JP2005185138A - Production method for formaldehyde dehydrogenation enzyme - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a production method which is useful for the industrial production of a formaldehyde dehydrogenation enzyme (EC. 1.2.1.1). <P>SOLUTION: This production method comprises transforming Escherichia coli with a recombinant plasmid vector integrated with a gene encoding the formaldehyde dehydrogenation enzyme (EC. 1.2.1.1), culturing the transformed Escherichia coli under proper conditions, and then recovering the expressed formaldehyde dehydrogenation enzyme. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for producing formaldehyde dehydrogenase using gene recombination technology.

  Formaldehyde is one of the causative substances that cause chronic allergic symptoms such as sick house syndrome. Formaldehyde dehydrogenase (hereinafter referred to as FDH) is expected to be applied in the field of environmental technology such as air purification as a means for decomposing and removing formaldehyde.

Among them, FDH called EC 1.2.1.1.1 based on the IUBMB nomenclature is particularly useful in that volatile odorous substances such as formic acid and methanol are not generated as decomposition products. This enzyme is glutathione-dependent FDH with NAD ⁺ as a coenzyme. This enzyme catalyzes a reaction in which formaldehyde is conjugated to glutathione to produce nonvolatile S-formylglutathione (Scheme 1).
Reaction formula 1:
HCHO + glutathione + NAD ⁺ → S-formyl glutathione + NADH + H ⁺

  The amino acid sequence of this FDH (EC 1.2.1.1) and the base sequence of the gene encoding it are known (GenBank Accesslon number: D38504).

  The present invention relates to a method for producing an FDH referred to as EC 1.2.1 (hereinafter referred to as “the FDH of the present invention”) based on the IUBMB nomenclature. Conventionally, obtaining FDH requires a complicated process of searching for and identifying microorganisms or cells that express the enzyme in large quantities, culturing them in large quantities, and then isolating and purifying the enzyme. And it was costly.

  Therefore, an object of the present invention is to provide an industrially useful production method capable of producing the FDH of the present invention in a larger amount and at a lower cost.

The present invention
(1) A method for producing formaldehyde dehydrogenase having the amino acid sequence of SEQ ID NO: 2,
A nucleic acid having the nucleotide sequence of SEQ ID NO: 1 is incorporated into a vector capable of expressing the nucleic acid to obtain an expression vector,
E. coli is transformed with the expression vector to obtain a transformant of E. coli,
Culturing the E. coli transformant under conditions suitable for expression of the nucleic acid;
Isolating the expressed formaldehyde dehydrogenase from the transformant of E. coli or a culture solution thereof;
(2) The production method of (1) above, wherein the vector capable of expressing a nucleic acid having the nucleotide sequence of SEQ ID NO: 1 is plasmid pQE30;
(3) The production method of (1) or (2) above, wherein in the expression vector, the nucleic acid having the nucleotide sequence of SEQ ID NO: 1 is incorporated between two different restriction enzyme sites;
(4) One of the two different restriction enzyme sites is BamHI, and the other is selected from the group consisting of SphI, SacI, KpnI, SmaI, XmaI, SalI, Pstl, HindllI, (1), (2 ) Or (3) production method;
(5) an expression vector incorporating a nucleic acid having the nucleotide sequence of SEQ ID NO: 1, capable of expressing formaldehyde dehydrogenase in E. coli;
(6) The expression vector according to (5), wherein the expression vector is a plasmid pQE30 into which a nucleic acid having the nucleotide sequence of SEQ ID NO: 1 has been incorporated;
(7) The expression vector of (5) or (6) above, wherein a nucleic acid having the nucleotide sequence of SEQ ID NO: 1 is incorporated between two different restriction enzyme sites;
(8) One of the two different restriction enzyme sites is BamHI, and the other is selected from the group consisting of SphI, SacI, KpnI, SmaI, XmaI, Sa1I, PstI, HindIII, (5), (6 ) Or (7) expression vector;
(9) A transformant of E. coli transformed with the expression vector of any one of (5) to (7) above;
(10) A formaldehyde dehydrogenase having the amino acid sequence of SEQ ID NO: 2 produced by the production method of any one of (1) to (4) above is provided.

  According to the production method of the present invention, a large amount of formaldehyde dehydrogenase can be produced at a low cost.

  The method for producing FDH of the present invention utilizes a gene recombination technique. Isolation of the gene encoding the FDH of the present invention, preparation of an expression vector containing the gene, preparation of a host cell transformed with the expression vector, culture of the host cell, etc. are conventional means in the art. For example, it can be performed according to a method described in Molecular Cloning (Cold Spring Harbor Publishing Co., 1989).

  The FDH of the present invention is an enzyme derived from E. coli. Therefore, the DNA carrying the genetic information of this enzyme can be obtained from genomic DNA extracted from E. coli. For example, E. coli such as JM109 strain is cultured and collected, then treated with protease, and genomic DNA of E. coli is extracted by ethanol precipitation.

  Next, a PCR reaction is performed using the extracted E. coli genomic DNA as a template to amplify the gene encoding the FDH of the present invention. The primer used for the PCR reaction is designed to amplify the open reading frame (nucleotide of SEQ ID NO: 1) based on the gene information (GenBank D38504) of the FDH of the present invention. Furthermore, appropriate nucleotides are added to the forward and reverse primers so that specific restriction enzyme sites are generated at both ends of the amplified fragment. Preferably, appropriate nucleotides are added to both primers so that different restriction enzyme sites are generated at both ends of the fragment so that the fragment is incorporated in a certain orientation when inserted into the vector. One skilled in the art can amplify the gene encoding the FDH of the present invention by performing a PCR reaction using conventional reagents and reaction conditions.

  The vector capable of expressing the gene encoding the FDH of the present invention is not particularly limited as long as it is suitable for expression in E. coli. In the present invention, “vector” means DNA used to transport heterologous DNA to a host. For example, pET (manufactured by Toyobo Co., Ltd.), pGEX (manufactured by Amersham), pBAD (manufactured by Invitrogen), pHAT (manufactured by Clontech), pQE (manufactured by Qiagen), etc. include plasmid vectors commonly used in the art. pQE is particularly preferable because it can be expressed by fusing six histidines to a target protein, and protein purification using this tag can be easily performed after protein expression.

  Integration of the gene encoding the FDH of the present invention into a vector can be carried out according to a conventional method. The gene and vector encoding FDH are each cleaved with a restriction enzyme and incubated under appropriate conditions for ligation. In the present invention, in particular, a vector in which a gene encoding the FDH of the present invention is incorporated so as to allow expression in a host is referred to as an “expression vector”.

  The expression vector obtained by the ligation is transduced into Escherichia coli using a conventional transformation method such as competent cell method or electroporation to obtain an E. coli transformant capable of expressing the FDH of the present invention. The transformant thus obtained is cultured after being cultured under conditions suitable for growth of the transformant and for expression of the FDH of the present invention. The collected cells are disrupted by sonication or freeze-thawing, or lysed with an enzyme such as lysozyme or an appropriate reagent, and then centrifuged to obtain a supernatant containing the desired FDH protein. obtain. Furthermore, the purified FDH protein of the present invention can be obtained by subjecting the centrifugal supernatant to a conventional purification means using a commercially available ion exchange resin, affinity resin, metal chelate column or the like.

  The present invention is specifically illustrated by the following examples.

1. Isolation of FDH gene of the present invention 1-1. Extraction of E. coli genomic DNA E. coli (JM109 strain) was cultured with shaking overnight at 37 ° C in 1.5 mL of LB medium. After culturing, the culture solution was collected by centrifugation at 5000 × g for 10 minutes, and E. coli genomic DNA was extracted using DNeasy Tissue Kit (Qiagen). That is, the bacterial pellet was resuspended in 180 μL of buffer ATL (attached to the kit), 20 μL of proteinase K (attached to the kit) was added, stirred, and incubated at 55 ° C. for 1 hour. After adding 200 μL of buffer AL (attached to the kit), the mixture was stirred and further incubated at 70 ° C. for 10 minutes. Next, after adding 200 μL of ethanol (99.5%) and stirring, this was transferred to a DNeasy mini column (attached to the kit) set in a 2 mL collection tube and centrifuged at 6000 × g for 1 minute. The DNeasy mini column was transferred to a new 2 mL collection tube, 500 μL of buffer AW (attached to the kit) was added, and centrifuged at 6000 × g for 1 minute. Furthermore, the DNeasy mini column was transferred to a new 2 mL collection tube, and 2500 μL of buffer AW was added, followed by centrifugation at 6000 × g for 3 minutes. Further, the DNeasy mini column was transferred to a new 1.5 mL collection tube, 200 μL of buffer AE (attached to the kit) was directly added to the membrane in the column, and incubated at room temperature for 1 minute. This was centrifuged at 6000 × g for 1 minute to elute the DNA. By this operation, approximately 4 μg (20 μg / mL) of E. coli genomic DNA could be recovered.

1-2. Amplification of FDH gene by PCR PCR reaction was carried out using E. coli genomic DNA obtained above as a template. ExTaq Polymerase (Takara Shuzo) was used for PCR. A forward primer and a reverse primer for amplifying the FDH gene were designed as follows. Specifically, the full length (SEQ ID NO: 1) of the open reading frame of the E. coli FDH gene sequence (GenBnak D38504) is amplified, and a primer is designed to add a BamHI restriction enzyme site on the forward side and a HindIII restriction enzyme site on the reverse side. did. The sequences of both primers are as follows. Primer synthesis was performed by SIGMA Genosys.
Primer sequence forward primer: 5′-cgggatccatgaaatcacgtgctgc-3 ′ (SEQ ID NO: 3)
Reverse primer: 5′-cgaagctttcagtaagcaattacgg-3 ′ (SEQ ID NO: 4)

The PCR reaction was performed using the following conditions.
Reaction cycle:
(94 ° C. × 30 seconds + 65 ° C. × 30 seconds + 72 ° C. × 60 seconds) 30 cycles reaction liquid composition:
Template DNA 10μL
10 × PCR buffer 5 μL
dNTPs mixture 4 μL
Forward primer 1μL
Reverse primer 1μL
H ₂ O 28.5 μL
ExTaq 0.5μL

  After completion of the PCR reaction, 5 μL of the reaction solution was electrophoresed on a 2% agarose gel to confirm the amplification of the desired 1128 bp band. The remaining reaction solution was purified by removing residual primers and unreacted components using GFX PCR DNA and Gel Band Purificatlon Kit (Amersham Biosciences). That is, the GFX column was set in a collection tube, 500 μL of capture buffer (attached to the kit) was added, the PCR product was added thereto, and the mixture was stirred by pipetting. Next, this was centrifuged at 10000 × g for 30 seconds, and the flow-through fraction was discarded. Then, 500 μL of wash buffer (attached to the kit) was placed in the column and washed again by centrifugation at 10000 × g for 30 seconds. The GFX column was set in a new collection tube, 50 μL of ultrapure water was placed in the column, incubated at room temperature for 1 minute, and then centrifuged at 10000 × g for 1 minute to collect the purified FDH gene PCR product.

2. Preparation of expression vector and E. coli transformant 2-1. Vector Preparation and Restriction Enzyme Treatment Plasmid pQE30 (Qiagen) was used as a vector for expressing the FDH gene of the present invention. pQE30 (vector) and the PCR product (insert) of the FDH gene obtained in Example 1 were cleaved with restriction enzymes BamHI and HindIII (both manufactured by Takara Shuzo Co., Ltd.), respectively.

  pQE30 (vector) and FDH gene PCR products (inserts) were each incubated at 37 ° C. for 1 hour. For Bekku, 5.5 μL of CIAP buffer and 2 μL of CIAP (both manufactured by Takara Shuzo Co., Ltd.) were added and incubated at 50 ° C. for 30 minutes to dephosphorylate the cut surface.

  The vector and the FDH gene insert were each treated with phenol / chloroform three times, and then applied to a G-25 spin column and eluted with 50 μL of TE buffer to remove the remaining phenol. By this operation, about 150 ng (30 ng / μL) of the vector and the FDH gene insert were recovered.

2-2. Ligation and transformation The vector obtained above and the FDH gene insert were incubated overnight at 16 ° C. in a solution having the following composition, and ligated with T4 ligase (Takara Shuzo).
composition:
Vector 2 μL
Insert 2μL
2 x buffer 5 μL
1 μL of T4 ligase

  E. coli competent cells were transformed with the expression vector obtained by ligation. That is, 2.5 μL of the ligation solution containing the expression vector obtained above was added to 25 μL of competent cells (JM109 strain: manufactured by Toyobo Co., Ltd.) thawed on ice, and gently mixed. After incubating on ice for 30 minutes, a heat shock was added at 42 ° C. for 30 seconds and placed on ice again for 2 minutes. 250 μL of SOL medium was added and pre-cultured at 37 ° C. for 1 hour, then plated on LB agar plates (with ampicillin) and incubated overnight at 37 ° C. to form colonies.

2-3. Confirmation of Insert The formed colony was cultured overnight at 37 ° C. in 3 mL of LB medium, and after collection, the plasmid was extracted using QIAprep Spin in Miniprep Kit (Qiagen). In order to confirm the size of the insert of the obtained plasmid, restriction enzyme treatment was performed with BamHl and HindIII, and 1.5% agarose gel electrophoresis was performed. As a result, a fragment of 1110 bp was confirmed, and the target FDH gene was It was confirmed that the incorporated Escherichia coli transformant was obtained.

3. Measurement of FDH expression and enzyme activity 3-1. Extraction and purification of expressed FDH The transformant of E. coli obtained in Example 2 was cultured overnight in an LB medium at 37 ° C. with shaking. In order to induce protein expression, IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a final concentration of 1 mM, and the cells were further cultured at 37 ° C. for 4 hours. The cells were collected by centrifugation at 3000 × g for 15 minutes, and then the pellets were frozen in a freezer at −40 ° C. After freezing, it was taken out and 1/20 volume of the Beaver (registered trademark) bacterial protein extraction reagent (Pierce) was added and incubated at room temperature for 10 minutes to dissolve the cells. Centrifugation at 27000 × g for 15 minutes to remove the insoluble matter, the glycerol for maintaining the stability of the enzyme and NaCl as a salt necessary for the purification to a final concentration of 40% and 300 mM, respectively. This was added as a crude extract.

  For purification of the FDH of the present invention, Ni 1 NTA spin kit (Qiagen) was used, and 500 μL of crude extract was used per spin column, and 200 μL was eluted twice to collect the FDH fraction. did.

  Through the above steps, approximately 50 μg of FDH could be recovered from 5 mL of the E. coli overnight culture solution (yield: 10 μg / 1 mL culture solution).

3-2. Enzyme activity measurement The activity of the FDH of the present invention obtained above was measured according to the method described in J. Biochem., 85, 1165 (1979). The principle of this enzyme activity measurement is shown by the following reaction formula. That is, NADH produced by the enzyme reaction of FDH reduces NTB (ditrotetrazolium blue) via PMS (phenazine methosulfate), and diform mazan is generated. The amount of diform mazan produced is measured by the increase in absorbance at 570 nm. The amount of enzyme that produces 1/2 μmol diformazan per minute was defined as 1 U (unit).
Reaction formula:
HCHO + NAD ⁺ + Glutathione → S-Formyl Glutathione + NADH + H ⁺
NADH + PMS → NAD ⁺ + PMS (Red)
2PMS (red) + NTB → 2PMS + diformazan

The reagents used for measuring the enzyme activity are as follows.
Substrate solution: 10 mM HCHO (in 50 mM phosphate buffer) + 0.5% (weight) Triton X-10, pH 7.5
NAD solution: 1.5% (weight) β-NAD (in H ₂ O)
GSH solution: 2 mg / mL (in H ₂ O)
PMS-NBT solution: 0.1 mg / mL PMS + 1 mg / mL NBT (in H ₂ O)
Enzyme solution: The purified enzyme fraction is appropriately diluted with a 50 mM phosphate buffer containing 40% (by weight) glycerol and 300 mM NaCl.

The activity was measured according to the following procedure.
(1) The above reagents are mixed in the following composition and preincubated for 5 minutes at 37 ° C.
Substrate solution 500 μL
NAD solution 100μL
GSH solution 100μL
PMS-NBT solution 100 μL
(2) Add 500 μL of the enzyme solution to the reaction solution to start the reaction.
(3) After reacting at 37 ° C. for 25 minutes, 3 mL of 0.3N HCl is added to stop the reaction.
(4) The absorbance of the solution at 570 nm is measured, and the difference from the blank is taken as ΔOD and converted to a unit of enzyme activity from the following formula.
U / mL = ΔOD × 4.3 (mL) × dilution ratio / 20.1 / 25 (min) /0.5 (mL) * 20.1 = 1/2 mmol molecular extinction coefficient of diformazan

  As a result of the measurement, the FDH of the present invention obtained in the above example had an activity of 4 U / mL.

  The production method of the present invention can be used for industrial production of formaldehyde dehydrogenase.

Claims (10)

A method for producing formaldehyde dehydrogenase having the amino acid sequence of SEQ ID NO: 2,
A nucleic acid having the nucleotide sequence of SEQ ID NO: 1 is incorporated into a vector capable of expressing the nucleic acid to obtain an expression vector,
E. coli is transformed with the expression vector to obtain a transformant of E. coli,
Culturing the E. coli transformant under conditions suitable for expression of the nucleic acid;
A production method comprising isolating the expressed formaldehyde dehydrogenase from the transformant of E. coli or a culture solution thereof. The production method according to claim 1, wherein a nucleic acid having the nucleotide sequence of SEQ ID NO: 1 is incorporated into a plasmid pQE30 to obtain an expression vector. The production method according to claim 1 or 2, wherein in the expression vector, the nucleic acid having the nucleotide sequence of SEQ ID NO: 1 is incorporated between two different restriction enzyme sites. One of the two different restriction enzyme sites is BamHI, and the other is selected from the group consisting of SphI, SacI, KpnI, SmaI, XmaI, Sa1I, PstI, HindIII. Manufacturing method. An expression vector incorporating a nucleic acid having a nucleotide sequence of SEQ ID NO: 1, capable of expressing formaldehyde dehydrogenase in E. coli. The expression vector according to claim 5, wherein the expression vector is a plasmid pQE30 into which a nucleic acid having the nucleotide sequence of SEQ ID NO: 1 has been incorporated. The expression vector according to claim 5 or 6, wherein a nucleic acid having a nucleotide sequence of SEQ ID NO: 1 is incorporated between two different restriction enzyme sites. 8. One of the two different restriction enzyme sites is BamH1, and the other is selected from the group consisting of SphI, SacI, KpnI, SmaI, XmaI, Sa1I, PstI, HindIII. Expression vector. A transformant of Escherichia coli transformed with the expression vector according to any one of claims 5 to 8. The formaldehyde dehydrogenase which has the amino acid sequence of sequence number 2 manufactured by the manufacturing method in any one of Claims 1-4.