JP3632246B2 - E. coli flavin reductase - Google Patents

Description

【図面の簡単な説明】
【図１】ニトロ還元酵素遺伝子の変異導入に用いられたＰＣＲ用のオリゴヌクレオチドプライマー（ＮＲ−ＮおよびＮＲ−Ｃ）。制限酵素切断部位は下線で示した。
【図２】本発明に係わる大腸菌の変異ニトロ還元酵素遺伝子を含有する本発明の組換えプラスミド構築工程を示す。
ｐＵＣプラスミドベクターに由来する部分は白色で、変異ニトロ還元酵素遺伝子の部分は斜め線で示した。
【図３】正常なニトロ還元酵素遺伝子とフラビン還元酵素遺伝子の塩基配列の相違部分とその周辺の構造を示す。
番号は正常なニトロ還元酵素およびフラビン還元酵素のアミノ酸配列の位置を示す。正常なニトロ還元酵素遺伝子とフラビン還元酵素活性遺伝子の塩基配列の相違部分は矢印で示した。
【図４】フラビン還元酵素の熱安定性試験の結果を示す。
酵素活性は４℃での活性を１００％として、各温度における活性を相対活性として％で表した。○は本発明のフラビン還元酵素で、□は発光細菌のフラビン還元酵素である。
【符号の説明】
ｌａｃＰ ラクトースオペロンのプロモーター
Ａｍｐｒ アンピシリン耐性遺伝子
ｐＵＣ１１８ プラスミドベクター
ｐＮＲ１ 正常なニトロ還元酵素遺伝子発現プラスミド
ｐＮＲ２４７ フラビン還元酵素遺伝子発現プラスミド
ＮｒｕＩ 制限酵素切断部位（ＴＣＧＣＧＡ）
ＰｓｔＩ 制限酵素切断部位（ＣＴＧＣＡＧ）
ＥｃｏＲＩ 制限酵素切断部位（ＧＡＡＴＴＣ）
ＸｂａＩ 制限酵素切断部位（ＴＣＴＡＧＡ）
ＨｉｎｃＩＩ 制限酵素切断部位（ＧＴＰｙＰｕＡＣ：Ｐｙはピリミジン、Ｐｕはプリンを表す）
ｎｒ 正常なニトロ還元酵素遺伝子の塩基配列
ｆｒ フラビン還元酵素遺伝子の塩基配列
ＮＲ 正常なニトロ還元酵素のアミノ酸配列
ＦＲ フラビン還元酵素のアミノ酸配列[0001]
[Industrial application fields]
The present invention relates to a flavin reductase gene of Escherichia coli, a flavin reductase, a recombinant DNA obtained by inserting the gene into a vector DNA, and a method for producing a flavin reductase using a microorganism containing the recombinant DNA.
[0002]
[Prior art]
Luminescent luciferase is a reduced flavin mononucleotide (hereinafter referred to as FMNH).₂And a long-chain fatty aldehyde as a substrate, in the presence of oxygen, an oxidized flavin mononucleotide (hereinafter referred to as FMN) and a long-chain carboxylic acid are produced, and the reaction that emits blue light at that time is catalyzed.
Luminescent bacterial luciferase is useful for measurement of various physiologically active substances using this luminescence as an index. When bacterial luciferase is used in a test tube or the like, FMNH as a substrate is used.₂Is immediately autoxidized and converted to FMN, so that flavin reductase must be present in the reaction system.
[0003]
Therefore, flavin reductase is an enzyme indispensable for a detection system using the luminescence of bacterial luciferase as an index. But luminous bacteriaVibrio  fischeriHowever, the flavin reductase was unstable and easily reduced in activity.
On the other hand, flavin reductaseVibrio  fischeriWas only known to have one enzyme. "Duane, W. and Hastings, JW (1975) Mol. Cell. Biochem. 6, 53-64"
Recently, the present inventorVibrio  fischeriWas found to be similar to nitroreductase at the amino acid sequence level and have a low level of nitroreductase activity (described in Japanese Patent Application No. 3-351717). In addition, the nitroreductase gene of Escherichia coli was isolated and its primary structure was clarified (described in Japanese Patent Application No. 5-340061).
[0004]
[Problems to be solved by the invention]
Therefore, a flavin reductase having higher stability and higher activity has been awaited for use in tests and diagnosis.
That is, the main object of the present invention is to develop a stable and highly active flavin reductase.
[0005]
[Means for Solving the Problems]
As a result of earnest research on the above problems, the present inventor succeeded in producing a flavin reductase gene encoding a stable and highly active flavin reductase by introducing mutations into the nitroreductase gene, and completed the present invention. did. That is, this application provides the following invention.
(1) Mutation treatment of wild-type nitroreductase gene derived from E. coliAs a method of contacting a drug asFlavin reductase gene obtained by performingAndThe wild type nitroreductase gene is the nfsB gene or the nfnB gene, and in the amino acid sequence encoded by the gene, the amino acid of phenylalanine at position 124 isMutated to serine amino acidCoding amino acid sequenceFlavin reductase gene.
(2) A flavin reductase gene comprising the base sequence represented by SEQ ID NO: 1 in the sequence listing.
(3)1A flavin reductase encoded by the flavin reductase gene according to Item.
(4)2The flavin reductase of SEQ ID NO: 2 encoded by the flavin reductase gene according to item 2.
(5) A recombinant DNA characterized by inserting a DNA whose base sequence is represented by SEQ ID NO: 1 in the sequence listing into a vector DNA.
(6)5A method for producing a flavin reductase, comprising culturing a microorganism belonging to the genus Escherichia having the ability to produce a flavin reductase in a medium, and collecting the flavin reductase from the culture.
[0006]
The configuration and effects of the present invention will be described in detail below.
The flavin reductase gene of the present invention is a gene obtained by modification based on a nitroreductase gene. For example, in the amino acid sequence of wild-type nitroreductase derived from E. coli, a flavin reductase gene that encodes an amino acid sequence in which the amino acid at position 124 is mutated to an amino acid other than phenylalanine. thisA flavin reductase gene that encodes an amino acid sequence in which the amino acid at position 124 is serine is preferable, and as such an example, a sequence that includes a 651 nucleotide chain in length and encodes a protein consisting of 217 amino acids. A gene containing the base sequence represented by number 1 can be shown as an example. The sequence type of this gene is genomic DNA.
The enzyme of the present invention has a flavin reducing activity, for example, an FMN reducing activity, and is a protein having an amino acid sequence represented by SEQ ID NO: 2, for example.
The recombinant DNA of the present invention is obtained by inserting a DNA whose base sequence is represented by SEQ ID NO: 1 into a vector DNA.
The method for producing the enzyme of the present invention includes, for example, culturing a microorganism modified with a recombinant DNA in which a DNA represented by SEQ ID NO: 1 is inserted into a vector DNA, and a protein comprising the amino acid sequence represented by SEQ ID NO: 2. Is to manufacture.
[0007]
As a premise for providing a thermostable and highly active flavin reductase by gene modification in the present invention, it is necessary to prepare a wild type nitroreductase gene and a recombinant DNA thereof. The type of wild-type nitroreductase gene or the like is used depending on the type of thermostable highly active flavin reductase gene that is intended to be provided. Any wild-type nitroreductase gene derived from E. coli can be used. For example, a nitroreductase gene that has already been cloned can be used. Such genes include E. colinfsB/nfnBNitro reductase gene "Japanese Patent Application No. 5-340061", Escherichia colinfsAThe nitroreductase gene “Japanese Patent Application No. 6-298936” is exemplified. These genes and the like are prepared according to already known methods. For example, the wild type E. coli gene and its recombinant DNA can be prepared by the method described in Japanese Patent Application No. 5-340061.
[0008]
Mutation treatment of the wild type nitroreductase gene can be performed by a generally known method according to the intended mutant form. That is, a method of contacting a wild-type nitroreductase gene or a recombinant DNA incorporating the gene with a mutagen agent, an ultraviolet irradiation method, a genetic engineering method, or a protein engineering method is widely used. be able to.
Examples of the mutagen used in the above mutation treatment include hydroxylamine, N-methyl-N′-nitro-N-nitrosoguanidine, nitrous acid, sulfurous acid, hydrazine, formic acid, 5-bromouracil and the like. Can do. The conditions for this contact action can be selected according to the type of drug used, and are not particularly limited as long as a desired mutation can be caused in the wild type nitroreductase gene. Usually, the desired mutation can be caused by contacting the drug with a concentration of 0.5-12 M, preferably at a reaction temperature of 20-80 ° C. for 10 minutes or more, preferably 10-180 minutes.
[0009]
Even in the case of performing ultraviolet irradiation, mutation can be caused according to the method described in “Contemporary Science, pp24-30, June 1989”.
As a protein engineering method, a method generally known as site-specific mutagenesis can be used. For example, Kramer method “Kramer W. et al. (1984) Nucleic Acids Res. 12, 9441-9456”, Eckstein method “Taylor, J. W. et al. (1985) Nucleic Acids Res. 87, 8749”. Kunkel method “Kunkel, TA (1985) Proc. Natl. Acad. Sci. USA, 82, 488-492”.
In addition to the above gene modification method, it is of course possible to directly synthesize a desired modified nitroreductase gene, that is, a heat-stable and highly active flavin reductase gene, by an organic synthesis method or an enzyme synthesis method. . The determination and confirmation of the base sequence of the desired flavin reductase gene obtained by the above method can be performed by, for example, the dideoxy modified method “Hattori, M., et al. and Sakaki, Y. et al. (1986) Anal. Biochem. 152, 232 ”and the like.
[0010]
The heat-stable highly active flavin reductase gene obtained as described above is incorporated into a vector such as a bacteriophage, a cosmid, or a plasmid used for transformation of prokaryotic cells or eukaryotic cells by a conventional method, A host corresponding to each vector can be transformed or transduced by a conventional method. For example, when selecting E. coli JM83, E. coli D1210, E. coli JM109, E. coli HB101 or the like as the host, “Sambrook, J. , Fritsch, E .; F. and Maniatis, T .; (1989) Molecular cloning: a laboratory manual. A transduced strain can be obtained by transduction according to the method described in Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
[0011]
Then, by screening for a strain having the ability to produce a highly active flavin reductase that is more heat stable than the above strain, the recombinant DNA containing the heat stable and highly active flavin reductase gene inserted into the vector DNA is included. And a highly active flavin reductase producing ability can be obtained. In order to obtain a novel recombinant DNA purified from the strain thus obtained, for example, the alkali method “Sambrook, J. et al. , Fritsch, E .; F. and Maniatis, T .; (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York ”or the like may be used.
[0012]
In order to obtain a DNA containing a highly active flavin reductase gene which is more stable than the recombinant DNA thus obtained, for example, a restriction enzyme such as EcoRI and XbaI is added to the recombinant DNA at a temperature. The reaction-terminated solution is allowed to act at 30-40 ° C. for 1-24 hours, preferably at 37 ° C. for 2 hours. , Fritsch, E .; F. and Maniatis, T .; (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York ".
[0013]
Using the obtained Escherichia coli strain containing the recombinant DNA obtained by inserting the heat-stable and highly active flavin reductase gene into the vector DNA and having the ability to produce a heat-stable and highly active flavin reductase as described above. In order to produce a heat-stable and highly active flavin reductase, this strain may be cultured by an ordinary solid culture method, but it is preferable to employ a liquid culture method as much as possible.
[0014]
Examples of the medium for culturing the above strain include one or more nitrogen sources such as yeast extract, tryptone, peptone, meat extract, corn steep liquor, soybean or wheat bran leachate, sodium chloride, phosphoric acid first Add one or more inorganic salts such as potassium, dibasic potassium phosphate, magnesium sulfate, magnesium chloride, ferric chloride, ferric sulfate or manganese sulfate, and if necessary, add saccharide raw materials, vitamins, etc. Those appropriately added are used.
[0015]
The initial pH of the medium is suitably adjusted to pH 7-9. The culture is preferably carried out by aeration and agitation culture, shaking culture, stationary culture, etc. at 30-42 ° C., preferably 37 ° C. for 4-24 hours, more preferably 37 ° C. for 12 hours. In order to collect a highly active flavin reductase which is heat-stable from the culture after completion of the culture, it can be obtained using a normal enzyme collecting means.
[0016]
For example, the bacterial cells are subjected to ultrasonic destruction treatment, grinding treatment, etc. by ordinary methods, or the enzyme is discharged using a lytic enzyme such as lysozyme, or shaken or left in the presence of toluene or the like. Thus, this enzyme can be excreted outside the cells by autolysis. Then, this solution is filtered, centrifuged, etc. to remove the solid part. If necessary, the nucleic acid is removed with streptomycin sulfate, protamine sulfate, manganese sulfate, etc., and then added with ammonium sulfate, alcohol, acetone, etc. Fractionate and collect the precipitate to obtain the crude enzyme.
[0017]
In order to obtain a purified enzyme preparation further from the above crude enzyme, for example, gel filtration using Sephadex, Ultrogel or biogel, adsorption elution using ion exchanger, electrophoresis using polyacrylamide gel, hydroxyapatite, etc. Adsorption elution method using sucrose, precipitation method such as sucrose density gradient centrifugation, affinity chromatography method, fractionation method using molecular sieve membrane or hollow fiber membrane, etc. are appropriately selected, and by combining these, A purified enzyme preparation can be obtained.
In this way, a desired thermostable highly active flavin reductase can be obtained.
[0018]
【Example】
Hereinafter, the present invention will be specifically described with reference to examples.
(1) Preparation of recombinant plasmid pNR1 DNA used as a template for PCR reaction
The Escherichia coli JM83 strain containing the plasmid pNR1 that can express the nitroreductase gene constructed by the method described in Japanese Patent Application No. 5-340061 is cultured at 37 ° C. overnight, and the alkaline method “Sambrook, J. et al. , Fritsch, E .; F. and Maniatis, T .; (1989) Molecular cloning: a laboratory manual. PNR1 plasmid DNA was prepared using Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. That is, 1.5 mL of the culture broth was centrifuged to collect bacterial cells, which were suspended in 0.1 mL of lysozyme solution (50 mM glucose, 10 mM EDTA, 25 mM Tris-HCl pH 8.4 mg / mL lysozyme solution) And reacted for 5 minutes to obtain a lysate. To this lysate, 0.2 mL of 0.2N sodium hydroxide / 1% sodium dodecyl sulfate (SDS) solution was added, and after mixing, the mixture was placed on ice for 5 minutes for alkali denaturation. Further, 0.15 mL of 3N sodium acetate pH 5.2 was added to the denatured product, mixed, and neutralized in ice for 5 minutes. The neutralized product was centrifuged, and after removing the precipitate, an equal volume of TE buffer (10 mM Tris-HCl pH 8.1 mM EDTA) saturated phenol was added to perform deproteinization, and then the aqueous layer was recovered. Two times the amount of ethanol was added to this, mixed, and then centrifuged at 12000 rpm for 10 minutes to obtain a precipitate. This precipitate was dissolved in 0.05 mL of TE buffer, and 0.001 mL of 0.5 mg / mL RNA-degrading enzyme A solution was added thereto. After mixing, the mixture was reacted at 37 ° C. for 30 minutes to decompose RNA. A 20% polyethylene glycol / 2.5M sodium chloride solution was added to the reaction solution, and the mixture was allowed to stand in ice for 1 hour, and then centrifuged at 12000 rpm for 10 minutes. The resulting precipitate was washed with 70% ethanol, dried, and dissolved in 0.05 mL of TE buffer to prepare recombinant pNR1 plasmid DNA.
[0019]
(2) Random mutation of nitroreductase gene by PCR
Using the prepared pNR1 plasmid DNA as a template, using an upstream synthetic oligonucleotide primer NR-N containing the start codon ATG and a downstream synthetic primer NR-C (shown in FIG. 1) containing the stop codon TAA, PCR method [Innis, M., et al. A. , Gelfand, D .; H. Sninsky, J .; J. et al. and White, T .; J. et al. (1990) PCR Protocols: A Guide to Methods And Applications. The coding region of the nitroreductase gene was amplified by “Academic Press”. That is, the concentration of dNTP which is a substrate of Taq DNA polymerase is set to 1 mM for dTTP, dCTP, and dGTP, 0.2 mM for dATP, and 0.5 mM MnCl.₂6.1 mM MgCl containing₂, And 25 cycles of PCR reaction were performed on 1 ng of the target plasmid. Agarose electrophoresis “Sambrook, J. et al. , Fritsch, E .; F. and Maniatis, T .; (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York "was followed by confirmation of target DNA amplification, and after deproteinization of this PCR reaction solution with phenol as described in (1) above, 1/10 amount of 3M sodium acetate pH 5.2 and 2.5 times amount of ethanol were added and left at a temperature of -70 ° C for 15 minutes, followed by centrifugation at 12000 rpm for 10 minutes to obtain a precipitate. This precipitate was dried and then dissolved in 0.05 mL of TE buffer to prepare a DNA fragment solution of a mutant nitroreductase gene.
[0020]
(3) Production of Escherichia coli having a plasmid capable of expressing a mutant nitroreductase gene
0.05 mL of a solution of the obtained mutant nitroreductase gene DNA fragment was mixed with 0.006 mL of Med buffer (10 mM Tris-HCl pH 7.5, 10 mM magnesium chloride, 1 mM dithiothreitol, 50 mM sodium chloride) and 10 units. Restriction enzymes XbaI and EcoRI were added, and the mixture was reacted at a temperature of 37 ° C. for 1 hour for cleavage. This digested product was treated at 70 ° C. to inactivate the restriction enzyme. This treated product was extracted with phenol in the same manner as in the method described in (1) above, and then ethanol precipitated in the same manner as in the method described in (2) above to obtain a precipitate. This precipitate was dried and then dissolved in 0.01 mL of TE buffer. In 0.005 mL of this solution, 0.001 mL of X10 ligation buffer (20 mM magnesium chloride, 660 mM Tris-HCl, pH 7.6, 10 mM ATP, 150 mM dithiothreitol), 1 unit of T4 DNA ligase and a DNA fragment of the mutant nitroreductase gene PUC118 plasmid “Vieira, J. C., cleaved with restriction enzymes XbaI and EcoRI in the same manner as described above. and Messaging, J.M. (1987) Methods Enzymology (Methods Enzymol.) 153, 3 ”was added in 0.001 mg, and water was added to make a total volume of 0.01 mL, followed by reaction at a temperature of 16 ° C. for 16 hours. Using the obtained reaction solution, “Sambrook, J. et al. , Fritsch, E .; F. and Maniatis, T .; (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York ", by the transformation method described in Escherichia coli JM83 strain" Messing, J. and Vieira, J. et al. (1982) Gene, 19, 269 ”was transformed, ampicillin resistance and beta-galactosidase activity were examined, and a transformed strain was obtained. FIG. 2 shows a construction process of a plasmid capable of expressing a mutant nitroreductase gene.
[0021]
(4) Selection of E. coli containing plasmid expressing flavin reductase gene
About 90 of the resulting transformants, the colonies were 0.05 mg / mL ampicillin-containing LB medium (containing 10 g of tryptone, 5 g of yeast extract and 10 g of sodium chloride per liter, and neutralized with sodium hydroxide. ) Sambrook, J. et al. , Fritsch, E .; F. and Maniatis, T .; (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York "was inoculated into 10 mL, and cultured at 37 ° C for 5 hours. The cells were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (abbreviated as SDS-PAGE) [Laemmli, U. K. (1970) Nature, 277, 680 ”to estimate the amount and size of the mutant nitroreductase expressed from the plasmid of the transformant compared to the expression protein pattern of E. coli without the plasmid. As a result, the expression level varied, and the size showed a molecular weight of 27,000.
[0022]
Next, 1.5 ml of the above culture solution was centrifuged at 10,000 rpm with respect to the transformed strain in which the expression of the mutant nitroreductase was confirmed, and the supernatant was removed. The cells were suspended in 0.5 ml of 50 mM potassium phosphate buffer (pH 7), and sonicated using a Branson Sonifier 250. This crushed material was centrifuged at 12,000 rpm and 4 ° C. for 30 minutes, and this supernatant was used as a crude enzyme extract. The extract was measured for flavin reductase activity and nitroreductase activity. The measurement of flavin reductase activity was performed using the method of Zenno et al., Zeno, S., using NADH as an electron donor and FMN as an electron acceptor. Saigo, K .; , Kanoh, H .; and Inouye, S .; (1994) Journal of Bacteriology (176, 3536). On the other hand, the measurement of nitroreductase activity was carried out using the method of Peterson et al., “Peterson, F. et al.” Using NADH as an electron donor and nitrofurazone as an electron acceptor. J. et al. Mason, R .; P. Hovsepian, J .; and Holtzman, J. et al. L. (1979) Journal of Biological Chemistry (J. Biol. Chem.), 254, 4009 ”. Further, the protein concentration was measured using a protein assay kit manufactured by Bio-Rad, using the standard protein as bovine plasma globulin and the dye binding method “Bradford, M. et al. M.M. (1976) Anal. Biochem. 72, 248 ”.
As a result, one transformant having a clearly higher flavin reductase activity than nitroreductase activity was obtained. The plasmid contained in this strain was named pNR247. That is, the mutant nitroreductase encoded by plasmid pNR247 was a flavin reductase.
[0023]
(5) Purification of flavin reductase encoded by plasmid pNR247
Based on (4) above, the JM83 strain containing pNR247 was cultured, and a crude enzyme extract was obtained from this culture. Starting from this extract, flavin reductase was purified at 4 ° C. as follows. The crude enzyme extract was dialyzed against 10 mM potassium phosphate buffer (pH 7). The dialyzate was applied to a Q Sepharose (Pharmacia LKB) anion exchange column (2.6 × 10 cm). After washing with 50 mM potassium phosphate buffer (pH 7), elution was performed using 50-500 mM potassium phosphate buffer (pH 7). This chromatography was performed at a flow rate of 2.0 ml / min. Flavin reductase activity was measured for each fraction. Next, the peak fraction of enzyme activity was dialyzed against 10 mM potassium phosphate buffer (pH 7), and then applied to a Blue Sepharose (Pharmacia LKB) column (1.0 × 10 cm). After washing with 20 mM potassium phosphate buffer (pH 7), elution was performed using 1 mM NADH · 20 mM potassium phosphate buffer (pH 7). The flow rate was 1.0 ml / min. Furthermore, ammonium sulfate was added to the peak fraction of enzyme activity so as to be 20%, and it was applied to a Phenyl Sepharose (Pharmacia LKB) column (1.0 × 15 cm). After washing with 20% ammonium sulfate / 50 mM potassium phosphate buffer (pH 7), elution was performed using 20-4% ammonium sulfate / 50 mM potassium phosphate buffer (pH 7). The flow rate was 1.0 ml / min. Finally, the peak fraction of enzyme activity was concentrated with Centricon 10 (Amicon) and applied to a Superose 12 (Pharmacia LKB) gel filtration column (1 × 30 cm). This chromatography was performed using a 100 mM potassium phosphate buffer (pH 7) at a flow rate of 0.2 ml / min. This enzyme activity fraction was used as a purified flavin reductase. When this sample was evaluated by the SDS-PAGE analysis described in (4) above, it was detected as a single band protein.
[0024]
Moreover, when the nitroreductase activity and the flavin reductase activity of this enzyme were measured, they were 9.55 mmol / mg protein and 1380 mmol / mg protein, respectively. On the other hand, the nitroreductase activity and the flavin reductase activity of the normal nitroreductase without the introduced mutation were 9.74 mmol / mg protein and 0.647 mmol / mg protein, respectively. It was. Therefore, the enzyme encoded by pNR247 is a flavin reductase, and nitroreductase could be converted to flavin reductase by introducing a mutation.
In addition, the method of Zenno et al. Saigo, K .; , Kanoh, H .; and Inouye, S .; (1994) J. Bacteriol., 176, 3536 ”.Vibrio  fischeriThe flavin reductase had a nitroreductase activity and a flavin reductase activity of 14.1 mmol / mg protein and 35.2 mmol / mg protein, respectively. From this, the flavin reductase obtained by modifying the nitroreductase obtained this time is the same as the nitroreductase of Escherichia coli.V.fischeriThe flavin reductase activity was higher than that of flavin reductase.
[0025]
(6) Determination of the base sequence of the flavin reductase gene encoded by the plasmid pNR247
PNR247 plasmid DNA having a flavin reductase gene was prepared using the alkaline method described in the above section (1). The base sequence of the inserted DNA fragment of the pNR247 plasmid was converted to the dideoxy modified method “Hattori, M. et al. and Sakaki, Y. et al. (1986) Anal. Biochem. 152, 232 ”. The base sequence is shown in SEQ ID NO: 1. The amino acid sequence predicted from the nucleotide sequence is shown in SEQ ID NO: 2. As shown in FIG. 3, when comparing the amino acid sequence of a normal nitroreductase with the amino acid sequence predicted from the obtained base sequence, the 124th phenylalanine (Phe) in the amino acid sequence was substituted with serine (Ser). It was.
[0026]
(7) Thermostability test of flavin reductase encoded by plasmid pNR247
The flavin reductase obtained in (5) above was placed in a 1.5 mL tube at a concentration of 1 mg / ml in a 1.5 mL tube, sealed, heated to 4 to 60 ° C. for 1 hour, and then treated ( The flavin reductase activity was measured by the method described in 4). For comparison, the method of Zenno et al. Saigo, K .; , Kanoh, H .; and Inouye, S .; (1994) Luminescent bacteria purified by J. Bacteriol., 176, 3536Vibrio  fischeriSimilarly, the thermal stability of the flavin reductase was also tested. These results are shown in FIG. The flavin reductase of the present invention is stable up to 40 ° C.Vibrio  fischeriThe flavin reductase was only stable up to 30 ° C. The flavin reductase of the present invention is a luminescent bacteriumVibrio  fischeriIt was found to be a more stable enzyme than flavin reductase.
[0027]
【The invention's effect】
According to the present invention, by introducing a mutation into the nitroreductase gene of E. coli, the nitroreductase and luminescent bacteria of E. coliV.fischeriIt has higher flavin reducing activity than flavin reductase ofV.fischeriThus, a new stable flavin reductase was able to be provided. And since the stable flavin reductase which has high flavin reduction activity by the method of this invention can be produced efficiently, this invention is very useful industrially.
[0028]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows oligonucleotide primers for PCR (NR-N and NR-C) used for introducing a mutation in a nitroreductase gene. Restriction enzyme cleavage sites are underlined.
FIG. 2 shows a process for constructing a recombinant plasmid of the present invention containing a mutant nitroreductase gene of Escherichia coli according to the present invention.
The part derived from the pUC plasmid vector is white and the part of the mutant nitroreductase gene is indicated by diagonal lines.
FIG. 3 shows the difference between the base sequences of the normal nitroreductase gene and the flavin reductase gene and the structure around it.
The numbers indicate the position of the amino acid sequences of normal nitroreductase and flavin reductase. The differences between the base sequences of the normal nitroreductase gene and the flavin reductase activity gene are indicated by arrows.
FIG. 4 shows the results of a thermal stability test for flavin reductase.
The enzyme activity was expressed as% with the activity at 4 ° C. as 100% and the activity at each temperature as the relative activity. ○ is the flavin reductase of the present invention, and □ is the flavin reductase of luminescent bacteria.
[Explanation of symbols]
lacP Lactose operon promoter
Ampr ampicillin resistance gene
pUC118 plasmid vector
pNR1 Normal nitroreductase gene expression plasmid
pNR247 Flavin reductase gene expression plasmid
NruI restriction enzyme cleavage site (TCGCGA)
PstI restriction enzyme cleavage site (CTGCAG)
EcoRI restriction enzyme cleavage site (GAATTC)
XbaI restriction enzyme cleavage site (TCTAGA)
HincII restriction enzyme cleavage site (GTPyPuAC: Py represents pyrimidine, Pu represents purine)
nr Nucleotide sequence of normal nitroreductase gene
base sequence of fr flavin reductase gene
NR Amino acid sequence of normal nitroreductase
Amino acid sequence of FR flavin reductase

Claims (6)

A flavin reductase gene obtained by performing a method of contacting a drug with a wild-type nitroreductase gene derived from E. coli as a mutation treatment , wherein the wild-type nitroreductase gene is an nfsB gene or an nfnB gene, A flavin reductase gene encoding an amino acid sequence in which the amino acid of phenylalanine at position 124 is mutated to an amino acid of serine in the encoded amino acid sequence. A flavin reductase gene comprising the base sequence represented by SEQ ID NO: 1 in the sequence listing. A flavin reductase encoded by the flavin reductase gene according to claim 1 . The flavin reductase of SEQ ID NO: 2 encoded by the flavin reductase gene according to claim 2 . A recombinant DNA characterized by inserting a DNA represented by SEQ ID NO: 1 in the sequence listing into a vector DNA. A method for producing a flavin reductase comprising culturing a microorganism belonging to the genus Escherichia comprising the recombinant DNA according to claim 5 and having the ability to produce flavin reductase in a medium, and collecting the flavin reductase from the culture. .

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