JP5332277B2 - Method for producing NADH and method for producing formate dehydrogenase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide useful applications of a formic dehydrogenase by utilizing the formic dehydrogenase which exhibits high specific activity unpredictable from conventional knowledge. <P>SOLUTION: It is elucidated that the formic dehydrogenase derived from Gibberella zeae exhibits preeminently high specific activity in the conventionally known formic dehydrogenases. As the formic dehydrogenase derived from Gibberella zeae, for example, proteins including specific amino acid sequence are included by utilization of the formic dehydrogenase derived from the Gibberella zeae can produce NADH (reduced type nicotinamide adenine dinucleotide). <P>COPYRIGHT: (C)2010,JPO&amp;INPIT

Description

  The present invention relates to a method for producing NADH using formate dehydrogenase and a method for producing formate dehydrogenase.

Oxidized nicotinamide adenine dinucleotide (NAD ⁺ ) is reduced to reduced nicotinamide adenine dinucleotide (NADH) by the catalytic reaction of formate dehydrogenase (EC.1.2.1.2) in the presence of formic acid and water. Thus, formate dehydrogenase is used in a system for regenerating NADH from NAD ⁺ . Conventionally, as formate dehydrogenase, formate dehydrogenase derived from Candida boidinii (ATCC32195) as described in Patent Document 1, for example, derived from Bacillus bacteria as disclosed in Patent Document 2 NAD ⁺ -dependent formate dehydrogenase, a formate dehydrogenase derived from Mycobacterium vaccae as disclosed in Patent Document 3, has been known.

  In addition, Non-Patent Document 1 discloses formic acid dehydrogenases derived from various microorganisms and plants in addition to these. However, as described in Non-Patent Document 1, formate dehydrogenase has a lower specific activity among various enzymes. In other words, it can be said that the NADH production method using the reduction reaction to NADH by formate dehydrogenase has poor productivity due to the low specific activity of formate dehydrogenase.

JP 2003-180383 A JP 2002-233395 A Japanese Patent Application No. 10-023896 BIOCHEMISTRY (Moscow) Vol. 69 No. 11 2004 pp. 1252-1267. (English translation of Biokhimiya, Vol. 69, No. 11, 2004, pp. 1537-1554.)

  Therefore, in view of the above-described circumstances, the present invention provides a method for producing NADH using formate dehydrogenase having a high specific activity that cannot be predicted from conventional knowledge, and also provides a high specific activity formate dehydrogenation. It aims at providing the useful manufacturing use of the manufacturing method of the enzyme, and the said formate dehydrogenase.

  As a result of intensive studies by the present inventors in order to achieve the above-described object, it has been clarified that the formic acid dehydrogenase derived from red mold exhibits a particularly high specific activity among the conventionally known formate dehydrogenases. Although the amino acid sequence of this red mold derived formate dehydrogenase has been entered in the database, this data is a deduced amino acid sequence from the base sequence information and is not actually isolated and the gene It was not cloned. In addition, the inventors of the present invention have intensively studied, and it is not possible to obtain a formic acid dehydrogenase derived from red mold according to a standard method, so that it can be obtained as an active form at all. Was successfully isolated for the first time as an enzyme exhibiting high specific activity.

The present invention based on the above findings includes the following.
(1) A method for producing NADH, wherein a formic acid dehydrogenase derived from red mold is allowed to act on a reaction system containing formic acid and NAD ⁺ .
In the method for producing NADH according to the present invention, the formic acid dehydrogenase derived from red mold is preferably a protein of any one of the following (a) to (c).
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 2 (b) comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 2, and formic acid and NAD ⁺ A protein having catalytic activity in a reaction in which carbon dioxide and NADH are produced using CO2 as a substrate (c) hybridizing under stringent conditions to a polynucleotide comprising a base sequence complementary to the base sequence shown in SEQ ID NO: 1 A protein having catalytic activity in a reaction using formic acid and NAD ⁺ as substrates and carbon dioxide and NADH as products, and in the method for producing NADH according to the present invention, the above-mentioned red mold derived formate dehydrogenase Is an amino acid sequence having 85% or more homology to the amino acid sequence shown in SEQ ID NO: 2. It is preferable that a protein-free.

  (2) culturing a host introduced with a vector in which a red mold-derived formate dehydrogenase gene is placed under the control of an inducible promoter, and inducing expression of the formate dehydrogenase gene when the logarithmic growth phase has passed, A method for producing formate dehydrogenase, comprising culturing at a temperature lower than the optimum growth temperature of the host and allowing the host to survive to obtain the formate dehydrogenase.

In the method for producing formate dehydrogenase according to the present invention, it is preferable that the formic acid dehydrogenase derived from red mold is a protein of any one of the above (a) to (c). Moreover, it is preferable that the above-mentioned red mold formate dehydrogenase is a protein comprising an amino acid sequence having 85% or more homology with the amino acid sequence shown in SEQ ID NO: 2.
Furthermore, in the method for producing formate dehydrogenase according to the present invention, when the logarithmic growth is exceeded, it is preferably a stationary phase. In the method for producing formate dehydrogenase according to the present invention, the temperature at which the host is able to survive and lower than the optimum growth temperature of the host can be 15 to 37 ° C.

  According to the method for producing NADH according to the present invention, NADH known as a very expensive substance can be produced with excellent productivity because a formate dehydrogenase having a very high specific activity is used. By applying the NADH production method according to the present invention, industrial production of NADH becomes possible.

  Moreover, according to the method for producing formate dehydrogenase according to the present invention, formic acid dehydrogenase derived from red mold can be produced in a state of showing very high specific activity. This red mold-derived formate dehydrogenase exhibits an exceptionally high specific activity as compared with conventionally known formate dehydrogenases, and thus can be widely used in NADH production methods and the like.

  Hereinafter, the present invention will be described in detail with reference to the drawings.

  In the present invention, it is based on the knowledge that red mold derived formate dehydrogenase is excellent in specific activity compared with conventionally known formate dehydrogenase. First, formic acid dehydrogenase derived from red mold is described.

Formic acid dehydrogenase derived from red mold The formate dehydrogenase that can be used in the present invention is formic acid dehydrogenase that is derived from red mold, that is, native to red mold. Here, in order to obtain red mold formate dehydrogenase, various conventionally known strains stored as red mold (scientific name: Gibberella zeae) can be used. For example, red mold stored as ATCC numbers 10910, 20271, 20272, 20274, 24689, 28106, or 48063 can be used for the American Type Culture Collection (ATCC). The ATCC may store the registered name as Fusarium graminearum, but if Gibberella zeae is registered as an alias, these can also be used. Gibberella zeae indicates a complete generation (Teleomorph) of Fusarium graminearum. In addition, red mold stored as NBRC Nos. 4474, 5269, 6608, 7160, 7520, 7772, 8850 or 9462 can be used in the Biological Genetic Resources Department (NBRC) of the National Institute for Product Evaluation and Technology (NBRC). .

  Moreover, you may acquire the formic acid dehydrogenase derived from a red mold using the mutant which introduce | transduced the mutation | mutation at random using the red mold as mentioned above as a wild type. Furthermore, formic acid dehydrogenase derived from red mold may be obtained using a red mold originally isolated from nature, not a strain stored in an organization such as ATCC or NBRC.

  As an example, the base sequence of the formate dehydrogenase gene and the amino acid sequence of formate dehydrogenase isolated from red mold conserved under NBRC No. 4474 are shown in SEQ ID NOs: 1 and 2, respectively. This base sequence and amino acid sequence are 100% identical with those registered as the registration number NW_059922 in the Genbank database. However, the base sequence and amino acid sequence registered under the registration number NW_059922 were only the coding region of the formate dehydrogenase gene as predicted by computer analysis, and the results of the experimental isolation of the formate dehydrogenase gene It was not identified as.

The formate dehydrogenase that can be used in the present invention is not limited to the amino acid sequence shown in SEQ ID NO: 2, for example, one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 are deleted, substituted, It may contain an added or inserted amino acid sequence and have catalytic activity in a reaction using formic acid and NAD ⁺ as substrates and carbon dioxide and NADH as products. Here, as the plurality of amino acids, for example, 1 to 30, preferably 1 to 20, more preferably 1 to 10, still more preferably 1 to 5, particularly preferably 1 to 3 means. In addition, amino acid deletion, substitution, or addition can be performed by modifying the base sequence encoding the gene by a technique known in the art. Mutation can be introduced into a base sequence by a known method such as Kunkel method or Gapped duplex method or a method according thereto, for example, a mutation introduction kit (for example, Mutant-) using site-directed mutagenesis. Mutations are introduced using K, Mutant-G (both trade names, manufactured by TAKARA) or the like, or LA PCR in vitro Mutagenesis series kit (trade name, manufactured by TAKARA).

Further, in the present invention, an amino acid sequence having homology of, for example, 85% or more, preferably 90% or more, more preferably 95% or more, and most preferably 98% or more with respect to the amino acid sequence shown in SEQ ID NO: 2. It is also possible to use a formate dehydrogenase having catalytic activity in a reaction containing carbon dioxide and NADH using formic acid and NAD ⁺ as substrates. Here, the homology value means a value obtained by default setting using a computer program in which the blast algorithm is implemented and a database storing gene sequence information.

Furthermore, in the present invention, a protein encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide complementary to a part or all of the base sequence shown in SEQ ID NO: 1, comprising formic acid A protein having catalytic activity in a reaction using carbon dioxide and NADH as products with NAD ⁺ as a substrate can be used as formate dehydrogenase. Here, hybridizing under stringent conditions means maintaining binding at 60 ° C. under 2 × SSC washing conditions. Hybridization can be performed by a conventionally known method such as the method described in J. Sambrook et al. Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory (1989).

  The formic acid-derived formate dehydrogenase described above has an excellent specific activity as compared with a conventionally known formate dehydrogenase. Examples of conventionally known formate dehydrogenase to be compared here include formate dehydrogenase derived from Neurospora crassa, derived from Saccharomyces cerevisiae, derived from Emericella nidulans, or derived from Candida boidinii. BIOCHEMISTRY (Moscow) Vol. 69 No. 11 2004 pp. 1252-1267. (English translation of Biokhimiya, Vol. 69, No. 11, 2004, pp. 1537-1554.) The specific activities of the enzymes are listed, and can be compared with the specific activities of these listed formate dehydrogenases. Compared to any conventionally known formate dehydrogenase, the specific activity of red mold formate dehydrogenase shows a value of 2 to 10 times or more. Among the conventionally known formate dehydrogenases, the specific activity of formate dehydrogenase derived from Paracoccus sp. 12-A is the highest. Compared with the specific activity of formate dehydrogenase derived from Paracoccus sp. 12-A, the specific activity of formate dehydrogenase derived from red mold is approximately doubled.

Production of red mold-derived formate dehydrogenase The aforementioned red mold-derived formate dehydrogenase cannot be obtained by a conventionally known protein production method. The method for producing formate dehydrogenase according to the present invention prepares a host into which a vector in which the above-described red mold derived formate dehydrogenase gene is placed under the control of an inducible promoter. Then, the host is cultured, and the expression of the formate dehydrogenase gene is induced when the logarithmic growth phase has passed. Next, formate dehydrogenase is expressed in the host by culturing at a temperature lower than the optimum growth temperature of the host and at which the host can survive.

  In the method for producing formate dehydrogenase according to the present invention, the inducible promoter is not particularly limited, and a conventionally known promoter can be used. For example, when E. coli is used as the above host, an inducible promoter exhibiting transcriptional activity in the presence of isopropyl-β-thiogalactopyranoside (IPTG) can be used. Examples of such promoters include Trp promoter, Lac promoter, Trc promoter, and Tac promoter. In addition, other promoters that exhibit transcriptional activity in the presence of inducers other than IPTG, and other promoters that exhibit transcriptional activity according to culture conditions such as medium components and temperature can also be used as inducible promoters.

  Moreover, in the method for producing formate dehydrogenase according to the present invention, the vector is not particularly limited as long as it can be replicated in the above-mentioned host, and any vector can be used. For example, when E. coli is used as the host, the vector may be a plasmid vector or a phage vector. Specific examples of the vector include pCDF series, pRSF series, and pET series.

  Further, the host is not particularly limited as long as it can be transcribed from a promoter incorporated in an expression vector. For example, when the expression vector is a pET (T7 promoter) system, E. coli BL21 (DE3) may be used. it can. As a technique for introducing the above-described vector into a host, various techniques generally known as transformation methods can be applied. As a specific method, for example, a calcium phosphate method, an electroporation method, a lipofection method, or the like can be applied.

  In particular, in the method for producing formate dehydrogenase according to the present invention, the host into which the vector has been introduced is cultured, and the expression of the formate dehydrogenase gene is induced when the logarithmic growth phase has passed. Before inducing the expression of the formate dehydrogenase gene, the culture conditions of the host are not limited in any way, and may be appropriately set in consideration of, for example, the optimum growth temperature and optimum growth pH of the host. However, the growth of the host is observed while continuing the culture, and the culture conditions are changed to satisfy the following conditions when the so-called logarithmic growth phase has passed. That is, as the condition 1, the expression of the formate dehydrogenase gene is induced, and as the condition 2, the culture is performed at a temperature lower than the optimum growth temperature of the host and at which the host can survive.

  Here, when the logarithmic growth phase has passed, the slope of the tangent line from the portion represented by the approximate straight line of the predetermined slope in the growth curve in which the horizontal axis represents the culture time and the vertical axis represents the logarithm of the number of cells. It means the point in time when it begins to decline. The culture curve can be prepared by measuring OD600 nm in the medium. Moreover, when inducing the expression of the formate dehydrogenase gene, it is preferable that the logarithmic growth phase is passed and the stationary phase is entered. Here, the stationary period is a period in which the slope of the tangent of the above-described growth curve is substantially zero.

  The optimum growth temperature of the host is known as a temperature range different for each host. For example, when E. coli B strain is used as the host, the optimum growth temperature is 37 ° C. For example, when Escherichia coli B strain is used as a host, the temperature at which growth is possible is 15 to 37 ° C. Therefore, when Escherichia coli B strain is used as a host, it means 15 to 37 ° C. as a temperature lower than the optimum growth temperature of the host and at which the host can survive. In particular, when Escherichia coli B strain is used as a host, it is preferable to continue the culture at about 20 ° C. after the logarithmic growth phase.

  When the logarithmic growth phase of the host is passed, the formate dehydrogenase gene is expressed by setting to the above temperature range, and a formate dehydrogenase having a very high specific activity is produced in the host. After culturing, since the target formate dehydrogenase is produced in the host, the cells or cells are disrupted to prepare a crude enzyme suspension. This crude enzyme suspension contains formate dehydrogenase having a very high specific activity. Therefore, the obtained crude enzyme suspension can be used as it is. Formate dehydrogenase can also be isolated and purified from the resulting crude enzyme suspension. At this time, general biochemical methods used for protein isolation and purification, such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography and the like, can be used alone or in appropriate combination. The isolated and purified formate dehydrogenase can be used in a state suspended in a buffer solution having a predetermined pH.

Utilization Form of Red Mold Formate Dehydrogenase The red mold derived formate dehydrogenase, which can be produced as described above, exhibits a particularly high specific activity compared to the conventionally known formate dehydrogenase. It can be used as an excellent alternative to any reaction system in which dehydrogenase is used. That is, in a reaction system in which a conventionally known formate dehydrogenase is used, by using the above-mentioned red mold formate dehydrogenase as the formate dehydrogenase, the reaction efficiency superior to that of the conventional enzyme reaction is achieved. Can be achieved.

For example, a utilization form of red mold formate dehydrogenase includes a NADH regeneration system using formate dehydrogenase. NADH is used in various enzyme reactions and converted to NAD ⁺ . NADH is used, for example, as a coenzyme when biologically synthesizing optical isomers in the chemical and pharmaceutical industries. The NADH regeneration system means that NAD ⁺ remaining in the reaction system is reduced to NADH, and NADH is recovered and used again for the enzyme reaction.

By the action of Fusarium-derived formate dehydrogenase as described above to a reaction system containing formic acid and NAD ^+, it can be synthesized NADH by reducing the NAD ^+.

As described above, NADH can be efficiently produced from NAD ⁺ contained in the reaction system by using formic acid dehydrogenase derived from red mold in the NADH regeneration system. In particular, formic acid dehydrogenase derived from red mold exhibits a very high specific activity compared to the conventionally known formate dehydrogenase. Can be manufactured. In other words, NADH production speed can be improved by using formic acid dehydrogenase derived from red mold compared to the case of using a conventionally known formate dehydrogenase.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to a following example.

[Example 1]
In this example, the formic acid dehydrogenase gene derived from red mold was cloned. First, as a red mold (Gibberella zeae), a stock preserved in NBRC of the National Institute for Product Evaluation and Technology was purchased and restored using a designated method. As a comparative control, Emericella nidulans purchased and restored in the same manner was used. These microorganism strains were cultured in a PD medium (composition: Potato Dextrose 24 g / L, adjusted to pH 7 and autoclave). The microorganisms prepared in this example are shown in Table 1.

Next, total RNA was prepared from the cells obtained by culturing the above-described microorganism strain using RNeasy Plant Mini Kit (manufactured by QIAGEN). Next, cDNA synthesis was performed using RNA PCR Kit (manufactured by TaKaRa) using the obtained total RNA as a template. The composition of the reaction solution is as follows. The reaction cycle was set to 99 ° C. (5 minutes) after 50 ° C. (2 hours), and then to 4 ° C.
(Reaction solution composition)
Final concentration
5 mM MgCl ₂
1X RT buffer
1mM dNTP mixture
0.5U RNase Inhibitor
0.25U AMV Reverse Transcriptase XL
0.125μM Oligo dT-Adaptor primer
5μg Total RNA
RNase free H ₂ O was added so that the liquid volume became 10 μl.

  Next, PCR was performed using the synthesized cDNA as a template and primers designed based on the base sequences stored in the database. The primer set shown in Table 2 was used for PCR using cDNA derived from Gibberella zeae. The primer sets shown in Table 3 were used for PCR using cDNA derived from Emericella nidulans.

In the PCR, Pyrobest DNA polymerase (TaKaRa) was used as an enzyme. The component composition in 50 μl of the reaction solution is as follows. The reaction cycle was maintained at 95 ° C for 1 minute, followed by 25 cycles of 95 ° C for 30 seconds, 60 ° C for 30 seconds and 72 ° C for 1 minute, and finally maintained at 72 ° C for 10 minutes. Maintained 4 ° C.
(Reaction solution composition)
1x Pyrobest buffer
200μM dNTPs mixture
2.5U Pyrobest DNA polymerase
50pmol Primer (forward)
50pmol Primer (reverse)
10 μl cDNA solution sterilized water was added to adjust the volume to 50 μl.

  Next, after confirming the size of the obtained PCR product by agarose gel electrophoresis, the PCR product purified from the agarose gel using MiniElute Gel Extraction Kit (manufactured by QIAGEN), pT7 Blue T-vector (manufactured by Novagen), Subcloning was performed using E. coli strain JM109 competent cell. The prepared plasmids are designated as GzFDH / pT7 and EnFDH / pT7. Here, Gz means Gibberella zeae and En means Emericella nidulans. The base sequence of the cloned PCR product was analyzed using Dye Terminator cycle sequence Kit, ABI PRIZM 3100 Genetic Analyzer (Applied Biosystems) and gene information processing program GENETYX-WIN (Software Development Co., Ltd.). The primers used for the base sequence analysis of the PCR product derived from Gibberella zeae are shown in Table 4, and the primers used for the base sequence analysis of the PCR product derived from Emericella nidulans are shown in Table 5.

  As a result, the base sequence of Gibberella zeae-derived formate dehydrogenase gene is shown in SEQ ID NO: 1, and the amino acid sequence of Gibberella zeae-derived formate dehydrogenase is shown in SEQ ID NO: 2. The amino acid sequence shown in SEQ ID NO: 2 obtained as a result of the analysis showed 100% agreement with the sequence published on the database (Genbank NW — 059922).

  Next, the isolated formate dehydrogenase gene was incorporated into an expression vector. Specifically, the prepared plasmids (GzFDH / pT7, EnFDH / pT7) were treated with restriction enzymes NdeI and EcoRI (37 ° C., 2 hours). The solution after the reaction was subjected to 0.8% agarose gel electrophoresis, and each formate dehydrogenase gene (about 1.2 kb) excised from the vector as an NdeI / EcoRI fragment was purified using Mini Elute Gel Extraction Kit (manufactured by QIAGEN). .

  Next, as with various FDH / pT7, TaKaRaligation kit Ver.2.1 (manufactured by TaKaRa) was used at the NdeI / EcoRI site of the gene expression vector pET23b (+) (manufactured by Novagen) treated with restriction enzymes NdeI and EcoRI. After introducing the formate dehydrogenase gene fragment, subcloning was performed using E. coli JM109 competent cell (TaKaRa). By colony PCR using Foward primer; pET-F (5'-ACT CAC TAT AGG GAG ACC ACA AC-3 ') and Reverse primer; pET-R (5'-TAT TGC TCA GCG GTG GCA G-3') After selecting a clone containing a DNA fragment of the desired size, the plasmid was purified using Plasmid mini prep kit (manufactured by QIAGEN). Furthermore, plasmids whose gene introduction was confirmed as planned by restriction enzyme mapping were designated as GzFDH / pET23b (+) and EnFDH / pET23b (+). The steps for constructing the expression vector described above are shown in FIG.

[Example 2]
In this example, the expression vector prepared in Example 1 was used to verify the expression of formate dehydrogenase in E. coli.

  Escherichia coli transformant containing the expression vector prepared in Example 1 was treated with 5 ml of LB-ampicillin medium (composition: Tryptone 10 g / L, Yeast Extract 5 g / L, NaCl 5 g / L and Ampicillin (manufactured by SIGMA) 50 mg / L). After overnight culture (37 ° C.), 1% inoculated into 100 ml of the same composition medium and aerobically shaken. Judging that the logarithmic growth phase has passed when the OD600 value reaches 0.5 to 0.9, add IPTG (isopropyl-1-thio-β-D-galactoside) (TaKaRa) to a final concentration of 1 mM. Then, the induction of formate dehydrogenase gene expression was started. Thereafter, the culture temperature was set to 20 ° C., and the culture was further continued for 16 hours.

  Next, the obtained culture broth was collected by centrifugation (13 krpm, 4 ° C., 10 min), suspended in 0.1 ml potassium phosphate buffer at 1 ml / 0.1 g (wet cell weight), and 1 g / 0.1 g ( (Bacterial wet weight) 0.1 mm diameter glass beads were added and the cells were crushed using a multi-bead shocker (manufactured by Yasui Kikai Co., Ltd.) to obtain a crude enzyme solution. The formate dehydrogenase activity of the crude enzyme solution was measured by measuring the increase in absorbance at 340 nm accompanying NADH production at 37 ° C in 0.1 M potassium phosphate buffer (pH 7.5) containing 162 mM sodium formate and 1.62 mM NAD. It was done by measuring. Protein quantification was performed by the Bradford method using Bovine serum albumin (BSA) (BioRad) as a standard protein. The results are shown in FIG.

  As shown in FIG. 2, in the crude enzyme solution derived from the transformant containing the formic acid dehydrogenase gene derived from red mold, an increase in absorbance at 340 nm accompanying NADH production was confirmed. On the other hand, an increase in absorbance at 340 nm was not confirmed in the crude enzyme solution derived from the transformant containing the formate dehydrogenase gene derived from Emericella nidulans. Similarly, an increase in absorbance at 340 nm was not confirmed in the E. coli crude enzyme solution transformed only with the vector not containing the formate dehydrogenase gene. From the above results, it was confirmed that according to the expression induction method shown in the present example, formic acid dehydrogenase derived from red mold can be produced in an E. coli expression system.

Example 3
In this example, the red mold-derived formate dehydrogenase obtained in Example 2 was purified, and the specific activity of the purified red mold-derived formate dehydrogenase was compared with a conventionally known formate dehydrogenase. .

  First, using the crude crushing solution containing formic acid dehydrogenase derived from red mold obtained in Example 2 as a sample, 5T of HiTrap Q FF column (manufactured by Amersham Biosciences) was used, and column purification was performed according to the procedure shown in FIG. did. Among the eluted fractions, 10 fractions (1/3 of the total) containing formate dehydrogenase were used as samples for secondary purification.

  The fraction containing formate dehydrogenase obtained above was desalted with Amicon Ultra-15 (30 kDa cut) and diluted to an appropriate amount (about 3 bed = 15 ml) with 10 mM KPB (pH 7.2). . Column purification was performed according to the procedure of FIG. 4 using 5 ml of Econo-Pac CHT-II column as the column for secondary purification. A fraction having a purity of 80% or more was obtained after secondary purification.

  The concentration of the obtained fraction was adjusted in order to compare the activity of formic acid dehydrogenase (GzFDH) derived from red mold in the formic acid decomposition reaction. As a control, Candida boidinii-derived FDH (CbFDH) (manufactured by Sigma) whose properties were known was used. CbFDH was dissolved in a potassium phosphate buffer and then purified by the same method as described above in this example as a sample. Each of the purified GzFDH and CbFDH was prepared to have a constant concentration after quantification by the Bradford method using BSA as a standard protein. Bio-Safe CBB G-250 stain (BioRad) was used as a Bradford staining solution.

For the two types of purified GzFDH and CbFDH whose concentrations were adjusted, the activity of the formic acid decomposition reaction was measured by the endpoint method, and the reaction rates were compared. The formic acid decomposition reaction by the FDH catalyst is represented by the following formula.
HCOO- + NAD + → CO ₂ + NADH

By adding Methoxy PMS (mPMS), which is an electron transfer material, and WST1, which is a redox coloring indicator (both manufactured by DOJINDO), the reaction proceeds as shown in the following formula, so yellow formazan is measured at an absorbance of 438 nm. By doing so, the amount of formic acid decomposition can be quantified. The extinction coefficient of yellow formazan is about 6 times that of NADH, and quantification with higher sensitivity than direct measurement of NADH is possible.
NADH + mPMS → NAD + + mPMS (reduced)
mPMS (reduced) + WST1 → mPMS + yellow formazan (37000 / M · cm, 438 nm)

  The composition of the reaction solution was 162 mM HCOONa, 1.62 mM NAD, 3.33 μg / ml mPMS, 0.33 mg / ml WST1, 0.02 mg / ml GzFDH or CbFDH and 100 mM potassium phosphate buffer (pH 7.5). The total volume was 100 μl. After mixing the reaction solution, the reaction was performed at 25 ° C. for 30 minutes, and then A430 was measured with a plate reader (manufactured by TECAN). A comparison of the amount of yellow formazan produced (reaction rate) after the reaction is shown in FIG. FIG. 6 shows a comparison with paper data (Biochemistry vol.69, No.11 2004 991252-1267) covering the properties of conventionally known formate dehydrogenase. From these results and a comparison with known data, it was shown that the red mold derived formate dehydrogenase obtained this time has the world's highest specific activity as compared with the conventionally known formate dehydrogenase.

[Comparative Example 1]
In Comparative Example 1, production of formic acid dehydrogenase derived from red mold was performed in the same manner as in Example 2 except that expression was induced in the logarithmic growth phase using the E. coli transformant containing the expression vector prepared in Example 1. Try. In Comparative Example 1, protein expression is confirmed as a result of the above expression, but formate dehydrogenase activity cannot be detected.

[Comparative Example 2]
In Comparative Example 2, using the Escherichia coli transformant containing the expression vector prepared in Example 1, expression induction was performed at the time when the logarithmic growth phase was passed, and then the culture was continued at 37 ° C. Similarly, production of formic acid dehydrogenase from red mold is attempted. In Comparative Example 2, protein expression is confirmed as a result of the above expression, but formate dehydrogenase activity cannot be detected.

FIG. 5 is a schematic diagram showing the steps of constructing GzFDH / pET23b (+) and EnFDH / pET23b (+). It is a characteristic view which shows the result of having detected the light absorbency of 340nm accompanying the production | generation of NADH by performing enzyme reaction using the crude enzyme liquid prepared in Example 2. FIG. 6 is a flowchart showing the procedure of column purification performed in Example 3. 4 is a flowchart showing a procedure of secondary purification performed in Example 3. It is a characteristic view which shows the result of having compared the reaction rate about refined GzFDH and CbFDH. It is a characteristic view which shows the result compared with the paper data (Biochemistry vol.69, No.11 2004 991252-1267) which covered the characteristic of conventionally well-known formate dehydrogenase about refined GzFDH and CbFDH.

Claims (4)

A method for producing NADH, wherein a formate dehydrogenase comprising the following protein (a) or (b) is allowed to act on a reaction system containing formic acid and NAD ⁺ .
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 2
(B) a protein comprising an amino acid sequence having 95% or more identity to the amino acid sequence shown in SEQ ID NO: 2 and having catalytic activity in a reaction using formic acid and NAD ⁺ as substrates and carbon dioxide and NADH as products. When a host into which a vector in which a formate dehydrogenase gene encoding the following protein (a) or (b) is placed is controlled under the control of an inducible promoter is cultured, and when the logarithmic growth phase has passed, Inducing gene expression, culturing at a temperature lower than the optimum growth temperature of the host and at which the host can survive,
A method for producing formate dehydrogenase, comprising obtaining the formate dehydrogenase.
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 2
(B) a protein comprising an amino acid sequence having 95% or more identity to the amino acid sequence shown in SEQ ID NO: 2 and having catalytic activity in a reaction using formic acid and NAD ⁺ as substrates and carbon dioxide and NADH as products. 3. The method for producing formate dehydrogenase according to claim 2 , wherein when the logarithmic growth phase is passed, it is a stationary phase. The method for producing formate dehydrogenase according to claim 2 , wherein the temperature at which the host is able to survive is lower than the optimum growth temperature of the host and is 15 to 37 ° C.

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