of the acyl Co-A synthetase gene expression vector used in the present invention, which is partially overlapped with FIG. 9.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI (C12N 15/09 ZNA C12R 1:38) (56) References Taylor DC, Weber N, Hogge LR, Underhier EW. , A simple en zymatic method for the preparation of the radiolabeled eru coyl-CoA and other long-chain fat, Anal. Biochem. , 1990, Vol. 184, No. 2, p. 311-316 (58) Fields surveyed (Int.Cl. ⁷ , DB name) C12N 15/00 C12N 9/00 BIOSIS (DIALOG) WPI (DIALOG)

Claims (5)

(57) [Claims] 1. The following amino acid sequence [Sequence 1] Incorporating a nucleotide sequence encoding the amino acid sequence represented by
Microbial you produce an acyl Co-A synthetase, wherein the recombinant plasmid I is a microorganism belongs Esshierihia coli transformed. 2. The following amino acid sequence [Sequence 2] Acyl Co-A sequence encoding the amino acid sequence represented by
DNA expressing insetase . 3. The following base sequence [Sequence 3] The DNA according to claim 2, which is a base sequence represented by: 4. The following amino acid sequence [Sequence 4] Incorporating a nucleotide sequence encoding the amino acid sequence represented by
Characterized in that the microorganism transformed with the recombinant plasmid that produces acyl Co-A synthetase is cultured in a medium, and then the acyl Co-A synthetase is collected from the culture. Method for producing synthetase. 5. The following amino acid sequence [Sequence 5] An acyl Co-A synthetase represented by: