JPH06303981A - Dna having genetic information on protein having formaldehyde dehydrogenase activity and production of formaldehyde dehydrogenase - Google Patents

Abstract

PURPOSE:To feed a large amount of a DNA having the non-glutathione- dependent formaldehyde dehydrogenase in a pure form at a low cost without producing other enzymes accompanying thereto in natural bacteria. CONSTITUTION:The DNA fragment contains a base sequence described in sequence No.1 of the sequence table and a recombinant vector has the DNA fragment. The transformant is obtained by transformation with the recombinant vector and the method for producing a formaldehyde dehydrogenase is characterized by culturing the transformant in a culture medium, producing the formaldehyde dehydrogenase and collecting the formaldehyde dehydrogenase. Although cleatininamide hydrolase, cleatininamidinohydrolase, sarcosine dehydrogenase, etc., are especially contained in the enzyme produced from natural bacteria and many steps are required for removing them, the steps for purification thereof can be simplified.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention has a genetic information of a protein having glutathione-independent formaldehyde dehydrogenase activity, which is used for creatine measurement, creatinine measurement, uric acid measurement or catalase measurement in body fluids.
The present invention relates to an NA fragment, a recombinant vector having the DNA fragment, a transformant transformed with the vector, and a method for producing formaldehyde dehydrogenase using the transformant.

[0002]

[Prior Art] Formaldehyde dehydrogenase (EC 1.2.
1.46) is an enzyme that catalyzes the oxidation of formaldehyde to formic acid. There are two types, glutathione-dependent and glutathione-independent. Glutathione-dependent formaldehyde dehydrogenase exists widely in the living world and is purified from many organisms from Escherichia coli to humans. On the other hand, the only glutathione-independent formaldehyde dehydrogenase to date is the enzyme produced by Pseudomonas bacteria. Formaldehyde dehydrogenase produced by a Pseudomonas bacterium is excellent in practical use in the measurement of creatinine, creatine, etc. in that glutathione is not required for activity expression. However, these Pseudomonas bacteria originally produce not only formaldehyde dehydrogenase but also creatinine, creatinine amide hydrolase, creatine amidinohydrolase, sarcosine dehydrogenase, etc. which are necessary for the measurement of creatine. Therefore, a large number of steps are required to purify formaldehyde dehydrogenase from the bacterium to obtain a pure product. Further, the productivity of formaldehyde dehydrogenase of Pseudomonas bacteria is not so sufficient, and it has been required to industrially produce a large amount of formaldehyde dehydrogenase.

[0003]

The object of the present invention is to isolate a DNA fragment carrying the genetic information of a protein having glutathione-independent formaldehyde dehydrogenase activity, clarify its molecular structure, and determine the formaldehyde dehydrogenase. Proteins having enzymatic activity are not produced by genetic engineering techniques in other bacteria that accompany them in natural bacteria,
It is to provide a means capable of mass-producing in a pure form at low cost.

[0004]

[Means for Solving the Problems] In order to achieve the above-mentioned object, the present inventors have established Pseudomonas putida as a glutathione-independent formaldehyde dehydrogenase-producing bacterium.
udomonas putida), and the chromosome D extracted from the cells
The formaldehyde dehydrogenase gene was successfully isolated from NA, and the entire structure of the DNA was determined. Furthermore, we succeeded in highly producing this formaldehyde dehydrogenase in transformants by a genetic engineering method, and made it possible to inexpensively supply a large amount of highly pure formaldehyde dehydrogenase.

That is, the present invention is a DNA fragment having the genetic information of a protein having glutathione-independent formaldehyde dehydrogenase activity, and an example of the DNA fragment contains the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing. Is mentioned.

The present invention is also a recombinant vector having the above DNA fragment.

Further, the present invention is a transformant transformed with the above recombinant vector.

Further, the present invention is a method for producing formaldehyde dehydrogenase, which comprises culturing the above transformant in a medium to produce formaldehyde dehydrogenase and collecting the formaldehyde dehydrogenase.

As the glutathione-independent formaldehyde dehydrogenase-producing bacterium of the present invention, any bacterium belonging to the genus Pseudomonas may be used. Etc. Pseudomonas putida PS-7 was used in the Example of this invention.

A medium for culturing these bacteria is a carbon source,
It contains a nitrogen source, inorganic ions, and if necessary, nitrates, phosphates and the like. As the carbon source, starch or starch hydrolyzate, sugar condensate, peptones and the like are used. As the nitrogen source, polypeptone, tryptone, meat extract, yeast extract and the like can be used. It is desirable to appropriately adjust the pH and temperature of the medium during the culture under aerobic conditions, but this is not always necessary depending on the transformed bacterium. The culture time is until the formaldehyde dehydrogenase activity of the culture reaches its maximum.

To purify formaldehyde dehydrogenase from the culture, the following methods have been conventionally used. A lysate containing formaldehyde dehydrogenase is prepared by centrifuging the culture to collect the cells and then lysing the cells. As a lysis method, for example, a treatment with a cell wall lysing enzyme such as lysozyme or β-glucanase, or a physical disruption method such as ultrasonic disruption or dynomill disruption is suitable. The lysate thus obtained is subjected to ammonium sulfate precipitation fractionation, desalted, and then applied to a carrier such as a cation exchange column, anion exchange column or hydroxylapatite column for fractionation by column chromatography. Pseudomonas bacteria, in addition to formaldehyde dehydrogenase,
It also produces creatinine, creatinine amidohydrolase, creatine amidinohydrolase, and sarcosine dehydrogenase, which are used for the measurement of creatine. For example, Pseudomonas putida PS-7 produces about 1 unit / ml creatinine amide hydrolase, about 1 unit / ml creatine amidinohydrolase, and about 0.5 unit / ml sarcosine dehydrogenase. If these enzymes are mixed in the purified preparation of formaldehyde dehydrogenase, an excess amount of the enzyme than the original set value will be present in the measured substance, which causes a measurement error. Therefore, a large number of steps are required to purify formaldehyde dehydrogenase from the bacterium to obtain a pure product. That is, it is necessary to consider a combination of two or more types of column chromatography and a combination with other purification methods.

The DNA having the genetic information of the protein having glutathione-independent formaldehyde dehydrogenase activity of the present invention (hereinafter, also referred to as formaldehyde dehydrogenase gene) can be extracted from Pseudomonas bacteria such as Pseudomonas putida. Well, it can also be synthesized. Examples of such a base sequence include the base sequence described in SEQ ID NO: 1 of the sequence listing or the base sequence encoding the amino acid sequence described in SEQ ID NO: 2 of the sequence listing. It should be noted that the DNA of the present invention is prepared by gene recombination technology at the specific site of the basic DNA
The formaldehyde dehydrogenase encoded by A does not change the basic characteristics, or those artificially mutated such as substitution, deletion, or insertion so as to improve the characteristics are included.

The DNA of the present invention is obtained by, for example, separating and purifying DNA of Pseudomonas putida, and then blunting or adhering the cut DNA and a linear expression vector with ultrasonic waves or restriction enzymes. At the end portion, a ligation ring is closed by a DNA ligase or the like to obtain a recombinant vector. The recombinant vector thus obtained is transferred to a replicable host microorganism, and then screened using the marker of the vector and formaldehyde dehydrogenase activity or hybridization with a probe specific to the formaldehyde dehydrogenase gene as an index. A microorganism carrying the recombinant vector is obtained. Culturing the microorganism, separating and purifying the recombinant vector from the cultured cells,
Then, the formaldehyde dehydrogenase gene may be collected from the recombinant vector.

Next, the method for collecting DNA will be described in more detail. DNA derived from a microorganism that is a gene donor is collected as follows. That is, for example, the above-mentioned bacteria that are donor microorganisms are cultivated in a liquid medium with aeration and stirring for about 1 to 3 days, the resulting culture is centrifuged to collect the cells, and then the cells are lysed to lyse the formaldehyde dehydrogenase gene. A lysate containing can be prepared. As a lysing method, for example, treatment with a cell wall lysing enzyme such as lysozyme or β-glucanase is performed, and if necessary,
Other enzymes such as protease and a surfactant such as sodium lauryl sulfate may be used in combination, and further freeze-thawing or French press treatment, which are physical disruption methods of cell walls, may be performed in combination with the above-mentioned lysis method. In order to separate and purify DNA from the lysate thus obtained, a method such as deproteinization treatment by phenol extraction, protease treatment, ribonuclease treatment, alcohol precipitation centrifugation etc. may be appropriately combined according to a conventional method. it can.

As a method for cleaving the DNA separated and purified from the microorganism, for example, ultrasonic treatment or restriction enzyme treatment can be carried out. However, since it facilitates the binding between the obtained microbial DNA fragment and the vector, there is a restriction. Enzymes, especially acting on specific nucleotide sequences, eg EcoRI, Hind
Type II restriction enzymes such as II and BamHI are suitable. Suitable vectors are those constructed for gene recombination from phages or plasmids capable of autonomous growth in host microorganisms. As the phage, for example, when Escherichia coli is used as the host microorganism, λgt · 10, λgt · 11 and the like can be used. As the plasmid, for example, pBR322, pUC18, etc. can be used when Escherichia coli is used as the host microorganism. It is desirable to obtain a vector fragment by cleaving such a vector with the same restriction enzyme as that used for cleaving the microbial DNA which is the above-mentioned formaldehyde dehydrogenase gene donor. The method of ligating the microbial DNA fragment with the vector fragment may be a method using a known DNA ligase. For example, after annealing the cohesive ends of the microbial DNA fragment and the vector fragment, a suitable D
A recombinant vector of a microbial DNA fragment and a vector fragment is prepared by using NA ligase. If necessary, after annealing, transfer to host microorganisms and
A ligase can also be used to create a recombinant vector.

The host microorganism may be any as long as the recombinant vector can stably and autonomously grow and can express the trait of the foreign DNA. For example, the host microorganism can be Escherichia coli W3110, Escherichia coli C60.
0, Escherichia coli JM109, Escherichia coli DH5 α, etc. can be used. As a method for transferring the recombinant vector to the host microorganism, for example, when the host microorganism is a microorganism belonging to the genus Escherichia, a method of transferring the recombinant DNA in the presence of calcium ions can be adopted. An electroporation method may be used. The transformant microorganism thus obtained is
It was found that a large amount of formaldehyde dehydrogenase can be stably produced by culturing in a nutrient medium.
The selection as to whether or not the target recombinant vector is transferred into the host microorganism may be carried out by searching for a microorganism capable of simultaneously expressing the drug resistance marker of the vector carrying the target DNA and the formaldehyde dehydrogenase, for example, the drug resistance marker. A microorganism that grows in a selective medium based on and produces formaldehyde dehydrogenase may be selected.

The nucleotide sequence of the formaldehyde dehydrogenase gene obtained by the above method is
e), 214, 1205 to 1210 (1981), using the dideoxy method, and the amino acid sequence of formaldehyde dehydrogenase was deduced from the nucleotide sequence. In this way, the recombinant vector carrying the once selected formaldehyde dehydrogenase gene is removed from the transformed microorganism,
Transfer to other host microorganisms is also easy. In addition, a DNA containing the formaldehyde dehydrogenase gene is excised by cutting with a restriction enzyme from a recombinant vector carrying the formaldehyde dehydrogenase gene,
It can also be easily transferred to a host microorganism by ligating it with a vector fragment obtained by cleaving it by the same method. Regarding the culture form of the host microorganism that is a transformant, the culture conditions may be selected in consideration of the nutritional physiological properties of the host. Usually, in most cases, liquid culture is performed, but industrially, aeration stirring culture is performed. Is advantageous. As the nutrient source of the medium, those usually used for culturing microorganisms can be widely used. The carbon source may be any assimilable carbon compound, for example, glucose, sucrose, lactose,
Maltose, fructose, molasses, pyruvic acid, etc. are used. Any available nitrogen compound may be used as the nitrogen source, and for example, peptone, meat extract, yeast extract, casein hydrolyzate, soybean meal alkali extract, etc. are used. In addition, salts such as phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese and zinc, specific amino acids, specific vitamins and the like are used as necessary.

The culturing temperature can be appropriately changed within the range where the bacterium grows and produces formaldehyde dehydrogenase, but in the case of Escherichia coli, it is preferably about 20 to 42 ° C. Although the culturing time varies slightly depending on the conditions, it may be completed at a suitable time in consideration of the time when the maximum yield of formaldehyde dehydrogenase is reached, and it is usually about 6 to 48 hours. The pH of the medium can be appropriately changed within the range in which the bacterium grows and produces formaldehyde dehydrogenase, but it is particularly preferably about pH 6.0 to 9.0. The culture solution containing the formaldehyde dehydrogenase-producing cells in the culture can be directly collected and used, but generally, when formaldehyde dehydrogenase is present in the culture solution, filtration and centrifugation are performed. It is used after separating the formaldehyde dehydrogenase-containing solution and the microbial cells by, for example,
When formaldehyde dehydrogenase is present in the microbial cells, the obtained culture is harvested by means such as filtration or centrifugation, and then the microbial cells are subjected to a mechanical method or an enzymatic method such as lysozyme. It is destroyed, and if necessary, a chelating agent such as EDTA and / or a surfactant is added to solubilize the formaldehyde dehydrogenase, and separated and collected as an aqueous solution.

The formaldehyde dehydrogenase-containing solution thus obtained is concentrated under reduced pressure, membrane concentration, salting out with ammonium sulfate, sodium sulfate or the like, or fractionation with a hydrophilic organic solvent such as methanol, ethanol or acetone. It may be precipitated by a precipitation method.
Further, heat treatment and isoelectric point treatment are also effective refining means.
Purified formaldehyde dehydrogenase can be obtained by gel filtration using an adsorbent or a gel filtration agent, adsorption chromatography, ion exchange chromatography, or affinity chromatography.

[0020]

EXAMPLES The present invention will be specifically described below with reference to examples. In the examples, formaldehyde dehydrogenase activity was measured as follows. That is, 21 mM phosphate buffer
(pH7.5), 4.2mM formaldehyde, 0.46mM NAD,
27 μM PMS, 0.1 mM NTB, 0.21% Triton X-10
NAD produced by reacting the enzyme at 37 ° C for 15 minutes in
NTB is reduced with H via PMS, and the absorbance of the produced diformazan at 570 nm is measured. One unit of enzyme activity was defined as the amount of enzyme that produced 1/2 micromol of diformazan per minute under this condition.

Example 1 Determination of amino acid sequence of glutathione-independent formaldehyde dehydrogenase Glutathione-independent formaldehyde dehydrogenase powder (manufactured by Toyobo) was purified by gel filtration chromatography on Superdex 200 column and hydroxyapatite column chromatography. . SDS-PAGE the final purified sample
, A nearly uniform band was obtained, and the molecular weight of the subunit was calculated to be about 42,000 daltons. After dialyzing this preparation against ammonia-formate buffer,
The amino acid sequence from the N-terminal of the enzyme which was freeze-dried and purified by the manual Edman degradation method was determined (SEQ ID NO: 3 in the sequence listing). Further, the peptide mixture obtained by decomposing the same sample with cyanogen bromide was separated and purified by reverse phase HPLC, and the amino acid sequence of the peptide fragment was determined by the manual Edman degradation method (SEQ ID NO: 4 in the sequence listing).

Example 2 Separation of chromosomal DNA Pseudomonas putida PS-7 chromosomal DNA was separated by the following method. The strain was treated with 100 ml of LB medium (1.0% polypeptone, 0.5% yeast extract, 0.5% sodium chloride (pH 7.2)).
After shaking culture at 37 ° C overnight, the cells were collected by centrifugation (8000 rpm, 10 minutes). After washing the cells with a solution containing 15 mM sodium citrate and 0.15 M sodium chloride, 20% sucrose, 1
5 ml of a solution containing mMEDTA and 50 mM Tris-HCl (pH 7.6)
The mixture was suspended in 0.5 ml of lysozyme solution (100 mg / ml) and kept at 37 ° C for 30 minutes. Then 11 ml of a solution containing 1% lauroyl sarcosine, 0.1 M EDTA (pH 9.6) was added. About 100% of 0.5% cesium chloride was added to this suspension with an ethidium bromide solution, the mixture was stirred and mixed, and the DNA was fractionated by ultracentrifugation at 55.000 rpm for 20 hours. The separated DNA was dialyzed against a solution (TE) containing 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA to obtain a purified DNA sample. Escherichia Collie DH5
The α competent cells were created by the method of Hanahan,
Used as host for library construction.

Example 3 Preparation of DNA Fragment Containing Formaldehyde Dehydrogenase Gene and Recombinant Vector Having the DNA Fragment Based on the amino acid sequence determined in Example 1, two types of probes (SEQ ID NO: 5 in the sequence listing) were prepared. , 6) was prepared, and the formaldehyde dehydrogenase gene fragment was amplified by the PCR method using the chromosomal DNA obtained in Example 2 as a template.
An amplified fragment of bp was obtained. Next, Southern hybridization of the chromosomal DNA obtained in Example 2 was carried out using the obtained fragment as a probe, and a specific band of 9 kbp EcoRI was detected. Therefore, the DNA of this part was extracted and EcoR
The pBR322 digested with I (manufactured by Toyobo) was reacted with T4-DNA ligase (manufactured by Toyobo) to ligate the DNA. The ligated DNA was transformed with Escherichia coli DH5α competent cells. The obtained colonies were screened by colony hybridization using a fragment amplified by the PCR method as a probe to obtain a recombinant vector having a 9 kbp EcoRI DNA fragment containing a formaldehyde dehydrogenase gene (designated E1).

Next, insert DNA from the recombinant vector E1
The fragment is digested with various restriction enzymes or digested with exonuclease III (Toyobo),
Subcloning into pBluescript to obtain a recombinant vector K3DN71 having an inserted DNA fragment of about 2.1 kbp was used to transform Escherichia coli DH5α. The insert DNA restriction map of recombinant vectors E1 and its derivative plasmids containing K3DN71 is shown in FIG.

Example 4 Determination of nucleotide sequence The inserted DNA fragment of the recombinant vector K3DN71 was cleaved with various restriction enzymes to prepare subclones. Various subclones can be sequenced according to standard methods
The base sequence was determined using (SEQUENASE VERSION 2.0 7-deaza-dGTP kit) (manufactured by Toyobo). The determined nucleotide sequence and amino acid sequence are shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing.

Example 5 Cultivation of transformants and production of formaldehyde dehydrogenase Formaldehyde dehydrogenase production medium (1.0% meat extract,
50 ml of 1.0% polypeptone, 0.5% NaCl (pH 7.5) 500m
The mixture was dispensed into a flask, autoclaved at 121 ° C. for 15 minutes, allowed to cool, and then an aseptically filtered antibiotic (50 mg / ml ampicillin (manufactured by Nacalai Tesque)) was added. This medium was inoculated with 5 ml of a culture solution of the K3DN71 recombinant, which had been shake-cultured at 30 ° C. for 18 hours in advance in a medium having the same composition as described above, and the mixture was aerated with stirring at 30 ° C. 20 hours after the start of culture, K3DN7
One recombinant showed a formaldehyde dehydrogenase activity of about 6.5 U / ml. This value is approximately 4 times the formaldehyde dehydrogenase activity of Pseudomonas putida PS-7 under the same conditions.
It was revealed that this formaldehyde dehydrogenase gene was efficiently expressed in Escherichia coli, which corresponds to 0 times.

[0027]

INDUSTRIAL APPLICABILITY According to the present invention, the nucleotide sequence and amino acid sequence of glutathione-independent formaldehyde dehydrogenase gene have been clarified, and formaldehyde dehydrogenase can be easily mass-produced in high purity by genetic engineering techniques. . In particular, the enzymes produced from natural bacteria include other enzymes, creatinine amide hydrolase, creatine amidinohydrolase, and sarcosine dehydrogenase, which require a large number of steps for their exclusion, but in the present invention, these It has become possible to simplify the purification process. Further, by using the glutathione-independent formaldehyde dehydrogenase gene of the present invention and various protein engineering techniques, it becomes possible to prepare a novel formaldehyde dehydrogenase having higher activity or higher stability.

[0028]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 1296 Sequence type: Nucleic acid (DNA) Number of strands: Double stranded Topology: Linear Sequence type: genomic DNA Origin organism name: Pseudomonas PUTIDA Strain name: PS -7 Sequence CCTTTGTATT CACAACAACA ACGGAGAATT ACGC ATG TCT GGT AAT CGT GGT 52 Met Ser Gly Asn Arg Gly 1 5 GTC GTT TAT CTC GGT TCG GGC AAG GTC GAA GTC CAG AAG ATC GAC TAC 100 Val Val Tyr Leu Gly Ser Gly Lys Val Glu Val Gln Lys Ile Asp Tyr 10 15 20 CCC AAA ATG CAG GAC CCT CGC GGC AAG AAG ATC GAG CAC GGG GTC ATC 148 Pro Lys Met Gln Asp Pro Arg Gly Lys Lys Ile Glu His Gly Val Ile 25 30 35 CTG AAA GTC GTC TCC ACC AAC ATC TGC GGC TCG GAC CAG CAC ATG GTG 196 Leu Lys Val Val Ser Thr Asn Ile Cys Gly Ser Asp Gln His Met Val 40 45 50 CGC GGT CGT ACC ACC GCC CAG GTA GGC CTC GTG CTT GGC CAC GAG ATC 244 Arg Gly Arg Thr Thr Ala Gln Val Gly Leu Val Leu Gly His Glu Ile 55 60 65 70 ACC GGT GAG GTG ATC GAG AAG GGC CGT GAC GTA GAG AAC CTG CAG ATC 292 Thr Gly Glu Val Ile Glu Lys Gly Arg Asp Val Glu Asn Leu Gln Ile 75 80 85 GGC GAC CTG GTT TCC GTA CCC TTC AAC GTG GCC TGC GGC CGC TGC CGT 340 Gly Asp Leu Val Ser Val Pro Phe Asn Val Ala Cys Gly Arg Cys Arg 90 95 100 TCC TGC AAG GAA ATG CAC ACC GGC GTT TGC CTG ACC GTC AAT CCG GCC 388 Ser Cys Lys Glu Met His Thr Gly Val Cys Leu Thr Val Asn Pro Ala 105 110 115 CGC GCT GGC GGC GCC TAC GGC TAT GTC GAC ATG GGC GAC TGG ACC GGT 436 Arg Ala Gly Gly Ala Tyr Gly Tyr Val Asp Met Gly Asp Trp Thr Gly 120 125 130 GGC CAG GCC GAG TAC TTG CTG GTG CCC TAC GCC GAC TTC AAC CTG CTC 484 Gly Gln Ala Glu Tyr Leu Leu Val Pro Tyr Ala Asp Phe Asn Leu Leu 135 140 145 150 AAG CTG CCG GAT CGC GAC AAG GCC ATG GAG AAG ATC CGT GAC CTG ACC 532 Lys Leu Pro Asp Arg Asp Lys Ala Met Glu Lys Ile Arg Asp Leu Thr 155 160 165 TGC CTT TCC GAC ATC CTG CCC ACC GGC TAC CAC GGC GCC GTA ACT GCC 580 Cys Leu Ser Asp Ile Leu Pro Thr Gly Tyr His Gly Ala Val Thr Ala 170 175 180 GGC GTT GGC CCG GGC AGC ACC GTG TAC GTG GCC GGT GCC GGC CCG GTC 628 Gly Val Gly Pro Gly Ser Thr Val Tyr Val Ala Gly Ala Gly Pro Val 185 190 195 GGC CTG GCC GCT GCC GCC TCC GCT CGC CTG CTG GGC GCC GCC GTG GTT 676 Gly Leu Ala Ala Ala Ala Ser Ala Arg Leu Leu Gly Ala Ala Val Val 200 205 210 ATC GTC GGC GAC CTC AAC CCC GCC CGA CTG GCC CAC GCC AAG GCG CAG 724 Ile Val Gly Asp Leu Asn Pro Ala Arg Leu Ala His Ala Lys Ala Gln 215 220 225 230 GGC TTC GAG ATC GCC GAC CTG TCG CTG GAC ACC CCG CTG CAC GAG CAG 772 Gly Phe Glu Ile Ala Asp Leu Ser Leu Asp Thr Pro Leu His Glu Gln 235 240 245 ATC GCT GCG CTG CTG GGA GAG CCG GAA GTG GAT TGC GCG GTG GAT GCG 820 Ile Ala Ala Leu Leu Gly Glu Pro Glu Val Asp Cys Ala Val Asp Ala 250 255 260 GTG GGC TTC GAA GCT CGC GGC CAC GGC CAC GAA GGT GCC AAG CAC GAA 868 Val Gly Phe Glu Ala Arg Gly His Gly His Glu Gly Ala Lys His Glu 265 270 275 GCC CCG GCC ACT GTG CTG AAC TCG CTC ATG CAA GTC ACC CGC GTG GCC 916 Ala Pro Ala Thr Val Leu Asn Ser Leu Met Gln Val Thr Arg Val Ala 280 285 290 GGC AAG ATC GGT ATC CCC GGC CTT TAC GTC ACC GAA GAT CCG GGT GCC 964 Gly Lys Ile Gly Ile Pro Gly Leu Tyr Val Thr Glu Asp Pro Gly Ala 295 300 305 310 GTG GAG CCC GCG GCG AAG ATC GGC AGC CTG AGT ATT CGC TTC GGC CTC 1012 Val Glu Pro Ala Ala Lys Ile Gly Ser Leu Ser Ile Arg Phe Gly Leu 315 320 325 GGC TGG GCG AAA TCC CAC AGC TTC CAC ACT GGC CAG ACC CCG GTG ATG 1060 Gly Trp Ala Lys Ser His Ser Phe His Thr Gly Gln Thr Pro Val Met 330 335 340 AAG TAC AAC CGC GCA CTC ATG CAA GCG ATC ATG TGG GAC CGC ATC AAT 1108 Lys Tyr Asn Arg Ala Leu Met Gln Ala Ile Met Trp Asp Arg Ile Asn 345 350 355 ATC GCC GAA GTG GTG GGC GTG CAG GTC ATC AGC CTG GAC GAC GCA CCG 1156 Ile Ala Glu Val Val Gly Val Gln Val Ile Ser Leu Asp Asp Ala Pro 360 365 370 CGT GGC TAT GGC GAG TTC GAT GCC GGC GTG CCG AAG AAG TTC GTC ATC 1204 Arg Gly Tyr Gly Glu Phe Asp Ala Gly Val Pro Lys Lys Phe Val Ile 375 380 385 390 GAC CCG CAC AAG ACC TTC AGC GCG GCC TGATCGACTG CAATACCCCC 1251 Asp Pro His Lys Thr Phe Ser Ala Ala 395 GACGCCGTGA TGTGACAGCC GCGGCGTCTG TCCCTCAAGG GGCCC 1296

SEQ ID NO: 2 Sequence length: 399 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin organism name: Pseudomonas PUTIDA Strain name: PS-7 Sequence Met Ser Gly Asn Arg Gly Val Val Tyr Leu Gly Ser Gly Lys Val Glu 1 5 10 15 Val Gln Lys Ile Asp Tyr Pro Lys Met Gln Asp Pro Arg Gly Lys Lys 20 25 30 Ile Glu His Gly Val Ile Leu Lys Val Val Ser Thr Asn Ile Cys Gly 35 40 45 Ser Asp Gln His Met Val Arg Gly Arg Thr Thr Ala Gln Val Gly Leu 50 55 60 Val Leu Gly His Glu Ile Thr Gly Glu Val Ile Glu Lys Gly Arg Asp 65 70 75 80 Val Glu Asn Leu Gln Ile Gly Asp Leu Val Ser Val Pro Phe Asn Val 85 90 95 Ala Cys Gly Arg Cys Arg Ser Cys Lys Glu Met His Thr Gly Val Cys 100 105 110 Leu Thr Val Asn Pro Ala Arg Ala Gly Gly Ala Tyr Gly Tyr Val Asp 115 120 125 Met Gly Asp Trp Thr Gly Gly Gln Ala Glu Tyr Leu Leu Val Pro Tyr 130 135 140 Ala Asp Phe Asn Leu Leu Lys Leu Pro Asp Arg Asp Lys Ala Met Glu 145 150 155 160 Lys Ile Arg Asp Leu Thr Cys Leu Ser Asp Ile Leu Pro Thr Gly Tyr 165 170 175 His Gly Ala Val Thr Ala Gly Val Gly Pro Gly Ser Thr Val Tyr Val 180 185 190 Ala Gly Ala Gly Pro Val Gly Leu Ala Ala Ala Ala Ser Ala Arg Leu 195 200 205 Leu Gly Ala Ala Val Val Ile Val Gly Asp Leu Asn Pro Ala Arg Leu 210 215 220 Ala His Ala Lys Ala Gln Gly Phe Glu Ile Ala Asp Leu Ser Leu Asp 225 230 235 240 Thr Pro Leu His Glu Gln Ile Ala Ala Leu Leu Gly Glu Pro Glu Val 245 250 255 Asp Cys Ala Val Asp Ala Val Gly Phe Glu Ala Arg Gly His Gly His 260 265 270 Glu Gly Ala Lys His Glu Ala Pro Ala Thr Val Leu Asn Ser Leu Met 275 280 285 Gln Val Thr Arg Val Ala Gly Lys Ile Gly Ile Pro Gly Leu Tyr Val 290 295 300 Thr Glu Asp Pro Gly Ala Val Glu Pro Ala Ala Lys Ile Gly Ser Leu 305 310 315 320 Ser Ile Arg Phe Gly Leu Gly Trp Ala Lys Ser His Ser Phe His Thr 325 330 335 Gly Gln Thr Pro Val Met Lys Tyr Asn Arg Ala Leu Met Gln Ala Ile 340 345 350 Met Trp Asp Arg Ile Asn Ile Ala Glu Val Val Gly Val Gln Val Ile 355 360 365 Ser Leu Asp Asp Ala Pro Arg Gly Tyr Gly Glu Phe Asp Ala Gly Val 370 375 380 Pro Lys Lys Phe Val Ile Asp Pro His Lys Thr Phe Ser Ala Ala 385 390 395

SEQ ID NO: 3 Sequence length: 10 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Origin Biological name: Pseudomonas PUTIDA Strain name: PS-7

SEQ ID NO: 4 Sequence length: 6 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Origin Biological name: Pseudomonas PUTIDA Strain name: PS-7

SEQ ID NO: 5 Sequence length: 20 Sequence type: Nucleic acid (DNA) Number of strands: Single-stranded Topology: Linear Sequence type: Synthetic DNA sequence MAWCGTGGTG TNGTNTAYYT 20

SEQ ID NO: 6 Sequence length: 20 Sequence type: Nucleic acid (DNA) Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence ARRTCiCKiA TYTTYTCCAT 20

[Brief description of drawings]

FIG. 1 shows a restriction map of insert DNAs of E1 and its derivative plasmids.

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Claims (5)

[Claims] 1. A DN having the genetic information of a protein having glutathione-independent formaldehyde dehydrogenase activity.
A fragment. 2. The DNA fragment according to claim 1, which contains the nucleotide sequence set forth in SEQ ID NO: 1 in the sequence listing. 3. A recombinant vector having the DNA fragment according to claim 2. 4. A transformant transformed with the recombinant vector according to claim 3. 5. A method for producing formaldehyde dehydrogenase, which comprises culturing the transformant according to claim 4 in a medium to produce formaldehyde dehydrogenase and collecting the formaldehyde dehydrogenase.