JP2005034086A - ACYL-CoA OXIDASE, GENE THEREOF, RECOMBINANT DNA, AND METHOD FOR PRODUCING ACYL-CoA OXIDASE - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain an acyl-CoA oxidase having excellent stability in the coexistence of an SH reagent. <P>SOLUTION: (1) The acyl-Co A oxidase exhibits ≥40% residual activity after the treatment at pH 7.5 at 37°C for 4 hr in the presence of 10 mM SH reagent. (2) Preferably, the acyl-Co A oxidase exhibits ≥60% residual activity after the treatment at pH 7.5 at 37°C for 4 hr in the presence of 10 mM SH reagent. (3) The acyl-CoA oxidase is composed of the following (a) or (b): (a) a protein having a specific amino acid sequence; (b) a protein having an amino acid sequence obtained by deleting, substituting or adding one or several amino acids from, for or to the amino acid sequence (a), and having the acyl-CoA oxidase activity. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to an acyl CoA oxidase excellent in stability against an SH reagent, a gene thereof, a recombinant DNA, and a method for producing an acyl CoA oxidase excellent in stability against an SH reagent.

Acyl CoA oxidase is an enzyme that oxidizes acyl coenzyme A (acyl CoA) and converts it into trans-2,3-dehydroacyl coenzyme A (enoyl CoA) and hydrogen peroxide, and is involved in β-oxidation of fatty acids in peroxisomes. . This enzyme is known to exist widely in nature such as mammals, plants and microorganisms.
For example, as for mammals, there are reports of rat origin (see Non-patent Document 1), mouse origin (see Non-patent Document 2), human origin (see Non-patent Document 3), and the like.
Known plant origins include pumpkin (see Non-Patent Document 4), cucumber (see Non-Patent Document 5), and Arabidopsis thaliana.
Moreover, as microorganism origin, Candida lipolytica (Yarrowia lipolytica) origin (refer patent document 1, nonpatent literature 6), Candida tropicalis origin (refer nonpatent literature 7), Candida maltosa origin (refer nonpatent literature 8), Macrophominaphase origin (Refer to Patent Document 2), derived from Cladospodium resinae, derived from Aspergillus candidus (refer to Patent Document 2), derived from the genus Monascus (refer to Patent Document 2), derived from Saccharomyces cerevisiae (refer to Patent Document 2), derived from the genus Arthrobacter (refer to Patent Document 2) From Dictyostelium discoidum is known. Some of these enzymes have already been successfully cloned and have been produced by recombinants.

Industrial usefulness of the enzyme includes, for example, measurement of free fatty acid or chemical conversion using a fatty acid β-oxidation system. Particularly in the field of clinical diagnosis, free fatty acids in serum are regarded as an index for diagnosis of diabetes, hyperlipidemia, hyperthyroidism, and severe liver damage. In recent years, it has become clear that free fatty acids act on GPR40, which is one of the orphan G protein receptors, to promote insulin secretion from pancreatic β cells, and attention has been paid to the functionality of free fatty acids in vivo. (See Non-Patent Document 9).
Several methods for measuring free fatty acids using enzymes are known.
A method using acyl CoA oxidase is widely used because of its high specificity and simplicity (see Patent Documents 3 and 4).
In this measurement, first, a first reagent containing coenzyme A (CoA), adenosine triphosphate (ATP), acyl CoA synthase and the like is added to free fatty acids in serum to produce acyl CoA. When a second reagent containing acyl CoA oxidase is added thereto, acyl CoA is oxidized and hydrogen peroxide is generated. When a peroxidase and a coloring reagent coexist in the second reagent, color develops due to the generated hydrogen peroxide, and thus the free fatty acid can be quantified by measuring this. The reaction formula is shown below.
Acyl CoA synthase free fatty acid + CoA + ATP → acyl CoA + PPi + AMP

Acyl CoA oxidase acyl CoA + O ₂ → enoyl CoA + H ₂ O ₂

In this measurement, it is known that CoA added excessively interferes with color development. To avoid this, SH such as N-ethylmaleimide (NEM), iodoacetic acid, iodoacetamide, etc. is contained in the second reagent. There has been reported a method in which a reagent is allowed to coexist and CoA is deactivated simultaneously with the addition of a second reagent (see Patent Documents 3 and 4). However, since the acyl CoA oxidase similarly formulated as the second reagent has a significantly reduced stability in the presence of the SH reagent, it has been difficult to prepare a liquefied reagent that can be used for a long time. Therefore, in order to develop a liquefaction reagent for measuring free fatty acids, there has been a strong demand for the development of an acyl CoA oxidase having excellent stability against the SH reagent.

Japanese Patent Publication No.59-15625 Japanese Patent Publication No.58-40466 Japanese Patent Publication No.57-29998 Japanese Patent Publication No.57-33955 Biochem. Biophys. Res. Commun. 217, 482 (1995) Eur. J. et al. Biochem. 267, 1254 (2000) Biochem. Biophys. Res. Commun. 198, 1113 (1994) J. et al. Biol. Chem. 273, 8301 (1998) J. et al. Biol. Chem. 261, 8570 (1986) J. et al. Biochem. 88, 1481 (1980) Biochem. Biophys. Res. Commun. 91, 108 (1979) Nucleic Acids Res. 16, 365 (1988) Nature 422, 173 (2003)

  The problem to be solved by the present invention is to provide an acyl CoA oxidase having excellent stability in the presence of an SH reagent, which can be used for various uses, particularly clinical diagnosis.

Accordingly, as a result of intensive studies for solving the above problems, the present inventors have found that Corynebacterium sp. Described in JP-A-11-155579. No. The present inventors have found that acyl-CoA oxidase derived from 2-3-1 (FERM BP-6183) can solve the above problems.
That is, the present invention provides the following inventions.
(1) An acyl CoA oxidase, which exhibits a residual activity of 40% or more when treated at 37 ° C. for 4 hours at pH 7.5 in the presence of 10 mM SH reagent.
(2) An acyl CoA oxidase that exhibits a residual activity of 60% or more after treatment at 37 ° C. for 4 hours at pH 7.5 in the presence of 10 mM SH reagent.
(3) The following acyl CoA oxidase (a) or (b).
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1, (b) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (a), and having an acyl CoA oxidase activity (4) A gene encoding an acyl CoA oxidase of (a) or (b) below:
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1, (b) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (a), and having an acyl CoA oxidase activity (5) an acyl CoA oxidase gene comprising the following DNA (a) or (b):
(A) DNA consisting of the base sequence shown in SEQ ID NO: 2
(B) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to DNA consisting of the base sequence of (a) and encodes a protein having acyl CoA oxidase activity
(6) A recombinant DNA, wherein the acyl CoA oxidase gene according to item (4) or (5) is inserted into a vector DNA.
(7) A transformant or transductant containing the recombinant DNA according to item (6).
(8) A method for producing an acyl CoA oxidase, comprising culturing the transformant or transductant according to item (7) in a medium and collecting acyl CoA oxidase from the culture.

  According to the present invention, an acyl CoA oxidase having excellent stability in the presence of an SH reagent is provided.

Hereinafter, the present invention will be described in detail.
Acyl CoA oxidase is widely distributed in nature, and the gene of this enzyme can be cloned from chromosomal DNA of various animals, plants, microorganisms, etc., and its biological resources are not particularly limited. , For example, microorganisms, more specifically Corynebacterium sp. No. Chromosomal DNA of 2-3-1 (FERM BP-6183) is used.
Chromosomal DNA can be prepared, for example, by the method described in Current Protocols in Molecular Biology (WILEY Interscience, 1989).
Primers for polymerase chain reaction (hereinafter abbreviated as PCR) are prepared based on the known amino acid sequence of acyl CoA oxidase. At this time, in consideration of codon degeneracy, a primer is prepared using a mixed base for a portion where a plurality of bases are degenerate. Using the prepared primer, PCR is performed using the previously obtained chromosomal DNA as a template. The amplified DNA fragment is incorporated into a vector DNA such as pT7Blue T Vector (Takara Shuzo Co., Ltd.) to obtain a recombinant plasmid. The nucleotide sequence of the inserted DNA in the plasmid is determined using, for example, a multicapillary DNA analysis system CEQ2000 (manufactured by Beckman Coulter), etc., and the nucleotide sequence correctly encodes the amino acid sequence of the peptide used for PCR primer design Is selected at both ends. The amplified DNA fragment obtained as described above is a part (partial gene) of the acyl CoA oxidase gene of the present invention. Then, based on the obtained DNA base sequence, a new PCR primer for inverse PCR is synthesized.

Next, the chromosomal DNA is completely digested with various restriction enzymes, and then subjected to ordinary agarose gel electrophoresis, and Southern blot analysis is performed using the partial gene as a probe. As a result, the chain length of the DNA fragment containing the acyl CoA oxidase gene can be determined.
Next, the chromosomal DNA is treated with the restriction enzyme used for Southern blot analysis, and then self-ligated. Using this as a template, inverse PCR is performed using the above-mentioned primers. When a plurality of DNA fragments are amplified, the amplified DNA fragment of a size estimated from the chain length obtained by the previous Southern blot analysis is a DNA fragment containing an acyl CoA oxidase gene. By determining and linking the base sequences of these DNA fragments, the entire base sequence of the acyl CoA oxidase gene can be determined. Next, the amino acid sequence of the polypeptide translated by the gene having the base sequence is determined.
This amino acid sequence is as represented by SEQ ID NO: 1. The gene encoding the amino acid sequence thus determined is the acyl CoA oxidase gene of the present invention.
In the amino acid sequence represented by SEQ ID NO: 1, all of the acyl CoA oxidase genes in which one or several amino acids have been deleted, substituted or added and which encode an amino acid sequence that brings about acyl CoA oxidase activity are all It is included in the present invention.
To obtain an acyl CoA oxidase gene encoding an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1 and bring about acyl CoA oxidase activity Any method can be used, for example, site-directed mutagenesis, which is a well-known technique for generating point mutations or deletion mutations in a gene; selectively cleaving the gene, and then removing or adding selected nucleotides. And a method of linking genes; an oligonucleotide mutagenesis method and the like.
These DNAs are highly likely to encode a polypeptide that provides acyl-CoA oxidase activity, and transformants can be produced and those having activity can be selected.

  In order to obtain a gene substantially identical to the acyl CoA oxidase gene of the present invention, DNA having the nucleotide sequence of SEQ ID NO: 2 or a complementary strand thereof, or a probe containing a part thereof is used under stringent conditions. Those that hybridize and encode a polypeptide having acyl-CoA oxidase activity can be selected. The stringent condition here is a condition in which only a specific hybrid is selectively formed and a signal is detected, but a non-specific hybrid is not formed. Such conditions vary slightly depending on the individual species, but can be easily determined by examining several salt concentrations or temperatures during hybridization and washing by conventional methods. As such a condition, for example, since a specific signal can be observed in item (3) of Examples described later, hybridization is performed using a DIG Easy Hyb reagent (manufactured by Roche Diagnostics) 37-42. Perform overnight at ° C. Washing is performed twice for 15 minutes using 0.5 × SSC and 0.1% SDS. The washing temperature is 45 ° C. or higher, preferably 52 ° C. or higher, more preferably 57 ° C. or higher. DNA that hybridizes under such conditions is highly likely to encode a peptide having acyl CoA oxidase activity, but also includes DNA having a mutation that loses acyl CoA oxidase activity. An amino acid sequence having a homology of 70% or more, preferably 80% or more, more preferably 90% or more of the amino acid sequence represented by SEQ ID NO: 1 is included in the present invention. A gene sequence having a homology of 70% or more, preferably 80% or more, more preferably 90% or more of the gene sequence shown in SEQ ID NO: 2 is also included in the present invention.

Since the recombinant DNA containing the acyl CoA oxidase gene obtained as described above does not have a promoter for high expression in Escherichia coli, the transformant strain containing the recombinant DNA has low acyl CoA oxidase productivity. Therefore, an acyl CoA oxidase high-producing strain is obtained by the following operation.
First, based on the base sequence obtained by the above operation, the acyl CoA oxidase gene includes the 5 ′ end or 3 ′ end, and about 15 base oligonucleotides before and after that (a total of 30 base oligonucleotides, 3 ′ end side). Is a complementary strand) and this is used as a primer. An NdeI site is incorporated into this synthetic primer, and a product amplified by PCR is digested by allowing NdeI to act so that a coding region can be obtained. That is, PCR is performed using the purified recombinant plasmid DNA obtained above and a synthetic primer as a template, and the resulting product is digested with NdeI to obtain the DNA of the region encoding acyl CoA oxidase. be able to.

  The obtained DNA is a vector DNA having a DNA sequence (see The Operan, p. 227, Cold Spring Harbor Laboratory, 1980) containing expression regions such as a promoter, an operator and a ribosome binding site derived from E. coli lactose operon. Insert into. The vector DNA used may be any, for example, plasmid DNA or bacteriophage DNA. Specifically, pUTE500K ', which is a pBR vector described in JP-A-08-205861, or pUTE300K' shown in item (5) of the embodiment obtained by improving this to a pUC system can be used. Using the obtained recombinant DNA, for example, Escherichia coli K-12, preferably Escherichia coli JM109 (manufactured by Toyobo Co., Ltd.), DH5α (manufactured by Takara Shuzo Co., Ltd.) and the like are transformed or transduced to obtain respective strains.

In order to produce acyl CoA oxidase using the transformant or transductant having the ability to produce acyl CoA oxidase obtained as described above, for example, a strain belonging to the genus Escherichia, it can be carried out as follows. it can. In order to culture the microorganism, it may be cultured by a normal solid culture method, but it is preferable to employ a liquid culture method as much as possible.
Examples of the culture medium for culturing the microorganism include yeast extract, peptone, meat extract, corn steep liquor, soybean or wheat straw leachate, one or more nitrogen sources, potassium dihydrogen phosphate, hydrogen phosphate One or more inorganic salts such as dipotassium, magnesium sulfate, ferric chloride, ferric sulfate or manganese sulfate are added, and further, saccharide raw materials, vitamins and the like are added as necessary.
In addition, it is appropriate to adjust the initial pH of the medium to 7 to 9, for example. The culture is preferably performed, for example, at 30 to 42 ° C., preferably around 37 ° C. for 6 to 24 hours, by aeration / agitation deep culture, shaking culture, stationary culture, or the like. In order to collect acyl CoA oxidase from the culture after completion of the culture, a normal enzyme collecting means can be used.

The cells are separated from the culture by, for example, filtration, centrifugation and the like, and washed. It is preferable to collect acyl CoA oxidase from the cells. In this case, the cells can be used as they are, but the cell walls using a cell wall lytic enzyme such as lysozyme, a method of destroying the cells using various disrupting means such as an ultrasonic crusher, a French press, or Dynamil. Acyl CoA oxidase is preferably collected from the cells by a method for dissolving the lysate, a method for extracting the enzyme from the cells using a surfactant such as Triton X-100, and the like.
In order to isolate acyl CoA oxidase from the crude enzyme solution thus obtained, a method commonly used for enzyme purification can be used. For example, an ammonium sulfate salting-out method, an organic solvent precipitation method, an ion exchange chromatography method, a gel filtration chromatography method, an adsorption chromatography method, an electrophoresis method or the like is preferably used in an appropriate combination.

The physicochemical properties of the obtained acyl CoA oxidase are as shown below.
(1) Action: 1 mol of enoyl CoA and 1 mol of hydrogen peroxide are produced from 1 mol of acyl CoA and 1 mol of oxygen.
(2) Substrate specificity: As shown in FIG. 1, it acts on acyl CoAs having various carbon numbers, and particularly works best on myristoyl CoA having 14 carbon atoms.
(3) Molecular weight: about 82,800 (calculated from amino acid sequence).
The “SH reagent” of the present invention refers to a reagent that reacts with an SH group, and is also referred to as a thiol reagent or a sulfhydryl reagent. For example, maleimide derivatives such as N-ethylmaleimide (NEM), iodoacetic acid, iodoacetamide, p-mercuribenzoic acid (PMB), 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) and the like can be mentioned. .
Further, “excellent stability to SH reagent” of the present invention means that the reaction conditions described in the activity measurement method and stability measurement method described below are 37 in the presence of 10 mM SH reagent at pH 7.5. It means that the residual activity ratio after 4 hours of treatment at 40 ° C. is 40% or more, preferably 60% or more of the activity before the treatment. An acyl CoA oxidase having excellent stability against an SH reagent is extremely useful in the industry because the storage stability of an enzyme-containing product in which the SH reagent coexists is significantly improved.

Various methods can be used as a method for measuring the activity of acyl CoA oxidase and a method for measuring stability. As an example, a method for measuring an acyl CoA oxidase activity and a method for measuring stability used in the present invention will be described below. .
(Method for measuring acyl CoA oxidase activity)
0.2 ml Tris-HCl buffer (pH 7.5) 0.9 ml, 0.1% palmitoyl CoA solution 0.1 ml, 15 mM Toos solution 0.04 ml, 150 U / ml peroxidase solution 0.04 ml, 1.76% 4-Aminoantipyrine solution 0.02 ml of the mixture was incubated at 37 ° C. for 5 minutes, and 0.05 ml of acyl CoA oxidase sample was added and mixed. A total volume of 1.15 ml was reacted at 37 ° C. for 3 minutes, and the change in absorbance at 555 nm of the reaction solution for 3 minutes from the start of the reaction was measured using a spectrophotometer. Enzyme 1U was defined as the amount of enzyme that produces 1 μmol of hydrogen peroxide per minute under the above measurement conditions.
(NEM stability measurement method)
Each acyl CoA oxidase is adjusted to a concentration of about 10 U / ml with 200 mM potassium phosphate buffer (pH 7.5) containing 0.1 mM FAD, and each enzyme solution contains 200 mM potassium phosphate containing 0.1 mM FAD and 20 mM NEM. An equal amount of a buffer solution (pH 7.5) was added to adjust the concentration of NEM to 10 mM. This enzyme solution (about 5 U / ml) was left at 37 ° C. for 4 hours. The stability of the sample was evaluated by measuring the enzyme activity of the sample before and after storage, and determining the residual activity ratio.

    Hereinafter, the present invention will be described more specifically with reference to examples.

Examples Cloning of acyl CoA oxidase gene (1) Corynebacterium sp. No. 2-3-1 Preparation of Chromosomal DNA Corynebacterium sp. No. 2-3-1 (FERM BP-6183) was added to LB agar medium [bacttripton 1% (W / V), yeast extract 0.5% (W / V), NaCl 0.5% (W / V) and Agar 1.4% (W / V)] and inoculated at 37 ° C. Bacteria grown on the surface of the medium were collected. From this bacterial cell, 100 μg of chromosomal DNA was obtained using GNOME DNA Isolation Kit (Funakoshi).

(2) Acquisition of partial fragment of acyl CoA oxidase gene 1
Next, using the international base sequence database of the Japan DNA Data Bank, the homology search of the amino acid sequences of various acyl CoA oxidases is performed, and the portions where the codons are degenerated are mixed based on the amino acid sequence portion having high homology. A plurality of PCR primers with bases were prepared. Using the chromosomal DNA prepared in Example (1) as a template, PCR was performed using Ex Taq DNA polymerase (Takara Shuzo). The PCR reaction was carried out for 30 cycles under the conditions of thermal denaturation 94 ° C., 2 minutes, annealing 58 ° C., 30 seconds, extension reaction 72 ° C., 1 minute using a PCR master cycler gradient (Eppendorf). As a result, the sense primer is 5′-TTYGCNATGACCNGARATHGGNCAYGG-3 ′ (26 mer, A is adenine, C is cytosine, G is guanine, T is thymine, H is adenine or cytosine or thymine, N is adenine or Cytosine or guanine or thymine, R represents adenine or guanine, Y represents thymine or cytosine, corresponding amino acid sequence: Phe Ala Met Thr Glu Ile Gly His Gly, antisense primer 5′-TGYTGCATNARNACNGTRTTTCTCTCTY 29mer, corresponding amino acid sequence: Glu Gly Asp Asn Thr Val Leu Met GlnGln), a gene fragment corresponding to about 0.9 kbp was amplified.
The amplified DNA fragment was recovered from the gel after 1% agarose gel electrophoresis and incorporated into pT7Blue T Vector (Takara Shuzo) to obtain a recombinant plasmid. The base sequence of the inserted DNA in the plasmid was determined using a multicapillary DNA analysis system CEQ2000 (manufactured by Beckman Coulter). As a result, the DNA fragment (841 bp) had base sequences at both ends that correctly encoded the amino acid sequence of the peptide used for PCR primer design. Further, in the amino acid sequence deduced from the determined base sequence, high homology was recognized with the internal amino acid sequence of known acyl CoA oxidase. Therefore, it was found that the obtained amplified DNA fragment was a part (partial gene) of the acyl CoA oxidase gene of the present invention.

(3) Corynebacterium sp. No. 2-3-1 Southern Blot Analysis of Chromosomal DNA Next, 2 μg of the chromosomal DNA prepared in Example (1) was digested with the restriction enzyme ClaI at 37 ° C. for 5 hours. The obtained restriction enzyme digested DNA was subjected to 0.7% agarose gel electrophoresis. After the electrophoresis, DNA was transferred to a nylon membrane (Hybond-N +, manufactured by Amersham Pharmacia Biotech) by Southern blotting. As a hybridization probe, PCR Dig labeling mix (manufactured by Roche Diagnostics) exists using the plasmid gene obtained in Example (2) as a template and the primers used in Example (2). The PCR amplified product below was used. PCR reaction conditions and the like were the same as in (2) above.
After washing the nylon membrane with 2 × SSC (0.3 M NaCl, 0.03 M sodium citrate; pH 7.0), hybridization was performed using the DIG system according to the user guide (Roche Diagnostics). Went. After the hybridization, the nylon membrane was washed with 2 × SSC containing 0.1% SDS (15 mM NaCl, 1.5 mM sodium citrate; pH 7.0) for 5 minutes at room temperature, twice, Performed twice with 0.1% SSC containing 1% SDS at 68 ° C. for 15 minutes.
As a result, a signal derived from the probe hybridized at a position of about 4.5 kbp was observed.

(4) Acquisition of partial fragment of acyl CoA oxidase gene 2
Based on the above results, 10 μg of ClaI-digested chromosomal DNA was separated by 1.0% agarose gel electrophoresis, and an agarose gel at a position corresponding to a size of about 4.5 kbp was cut out. A DNA fragment was extracted and purified from the gel by GENE CLEAN II (Funakoshi), and the DNA fragment was self-ligated using T4 DNA Ligase (Roche). Sense primer 5′-GGACCTGGATGGTTAGTTCACGTTC-3 ′ (25 mer) and antisense primer 5′-GGTGGCAATGCTGGCAACGTCCGAG-3 ′ (25 mer) prepared based on the partial gene sequence of Example (2) using the above ligation product as a template ) Was used to perform inverse PCR. Using Ex Taq DNA polymerase (Takara Shuzo), 30 cycles were performed under conditions of heat denaturation 94 ° C., 30 seconds, annealing 67 ° C., 30 seconds, extension reaction 72 ° C., 7 minutes. As a result, amplified DNA (about 3.5 kbp) could be obtained.
This amplified DNA fragment was recovered from the gel after 1% agarose gel electrophoresis and incorporated into pT7Blue T Vector (Takara Shuzo) to obtain a recombinant plasmid. The base sequence of the inserted DNA in the plasmid was determined using a multicapillary DNA analysis system CEQ2000 (manufactured by Beckman Coulter). The amino acid sequence deduced from the determined base sequence was found to have high homology with the internal amino acid sequence of known acyl CoA oxidase, and it was found that TAG as a stop codon was included.

(5) Cloning of full-length acyl CoA oxidase gene and acquisition of acyl CoA oxidase producing strain First, based on the base sequence obtained by the above operation, an oligonucleotide containing the 5 'end or 3' end of the acyl CoA oxidase gene (total A 30-base oligonucleotide and a complementary strand on the 3 ′ end side were designed. An NdeI site was incorporated into this primer, and the product amplified by PCR was digested with NdeI to obtain a coding region. That is, 5′-ATCGGTCTCGTCACATATGCCGGGTGAGATG-3 ′ (30 mer) was synthesized as a sense primer, and 5′-GGCCGTCAACGGACATATGCTAACGGGCT-3 ′ (30 mer) was synthesized as an antisense primer. Using the chromosomal gene prepared in Example (1) as a template, using a synthetic primer and KOD Plus polymerase (manufactured by Toyobo Co., Ltd.), heat denaturation 94 ° C., 15 seconds, annealing 56 ° C., 30 seconds, extension reaction 68 ° C., 4 minutes 30 cycles were performed under the conditions of As a result, amplified DNA (about 2.3 kbp) was obtained, and this was digested with NdeI to obtain DNA in a region encoding acyl CoA oxidase.
The pBR system possessing a DNA sequence (see The Operan, p. 227, Cold Spring Harbor Laboratory, 1980) containing an expression region such as a promoter, an operator, and a ribosome binding site derived from the E. coli lactose operon. The vector pUTE500K ′ (described in JP-A-08-205861) was inserted into the NdeI site of pUTE300K ′ modified to the pUC system to obtain a recombinant plasmid DNA pACO-C3002L. D. M.M. According to the method of Morrison (Methods in Enzymology, 68, p. 326-331, 1979), E. coli JM109 (Toyobo Co., Ltd.) was transformed with the recombinant plasmid DNA pACO-C3002L, and the transformed strain E. coli JM109 (pACO-C3002L) was obtained. The recombinant plasmid pACO-C3002L was extracted from the cells using QIAGEN tip-100 (Qiagen) and purified to obtain 100 μg of the recombinant plasmid.
The base sequence of the inserted DNA in the plasmid was determined using a multicapillary DNA analysis system CEQ2000 (manufactured by Beckman Coulter). By carrying out PCR of multiple lots using chromosomal DNA as a template, it was confirmed that no error was contained. The determined nucleotide sequence of the acyl CoA oxidase gene is shown in SEQ ID NO: 2, and the amino acid sequence of the polypeptide translated from the DNA sequence is shown in SEQ ID NO: 1, respectively. It was found that the ORF of the acyl CoA oxidase gene inserted in pACO-C3002L consists of 2271 bp and 757 amino acids. Note that pACO-C3002L is deposited as FERM BP-8428 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology.
The obtained E. coli JM109 (pACO-C3002L) was mixed with LB-IPTG-amp medium [Bactotryptone 1% (W / V), Yeast extract 0.5% (W / V), NaCl 0. 5% (W / V), isopropyl-β-D-thiogalactopyranoside (1 mM) and ampicillin (50 μg / ml)] after shaking culture for 10 hours, the acyl CoA oxidase activity was measured. 0.02 U / ml.

(6) Method for producing acyl CoA oxidase E. coli JM109 (pACO-C3002L) was cultured in 50 ml of medium by the method of Example (5) to produce acyl CoA oxidase. A purified acyl-CoA oxidase solution was obtained from the main culture solution by the method described in Japanese Patent Publication No. 58-40466.

(7) Evaluation of stability of acyl CoA oxidase to NEM Recombinant acyl CoA oxidase prepared in Example (6) and commercially available purified Arthrobacter sp. The derived acyl CoA oxidase (Sigma) was adjusted to a concentration of about 10 U / ml with 200 mM potassium phosphate buffer (pH 7.5) containing 0.1 mM FAD, and each enzyme solution contained 0.1 mM FAD and 20 mM NEM. An equal amount of 200 mM potassium phosphate buffer (pH 7.5) was added to adjust the concentration of NEM to 10 mM. The enzyme solution (about 5 U / ml) was allowed to stand at 37 ° C., and the enzyme activity of the sample before and after storage was measured to determine the residual activity ratio, thereby evaluating the stability. As shown in FIG. 2, Corynebacterium sp. No. 2-3-1-derived acyl-CoA oxidase is commercially available purified Arthrobacter sp. The stability in the presence of NEM was higher than that of the derived acyl-CoA oxidase, and a residual activity of about 65% was observed even after standing for 4 hours.

  The acyl CoA oxidase of the present invention, which is excellent in stability in the presence of an SH reagent, can be advantageously used in a measurement kit as a diagnostic enzyme or the like and is industrially useful.

The figure which shows the substrate specificity of this invention acyl-CoA oxidase. The figure which shows the stability with respect to NEM of this invention acyl CoA oxidase.

Claims (8)

An acyl CoA oxidase which exhibits a residual activity of 40% or more after treatment at 37 ° C. for 4 hours at pH 7.5 in the presence of 10 mM SH reagent. An acyl CoA oxidase which exhibits a residual activity of 60% or more after treatment at 37 ° C. for 4 hours at pH 7.5 in the presence of 10 mM SH reagent. The acyl CoA oxidase of the following (a) or (b).
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1, (b) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (a), and having an acyl CoA oxidase activity Protein The gene which codes the acyl CoA oxidase of the following (a) or (b).
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1, (b) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (a), and having an acyl CoA oxidase activity Protein An acyl CoA oxidase gene comprising the following DNA (a) or (b):
(A) DNA consisting of the base sequence shown in SEQ ID NO: 2
(B) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to DNA consisting of the base sequence of (a) and encodes a protein having acyl CoA oxidase activity A recombinant DNA, wherein the acyl CoA oxidase gene according to claim 4 or 5 is inserted into a vector DNA. A transformant or transductant comprising the recombinant DNA according to claim 6. A method for producing an acyl CoA oxidase, comprising culturing the transformant or transductant according to claim 7 in a medium and collecting acyl CoA oxidase from the culture.

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