# Patent

## Title
Mutant formate dehydrogenase, gene encoding the same, and method for producing NADH

## Abstract

Durability of formate dehydrogenase is improved with the use of formate dehydrogenase exhibiting high specific activity that is unpredictable from conventional findings. A specific amino acid substitution is introduced into Gibberella zeae-derived formate dehydrogenase. Mutant formate dehydrogenase exhibits durability that is extremely superior to that of wild-type formate dehydrogenase. Thus, the productivity of NADH that is produced using the mutant formate dehydrogenase can be improved.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a National Stage of International Application No. PCT/JP2009/063743 filed Aug. 3, 2009 the contents of all of which are incorporated herein by reference in their entirety.
TECHNICAL FIELD
The present invention relates to mutant formate dehydrogenases having substitutional mutations at specific sites in a wild-type formate dehydrogenase, genes encoding the mutant formate dehydrogenases, and a method for producing NADH.
BACKGROUND ART
Formate dehydrogenase (EC.1.2.1.2) reduces NAD+ to reduced nicotinamide adenine dinucleotide (NADH) and oxidizes formic acid to carbon dioxide in the presence of oxidized nicotinamide adenine dinucleotide (NAD+), formic acid, and water. Based on the enzyme reaction, formate dehydrogenase is used for a system for regeneration of NADH from NAD+. Conventionally known examples of formate dehydrogenase include Candida boidinii (ATCC32195)-derived formate dehydrogenase as described in JP Patent Publication (Kokai) No. 2003-180383 A, NAD+-dependent-formate dehydrogenase from bacteria of the genus Bacillus as disclosed in JP Patent Publication (Kokai) No. 2002-233395 A, and Mycobacterium vaccae-derived formate dehydrogenase as disclosed in JP Patent Application No. H10-023896 (1998).
Also, the English translation of BIOCHEMISTRY (Moscow), Vol. 69, No. 11, 2004, pp. 1252-1267 (Biokhimiya, Vol. 69, No. 11, 2004, pp. 1537-1554) discloses formate dehydrogenase from various microorganisms or plants in addition to the above examples. However, as described in this document, the specific activity of formate dehydrogenase is not so significant compared with that of various enzymes. In other words, a method for producing NADH using a formate dehydrogenase reduction reaction to result in NADH can be said to result in poor productivity because of the low specific activity of formate dehydrogenase.
Various research findings regarding formate dehydrogenase have been accumulated to date, and functional alterations by site-directed mutagenesis have been reported (Biomolecular Engineering, 23, (2006) 98-110). However, all conventionally known formate dehydrogenases have low specific activity and low durability. Thus, the use of the formate dehydrogenase must be evaluated as insufficient for NADH production.
DISCLOSURE OF THE INVENTION
Problem to be Solved by the Invention
In view of the above circumstances, the present inventors have applied for a patent concerning formate dehydrogenase exhibiting very high specific activity unpredictable from conventional findings and a method for producing NADH using the formate dehydrogenase (JP Patent Application No. 2008-100448). Hence, an object of the present invention is to provide, as a result of a search for a useful substitutional mutation in the formate dehydrogenase according to this patent application and conventionally known formate dehydrogenases, mutant formate dehydrogenase having better durability than conventionally known mutant formate dehydrogenases, a gene encoding the mutant formate dehydrogenase, and a method for producing NADH using the mutant formate dehydrogenase.
Means for Solving Problem
As a result of intensive studies in order to attain the above object, the present inventors have found that amino acid substitutional mutations at specific sites in the formate dehydrogenase according to the above patent application and in conventionally known formate dehydrogenases significantly improve the durability of formate dehydrogenase, and thus have completed the present invention.
Specifically, the mutant formate dehydrogenase according to the present invention comprises any one amino acid sequence of the following amino acid sequences A) to C) and thus has improved durability:

    A) an amino acid sequence that has substitutions of amino acid residues corresponding to the 99th valine, the 153rd valine, the 155th histidine, and the 298th asparagine with other amino acids;
    B) an amino acid sequence that has substitutions of amino acid residues corresponding to the 73rd glycine, the 99th valine, the 153rd valine, and the 155th histidine with other amino acids; and
    C) an amino acid sequence that has substitutions of amino acid residues corresponding to the 73rd glycine, the 99th valine, the 153rd valine, and the 159th glutamine with other amino acids,
    in the formate dehydrogenase consisting of the amino acid sequence shown in SEQ ID NO: 2.


Also, the mutant formate dehydrogenase according to the present invention preferably comprises an amino acid sequence that has further substitutions of amino acid residues corresponding to 1 to 3 amino acids selected from the group consisting of the 31st isoleucine, the 50th arginine, the 60th glutamic acid, the 64th isoleucine, the 73rd glycine, the 82nd lysine, the 136th isoleucine, the 159th glutamine, the 239th methionine, the 286th aspartic acid, the 287th histidine, the 293rd glutamic acid, and the 343rd asparagine with other amino acids, with respect to the above amino acid sequence A).
Furthermore, the mutant formate dehydrogenase according to the present invention preferably comprises an amino acid sequence that has further substitutions of amino acid residues corresponding to 1 to 3 amino acids selected from the group consisting of the 31st isoleucine, the 50th arginine, the 60th glutamic acid, the 64th isoleucine, the 82nd lysine, the 136th isoleucine, the 159th glutamine, the 239th methionine, the 286th aspartic acid, the 287th histidine, the 293rd glutamic acid, the 298th asparagine, and the 343rd asparagine with other amino acids, with respect to the above amino acid sequence B).
Furthermore, the mutant formate dehydrogenase according to the present invention preferably comprises an amino acid sequence that has further substitutions of amino acid residues corresponding to 1 to 3 amino acids selected from the group consisting of the 31st isoleucine, the 50th arginine, the 60th glutamic acid, the 64th isoleucine, the 82nd lysine, the 136th isoleucine, the 155th histidine, the 239th methionine, the 286th aspartic acid, the 287th histidine, the 293rd glutamic acid, the 298th asparagine, and the 343rd asparagine with other amino acids, with respect to the above amino acid sequence C).
Here, amino acids after substitutional mutation are not particularly limited. For example, an amino acid after substitution of the above amino acid corresponding to the 31st isoleucine can be leucine, an amino acid after substitution of the above amino acid corresponding to the 50th arginine can be glycine, an amino acid after substitution of the above amino acid corresponding to the 60th glutamic acid can be valine, an amino acid after substitution of the above amino acid corresponding to the 64th isoleucine can be valine, an amino acid after substitution of the above amino acid corresponding to the 73rd glycine can be alanine, an amino acid after substitution of the above amino acid corresponding to the 82nd lysine can be arginine, an amino acid after substitution of the above amino acid corresponding to the 99th valine can be isoleucine, an amino acid after substitution of the above amino acid corresponding to the 136th isoleucine can be valine, an amino acid after substitution of the above amino acid corresponding to the 153rd valine can be isoleucine, an amino acid after substitution of the above amino acid corresponding to the 155th histidine can be glutamine or lysine, an amino acid after substitution of the above amino acid corresponding to the 159th glutamine can be leucine or asparagine, an amino acid after substitution of the above amino acid corresponding to the 239th methionine can be leucine, amino acid after substitution of the above amino acid corresponding to the 286th aspartic acid can be phenylalanine, an amino acid after substitution of the above amino acid corresponding to the 287th histidine can be glutamine or proline, an amino acid after substitution of the above amino acid corresponding to the 293rd glutamic acid can be aspartic acid, an amino acid after substitution of the above amino acid corresponding to the 298th asparagine can be tyrosine, and an amino acid after substitution of the above amino acid corresponding to the 343rd asparagine can be arginine.
Also, the method for producing NADH according to the present invention comprises causing the above-mentioned mutant formate dehydrogenase according to the present invention to act on a reaction system containing formic acid and NAD+.
Effects of the Invention
The mutant formate dehydrogenase according to the present invention has a feature such that the durability is significantly improved compared with formate dehydrogenase before mutation thereof due to a novel substitutional mutation(s). With the use of the mutant formate dehydrogenase according to the present invention, NADH known as a very expensive substance can be produced with good productivity. NADH production using the mutant formate dehydrogenase according to the present invention enables industrial production of NADH.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1-1 to 1-2 are characteristic diagrams showing the results of multiple alignment analysis for Gibberella zeae-derived FDH (SEQ ID NO: 2), Pseudomonas sp. 101-derived FDH (SEQ ID NO: 3),Mycobacterium vaccae N10.-derived FDH (SEQ ID NO: 4), Candida boidini-derived FDH (SEQ ID NO: 6), Candida methylica-derived FDH (SEQ ID NO: 8), Saccharomyces cerevisiae-derived FDH (SEQ ID NO: 10), and Paracoccus sp.12-A-derived FDH (SEQ ID NO: 12), as well as a novel mutation(s) according to the present invention and a known mutation(s).
FIG. 2 is a schematic diagram showing the steps for constructing GzFDH/pET23b(+).
FIG. 3 is a characteristic diagram showing the remaining activity of wild-type FDH and quintuple mutant formate dehydrogenase denoted with the plasmid name “5B-2” at 37° C. after 500 hours.
FIG. 4 is a characteristic diagram showing the results of evaluating the remaining activity of quadruple mutant formate dehydrogenase with an acceleration test.
FIG. 5 is a characteristic diagram showing the results of evaluating the remaining activity of mutant formate dehydrogenases prepared in Examples by an acceleration test.
FIG. 6 is a characteristic diagram showing the results of evaluating the remaining activity of mutant formate dehydrogenases prepared in Examples by an acceleration test.
FIG. 7 is a characteristic diagram showing the results of evaluating the remaining activity of mutant formate dehydrogenases prepared in Examples by an acceleration test.
FIG. 8 is a characteristic diagram showing the results of evaluating the remaining activity of mutant formate dehydrogenases prepared in Examples by an acceleration test.
FIG. 9 is a characteristic diagram showing the results of evaluating the remaining activity of mutant formate dehydrogenases prepared in Examples by an acceleration test.
FIG. 10 is a characteristic diagram showing the results of evaluating the remaining activity of mutant formate dehydrogenases prepared in Examples by an acceleration test.
FIG. 11 is a characteristic diagram showing the results of evaluating the remaining activity of mutant formate dehydrogenases prepared in Examples by an acceleration test.
FIG. 12 is a characteristic diagram showing the results of evaluating the remaining activity of mutant formate dehydrogenases prepared in Examples by an acceleration test.
FIG. 13 is a characteristic diagram showing the results of evaluating the remaining activity of mutant formate dehydrogenases prepared in Examples by an acceleration test.
FIG. 14 is a characteristic diagram showing the results of evaluating the remaining activity of mutant formate dehydrogenases prepared in Examples by an acceleration test.
FIG. 15 is a characteristic diagram showing the results of evaluating the specific activity of mutant formate dehydrogenases prepared in Examples.
FIG. 16 is a characteristic diagram showing the results of evaluating the specific activity of mutant formate dehydrogenases prepared in Examples.



BEST MODE FOR CARRYING OUT THE INVENTION
The present invention will be described in detail as follows, referring to the drawings.
Formate Dehydrogenase
The mutant formate dehydrogenase according to the present invention has an amino acid sequence that has substitutions of specific amino acid residues in a formate dehydrogenase. Here, formate dehydrogenase is not particularly limited, as long as it has amino acid residues to be substituted as specifically described later and formate dehydrogenase activity. Specifically, formate dehydrogenase is an enzyme, which is classified into EC1.2.1.2 under the enzyme classification, oxidizes formic acid ions to carbon dioxide, and has activity of catalyzing a reaction for reducing NAD+ ions to NADH.
The formate dehydrogenase may be a plant-derived enzyme, an animal-derived enzyme, or a microorganism-derived enzyme. Examples of the microorganism-derived formate dehydrogenase include various formate dehydrogenases as disclosed in the overview concerning formate dehydrogenase (Biomolecular Engineering 23 (2006) 98-110). More specifically, the present invention can be applied to Staphylococcus aureus-derived formate dehydrogenase (Baba, T. et al., Lancet 359, 1819-1827, 2002), Mycobacterium avium subsp. paratuberculosis str.k10 (Li et al., Proc. Natl. Acad. Sci. U.S.A. 102, 12344-12349, 2005), Bordetella-derived formate dehydrogenase (Parkhill et al., Nat. Genet. 35, 32-40., 2003), Legionella-derived formate dehydrogenase (Chien et al., Science 305, 1966 v 1968, 2004, and Cazalet et al., Nat. Genet. 36, 1165-1173, 2004), Francisella tularensis subsp. tularensis SCHU S4-derived formate dehydrogenase (Larsson et al., Nat. Genet. 37, 153-159, 2005), Histoplasma capsulatum-derived formate dehydrogenase (Hwang et al., Mol. Biol. Cell 14, 2314-2326, 2003), Cryptococcus neoformans var. neoformans JEC21 (Loftus et al., Science 307, 1321-1324, 2005), and the like revealed by recent genomic analysis.
The present invention can also be applied to Pseudomonas sp. 101-derived formate dehydrogenase, Mycobacterium vaccae N10.-derived formate dehydrogenase, Candida boidini-derived formate dehydrogenase, Candida methylica-derived formate dehydrogenase, Saccharomyces cerevisiae-derived formate dehydrogenase, and Paracoccus sp. 12-A-derived formate dehydrogenase disclosed in the above overview.
In particular, the present invention is preferably applied to Gibberella zeae-derived formate dehydrogenase that has been previously applied for a patent (JP Patent Application No. 2008-100448). Gibberella zeae-derived formate dehydrogenase exhibits specific activity significantly better than that of the above-mentioned conventionally known formate dehydrogenases. The nucleotide sequence of a gene encoding Gibberella zeae-derived formate dehydrogenase is shown in SEQ ID NO: 1. The amino acid sequence of Gibberella zeae-derived formate dehydrogenase is shown in SEQ ID NO: 2. To obtain formate dehydrogenase, conventionally known various microbial strains that have been preserved as Fusarium (scientific name: Gibberella zeae) can be used. For example, Fusarium strains preserved at the American Type Culture Collection (ATCC) under ATCC No. 10910, No. 20271, No. 20272, No. 20274, No. 24689, No. 28106, or No. 48063 can be used. Also, Fusarium strains are preserved at the ATCC under registration name of Fusarium graminearum. If Gibberella zeae is registered under another name, this can also be used herein. In addition, Gibberella zeae to be used herein indicates Fusarium graminearum at the perfect stage (teleomorph). Also, Fusarium preserved at the NITE Biological Resource Center (NBRC) under NBRC No. 4474, No. 5269, No. 6608, No. 7160, No. 7520, No. 7772, No. 8850, or No. 9462 can also be used herein. Moreover, formate dehydrogenase may also be obtained without using microbial strains preserved at institutions such as ATCC and NBRC, but instead using Fusarium independently isolated from nature.
The amino acid sequence of Pseudomonas sp. 101-derived formate dehydrogenase is shown in SEQ ID NO: 3. The amino acid sequence of Mycobacterium vaccae N10.-derived formate dehydrogenase is shown in SEQ ID NO: 4. The nucleotide sequence of a gene encoding Candida boidini-derived formate dehydrogenase is shown in SEQ ID NO: 5. The amino acid sequence of Candida boidini-derived formate dehydrogenase is shown in SEQ ID NO: 6. The nucleotide sequence of a gene encoding Candida methylica-derived formate dehydrogenase is shown in SEQ ID NO: 7. The amino acid sequence of Candida methylica-derived formate dehydrogenase is shown in SEQ ID NO: 8. The nucleotide sequence of a gene encoding Saccharomyces cerevisiae-derived formate dehydrogenase is shown in SEQ ID NO: 9. The amino acid sequence of Saccharomyces cerevisiae-derived formate dehydrogenase is shown in SEQ ID NO: 10. The nucleotide sequence of a gene encoding Paracoccus sp. 12-A-derived formate dehydrogenase is shown in SEQ ID NO: 11. The amino acid sequence of Paracoccus sp. 12-A-derived formate dehydrogenase is shown in SEQ ID NO: 12.
Formate dehydrogenase that can be used in the present invention is not limited to the one consisting of the amino acid sequence shown in SEQ ID NO: 2, 3, 4, 6, 8, 10, or 12. An example thereof to be used herein may be formate dehydrogenase that comprises an amino acid sequence that has a deletion, a substitution, an addition, or an insertion of 1 or a plurality of amino acids excluding amino acid residues to be substituted as described in detail later, with respect to the amino acid sequence shown in SEQ ID NO: 2, 3, 4, 6, 8, 10, or 12, and has catalytic activity in a reaction wherein formic acid and NAD+ are substrates and carbon dioxide and NADH are products. Here, the term “a plurality of amino acids” refers to, for example, 1 to 30, preferably 1 to 20, more preferably, 1 to 10, further more preferably 1 to 5, and particularly preferably 1 to 3 amino acids. In addition, deletion, substitution, or addition of amino acids can be performed by altering the nucleotide sequence encoding the above gene by a technique known in the art. Mutation can be introduced into a nucleotide sequence by known techniques such as Kunkel method or Gapped duplex method or methods according thereto. For example, mutation is introduced using a mutagenesis kit using site-directed mutagenesis (e.g., Mutant-K and Mutant-G (both are commercial names, TAKARA)) or a LA PCR in vitro Mutagenesis series kit (trade name, TAKARA).
Also, formate dehydrogenase that can be used in the present invention comprises an amino acid sequence that has 85% or more, preferably 90% or more, more preferably 95% or more, and most preferably 98% or more homology, for example, with the amino acid sequence shown in SEQ ID NO: 2, 3, 4, 6, 8, 10, or 12, and has catalytic activity in a reaction wherein formic acid and NAD+ are substrates and carbon dioxide and NADH are products. Here, the value of homology refers to a value that is found using a database with default setting, in which a computer program with blast algorithm mounted therein and gene sequence information are stored.
Furthermore, a protein that can be used as formate dehydrogenase in the present invention is a protein that is encoded by polynucleotide hybridizing under stringent conditions to polynucleotide complementary to a portion of or the entire nucleotide sequence shown in SEQ ID NO: 1, 5, 7, 9, or 11 and has catalytic activity in a reaction in which formic acid and NAD+ are substrates and carbon dioxide and NADH are products. Here, the expression, “hybridizing under stringent conditions” means that binding is maintained under washing conditions of 60° C. and 2×SSC. 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).
Substitutional Mutation
The mutant formate dehydrogenase according to the present invention is prepared by substituting a plurality of predetermined amino acid residues in the above-mentioned formate dehydrogenase, having significantly improved durability compared with that of formate dehydrogenase before amino acid substitution. Here, amino acid residues to be substituted can be specified using numerical values counted from the N-terminus on the basis of Gibberella zeae-derived formate dehydrogenase consisting of the amino acid sequence shown in SEQ ID NO: 2. However, amino acid residues to be substituted, which are specified with specific numerical values found on the basis of the amino acid sequence shown in SEQ ID NO: 2 are represented by different numerical values depending on the types of formate dehydrogenase. Therefore, in the case of expression, “Xth (or Xrd) amino acid residue (at position X) in the amino acid sequence shown in SEQ ID NO: 2,” an amino acid residue (corresponding to the Xth or Xrd amino acid residue) in formate dehydrogenase that has an amino acid sequence differing from the amino acid sequence shown in SEQ ID NO: 2 is represented by a numerical value differing from “X.”
An amino acid residue (in an amino acid sequence differing from the amino acid sequence shown in SEQ ID NO: 2) corresponding to such a predetermined amino acid residue in the amino acid sequence shown in SEQ ID NO: 2 can be specified by multiple alignment analysis for a plurality of amino acid sequences including the amino acid sequence shown in SEQ ID NO: 2. Multiple alignment analysis can be easily performed by persons killed in the art using a CLUSTAL W (1.83) multiple sequence alignment program (that can be used with DDBJ of the National Institute of Genetics clustalw.ddbj.nig.ac.jp/top-j.html)), for example. But the example thereof is not limited thereto. In addition, with the use of a pair-wise alignment analysis method, another different amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 2, amino acid residues corresponding to predetermined amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 can also be specified in the different amino acid sequence.
FIG. 1 shows the results of multiple alignment analysis for Gibberella zeae-derived formate dehydrogenase (SEQ ID NO: 2), Pseudomonas sp. 101-derived formate dehydrogenase (SEQ ID NO: 3), Mycobacterium vaccae N10.-derived formate dehydrogenase (SEQ ID NO: 4), Candida boidini-derived formate dehydrogenase (SEQ ID NO: 6), Candida methylica-derived formate dehydrogenase (SEQ ID NO: 8), Saccharomyces cerevisiae-derived formate dehydrogenase (SEQ ID NO: 10), and Paracoccus sp. 12-A-derived formate dehydrogenase (SEQ ID NO: 12). In addition, formate dehydrogenases other than these specific formate dehydrogenases can also be similarly subjected to multiple alignment analysis, so that they can be compared with Gibberella zeae-derived formate dehydrogenase (SEQ ID NO: 2).
In the following explanation, amino acids to be substituted are denoted on the basis of the amino acid sequence shown in SEQ ID NO: 2; that is, Gibberella zeae-derived formate dehydrogenase. However, as described above, it should be noted that numerical values representing the positions of amino acids differ for different formate dehydrogenases. Examples of the mutant formate dehydrogenase according to the present invention include quadruple mutant formate dehydrogenase, which has substitutional mutations of amino acid residues at 4 positions described later and mutant formate dehydrogenase, which has other substitutional mutations at 1 to 3 positions of the quadruple mutant formate dehydrogenase.
Quadruple Mutant Formate Dehydrogenase
The quadruple mutant formate dehydrogenase according to the present invention has the amino acid sequence shown in any one of the following A) to C):

    A) an amino acid sequence having substitutions of the 99th valine, the 153rd valine, the 155th histidine, and the 298th asparagine with other amino acids;
    B) an amino acid sequence having substitutions of the 73rd glycine, the 99th valine, the 153rd valine, and the 155th histidine with other amino acids; and
    C) an amino acid sequence having substitutions of the 73rd glycine, the 99th valine, the 153rd valine, and the 159th glutamine with other amino acids.


In the above amino acid sequences A) to C), examples of “other amino acids” are not particularly limited and may be any amino acids, as long as the durability of formate dehydrogenase after mutation is significantly improved compared with that of formate dehydrogenase before mutation. Durability can be evaluated with remaining activity found after an object is left to stand at 37° C. for 500 hours. In addition, durability may also be evaluated with an acceleration test, by which an object is left to stand at 37° C. for 500 hours (e.g., under conditions wherein an object is left to stand at 52° C. to 58° C. for 30 minutes to 50 minutes). Also, the expression “durability is improved” means that remaining activity after mutagenesis is statistically significantly higher than that of wild-type formate dehydrogenase before mutagenesis. Here, the enzyme activity of mutant formate dehydrogenase and the same of formate dehydrogenase before substitutional mutation can be determined by appropriately using conventionally known techniques. For example, through direct or indirect measurement of the amount of NADH generated according to the following formula (reaction formula) or the amounts of components consumed according to the following reaction formula, the enzyme activity of formate dehydrogenase contributing to the following formula can be measured.

HCOO−+NAD+→CO2+NADH

In particular, in the above amino acid sequences A) to C), a substitutional mutation of the 73rd glycine to alanine, a substitutional mutation of the 99th valine to isoleucine, a substitutional mutation of the 153rd valine to isoleucine, a substitutional mutation of the 155th histidine to glutamine or lysine, a substitutional mutation of the 159th glutamine to leucine, and a substitutional mutation of the 298th asparagine to tyrosine are preferred.
In addition, as described above, amino acids specifically preferable as amino acids after substitution are exemplified, but the examples of amino acids after substitution are not limited to the above examples. As also described in reference (1) (“McKee Biochemistry (Biochemistry: the molecular basis of life)” 3rd ed., Chapter 5 Amino Acid.Peptide.Protein 5.1 Amino acid, Edition: Atsushi Ichikawa, supervised translation: Shinichi Fukuoka, publisher: Ryosuke Sone, publishing office: Kagaku-Dojin Publishing Company, Inc., ISBN4-7598-0944-9), it is well known that amino acids are classified based on side chains having similar properties (e.g., chemical properties and physical size). It is also well known that molecular evolutional substitution takes place with high frequency between amino acid residues that are classified in a given group, while the protein activity is maintained. Based on this concept, score matrix (BLOSUM) for substitutional mutation of amino acid residues has been proposed as in FIG. 2 of reference (2): Henikoff S., Henikoff J. G., Amino-acid substitution matrices from protein blocks, Proc. Natl. Acad. Sci. U.S.A., 89, 10915-10919 (1992) and is broadly used. Reference (2) is based on the finding such that substitution between amino acids having similar chemical properties of side chains cause less structural or functional changes on the entire protein. According to references (1) and (2) above, amino acid side chain groups to be taken into consideration for multiple alignment may be determined based on indices such as chemical properties, physical size, and the like. These groups are shown by score matrix (BLOSUM) disclosed in reference (2) as groups of amino acids having the score of 0 or more and amino acids preferably having the score of 1 or more.
Based on the above findings, amino acids having properties analogous to each other can be classified into the following 8 groups. Therefore, amino acids after substitution are preferably amino acids that are classified in groups including the above exemplified amino acids. For example, the 99th valine in Gibberella zeae-derived formate dehydrogenase is preferably substituted with isoleucin, or may be substituted with leucine, methionine, or valine that is classified in the following 1) aliphatic hydrophobic amino acid group in which isoleucine is classified. Similarly, the 155th histidine in Gibberella zeae-derived formate dehydrogenase is preferably substituted with glutamine or lysine, or may be substituted with glutamic acid or arginine that is classified in the following 6) group of methylene group=polar group in which glutamine and lysine are classified. Also, the 159th glutamine in Gibberella zeae-derived formate dehydrogenase is preferably substituted with leucine, or may be substituted with isoleucine, methionine, or valine that is classified in the following 1) aliphatic hydrophobic amino acid group in which leucine is classified. Furthermore, the 298th asparagine in Gibberella zeae-derived formate dehydrogenase is preferably substituted with tyrosine, or may be substituted with phenylalanine or tryptophan that is classified in the following 7) aromatic group in which tyrosine is classified or with histidine that is classified in the following 8) ring & polar group.
1) Aliphatic Hydrophobic Amino Acid Group (ILMV Group)
This group is a group of neutral non-polar amino acids (described in reference (1) above) having aliphatic hydrophobic side chains, which is composed of V (Val, valine), L (Leu, leucine), I (Ile, isoleucine), and M (Met, methionine). Of those classified as neutral non-polar amino acids as in reference (1), FGACWP is not included in the “aliphatic hydrophobic amino acid group” because of the following reasons. G (Gly, glycine) or A (Ala, alanine) is of the same size as or smaller than a methyl group and thus has a weak non-polar effect. C (Cys, cysteine) may play an important role in S—S linkage, and, has a feature such that it forms a hydrogen bond with an oxygen atom or a nitrogen atom. F (Phe, phenylalanine) and W (Trp, tryptophan) have side chains with particularly high molecular weights, and thus have a strong aromatic effect. P (Pro, proline) has a strong imino acid effect, so as to fix the angle of the main chain of the polypeptide.
2) Group Having Hydroxymethylene Group (ST Group)
This group is a group of neutral polar amino acids having hydroxymethylene groups in the side chains, which is composed of S (Ser, serine) and T (Thr, threonine). Hydroxyl groups existing in S and T side chains are sugar-binding sites, so that the sites are often important for polypeptides (proteins) to have specific activity.
3) Acidic Amino Acid Group (DE Group)
This group is a group of amino acids having acidic carboxyl groups in the side chains, which is composed of D (Asp, aspartic acid) and E (Glu, glutamic acid).
4) Basic Amino Acid Group (KR Group)
This group is a group of basic amino acids, which is composed of K (Lys, lysine) and R (Arg, arginine). These K and R are positively charged within a wide pH range and thus have basic properties. Meanwhile, H (His, histidine) that is classified as basic amino acid is almost never ionized at pH 7 and thus is not classified in this group.
5) Methylene Group=Polar Group (DHN Group)
This group is characterized in that methylene groups bind as side chains to all carbon elements at position a beyond which a polar group is present. The group is also characterized in that the physical sizes of methylene groups as non-polar groups closely resemble to each other, which is composed of N (Asn, asparagine, and the polar group is an amide group), D (Asp, aspartic acid, and the polar group is a carboxyl group), and H (His, histidine, and the polar group is an imidazole group).
6) Dimethylene Group=Polar Group (EKQR Group)
This group is characterized in that linear hydrocarbons each having carbon chain length longer than a dimethylene group bind as side chains to all carbon elements at position a beyond which a polar group is present, and the physical sizes of dimethylene groups as non-polar groups closely resemble from each other. The group is composed of E (Glu, glutamic acid, and the polar group is a carboxyl group), K (Lys, lysine, and the polar group is an amino group), Q (Gln, glutamine, and the polar group is an amide group), and R (Arg, arginine, and the polar groups are an imino group and an amino group).
7) Aromatic Group (FYW Group)
This group is a group of aromatic amino acids having benzene nuclei in side chains and is characterized by chemical properties peculiar to the aromatic group. The group is composed of F (Phe, phenylalanine), Y (Tyr, tyrosine), and W (Trp, tryptophan).
8) Ring & Polar Group (HY Group)
This group is a group of amino acids having ring structures in side chains, in addition to polarity, which is composed of H (H, histidine, and both ring structure and polar group are imidazole groups), Y (Tyr, tyrosine, and the ring structure is a benzene nucleus and the polar group is a hydroxyl group).
Quintuple, Hexatic, and Septuple Mutant Formate Dehydrogenases
Quintuple, hexatic, and septuple mutant formate dehydrogenases can be obtained by further introducing 1 to 3 substitutional mutations to the above-mentioned quadruple mutant formate dehydrogenase. Examples of further 1 to 3 substitutional mutations include amino acids at positions other than mutation positions in the above amino acid sequences A) to C), such as the 31st isoleucine, the 50th arginine, the 60th glutamic acid, the 64th isoleucine, the 73rd glycine, the 82nd lysine, the 136th isoleucine, the 155th histidine, the 159th glutamine, the 239th methionine, the 286th aspartic acid, the 287th histidine, the 293rd glutamic acid, the 298th asparagine, and the 343rd asparagine in the amino acid sequence shown in SEQ ID NO: 2.
Amino acid residues listed herein are not particularly limited, as long as the durability of formate dehydrogenase after mutation is significantly improved compared with the same of wild-type formate dehydrogenase, and may be substituted with any amino acid. Particularly, a substitutional mutation of the 31st isoleucine to leucine, a substitutional mutation of the 50th arginine to glycine, a substitutional mutation of the 60th glutamic acid to valine, a substitutional mutation of the 64th isoleucine to valine, a substitutional mutation of the 73rd glycine to alanine, a substitutional mutation of the 82nd lysine to arginine, a substitutional mutation of the 136th isoleucine to valine, a substitutional mutation of the 155th histidine to glutamine or lysine, a substitutional mutation of the 159th glutamine to leucine or asparagine, a substitutional mutation of the 239th methionine to leucine, a substitutional mutation of the 286th aspartic acid to phenylalanine, a substitutional mutation of the 287th histidine to glutamine or proline, a substitutional mutation of the 293rd glutamic acid to aspartic acid, a substitutional mutation of the 298th asparagine to tyrosine, and a substitutional mutation of the 343rd asparagine to arginine are preferred. In addition, preferable amino acid types of the amino acid residues listed herein after substitutional mutation are disclosed. In a manner similar to the above, amino acid types after substitutional mutation can be appropriately selected based on the above 8 groups in which amino acids having analogous properties are classified.
Also, in particular, an example of the quintuple mutant formate dehydrogenase is a mutant formate dehydrogenase having substitutional mutation (abbreviated as G73A using single character codes for amino acids, and the same applies to the following) of the 73rd glycine to alanine, V99I, V153I, H155Q, and N298Y. The quintuple mutant formate dehydrogenase having these G73A, V99I, V153I, H155Q, and N298Y can exhibit good durability as defined above while maintaining specific activity equivalent to that of a wild-type formate dehydrogenase.
Furthermore, an example of the quintuple mutant formate dehydrogenase is a mutant formate dehydrogenase having G73A, V99I, V153I, H155Q, and H287Q. The quintuple mutant formate dehydrogenase having these G73A, V99I, V153I, H155Q, and H287Q can also exhibit good durability.
Furthermore, an example of the quintuple mutant formate dehydrogenase is a mutant formate dehydrogenase having G73A, V99I, V153I, H155K, and N298Y. The quintuple mutant formate dehydrogenase having these G73A, V99I, V153I, H155K, and N298Y can also exhibit good durability. In addition, as described in Examples below, the quintuple mutant formate dehydrogenase having G73A, V99I, V153I, H155K, and N298Y was compared with the quintuple mutant formate dehydrogenase having G73A, V99I, V153I, H155Q, and N298Y in terms of durability. As a result, the quintuple mutant formate dehydrogenase having G73A, V99I, V153I, H155K, and N298Y exhibited better durability. Therefore, it can be understood that lysine is more preferable than glutamine as amino acid after substitutional mutation of the 155th histidine.
Furthermore, examples of the quintuple mutant formate dehydrogenase include a mutant formate dehydrogenase having G73A, V99I, V153I, Q159L, and N298Y, and a mutant formate dehydrogenase having V99I, V153I, H155Q, H287Q, and N298Y. These quintuple mutant formate dehydrogenases can also exhibit durability better than that of the wild-type formate dehydrogenase, in a manner equivalent to that of the quintuple mutant formate dehydrogenase having G73A, V99I, V153I, H155Q, and N298Y.
Furthermore, examples of the quintuple mutant formate dehydrogenase include a mutant formate dehydrogenase having E60V, G73A, V99I, V153I, and H155Q, and a mutant formate dehydrogenase having E60V, V99I, V153I, H155Q, and N298Y. These quintuple mutant formate dehydrogenases can also exhibit higher degrees of durability than wild-type formate dehydrogenase.
Meanwhile, a preferable example of the hexatic mutant formate dehydrogenase is prepared by further introducing one substitutional mutation selected from M239L, K82R, H287Q, E60V, I31L, I136V, and E293D into the quintuple mutant formate dehydrogenase having G73A, V99I, V153I, H155Q, and N298Y. The hexatic mutant formate dehydrogenase can exhibit a higher degree of durability than the quintuple mutant formate dehydrogenase having G73A, V99I, V153I, H155Q, and N298Y. Also, a hexatic mutant formate dehydrogenase having G73A, V99I, V153I, H155Q, M239L, and N298Y, a hexatic mutant formate dehydrogenase having E60V, G73A, V99I, V153I, H155Q, and N298Y, and a hexatic mutant formate dehydrogenase having G73A, V99I, V153I, H155K, M239L, and N298Y can exhibit not only a high degree of durability as described above, but also better specific activity than the wild-type formate dehydrogenase.
Also, an example of the hexatic mutant formate dehydrogenase is the one prepared by introducing M239L into the above quintuple mutant formate dehydrogenase having G73A, V99I, V153I, Q159L, and N298Y. Through introduction of M239L, specific activity can be improved while maintaining durability equivalent to that of the above quintuple mutant formate dehydrogenase having G73A, V99I, V153I, Q159L, and N298Y.
Furthermore, an example of the hexatic mutant formate dehydrogenase is the one prepared by introducing H287P into the quintuple mutant formate dehydrogenase having G73A, V99I, V153I, H155Q, and N298Y. However, when compared with the hexatic mutant formate dehydrogenase prepared by introducing H287Q into the quintuple mutant formate dehydrogenase having G73A, V99I, V153I, H155Q, and N298Y, as described above, decreased specific activity was observed. Therefore, it can be understood that as an amino acid after substitutional mutation of the 287th histidine, glutamine is more preferable than proline.
Furthermore, an example of the hexatic mutant formate dehydrogenase is the one prepared by introducing R50G or N343R into the quintuple mutant formate dehydrogenase having G73A, V99I, V153I, H155Q, and N298Y. These hexatic mutant formate dehydrogenases can also exhibit durability equivalent to that of the quintuple mutant formate dehydrogenase having G73A, V99I, V153I, H155Q, and N298Y.
Furthermore, an example of the hexatic mutant formate dehydrogenase is the one prepared by introducing K82R or Q159L into the quintuple mutant formate dehydrogenase having G73A, V99I, V153I, H155K, and N298Y. The hexatic mutant formate dehydrogenase can exhibit durability better than that of the quintuple mutant formate dehydrogenase having G73A, V99I, V153I, H155K, and N298Y.
Furthermore, examples of the hexatic mutant formate dehydrogenase include a mutant formate dehydrogenase having I64V, G73A, V99I, V153I, H155Q, and N298Y and a mutant formate dehydrogenase having G73A, V99I, V153I, H155Q, Q159N, and N298Y. These quintuple mutant formate dehydrogenases can also exhibit durability better than that of wild-type formate dehydrogenase.
Meanwhile, an example of the septuple mutant formate dehydrogenase is the one prepared by introducing K82R into the above hexatic mutant formate dehydrogenase having G73A, V99I, V153I, H155Q, M239L, and N298Y. The septuple mutant formate dehydrogenase can exhibit both durability and specific activity better than those of the above hexatic mutant formate dehydrogenase having G73A, V99I, V153I, H155Q, M239L, and N298Y.
Also, examples of the septuple mutant formate dehydrogenase include a septuple mutant formate dehydrogenase having G73A, K82R, V99I, V153I, H155Q, H287Q, and N298Y, a septuple mutant formate dehydrogenase having G73A, V99I, V153I, H155Q, Q159L, M239L, and N298Y, and a septuple mutant formate dehydrogenase having G73A, V99I, V153I, H155Q, Q159L, H287Q, and N298Y. These septuple mutant formate dehydrogenases can also exhibit durability better than that of wild-type formate dehydrogenase.
Furthermore, examples of the septuple mutant formate dehydrogenase include a septuple mutant formate dehydrogenase having G73A, V99I, V153I, H155Q, D286F, H287P, and N298Y and a septuple mutant formate dehydrogenase having G73A, V99I, V153I, H155Q, D286F, H287P, and N298Y. These septuple mutant formate dehydrogenases can also exhibit durability better than that of wild-type formate dehydrogenase.
Production of Fusarium-derived Formate Dehydrogenase
Among the above examples of the mutant formate dehydrogenase according to the present invention, a mutant formate dehydrogenase derived from Fusarium (Gibberella zeae)-derived formate dehydrogenase exhibits very high specific activity even before substitutional mutation. Therefore, a mutant formate dehydrogenase derived from Fusarium-derived formate dehydrogenase is preferable since the durability and/or specific activity thereof are significantly higher than those of mutant formate dehydrogenases derived from other organisms-derived formate dehydrogenases.
Here, the Fusarium-derived formate dehydrogenase cannot be obtained by conventionally known methods for protein production. Therefore, obtainment of the Fusarium-derived mutant formate dehydrogenase by conventionally known methods for protein production is also difficult. The Fusarium-derived mutant formate dehydrogenase can be produced according to techniques described below. Specifically, a method for producing the Fusarium-derived formate dehydrogenase comprises preparing a host by introducing a vector in which a Fusarium-derived formate dehydrogenase gene is arranged under control of an inducible promoter, culturing the host, inducing the expression of the formate dehydrogenase gene after the logarithmic growth phase, culturing the host at a temperature that is lower than the optimum temperature for the growth of the host and allows the survival of the host, and thus causing the expression of the formate dehydrogenase within the host.
As an inducible promoter to be used in the method for producing the Fusarium-derived formate dehydrogenase, any conventionally known promoter can be used without particular limitation. For example, when Escherichia 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 a promoter include Trp promoter, Lac promoter, Trc promoter, and Tac promoter. Moreover, another promoter exhibiting transcriptional activity in the presence of an inducing substance other than IPTG and another promoter exhibiting transcriptional activity according to culture conditions of medium components, temperature, and the like can also be used as inducible promoters.
Also, any vector can be used without particular limitation in the method for producing the Fusarium-derived formate dehydrogenase, as long as it is replicable within the above host. For example, when Escherichia coli is used as the above host, a vector may be either a plasmid vector or a phage vector. Specific examples of a vector include pCDF series, pRSF series, and pET series.
Furthermore, a host to be used herein is not particularly limited, as long as it enables transcription from a promoter incorporated into an expression vector. For example, when an expression vector is a pET (T7 promoter)-based vector, Escherichia coli BL21 (DE3) can be used. As techniques for introducing the above vector into a host, various techniques generally known as transformation methods can be applied. As specific techniques, for example, a calcium phosphate method, electroporation, lipofection, and the like can be applied.
Particularly in the method for producing the Fusarium-derived formate dehydrogenase, a host into which a vector has been introduced is cultured, and then the expression of the formate dehydrogenase gene is induced after the logarithmic growth phase. Culture conditions for a host before induction of the expression of the formate dehydrogenase gene are not particularly limited. For example, the culture conditions may be appropriately determined in view of the optimum temperature and the optimum pH for the growth of the relevant host. However, the growth of the host is observed while continuing culture. At a time point after the logarithmic growth phase, the culture conditions are changed so as to satisfy the following requirements: Requirement 1 is to induce the expression of the formate dehydrogenase gene and requirement 2 is to culture the host at a temperature that is lower than the optimum temperature for the growth of the host, but allows the survival of the host.
Here, the expression “after the logarithmic growth phase” refers to the time point at which the tangential slope begins to decrease from a portion that is an approximately straight line with a predetermined slope on a growth curve that is created by plotting the times for culture on the horizontal axis and a logarithmic scale representing the number of cells on the vertical axis. In addition, a culture curve can be created by measuring OD600 nm in a medium. Also, when the expression of the formate dehydrogenase gene is induced, the expression is preferably induced when the growth has passed the logarithmic growth phase and then entered the stationary phase. Here, “stationary phase” refers to a period during which the tangential slope of the above growth curve becomes almost 0.
Also, the optimum temperature for the growth of a host is known to fall within a temperature range that differs depending on host. For example, when an Escherichia coli B strain is used as a host, the optimum growth temperature is 37° C. Specifically, when an Escherichia coli B strain is used as a host, a temperature range that enables the growth of E. coli is between 15° C. and 37° C. Therefore, when the Escherichia coli B strain is used as a host, a range of temperatures that are lower than the optimum temperature for the growth of the host and allow the survival of the host is a temperature range between 15° C. and 37° C. In particular, when the Escherichia coli B strain is used as a host, when the growth has passed the logarithmic growth phase, culture is preferably continued at a culture temperature of about 20° C.
The above temperature range is employed when the growth of the host has passed the logarithmic growth phase, so that the formate dehydrogenase gene is expressed and formate dehydrogenase exhibiting very high specific activity is generated within the host. After culture, the target formate dehydrogenase is produced within the host. Microorganisms or cells are then disrupted and then a crude enzyme suspension is prepared. The crude enzyme suspension contains the formate dehydrogenase exhibiting very high specific activity. Therefore, the thus obtained crude enzyme suspension can be directly used. In addition, the formate dehydrogenase can also be isolated and purified from the thus obtained crude enzyme suspension. At this time, general biochemical methods to be employed for protein isolation and purification can be used independently or appropriately in combination, such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, and affinity chromatography. The thus isolated and purified formate dehydrogenase can be used in a form suspended in a buffer or the like with predetermined pH.
Utilization Form of Mutant Formate Dehydrogenase
The above explained mutant formate dehydrogenase exhibits durability significantly higher than that of formate dehydrogenase before mutagenesis, so that it can be used in any reaction system as a good alternative for conventionally known formate dehydrogenase used therein. An example of a utilization form of the mutant formate dehydrogenase is an NADH regeneration system. NADH is used in various enzyme reactions and then converted to NAD−. NADH is used as a coenzyme when an optical isomer is biologically synthesized in the fields of chemical industry and pharmaceutical industry, for example. The term “NADH regeneration system” refers to a system by which NAD+ remaining in a reaction system is reduced to result in NADH, NADH is recovered, and then NADH is used again in the above enzyme reaction. The above mutant formate dehydrogenase is caused to act on a reaction system containing formic acid and NAD+, so that the NAD+ can be reduced and NADH can be synthesized.
As described above, the mutant formate dehydrogenase is used for the NADH regeneration system, so that NADH can be efficiently produced from NAD+ contained in the reaction system. In particular, the mutant formate dehydrogenase exhibits durability significantly better than that of conventionally known formate dehydrogenase. Moreover, the mutant formate dehydrogenase can maintain high activity for a period longer than formate dehydrogenase before mutagenesis. Accordingly, through the use of the mutant formate dehydrogenase, NADH productivity can be significantly improved compared with a case in which conventionally known formate dehydrogenase is used.
EXAMPLES
The present invention is hereafter described in greater detail with reference to the following examples, although the technical scope of the present invention is not limited thereto.
Example 1
In this Example, cloning of a Fusarium-derived formate dehydrogenase gene was performed, random mutations were introduced into the gene, and then the durability and specific activity of the thus obtained formate dehydrogenase derived from the mutated genes were evaluated.
(1) Reagents
Reagents used herein were produced by NACALAI TESQUE, INC., unless otherwise specified.


    
    
        Potassium phosphate buffer (KPB) pH 7.5
    
    
    <Solution A> 0.5 M KH2PO4 13.6 g/200 ml
    <Solution B> 0.5 M K2HPO4 26.13 g/300 ml
    0.5 M KPB (pH 7.5) was prepared by mixing Solution A with Solution B in the following proportion.
    [Solution A 16 ml]+[Solution B 84 ml]→100 ml
    
        EcoPro T7 system (Novagen)
    
    
    Lysate, Methionine
    
        1.62 M sodium formate (formic acid Na)
    
    
    0.5 M KPB (5.5 g/50 ml, pH 7.5) was prepared and then sterilized with a 0.22-μm filter (Millipore).
    
        16.2 mM NAD
    
    
    0.5 M KPB (581 mg/50 ml, pH 7.5) was prepared and then sterilized with a 0.22-μm filter (Millipore).
    
        mPMS (DOJINDO)
    
    
    Methoxy PMS was prepared to 0.5 mg/ml with distilled water.
    
        WST1 (DOJINDO)
    
    
    WST1 was prepared to 8 mg/ml with distilled water.
    
        PD medium
    
    
    Potato dextrose broth (24 g/L, Difco) was adjusted to pH 7, autoclaved, and then used.
    
        LB medium
    
    
    LB Broth (20 g/L, Difco) was autoclaved and then used. Before use thereof, ampicillin (SIGMA) was added to the LB medium to 50 μg/ml.
    
        100 mM MgCl2 
    
    
    MgCl2.6H2O (2.03 g/100 ml) was autoclaved and then used.
    
        ExTaq Polymerase [5 U/μl] (Takara Bio Inc.)
    
    
    10× Buffer
    
        KOD-Plus-: KOD-Plus-Polymerase [1 U/μl] (TOYOBO)
    
    
    25 mM MgSO4 2 mM dNTP, 10× Buffer
    Pyrobest DNA polymerase (Takara Bio Inc.)
    
        Triton X-100
        100 mM dATP, dCTP, dGTP, dTTP (Takara Bio Inc.)
        RNeasy Plant Mini Kit (QIAGEN)
        RNA PCR Kit (Takara Bio Inc.)
        MinElute Gel Extraction Kit (QIAGEN)
        MinElute PCR Purification Kit (QIAGEN)
        BigDyeTerminator v3.1 (ABI)
        dH2O: DNase/RNase Free Distilled Water (Invitrogen)
        Restriction enzyme Nde I/EcoR I (Takara Bio Inc.)
        DNA Ligation kit ver2.1, solution I (Takara Bio Inc.)
        JM109 Competent Cells (Takara Bio Inc.)
        pET-23b(+) vector (Novagen)
        pT7 Blue T-vector (Novagen)
        KOD-Plus-Mutagenesis Kit (TOYOBO)

(2) Cloning of Gibberella Zeae FDH Gene

(2-1) Microbial Strain

    
    


A strain (NBRC No. 4474) of Gibberella zeae preserved at the NITE Biological Resource Center (hereinafter, referred to as NBRC), which is a related organization of the Incorporated Administrative Agency, National Institute of Technology (NITE) was purchased, regenerated by a specified method, and then cultured using a PD (Potato Dextrose) medium.
(2-2) Isolation of Formate Dehydrogenase Gene
(2-2-1) Amplification of Formate Dehydrogenase Gene
Cells obtained by culturing according to the method in 2-1 were prepared using an RNeasy Plant Mini Kit (QIAGEN), so that total RNA (containing mRNA, rRNA, tRNA, and the like) was prepared. First, cDNA synthesis was performed using an RNA PCR Kit (Takara Bio Inc.) and total RNA as a template. Table 1 shows the composition of the reaction solution.







TABLE 1





(Composition of reaction solution)


Final concentration


















5 mM
MgCl2



1X
RT buffer



1 mM
dNTP mixture



0.5U
RNase Inhibitor



0.25U
AMV Reverse Transcriptase XL



0.125 μM
Oligo dT-Adpoter primer



5 μg
Total RNA










RNase free H2O was added, so that the liquid volume was 10 μl.

A cDNA synthesis reaction was performed using the reaction solution with the above composition and a reaction cycle of 50° C. for 2 hours, 99° C. for 5 minutes, and then 4° C.
Next, PCR was performed using the thus synthesized cDNA as a template and Pyrobest DNA polymerase. Table 2 shows the composition of components in 50 μl of the reaction solution.







TABLE 2





(Composition of reaction solution)


















1x
Pyrobest buffer



200 μM
dNTPs mixture



2.5U
Pyrobest DNA polymerase



50 pmol
Primer(forward)



50 pmol
Primer(reverse)



10 μl
cDNA solution










Sterile water was added, so that the liquid volume was 50 μl.

PCR was performed using the reaction solution with the above composition by, after 95° C. for 1 minute, repeating 25 reaction cycles each consisting of 95° C. for 30 seconds, 60° C. for 30 seconds, and 72° C. for 1 minute, followed by 72° C. for 10 minutes and then 4° C. In addition, Gib FDH1-F-Nde I (forward): CGC CAT ATG GTC AAG GTT CTT GCA GTT C (SEQ ID NO: 13) and Gib FDH1-R (reverse): CTA TTT CTT CTC ACG CTG ACC (SEQ ID NO: 14) were used as a primer pair in this PCR.
(2-2-2) Cloning and Structural Analysis of Formate Dehydrogenase Gene
The sizes of various PCR products obtained were confirmed by agarose gel electrophoresis. A PCR product purified from agarose gel using a MinElute Gel Extraction Kit (QIAGEN) was subcloned (GzFDH/pT7) using a pT7 Blue T-vector (Novagen) and JM109 competent cells (Takara Bio Inc.) (see FIG. 2). The isolated formate dehydrogenase gene sequence was 100% consistent (amino acid level) with the sequence (Genbank No. XP—386303) disclosed in the database.
(2-2-3) Construction of Vector for Expression of Formate Dehydrogenase Gene
The plasmid (FDH/pT7) prepared in 2-2-2 was treated with restriction enzymes Nde I/EcoR I. Table 3 shows the composition of the reaction solution. Treatment with restriction enzymes was performed under reaction conditions of 37° C. for 2 hours.







TABLE 3





(Composition of reaction solution)


Final concentration


















1X
Universal buffer H



25U
NdeI



25U
EcoRI



5 μg
GzFDH/pT7










Sterile water was added, so that the total volume was 20 μl.

The solution after reaction was subjected to 0.8% agarose gel electrophoresis, a formate dehydrogenase gene (about 1.1 kb) excised from the vector as an Nde I/EcoR I fragment was purified using a MinElute Gel Extraction Kit (QIAGEN). Next, a vector pET23b(+) (Novagen) for gene expression was treated with restriction enzymes in a manner similar to that for FDH/pT7. The above formate dehydrogenase gene fragment was introduced to the Nde I/EcoR I site of the pET23b(+) vector using a DNA Ligation kit Ver.2.1 (Takara Bio Inc.). Subcloning (FDH/pET23b(+)) was then performed using JM109 competent cells (Takara Bio Inc.) (see FIG. 2).
(3) Preparation of Mutant Formate Dehydrogenase by Site-directed Mutagenesis
(3-1) Multiple Mutant Formate Dehydrogenase
Mutations were site-specifically introduced to FDH using a KOD-Plus-Mutagenesis Kit according to the protocols. Table 4 shows the thus prepared mutants and combinations of templates and primers used for preparation. Table 5 shows the sequences of primers used herein.












TABLE 4









Prepared
Site for
Template
Primer













plasmid
mutagenesis
plasmid
Forward
Reverse
















1 mutation
5-O11-B
V99I
WT
5-O11-B-F
5-O11-B-R


Triple
3M-4
V153I/H155Q
5-O11-B
LoopF-155
LoopR-153



3M-2
V153I/Q159L
5-O11-B
LoopF-159
LoopR-153


Quadruple
19K-2
N298Y
3M-4
298NY-F
298NY-R



4A-1
G73A
3M-2
73GA-F
73GA-R



15H-5
G73A
3M-4
73GA-F
73GA-R



16F-2
H287Q
3M-4
H287Q-F
H287Q-R



9M-2
E60V
3M-4
E60V-F
E60V-R


Quintuple
5A-2
N298Y
4A-1
298NY-F
298NY-R



5B-2
N298Y
15H-5
298NY-F
298NY-R



5B-4
G73A
16F-2
73GA-F
73GA-R



5B-5
N298Y
16F-2
298NY-F
298NY-R



5B-6
G73A
9M-2
73GA-F
73GA-R



5B-7
N298Y
9M-2
298NY-F
298NY-R



5C-2
Q155K
5B-2
Q155K-F
5B-2-155-R


Hexatic
6A-1
M239L
5A-2
6-O11-B-F
6-O11-B-R



6B-1
M239L
5B-2
6-O11-B-F
6-O11-B-R



6B-2
G73A
5B-5
73GA-F
73GA-R



6B-3
G73A
5B-7
73GA-F
73GA-R



6B-4
H287P
5B-2
H287P-F
286-R



6B-5
I31L
5B-2
I31L-F
I31L-R



6B-6
R50G
5B-2
R50G-F
R50G-R



6B-7
I136V
5B-2
I136V-F
I136V-R



6B-8
E293D
5B-2
E293D-F
E293D-R



6B-9
N343R
5B-2
N343R-F
N343R-R



6B-10
I64V
5B-2
I64V-F
I64V-R



6B-11
K82R
5B-2
K82R-F
K82R-R



6B-12
Q159N
5B-2
Q159N-F
5B-2-155-R



6C-1
Q155K
6B-1
Q155K-F
5B-2-155-R



6C-2
Q155K
6B-2
Q155K-F
5B-2-155-R



6D-1
Q155K/Q159L
5B-2
Q155KA156VQ159L-F
5B-2-155-R


Septuple
7B-1
D286F/H287P
5B-2
D286FH287P-F
286-R



7B-2
D286F/H287Q
5B-2
D286FH287Q-F
286-R



7B-3
K82R
6B-1
K82R-F
K82R-R



7B-4
K82R
6B-2
K82R-F
K82R-R



7D-1
Q155K/Q159L
6B-1
Q155KA156VQ159L-F
5B-2-155-R



7D-2
Q155K/Q159L
6B-2
Q155KA156VQ159L-F
5B-2-155-R


















TABLE 5





Primer
Sequence
SEQ ID NO:







298NY-F
TACGCCAAGAACAACTGGGGC
SEQ ID NO: 15


 


298NY-R
TCGCAGAGGGTGCTCCTTGGGA
SEQ ID NO: 16


 


73GA-F
CTTACCTGACCGCTGAGCGTCTG
SEQ ID NO: 17


 


73GA-R
CAGGGTGGAAGGGAGTGGTGAT
SEQ ID NO: 18


 


Q155K-F
AAAGCTGCTAAGCAGGAGTTCGA
SEQ ID NO: 19


 


5B-2-155-R
GGCGATGTCCCACTCACCAGCCT
SEQ ID NO: 20


 


6-O11-B-F
TTGTTCAACAAGGACCTCATCTCCAA
SEQ ID NO: 21


 


6-O11-B-R
ACCCTTGGTCTTCTCGTGGAGAG
SEQ ID NO: 22


 


H287P-F
GACCCACAGCCCGCTCCCAAGGAGCA
SEQ ID NO: 23


 


286-R
CCAGACATCACCACCGTAACCGGCGA
SEQ ID NO: 24


 


I31L-F
CTCCGCAAGTGGCTCGAGGACCAA
SEQ ID NO: 25


 


I31L-R
GCCGAGCTCGTTCTCAGTTGTT
SEQ ID NO: 26


 


R50G-F
GGTGAGGGTTCCAAATTCGACGA
SEQ ID NO: 27


 


R50G-R
GTCCTTGTCGGAAGTGGTGA
SEQ ID NO: 28


 


I136V-F
GTCCGCAACTTCGTCCCTGCCCA
SEQ ID NO: 29


 


I136V-R
GAGGACGAGGATGGTCATGAGA
SEQ ID NO: 30


 


E293D-F
TCACCCTCTGCGATACGCCAAGA
SEQ ID NO: 31


 


E293D-R
TCCTTGGGAGCGGGCTGGTGGT
SEQ ID NO: 32


 


N343R-F
GACCTCACGACCTCATTGTCCACCA
SEQ ID NO: 33


 


N343R-R
TGTAGTCGTGGCGACCAGACAGGT
SEQ ID NO: 34


 


I64V-F
GTCATCATCACCACTCCCTTCCA
SEQ ID NO: 35


 


I64V-R
CTCAGCATCCTCGAGCTCCT
SEQ ID NO: 36


 


K82R-F
GGGCCAAGAAGCTCAAGCTCGCT
SEQ ID NO: 37


 


K82R-R
TGGCCAGACGCTCAGCGGTCA
SEQ ID NO: 38


 


Q155KA156VQ159L-F
AAAGCGGCTAAGCTGGAGTTCGACCTTGA
SEQ ID NO: 39


 


5-O11-B-F
ATCGACCTCAACGCTGCCAACAAGAC
SEQ ID NO: 40


 


5-O11-B-R
GTGGTCGGAGCCGATACCAGCGGTA
SEQ ID NO: 41


 


LoopF-155
aGCTGCTAAGCAGGAGTTCGACCTTGAG
SEQ ID NO: 42


 


H287Q-F
ACAGCCCGCTCCCAAGGAGCA
SEQ ID NO: 43


 


H287Q-R
TGGTCCCAGACATCACCACCGT
SEQ ID NO: 44


 


E60V-F
TGGATGCTGAGATCATCATCACCA
SEQ ID NO: 45


 


E60V-R
CGAGCTCCTCGTCGAATTTGGAA
SEQ ID NO: 46


 


Q159N-F
CAAGCTGCTAAGAATGAGTTCGACCTTGA
SEQ ID NO: 47


 


D286FH287P-F
TTCCCACAGCCCGCTCCCAAGGAGCA
SEQ ID NO: 48


 


D286FH287Q-F
TTCCAACAGCCCGCTCCCAAGGAGCA
SEQ ID NO: 49









In addition, “WT” in the “Template plasmid” column in Table 4 denotes “wild-type FDH.” For example, V99I is mutant FDH resulting from substitution of the 99th valine in FDH with isoleucine.
Specifically, an independent mutant formate dehydrogenase gene was first prepared by introducing a V99I mutation into wild-type FDH, and then a V153I/H155Q mutation or a V153I/Q159L mutation were introduced into the gene, so as to prepare a triple mutant formate dehydrogenase gene. An N298Y mutation, a G73A mutation, an H287Q mutation, or an E60V mutation was then introduced into the triple mutant formate dehydrogenase gene, so that a quadruple mutant formate dehydrogenase was prepared.
Of the thus prepared quadruple mutant formate dehydrogenases, 3 types of quadruple mutant formate dehydrogenase genes shown in Table 6 were used for “(4) Evaluation of the properties of recombinant FDH” described later.









TABLE 6









Mutation position













Plasmid name
73
99
153
155
159
298





19K-2

V99I
V153I
H155Q

N298Y


15H-5
G73A
V99I
V153I
H155Q


4A-1
G73A
V99I
V153I

Q159L









Also, quintuple-to-septuple mutant formate dehydrogenase genes were similarly prepared. Quintuple-to-septuple mutant formate dehydrogenase genes shown in Table 7 were used for (4) Evaluation of the properties of recombinant FDH described later.









TABLE 7







Number of
Plasmid
Mutation position


















mutations
name
31
50
60
64
73
82
99
136
153
155





Quintuple
5B-2




G73A

V99I

V153I
H155Q



5B-4




G73A

V99I

V153I
H155Q



5C-2




G73A

V99I

V153I
Q155K



5A-2




G73A

V99I

V153I



5B-5






V99I

V153I
H155Q



5B-6


E60V

G73A

V99I

V153I
H155Q



5B-7


E60V



V99I

V153I
H155Q


Hexatic
6B-1




G73A

V99I

V153I
H155Q



6B-11




G73A
K82R
V99I

V153I
H155Q



6B-2




G73A

V99I

V153I
H155Q



6B-3


E60V

G73A

V99I

V153I
H155Q



6B-5
I31L



G73A

V99I

V153I
H155Q



6B-7




G73A

V99I
I136V
V153I
H155Q



6B-8




G73A

V99I

V153I
H155Q



6C-1




G73A

V99I

V153I
Q155K



6A-1




G73A

V99I

V153I



6B-4




G73A

V99I

V153I
H155Q



6B-6

R50G


G73A

V99I

V153I
H155Q



6B-9




G73A

V99I

V153I
H155Q



6C-2




G73A
K82R
V99I

V153I
Q155K



6D-1




G73A

V99I

V153I
Q155K



6B-10



I64V
G73A

V99I

V153I
H155Q



6B-12




G73A

V99I

V153I
H155Q


Septuple
7B-3




G73A
K82R
V99I

V153I
H155Q



7B-4




G73A
K82R
V99I

V153I
H155Q



7D-1




G73A

V99I

V153I
Q155K



7D-2




G73A

V99I

V153I
Q155K



7B-1




G73A

V99I

V153I
H155Q



7B-2




G73A

V99I

V153I
H155Q














Number of
Plasmid
Mutation position

















mutations
name
159
239
286
287
293
298
343







Quintuple
5B-2





N298Y




5B-4



H287Q




5C-2





N298Y




5A-2
Q159L




N298Y




5B-5



H287Q

N298Y




5B-6




5B-7





N298Y



Hexatic
6B-1

M239L



N298Y




6B-11





N298Y




6B-2



H287Q

N298Y




6B-3





N298Y




6B-5





N298Y




6B-7





N298Y




6B-8




E293D
N298Y




6C-1

M239L



N298Y




6A-1
Q159L
M239L



N298Y




6B-4



H287P

N298Y




6B-6





N298Y




6B-9





N298Y
N343R




6C-2





N298Y




6D-1
Q159L




N298Y




6B-10





N298Y




6B-12
Q159N




N298Y



Septuple
7B-3

M239L



N298Y




7B-4



H287Q

N298Y




7D-1
Q159L
M239L



N298Y




7D-2
Q159L


H287Q

N298Y




7B-1


D286F
H287P

N298Y




7B-2


D286F
H287Q

N298Y










(4) Evaluation of the Properties of Recombinant FDH

(4-1) Synthesis of Recombinant FDH by Escherichia Coli S30

(4-1-1) Preparation of Template

PCR was performed using the plasmids constructed in the previous section (3-1) as templates. The resulting amplification products were used as templates for translation reaction. For PCR, Single-F and Single-R1 were used as a primer pair. The composition of the solution for PCR is shown in Table 8.







(SEQ ID NO: 50)


Single-F: 5′ -CGA TCC CGC GAA ATT AAT ACG ACT-3′


 


(SEQ ID NO: 51)


Single-R1: 5′ -TCC GGA TAT AGT TCC TCC TTT CAG-3′
















TABLE 8









10XBuffer
5 μl



dNTP (2mMeach)
5 μl



MgSO4 (25 mM)
2 μl



Single-F (10 pmol/μl)
1.5 μl  



Single-R1 (10 pmol/μl)
1.5 μl  



KOD-Plus-DNA Polymerase (1 U/μl)
1 μl



Plasmid DNA
0.3 μl  



dH2O
33.7 μl  




50 μl 










PCR was performed using a reaction solution with the above composition under conditions of: 94° C. for 2 minutes, and then 30 reaction cycles each consisting of 94° C. for 15 seconds, and 68° C. for 1 minute and 30 seconds, followed by 68° C. for 2 minutes and then 4° C. An about 1.4-kbp fragment amplified by PCR was purified using a MinElute PCR Purification Kit and then used for translation reaction.
(4-1-2) Cell-free Translation Reaction
The translation reaction was performed using the DNA fragment purified in 4-1-1 as a template and an EcoPro T7 system (Novagen). The method was performed according to the protocols for the kit.
(4-2) Evaluation of Durability in S30 (Acceleration Test)
The durability of mutant enzymes in S30 (Escherichia coli crude extract) were evaluated with an acceleration test. The procedures are as described below. The translation product prepared in 4-1-2 was dispensed to PCR tubes (1.8 μl each) and then heated with a thermal cycler at 52° C., 56° C., or 57° C. Tubes were removed every 0, 25, and 50 minutes after the initiation of heating and then exposed briefly to and cooled with ice. Subsequently, 98 μl of the resultants were added to each tube containing a reagent for activity measurement with the composition shown in Table 9. The resultants were then heated with a thermal cycler at 37° C. (for about 30 minutes). Tubes were removed and then exposed briefly to ice to stop the reaction.







TABLE 9





<Reagent for activity measurement>


















1.62M Sodium formate
10 μl



16.2 mM NAD
10 μl



 100 mM KPB(pH 7.5)
74.63 μl  



 0.5 mg/ml mPMS
0.67 μl  



  8 mg/ml WST1
2.7 μl 










In addition, a formic acid degradation reaction mediated by an FDH catalyst is represented by the following formula.

HCOO−+NAD+→CO2+NADH

Methoxy PMS (mPMS), which is an electron transfer substance and WST1, which is an oxidation-reduction coloring indicator (both produced by DOJINDO), were added, so that the reaction proceeds as per the following formula. Hence, measurement of yellow formazan with absorbance at a wavelength of 438 nm enables the determination of the amount of degraded formic acid. In addition, the absorption coefficient of yellow formazan was about 6 times that of NADH, enabling determination with higher sensitivity than that possible with direct measurement of NADH.

NADH+mPMS→NAD++mPMS (reduced form)

mPMS (reduced form)+WST1→mPMS+yellow formazan (37000/M·cm, 438 nm)

Yellow formazan was determined by measurement of absorbance at 430 nm using a plate reader (Spectrafluor Plus: TECAN), so that the activity of FDH having a plurality of amino acid mutations prepared in 4-1-2 could be evaluated.
(4-3) Overexpression and Purification of Recombinant FDH in Escherichia Coli 
(4-3-1) Expression Induction
G. zeae-derived FDH (wild-type and mutant) was expressed as described below. As microbial strains, an Escherichia coli BL21 (DE3) strain prepared by introducing the recombinant plasmid 5B-2 constructed in the previous (3-1) and a strain prepared by introducing wild-type FDH were used. Pre-culture was performed using an LB medium (5 ml) at 37° C. for 22 hours. Cells were inoculated to an LB medium (500 ml), so as to obtain a 1.5% culture solution for pre-culture, and then main culture was performed at 37° C. for 4 hours (O.D.600: 3-). Also, as induction culture for inducing protein expression, IPTG was added to a final concentration of 1 mM, and then culture was performed at 20° C. for 16 hours (O.D.600: 3.5-4.1).
After culture, the culture solution was briefly exposed to ice to cool the solution and then cells were collected. The culture solution was divided into two 500-ml tubes, followed by centrifugation at 5200 rpm for 10 minutes at 4° C. The supernatants were discarded, 60 ml of 10 mM KPB (pH7.5) was added to cells for suspension, 120 ml of the suspension was separated into three disruption tubes only for a 50-ml multibead shocker (Yasui Kikai Corporation), and then centrifugation was performed again at 5600 rpm for 5 minutes at 4° C., so that cells were obtained.
(4-3-2) Disruption of Cells Subjected Expression Induction
Cells obtained in (4-3-1) were disrupted while cooling, so that a crude extract was prepared. After cells were collected, the wet cell weight was measured. 10 mM KPB (pH 7.5) was added in an amount 10 times that of wet cells (10 ml per g of wet cell weight) and 0.1-mm glass beads only for a multibead shocker (Yasui Kikai Corporation) were added in an amount equivalent to that of KPB (10 g per 10 ml of KPB). A multibead shocker (Yasui Kikai Corporation) was used for disruption.
A crude extract was prepared according to the procedures. First, a step that involves shaking at 6000 rpm for 120 seconds and after 60 seconds of interval and shaking again under the same conditions was repeated 3 times. Subsequently, centrifugation was performed at 6000 rpm for 20 minutes at 4° C. and then the supernatant was recovered. Centrifugation was then performed at 6000 rpm for 10 minutes at 4° C. and then the supernatants were filtered using a 0.45-μm filter (Millipore), so that a crude extract was prepared.
(4-3-3) Ion Exchange Column
The crude extract prepared in the above section 4-3-2 was fractionated using a HiTrap Q FF column (5 ml) (GE HEALTHCARE BIOSCIENCE). The procedures are as follows. First, 25 ml of 10 mM KPB (pH 7.5) was supplied to the column at a flow rate of 1 ml/min and then equilibration was performed. Next, the crude extract was supplied at a flow rate of 1 ml/min for binding of protein components. Next, 25 ml of 10 mM KPB (pH 7.5) was supplied to the column at a flow rate of 1 ml/min and then washing was performed. Next, with 10 mM KPB (pH 7.5) as a base, NaCl (10 ml) with a gradient from 0 mM to 200 mM was supplied to the column at a flow rate of 0.5 ml/min, and thus elution was performed. In addition, fractionation was performed so that the amount of each fraction was 0.5 ml. Finally, 15 ml of 10 mM KPB (pH 7.5) and 500 mM NaCl was supplied to the column at a flow rate of 1 ml/min and then washing was performed.
Of fractions obtained by the elution step, fractions for which peaks of FDH activity had been observed were used as samples for secondary purification using a hydroxy apatite column.
(4-3-4) Hydroxy Apatite Column
Secondary purification was performed using the FDH activity-containing fractions obtained in the previous section 4-3-3. The fractions were mixed together to result in a single fraction. The fraction was subjected to 30 minutes of centrifugation using an Amicon Ultra-15 (30 kDa cut) (Millipore) at 5000 rpm and 4° C. After demineralization, the resultant was diluted to about 15 ml with mM KPB (pH 7.2). An Econo-Pac CHT-II column (5 ml) (BIO-RAD) was used for secondary purification. Procedures are as described below.
First, 30 ml of 1 mM KPB (pH 7.2) was supplied to the column at a flow rate of 1 ml/min and then equilibration was performed. After demineralization, a sample was supplied to the column at a flow rate of 0.5 ml/min (circulated twice) for binding of protein components. Next, 10 ml of 1 mM KPB (pH 7.2) was supplied to the column at a flow rate of 1 ml/min, and then washing was performed. Next, 10 ml each of 1 mM to 10 mM KPB (pH 6.8) was supplied to the column at a flow rate of 0.5 ml/min, and then elution was performed. In addition, fractionation (total of 49 fractions) was performed so that the amount of each fraction was 0.5 ml. Finally, 5 ml of 500 mM KPB (pH 6.8) was supplied to the column at a flow rate of 1 ml/min and then washing was performed.
(4-3-5) Measurement of Purified Protein Concentration
Determination of protein concentration after purification was performed by the Bradford method using a Protein Assay reagent. Next, on the basis of this value, wild-type FDH and mutant FDH (5B-2) were each adjusted with 1 mM MOPS (pH7.5) so that the protein concentration of each thereof was 0.067 mg/ml.
(4-4) Measurement of Durability of FDH Mutant
Wild-type FDH and mutant FDH (5B-2) were each adjusted to have a concentration of 0.067 mg/ml. Each of the thus obtained enzyme solutions was dispensed at 5 μl per 0.2-ml tube (BIO-RAD) to 0.7M MOPS (pH 7.5), 10 mg/ml BSA, and 0.3 M trehalose, and then the remaining activity was measured after 500 hours of heating with a thermal cycler (BIO-RAD) at 37° C.
(5) Summary of the Results
FIG. 3 shows the results of the durability test performed in (4-4) above. As shown in FIG. 3, it was revealed that the quintuple mutant formate dehydrogenase (G73A, V99I, V153I, H155Q, and N298Y) denoted with the plasmid name “5B-2” could maintain 80% or more activity even after a time period during which the wild-type formate dehydrogenase almost completely lost such activity. As described above, the quintuple mutant formate dehydrogenase denoted with the plasmid name “5B-2” can be said to be an enzyme having extremely high durability.
Also, FIG. 4 shows the results of an acceleration test for durability (performed in (4-2) above) for the 3 types of quadruple mutant formate dehydrogenase obtained in (3-1) above. Moreover, FIG. 5 shows the results of measuring specific activity of these 3 types of quadruple mutant formate dehydrogenase and wild-type formate dehydrogenase. As shown in FIG. 5, the 3 types of quadruple mutant formate dehydrogenase obtained in (3-1) above exhibited specific activity almost equivalent to that of the wild-type. In contrast, as shown in FIG. 4, the 3 types of quadruple mutant formate dehydrogenase obtained in (3-1) above were found to have significantly improved durability compared with the wild-type.
FIG. 6 to FIG. 14 show the results of an acceleration test for durability performed for the quintuple mutant formate dehydrogenase, the hexatic mutant formate dehydrogenase, and the septuple mutant formate dehydrogenase obtained in (3-1) above and the wild-type formate dehydrogenase. Also, FIG. 15 and FIG. 16 show the results of measuring the specific activity of the quintuple mutant formate dehydrogenase, the hexatic mutant formate dehydrogenase, and the septuple mutant formate dehydrogenase obtained in (3-1) above and the wild-type formate dehydrogenase. In addition, FIG. 15 and FIG. 16 show the results as relative values when the specific activity of the wild-type formate dehydrogenase is designated as 1. Also, FIG. 15 shows the results for mutant formate dehydrogenases that exhibited specific activity equivalent to that of the wild-type formate dehydrogenase are shown together. Also, FIG. 16 mainly shows the results of mutant formate dehydrogenases that exhibited specific activity slightly inferior to that of the wild-type formate dehydrogenase.
As shown in FIG. 6 to FIG. 9, it was revealed that the mutant formate dehydrogenases denoted with plasmid names 5B-2, 5B-4, 5C-2, 6B-1, 6B-11, 6B-2, 6B-3, 6B-5, 6B-7, 6B-8, 6C-1, and 7B-3 exhibited good durability while exhibiting specific activity equivalent to that of the wild-type formate dehydrogenase.
Also, as shown in FIG. 10 to FIG. 12, it was revealed that the mutant formate dehydrogenases denoted with 5A-2, 5B-5, 6A-1, 6B-4, 6B-6, 6B-9, 6C-2, 6D-1, 7B-4, 7D-1, and 7D-2 exhibited good durability while exhibiting specific activity slightly inferior to that of the wild-type formate dehydrogenase.
Furthermore, as shown in FIG. 13 and FIG. 14, it was revealed that the mutant formate dehydrogenases denoted with the plasmid names 5B-6, 5B-7, 6B-10, 6B-12, 7B-1, and 7B-2 exhibited higher degrees of durability than the wild-type formate dehydrogenase, although they were inferior to the above mutant formate dehydrogenases in terms of specific activity and/or durability.
Sequence Listing

## Claims

1. A mutant formate dehydrogenase, having improved durability and comprising any one of the following amino acid sequences:
A) an amino acid sequence having a substitution of the 99th valine with isoleucine, a substitution of the 153rd valine with isoleucine, a substitution of the 155th histidine with glutamine, and a substitution of the 298th asparagine with tyrosine;
B) an amino acid sequence having a substitution of 73rd glycine with alanine, a substitution of the 99th valine with isoleucine, a substitution of the 153rd valine with isoleucine, and a substitution of the 155th histidine with glutamine; and
C) an amino acid sequence having a substitution of the 73th glycine with alanine, a substitution of the 99th valine with isoleucine, a substitution of the 153rd valine with isoleucine, and a substitution of the 159th glutamine with leucine;
in the formate dehydrogenase consisting of the amino acid sequence shown in SEQ ID NO: 2.

2. The mutant formate dehydrogenase according to claim 1, further having 1 to 3 substitutional mutations selected from the group consisting of, with respect to the amino acid sequence A):
a substitution of the 31st isoleucine with leucine, a substitution of the 50th arginine with glycine, a substitution of the 60th glutamic acid with valine, a substitution of the 64th isoleucine with valine, a substitution of the 73rd glycine with alanine, a substitution of the 82nd lysine with arginine, a substitution of the 136th isoleucine with valine, a substitution of the 159th glutamine with leucine or asparagine, a substitution of the 239th methionine with leucine, a substitution of the 286th aspartic acid with phenylalanine, a substitution of the 287th histidine with glutamine or proline, a substitution of the 293rd glutamic acid with aspartic acid, and a substitution of the 343rd asparagine with arginine.

3. The mutant formate dehydrogenase according to claim 1, further having 1 to 3 substitutional mutations selected from the group consisting of, with respect to the amino acid sequence B):
a substitution of the 31st isoleucine with leucine, a substitution of the 50th arginine with glycine, a substitution of the 60th glutamic acid with valine, a substitution of the 64th isoleucine with valine, a substitution of the 82nd lysine with arginine, a substitution of the 136th isoleucine with valine, a substitution of the 159th glutamine with leucine or asparagine, a substitution of the 239th methionine with leucine, a substitution of the 286th aspartic acid with phenylalanine, a substitution of the 287th histidine with glutamine or proline, a substitution of the 293rd glutamic acid with aspartic acid, a substitution of the 298th asparagine with tyrosine, and a substitution of the 343rd asparagine with arginine.

4. The mutant formate dehydrogenase according to claim 1, further having 1 to 3 substitutional mutations selected from the group consisting of, with respect to the amino acid sequence C):
a substitution of the 31st isoleucine with leucine, a substitution of the 50th arginine with glycine, a substitution of the 60th glutamic acid with valine, a substitution of the 64th isoleucine with valine, a substitution of the 82nd lysine with arginine, a substitution of the 136th isoleucine with valine, a substitution of the 155th histidine with glutamine or lysine, a substitution of the 239th methionine with leucine, a substitution of the 286th aspartic acid with phenylalanine, a substitution of the 287th histidine with glutamine or proline, a substitution of the 293rd glutamic acid with aspartic acid, a substitution of the 298th asparagine with tyrosine, and a substitution of the 343rd asparagine with arginine.

5. A gene encoding the mutant formate dehydrogenase according to claim 1.

6. A method for producing NADH, comprising contacting the mutant formate dehydrogenase according to claim 1 with a reaction system containing formic acid and NAD+.