WO2015064517A1

WO2015064517A1 - Formate dehydrogenase and use thereof

Info

Publication number: WO2015064517A1
Application number: PCT/JP2014/078442
Authority: WO
Inventors: 智行中川; 光裕水口; 達也澤野
Original assignee: 国立大学法人岐阜大学; オリエンタル酵母工業株式会社
Priority date: 2013-10-28
Filing date: 2014-10-27
Publication date: 2015-05-07
Also published as: JPWO2015064517A1

Abstract

The purpose of the present invention is to provide: a formate dehydrogenase having a high specific activity; and a method for producing a formate dehydrogenase with high efficiency without using methanol. The present invention relates to: a novel formate dehydrogenase; a gene encoding the formate dehydrogenase; a method for producing the formate dehydrogenase; and a method for regenerating a coenzyme using the formate dehydrogenase.

Description

Formate dehydrogenase and its utilization

The present invention relates to formate dehydrogenase, a gene encoding the enzyme, and production and use of the enzyme.

When producing an active pharmaceutical ingredient intermediate, reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) is required as a coenzyme. However, since these coenzymes are expensive, regeneration of these coenzymes is indispensable for efficiently producing an active pharmaceutical ingredient intermediate.

Conventionally, in order to reduce a coenzyme, examples using glucose dehydrogenase as described in Patent Document 1 and examples using formate dehydrogenase as described in Patent Document 2 have been reported. . However, since glucose dehydrogenase converts glucose to gluconic acid, there is a problem that the same amount of gluconic acid as that of the intended drug substance intermediate is produced.

On the other hand, formate dehydrogenase (EC.1.2.1.2) is oxidized nicotinamide adenine dinucleotide (NAD ^+), in the presence of formic acid and water, the NAD ⁺ reduced nicotinamide adenine dinucleotide ( NADH) and formic acid is oxidized to carbon dioxide. Therefore, an active pharmaceutical ingredient intermediate can be made without producing a by-product.

However, the disadvantage of using formate dehydrogenase is that the amount of the enzyme added when regenerating the coenzyme increases because of the low specific activity of the enzyme. For example, Candida bodinini (ATCC 32195) -derived formate dehydrogenase as described in Patent Document 3 has a low specific activity and is not sufficient for regeneration of coenzymes using formate dehydrogenase. is there.

In addition, when formate dehydrogenase is expressed in a methanol-utilizing yeast such as Pichia methanolica, it is necessary to induce it with methanol, and in the case of mass production, an explosion-proof facility is required, which is expensive to manufacture. There were disadvantages.

Japanese Patent Laid-Open No. 2000-236883 JP 2002-233395 A JP 2003-180383 A

The present invention has been made in view of the above points, and provides a formate dehydrogenase having a high specific activity and an object of providing a method for efficiently producing formate dehydrogenase without using methanol. And

In view of the above problems, the present inventors have extensively searched for microorganisms having formate dehydrogenase activity, and as a result, have found a Pichia methanolica strain that highly produces formate dehydrogenase having excellent properties. Then, formate dehydrogenase was isolated and purified from the Pichia methanolica strain, and the formate dehydrogenase gene was successfully isolated and expressed in the host microorganism. And, the formate dehydrogenase has a high specific activity, and the formate dehydrogenase can be produced inexpensively and efficiently without using methanol unlike the yeast expression system by expressing the enzyme in E. coli. I found. Furthermore, it has been found that this formate dehydrogenase is useful for coenzyme regeneration.

That is, the present invention includes the following.

(1) The following protein (a), (b) or (c):
(A) a protein comprising the amino acid sequence of SEQ ID NO: 1,
(B) a protein having at least 70% identity with the amino acid sequence of SEQ ID NO: 1 and having formate dehydrogenase activity;
(C) A protein having a formate dehydrogenase activity, comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence of SEQ ID NO: 1.

(2) A gene encoding the protein according to (1).

(3) A gene comprising the following DNA (d), (e) or (f):
(D) DNA consisting of the base sequence of SEQ ID NO: 2,
(E) DNA encoding a protein having 70% or more identity with the base sequence of SEQ ID NO: 2 and having formate dehydrogenase activity,
(F) DNA encoding a protein having a formate dehydrogenase activity, comprising a base sequence in which one or several bases are deleted, substituted, inserted or added in the base sequence of SEQ ID NO: 2.

(4) A recombinant vector comprising the gene according to (2) or (3).

(5) A transformant obtained by introducing the gene according to (2) or (3) into a host.

(6) The transformant according to (5), wherein the host is Escherichia coli.

(7) A method for producing formate dehydrogenase, comprising culturing the transformant according to (5) or (6) and collecting a protein having formate dehydrogenase activity from the culture.

(8) A method for regenerating a coenzyme by allowing the protein according to (1), the transformant according to (5) or (6) or a processed product thereof to coexist in an enzymatic reduction reaction system.

This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2013-223317 which is the basis of the priority of the present application.

The formate dehydrogenase of the present invention is characterized by high specific activity. By using the formate dehydrogenase according to the present invention, an expensive coenzyme can be regenerated with a small amount of enzyme. In addition, by expressing the formate dehydrogenase gene of the present invention in E. coli, it is possible to produce formate dehydrogenase at low cost and efficiently without using methanol. Furthermore, the formate dehydrogenase of the present invention can efficiently regenerate the coenzyme in the reduction reaction of alcohol, amine, aldehyde, etc., and can greatly reduce the amount of expensive coenzyme used.

The construction diagram of plasmid pTRP-pmfdh is shown. It is a graph which shows the result of having measured the pH stability of recombinant PmFDH. It is a graph which shows the result of having measured the optimum pH of recombinant PmFDH. It is a graph which shows the result of having measured the temperature stability of recombinant PmFDH. It is a graph which shows the result of having measured the optimal temperature of recombinant PmFDH. It is a graph which shows the result of having measured the temperature stability in 50 degreeC of recombinant PmFDH.

Formate dehydrogenase (enzyme number EC 1.2.1.2) is a reaction that produces carbon dioxide and reduced nicotinamide adenine dinucleotide (NADH) from formic acid and oxidized nicotinamide adenine dinucleotide (NAD ⁺ ). It is an enzyme that catalyzes. Formate dehydrogenase is a useful enzyme that has advantages such as the availability of inexpensive formic acid and the by-product of carbon dioxide that does not accumulate in the system when used for coenzyme regeneration in NADH-dependent enzyme reactions. .

In one embodiment, the present invention relates to a protein having formate dehydrogenase activity, that is, formate dehydrogenase. Examples of the protein having formate dehydrogenase activity of the present invention include a protein consisting of the amino acid sequence of SEQ ID NO: 1. A protein functionally equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 1, for example, 70% or more, preferably 80% or more, more preferably 90% or more, more preferably 95% or more, of the amino acid sequence of SEQ ID NO: 1, A protein having an amino acid sequence having an identity of 99% or more and having formate dehydrogenase activity is also included in the protein of the present invention. Furthermore, the protein of the present invention includes a protein having an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence of SEQ ID NO: 1 and having formate dehydrogenase activity. . The term “several” refers to 2 to 5, preferably 2 to 3. “Amino acid sequence in which one or several amino acids have been deleted, substituted, inserted or added” is an amino acid deleted, substituted, inserted or added by methods well known to those skilled in the art, such as partial-directed mutagenesis. Can be obtained.

Formate dehydrogenase activity is measured by measuring the increase in absorbance at 340 nm accompanying NADH production at 25 ° C. in 0.1 M phosphate buffer (pH 7.5) containing 20 mM sodium formate and 1 mM NAD ^+. Can be implemented.

The protein having formate dehydrogenase activity is 10% or more, preferably 40% or more, more preferably 60% or more when a protein comprising the amino acid sequence of SEQ ID NO: 1 is used under the activity measurement conditions as described above. More preferably, it refers to a protein exhibiting an activity of 80% or more.

The protein of the present invention can be obtained preferably from Pichia methanolica, for example, Pichia methanolica IAM 12901 strain. This strain has been preserved in the IAM Culture Collection, Institute for Molecular Cell Biology, University of Tokyo, and has now been transferred to the RIKEN BioResource Center Microbial Materials Development Department (JCM).

The protein having formate dehydrogenase activity of the present invention may be a natural enzyme obtained from a microorganism as described above, or may be a recombinant enzyme produced using a gene recombination technique. .

The present invention also relates to a gene encoding formate dehydrogenase (formate dehydrogenase gene). Examples of the gene encoding the formate dehydrogenase of the present invention include a gene consisting of the nucleotide sequence of SEQ ID NO: 2. Genes include nucleic acids and single-stranded, double-stranded or triple-stranded DNA or RNA. A gene functionally equivalent to the gene consisting of the nucleotide sequence of SEQ ID NO: 2, for example, 70% or more, preferably 80% or more, more preferably 90% or more, more preferably 95% or more, of the nucleotide sequence of SEQ ID NO: 2, A gene encoding a protein consisting of a base sequence having an identity of 99% or more and having formate dehydrogenase activity is also included in the gene of the present invention. Furthermore, a gene encoding a protein consisting of a base sequence in which one or several bases are deleted, substituted, inserted or added in the base sequence of SEQ ID NO: 2 and having formate dehydrogenase activity is also a gene of the present invention. Is included. The term “several” refers to 2 to 5, preferably 2 to 3.

A gene functionally equivalent to the gene consisting of the base sequence of SEQ ID NO: 2 hybridizes under stringent conditions with a gene consisting of a base sequence complementary to the gene consisting of the base sequence of SEQ ID NO: 2, and formic acid A gene encoding a protein having dehydrogenase activity is included. The stringent condition means a condition in which a specific hybrid is formed and a non-specific hybrid is not formed, and a highly stringent condition is preferable. Highly stringent conditions are conditions in which, for example, washing at 65 ° C., 0.1 × SSC and 0.1% SDS is performed after washing after hybridization.

A method for obtaining a desired gene by cloning is well known in the field of molecular biology. For example, a suitable genomic library can be prepared by restriction endonuclease digestion and screened using a probe complementary to the desired gene sequence. Once the sequence is isolated, the DNA can be amplified using standard amplification methods such as polymerase chain reaction (PCR) to obtain an amount of DNA suitable for transformation (gene transfer).

Methods for preparing a genomic DNA library used for gene cloning, hybridization, PCR, plasmid preparation, DNA cleavage and ligation, transformation, and the like are described in, for example, Sambrook, J et al. , Molecular Cloning 2nd ed. , 9.47-9.58, Cold Spring Harbor Lab. press (1989).

The method for producing formate dehydrogenase of the present invention is characterized by culturing a transformant obtained by introducing the formate dehydrogenase gene into a host, and collecting a protein having formate dehydrogenase activity from the culture. To do. A transformant obtained by introducing a formate dehydrogenase gene into a host can be produced by a method known in the art. In the present invention, gene introduction includes all cases where a polynucleotide having a gene function is introduced into a host as a recombinant nucleic acid. Polynucleotides include nucleic acids and single, double or triple stranded DNA or RNA. The introduction of a gene includes a case of introduction by a recombinant vector and a case of introduction by homologous recombination using a nucleic acid synthesized by PCR or the like.

The type of host into which the gene is to be introduced is not limited, and unicellular eukaryotes such as bacteria, fungi, various yeasts, or live cells of animals or plants can be arbitrarily selected. E. coli is preferred. As the host E. coli, an appropriate one is selected from E. coli K-12 strains usually used for genetic engineering. Representative examples include JM105 and JM109, but DH5 or BL21, N99cI ⁺ used in an inducible expression system may be used.

The gene is more preferably introduced by an expression vector that enhances the expression of the gene. An expression vector is obtained by fusing a gene to be introduced with various DNA fragments or RNA fragments that enhance the expression. Preferably, the expression vector may contain a transcription promoter, transcription terminator, and selection marker for constitutively or inducibly expressing the gene. If desired, a gene that controls a cis element such as an enhancer, an operator, or a promoter may be contained.

Examples of the vector include, but are not limited to, plasmids frequently used when E. coli is used as a host, pUC18, pUC19, pUC118, pUC119, pSC101, pBR322, pHSG298, pVC18, pVC19, pTrc99A, pMal-c2, pGEX2T, pTV118N, pTV119N, pTRP and the like can be preferably used. YEp13, YEp24, YCp50, pRS414, pRS415, pRS404, pAUR101, pKG1, etc. often used when cerevisiae is used as a host, and plasmids pUB110, pC194, etc. often used when Bacillus subtilis is used as a host are also used. it can. Further, pBI122, pBI1101 and other various types can be used without limitation.

Examples of transcription promoters for expression in E. coli include tryptophan synthase (trp), lactose operon (lac), tac and trc promoters fused with these, λ phage PL and PR promoters, T7 phage promoters, and the like. It is done. However, if the promoter is too strong, the target protein is excessively expressed in E. coli, and as a result, the target protein tends to form inclusion bodies, making subsequent separation and purification steps difficult. Therefore, it is necessary to select an optimal promoter so as to enable expression in the soluble fraction or secretion of the protein into the culture supernatant. In view of such points, tryptophan promoter (trp) is desirable. Examples of the expression vector having a tryptophan promoter (trp) include pTRP (Clinica Chimica Acta 237, 43-58 (1995)).

Examples of selectable markers include formaldehyde resistance markers, drug resistance markers such as kanamycin, ampicillin, tetracycline, chloramphenicol, and auxotrophic markers such as leucine, histidine, lysine, methionine, arginine, tryptophan, and uracil. It is not limited to this.

As a general method for constructing a recombinant vector, for example, a method of incorporating a gene fragment prepared by PCR or the like into a recombinant vector using an appropriate restriction enzyme and ligase can be mentioned. Preferably, a recombinant vector can be obtained by performing a ligation reaction under defined conditions using a commercially available ligation kit such as Ligation High (Toyobo Co., Ltd.). If necessary, these vectors are purified by a boil method, an alkaline SDS method, a magnetic bead method and a commercially available kit using these principles, and further concentrated by an ethanol precipitation method, a polyethylene glycol precipitation method or the like. It can be concentrated by means.

The gene introduction method is not particularly limited, and examples thereof include an electric pulse method, a competent cell method, a calcium chloride method, a protoplast method, a particle gun method, and an electroporation method. Specifically, the Hanahan method or the like can be used for gene introduction into E. coli, and the lithium ion method or the like can be used for gene introduction into yeast.

In the method of inserting a target gene at an arbitrary position on the genome by homologous recombination, the target gene is inserted into a sequence homologous to the sequence on the genome together with a promoter, and this DNA fragment is introduced into the cell by electroporation. Can be carried out by causing homologous recombination. At the time of introduction into the genome, a strain in which homologous recombination has occurred can be easily selected by using a DNA fragment in which a target gene and a selection marker gene are linked. In addition, a gene linked to a drug resistance gene and a gene that becomes lethal under specific conditions is inserted into the genome by homologous recombination by the above method, and then becomes lethal under specific conditions with the drug resistance gene. The target gene can also be introduced using homologous recombination in the form of replacing the gene.

Generally, it is recognized in the production method of the target product using the transformant. In the production method of formate dehydrogenase of the present invention, the selection of the transgene, the selection of the host to be introduced, the expression vector Factors such as selection of introduction means and construction method of DNA or RNA suitable for it, selection of medium or additive type and concentration, culture condition or growth condition of transformant, etc. are the production of formate dehydrogenase. May affect quantity.

The method of culturing the transformant in a medium is performed according to a normal method used for host culture. As a medium for culturing transformants obtained using microorganisms such as Escherichia coli and yeast as a host, the medium contains a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by the microorganisms. As long as the medium can be used, either a natural medium or a synthetic medium may be used.

As the carbon source, any carbon compound that can be assimilated may be used. For example, polyols such as glycerin, or organic acids such as pyruvic acid, succinic acid, or citric acid can be used. The nitrogen source may be any available nitrogen compound. For example, peptone, meat extract, yeast extract, casein hydrolyzate, soybean meal alkaline extract, or ammonia or a salt thereof can be used. In addition, salts such as phosphate, carbonate, sulfate, magnesium, calcium, potassium, iron, manganese, zinc, a specific amino acid, a specific vitamin, an antifoaming agent, and the like may be used as necessary. In addition, a protein expression inducer such as isopropyl-β-D-thiogalactopyranoside may be added to the medium as necessary.

The culture is usually carried out under aerobic conditions such as shaking culture or aeration and agitation culture, preferably at 0 to 40 ° C., more preferably at 10 to 37 ° C., particularly preferably at 15 to 37 ° C. During the culturing period, the pH of the medium can be changed as appropriate as long as the growth of the host is possible and the activity of the produced formate dehydrogenase is not impaired, but it is preferably in the range of about pH 4-8. The pH is adjusted using an inorganic or organic acid, an alkaline solution, or the like. During culture, an antibiotic such as ampicillin or tetracycline may be added to the medium as necessary.

Subsequently, formate dehydrogenase expressed solubilized by culturing the transformant is collected from the culture. The culture includes a culture solution, a culture supernatant, cultured cells, cultured cells, cells or disrupted cells. The collection method is not only extraction from cells or disrupted cells or cells that are usually performed, but in some cases it can also be extracted directly from the culture solution using an appropriate extraction solvent. Depending on the type of host used, at least part of the formate dehydrogenase may remain in the host cell or on the cell surface, but various known operations such as disruption of the cell membrane or cell wall and extraction with an appropriate extraction solvent may be performed. After that, it can be collected.

When the host's logarithmic growth phase has passed, the formate dehydrogenase gene is expressed by setting to the above temperature range, and formate dehydrogenase exhibiting a very high specific activity in the host can be produced. After culturing, the target formate dehydrogenase is produced in the host, so 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 may 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.

Coenzyme can be regenerated by coexisting the above protein, a transformant producing the protein, or a processed product thereof in the enzymatic reduction reaction system. That is, the coenzyme can be efficiently regenerated by allowing the formate dehydrogenase obtained in the present invention to coexist in the enzymatic reduction reaction system. The treated product of the transformant means, for example, a crude extract, a crude enzyme suspension, a cultured cell freeze-dried organism, an acetone-dried organism, or a ground product of these cells. Furthermore, they may be used by immobilizing them with a known means in the form of enzymes or cells. Immobilization can be carried out by methods well known to those skilled in the art (for example, crosslinking method, physical adsorption method, entrapment method, etc.). The reaction is carried out at 10 ° C. to 70 ° C., preferably 20 ° C. to 60 ° C., pH 4 to 10, preferably pH 5.5 to 9.5. The substrate concentration is 0.1% to 90% (w / v), but the substrate can also be added continuously. The reaction can be carried out batchwise or continuously. The reaction can also be performed using an immobilized enzyme, a membrane reactor, or the like. As described above, according to the present invention, in the reduction reaction for enzymatically producing alcohol, amine, aldehyde, etc., the coenzyme can be efficiently regenerated and the amount of expensive coenzyme used can be greatly reduced. Can do.

Next, the present invention will be described with reference to examples. The technical scope of the present invention is not limited by the following examples. In the examples, the protein according to the invention is called “recombinant PmFDH”.

[Example 1: Construction of recombinant PmFDH expression system]
The amino acid sequences of formate dehydrogenase (FDH) are aligned, and the FDH sequences of methylotrophic yeasts Candida voyinii (Accession No. AJ245934) and budding yeast Saccharomyces cerevisiae (Accession No. Z75296.1) are aligned and homologous. Two sites, TPFHPAY and DYPRQDII, were found as high sequences. Based on both amino acid sequences, two types of PCR primers, FDH-inf (5′-GGTAAGCACGCTGCTGGATGAA-3 ′) (SEQ ID NO: 3) and FDH-inr (5′-TAATATCTTGTGGTCTGTAATC-3 ′) (SEQ ID NO: 4) were prepared. did. The genomic DNA of Pichia methanolica used as a template was prepared using Gen Torukun ^™ (for yeast) High Recovery (manufactured by Takara Bio Inc.). As a result of PCR using both the prepared primers, an amplified fragment of about 1,000 bp could be obtained.

PCR reaction (per 50 μL) with 1U KOD plus polymerase (manufactured by Toyobo), 10 pg template DNA, 0.3 μM sense primer, 0.3 μM antisense primer, 0.2 mM dNTP Mix, 1 mM MgSO ₄ , 10 × buffer 94 ° C./15 seconds (denaturation), 50 ° C./30 seconds (annealing), and 68 ° C./1 minute 30 seconds (extension) were repeated 30 cycles.

A full-length Pichia methanolica-derived FDH sequence was obtained from the internal sequence of the obtained FDH by using DNA Walking SpeedUP ^™ Premix Kit (manufactured by Seegene). Specifically, PCR primers, pmfdh-wkf1 (5′-GGAGAACCCATGAGAACA-3 ′: for 1st PCR) (SEQ ID NO: 5) and pmfdh-wkf2 (5′-ATGAGAAACAAATACGGTGCCC-3 ′: 2nd PCR for N-terminal sequence analysis (SEQ ID NO: 6) for pmfdh-wkr (5'-CCTTGAAATGTAACCTGGGTG-3 ': 1st PCR) (SEQ ID NO: 7) and pmfdh-wkr2 (5'-TAATGTCGCATCATGGATT-3': 2nd PCR (SEQ ID NO: 8) was used.

The base sequence of the FDH gene derived from Pichia methanolica thus obtained is shown in SEQ ID NO: 2, and the amino acid sequence is shown in SEQ ID NO: 1.

In order to express the FDH gene derived from Pichia methanolica in E. coli, a plasmid pTRP-pmfdh into which the FDH gene was inserted was prepared according to the construction diagram shown in FIG. First, PCR was performed using Pichia methanolica chromosomal DNA as a template and the primers shown below to amplify the FDH gene, and an approximately 1.1 kb DNA fragment of the FDH gene was confirmed by agarose electrophoresis.
Sense primer (SacI-pmfdh):
5′-GAGCTCCATGAAGGTCGTTTTTAGTT-3 ′ (SEQ ID NO: 9)
Antisense primer (pmfdh-SalI):
5′-GTCGACTTAGACACTTCTTTGTCAGCA-3 ′ (SEQ ID NO: 10)

PCR reaction (per 50 μL) with 1U KOD plus polymerase (manufactured by Toyobo), 10 pg template DNA, 0.3 μM sense primer, 0.3 μM antisense primer, 0.2 mM dNTP Mix, 1 mM MgSO ₄ , 10 × buffer 94 ° C./2 minutes (denaturation), 55 ° C./30 seconds (annealing), and 68 ° C./1 minute 30 seconds (elongation) were repeated 30 cycles.

The PCR products of the obtained FDH gene were respectively treated with restriction enzymes SacI and SalI (Takara Bio), and after agarose electrophoresis, GFX ^™ PCR DNA and Gel Band Purification Kit (GE Healthcare Japan) It was collected. The recovered fragment was introduced into an expression vector pTRP (2.9 kb) previously treated with restriction enzymes SacI and SalI (manufactured by Takara Bio Inc.) using LigaFast ^™ Rapid DNA Ligation System (manufactured by Promega). Next, E. coli HB101 strain was transformed to obtain recombinant pTRP-pmfdh / HB101.

[Example 2: Flask culture of recombinant PmFDH]
Production of recombinant PmFDH derived from Pichia methanolica by batch culture of transformants was carried out by the following method.

Escherichia coli HB101 strain transformed with plasmid pTRP-pmfdh was subjected to batch culture for 9 hours at 37 ° C. and 160 rpm using 2 L of LB medium containing 50 μg / mL ampicillin. Cell growth was examined by measuring the absorbance of the culture at 600 nm. As a result, a batch culture of Escherichia coli HB101 strain transformed with the plasmid pTRP-pmfdh could be obtained.

In order to confirm the expression level, this culture solution was centrifuged (8,000 rpm, 15 minutes, 4 ° C., Model HP-26XP Centrifuge manufactured by Beckman Coulter), and the wet cells were collected. Then, 5 g of the obtained wet cells were suspended in 3 mL of extraction buffer (10 mM phosphate buffer, pH 7.5), and after ultrasonic disruption (MISSONIX Astrason ULTRASONIC PROCESSOTR XL) and centrifuged (18,000 × g, 15 minutes, 4 ° C., Model HP-26XP (manufactured by Beckman Coulter, Inc.) and a crude extract were prepared.

The activity of recombinant PmFDH was measured by the following method. After adding recombinant PmFDH to a reaction solution composed of phosphate buffer (0.1 M, pH 7.5), NAD ⁺ (1 mM, manufactured by Oriental Yeast Co., Ltd.), sodium formate (20 mM, manufactured by Nacalai Tesque), The absorbance change at 340 nm was measured at 25 ° C., and the formate dehydrogenase activity of the recombinant PmFDH was calculated. The production amount of 1 μmol of NADH per minute under these reaction conditions was defined as 1 unit, which was defined as formate dehydrogenase activity. When the formate dehydrogenase activity of the obtained enzyme solution was measured, an activity of 0.24 U / mL per 1 mL of the medium was confirmed.

[Example 3: Purification of recombinant PmFDH]
For mass purification, the wet cells obtained from 6 L of the culture solution are suspended in 10 mM phosphate buffer (pH 7.5), and then the cells are disrupted by ultrasonic disruption. Was centrifuged (18,000 × g, 15 minutes, 4 ° C., Model HP-26XP Centrifuge manufactured by Beckman Coulter) to prepare a crude extract. Purification was performed by the following method using the obtained crude extract.

It was passed through DEAE-Cellulofine (manufactured by Chisso) equilibrated with buffer A (10 mM phosphate buffer, pH 7.5), washed with buffer A, and the target protein was eluted with a linear gradient of 0-0.5 M NaCl. . The obtained target fraction was finally dialyzed against 10 mM phosphate buffer (pH 7.5) to obtain a purified preparation of recombinant PmFDH having a specific activity of 3.3 U / mg protein.

[Example 4: Evaluation of basic enzymatic performance of recombinant PmFDH]
The basic enzymatic performance of the recombinant PmFDH obtained in Example 3 was evaluated. Formate dehydrogenase activity was measured in the same manner as in Example 2 except for the conditions specifically described.

pH stability:
Using the following buffers, the residual activity after 7 days at 30 ° C. was measured.
pH 4.5 to 6.0: 0.1 M citrate-NaOH buffer pH 6.0 to 7.5: 0.1 M phosphate (K-Pi) buffer pH 6.0 to 7.5: 0.1 M PIPES-NaOH buffer pH 7.5 to 9.0: 0.1 M Tris-HCl buffer pH 9.0 to 9.5: 0.1 M glycine-NaOH buffer

The results are shown in FIG. The activity on day 0 in each buffer was defined as 100 and expressed as relative activity.

Optimum pH:
Formate dehydrogenase activity was measured using the following buffers.
pH 4.5 to 6.0: 0.1 M citrate-NaOH buffer pH 6.0 to 7.5: 0.1 M phosphate (K-Pi) buffer pH 6.0 to 7.5: 0.1 M PIPES-NaOH buffer pH 7.5 to 9.0: 0.1 M Tris-HCl buffer pH 9.0 to 9.5: 0.1 M glycine-NaOH buffer

The results are shown in FIG. The activity in 0.1 M PIPES-NaOH buffer (pH 7.5) was defined as 100 and expressed as relative activity.

Temperature stability:
After incubation for 20 minutes at temperatures of 4, 25, 30, 37, 40, 45, 50, 60, and 70 ° C., formate dehydrogenase activity was measured. The results are shown in FIG. The activity at 4 ° C. was defined as 100 and expressed as relative activity.

Optimal temperature:
Formate dehydrogenase activity was measured at temperatures of 25, 30, 37, 40, 45, 50, 55, 60, and 70 ° C. The results are shown in FIG. The activity at 50 ° C. was defined as 100 and expressed as relative activity.

Temperature stability at 50 ° C:
Residual activity when reacted at 50 ° C. for 24 hours was measured. The results are shown in FIG. The activity at 0 hours was defined as 100 and expressed as relative activity.

The basic enzymatic performance of the recombinant PmFDH obtained from the above results is summarized in Table 1 below.

[Example 5: Ethanol production by recombinant PmFDH]
Using the recombinant PmFDH obtained in Example 3, it was confirmed whether ethanol could be produced from acetaldehyde when NAD ⁺ was used as a coenzyme. Specifically, acetaldehyde (2%, manufactured by Merck & Co.) and alcohol dehydrogenase (10 U / mL, manufactured by Oriental Yeast Co., Ltd.), NAD ⁺ (0.3 mM, manufactured by Oriental Yeast Co., Ltd.), sodium formate (100 mM) Recombinant PmFDH was added to a reaction solution composed of Nacalai Tesque, Inc.) and phosphate buffer (250 mM, pH 7.0) and reacted at 30 ° C. for 24 hours. The produced ethanol is 99.5% ethanol (manufactured by Wako Pure Chemical Industries, Ltd.) as a standard product, alcohol oxidase (0.2 U / mL, manufactured by Sigma) and horseradish peroxidase (5 U / mL, manufactured by Oriental Yeast Co., Ltd.) ), 4-aminoantipyrine (0.04%, manufactured by Wako Pure Chemical Industries, Ltd.), phenol (0.24%, manufactured by Wako Pure Chemical Industries, Ltd.) and a change in absorbance at 500 nm at 30 ° C. Calculated from

As a result, ethanol was not produced in the reaction solution to which recombinant PmFDH was not added, but 0.72% ethanol (conversion rate: 35%) could be produced in the system to which recombinant PmFDH was added. In addition, when NADH (0.3 mM, manufactured by Oriental Yeast Co., Ltd.) was added instead of NAD ⁺ , sodium formate, and recombinant PmFDH, ethanol production was 0.02%.

From these results, it was confirmed that the recombinant PmFDH of the present invention can efficiently regenerate the coenzyme NAD ⁺ in the enzymatic reduction reaction from acetaldehyde to ethanol.

The present invention makes it possible to produce formate dehydrogenase efficiently. Further, the formate dehydrogenase prepared by the present invention is extremely useful as a coenzyme regeneration system enzyme.

All publications, patents and patent applications cited in this specification shall be incorporated into this specification as they are.

Claims

The following protein (a), (b) or (c):
(A) a protein comprising the amino acid sequence of SEQ ID NO: 1,
(B) a protein having at least 70% identity with the amino acid sequence of SEQ ID NO: 1 and having formate dehydrogenase activity;
(C) A protein having a formate dehydrogenase activity, comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence of SEQ ID NO: 1.
A gene encoding the protein according to claim 1.
A gene comprising the following DNA (d), (e) or (f):
(D) DNA consisting of the base sequence of SEQ ID NO: 2,
(E) DNA encoding a protein having 70% or more identity with the base sequence of SEQ ID NO: 2 and having formate dehydrogenase activity,
(F) DNA encoding a protein having a formate dehydrogenase activity, comprising a base sequence in which one or several bases are deleted, substituted, inserted or added in the base sequence of SEQ ID NO: 2.
A recombinant vector comprising the gene according to claim 2 or 3.
A transformant obtained by introducing the gene according to claim 2 or 3 into a host.
The transformant according to claim 5, wherein the host is Escherichia coli.
A method for producing formate dehydrogenase, comprising culturing the transformant according to claim 5 or 6 and collecting a protein having formate dehydrogenase activity from the culture.
A method for regenerating a coenzyme by allowing the protein of claim 1 or the transformant of claim 5 or 6 or a processed product thereof to coexist in an enzymatic reduction reaction system.