Formate dehydrogenase mutant and use thereof

Abstract

To provide a formate dehydrogenase mutant and a use thereof which enable enhancement of production efficiency for high value-added chemicals dependent on an NADH regeneration system through efficient regeneration of NADH, and reduction of production cost for the high value-added chemicals.SOLUTION: The amino acid sequence of the formate dehydrogenase mutant is obtained by mutation of at least one of N24D, K185S, M334I, and V377T from the amino acid sequence of the wild-type formate dehydrogenase shown in a specific sequence. The purpose is to improve the catalytic activity of reducing oxidized coenzyme I to reduced coenzyme I.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the field of biotechnology, and in particular to a formate dehydrogenase mutant and its use. Regarding. [Background technology]

[0002] In the prior art, formate dehydrogenase (FDH ) is often used to regenerate NADH. FDH is a D-2-hydroxy acid dehydrogenase. It belongs to the class of enzymes that catalyze the oxidation of formate to produce carbon dioxide and simultaneously produce oxidized cofactors. Enzyme I (Oxidized form of nicotinamide adenine dinucleotide, NAD + ) can be reduced to NADH, and NADH can be recycled In addition, in the regeneration process of NADH with FDH intervention, The only reactant is carbon dioxide. Carbon dioxide easily escapes from the reaction system, so it is difficult to activate the enzyme in the reaction system. It does not affect the activity or product isolation and purification. However, the activity of NADH regenerated via existing FDH is low, and the NADH regeneration cycle The production efficiency of high-value-added chemicals that depend on this system is reduced, resulting in high production costs. Summary of the Invention

[0003] The present invention adopts the following technical solutions. In a first aspect, the present invention provides a formate dehydrogenase mutant, The amino acid sequence is the same as that of wild-type formate dehydrogenase shown in SEQ ID NO:1. by at least one of the following mutations: N24D, K185S, M334I, and V377T. Obtained. Here, N24D, K185S, M334I and V377T are all standard amino acids. This is a standard substitution notation using typical single-letter codes. The above-mentioned N24D has the amino acid sequence of the formate dehydrogenase mutant shown in SEQ ID NO: 1. The asparagine (N) at position 24 of the N-terminus is replaced with aspartic acid (D). . The above K185S is the amino acid sequence of the formate dehydrogenase mutant shown in SEQ ID NO: 1 The lysine (K) at position 185 of the N-terminus is replaced with serine (S). The above M334I has the amino acid sequence of the formate dehydrogenase mutant shown in SEQ ID NO: 1 The methionine (M) at position 334 of the N-terminus is replaced with isoleucine (I). . The above V377T is the amino acid sequence of the formate dehydrogenase mutant shown in SEQ ID NO: 1 The valine (V) at position 377 of the N-terminus is replaced with threonine (T). In an embodiment of the first aspect, at least one of N24D, K185S, M334I, and V377T is present. The formate dehydrogenase mutant obtained by mutation with one can be: 1. Formate dehydrogenase mutant, amino acid sequence of formate dehydrogenase mutant, SEQ ID NO. :1 is obtained by N24D mutation from the amino acid sequence of wild-type formate dehydrogenase shown in The amino acid sequence of the formate dehydrogenase mutant is shown in SEQ ID NO:2. Second formate dehydrogenase mutant, amino acid sequence of formate dehydrogenase mutant, SEQ ID NO. :1 is obtained by the K185S mutation from the wild-type formate dehydrogenase amino acid sequence shown in The amino acid sequence of the 2 formate dehydrogenase mutant is shown in SEQ ID NO:3. 3. Formate dehydrogenase mutant, amino acid sequence of formate dehydrogenase mutant, SEQ ID NO. : The wild-type formate dehydrogenase shown in 1 is obtained by the M334I mutation, The amino acid sequence of the three formate dehydrogenase mutants is shown in SEQ ID NO:4. 4. Formate dehydrogenase mutant, amino acid sequence of formate dehydrogenase mutant, SEQ ID NO. : The wild-type formate dehydrogenase shown in 1 is obtained by the V377T mutation, The amino acid sequence of the four formate dehydrogenase mutants is shown in SEQ ID NO:5. 5 formate dehydrogenase mutant, amino acid sequence of formate dehydrogenase mutant, SEQ ID NO : The amino acid sequence of wild-type formate dehydrogenase shown in 1 is N24D mutation and K185S mutation. The amino acid sequence of the fifth formate dehydrogenase mutant is set forth in SEQ ID NO:6. is shown. 6 formate dehydrogenase mutant, amino acid sequence of formate dehydrogenase mutant, SEQ ID NO : The amino acid sequence of wild-type formate dehydrogenase shown in 1 is N24D mutation and M334I mutation. The amino acid sequence of the sixth formate dehydrogenase mutant is set forth in SEQ ID NO:7. is shown. Formate dehydrogenase mutant 7, amino acid sequence of formate dehydrogenase mutant, SEQ ID NO : The amino acid sequence of wild-type formate dehydrogenase shown in 1 is N24D mutation and V377T mutation. The amino acid sequence of the seventh formate dehydrogenase mutant is set forth in SEQ ID NO:8. is shown. Formate dehydrogenase mutant 8, amino acid sequence of formate dehydrogenase mutant, SEQ ID NO The amino acid sequence of wild-type formate dehydrogenase shown in 1 was analyzed using the K185S and M334I mutations. The amino acid sequence of the eighth formate dehydrogenase mutant obtained by the mutation is SEQ ID NO:9 As shown in. 9 formate dehydrogenase mutant, amino acid sequence of formate dehydrogenase mutant, SEQ ID NO The amino acid sequence of wild-type formate dehydrogenase shown in 1 was analyzed using the K185S and V377T mutations. The amino acid sequence of the 9th formate dehydrogenase mutant obtained by the mutation is SEQ ID NO:1 Shown as 0. 10th formate dehydrogenase mutant, amino acid sequence of formate dehydrogenase mutant, SEQ ID N The amino acid sequence of wild-type formate dehydrogenase shown in O:1 contains the M334I mutation and V377T The amino acid sequence of the 10th formate dehydrogenase mutant obtained by the mutation is SEQ ID NO: :11. Formate dehydrogenase mutant 11, amino acid sequence of formate dehydrogenase mutant, SEQ ID N The amino acid sequence of wild-type formate dehydrogenase shown in O:1 contains the N24D mutation, K185S mutation, and The amino acid sequence of the 11th formate dehydrogenase mutant is SE Shown in Q ID NO:12. 12th formate dehydrogenase mutant, amino acid sequence of formate dehydrogenase mutant, SEQ ID N The amino acid sequence of wild-type formate dehydrogenase shown in O:1 contains the N24D mutation, K185S mutation, and The amino acid sequence of the 12th formate dehydrogenase mutant is SE Shown in Q ID NO:13. 13th formate dehydrogenase mutant, amino acid sequence of formate dehydrogenase mutant, SEQ ID N O: The amino acid sequence of wild-type formate dehydrogenase shown in 1 is similar to that of the K185S and M334I mutations. and V377T mutations, and the amino acid sequence of the 13th formate dehydrogenase mutant is S Shown in EQ ID NO:14. 14th formate dehydrogenase mutant, amino acid sequence of formate dehydrogenase mutant, SEQ ID N From the amino acid sequence of wild-type formate dehydrogenase shown in O:1, the N24D mutation, M334I mutation, and The amino acid sequence of the 14th formate dehydrogenase mutant is SE Shown in Q ID NO:15. 15th formate dehydrogenase mutant, amino acid sequence of formate dehydrogenase mutant, SEQ ID N The amino acid sequence of wild-type formate dehydrogenase shown in O:1 is N24D mutation, K185S mutation, The 15th formate dehydrogenase mutant, obtained by the M334I and V377T mutations, The amino acid sequence is shown in SEQ ID NO:16. In a second aspect, the present invention further provides a nucleic acid encoding the formate dehydrogenase mutant. do. In a third aspect, the present invention further provides an expression vector comprising the nucleic acid described above. In a fourth aspect, the present invention relates to a method for transforming or transfecting the above expression vector. Further provided is a host cell that In a fifth aspect, the present invention further provides a method for preparing the above-mentioned formate dehydrogenase mutant. In a sixth aspect, the present invention relates to the formate dehydrogenase mutant, the nucleic acid, the expression vector, the host cell The oxidized coenzyme I in the cells or the culture prepared by the above-mentioned preparation method is reduced to the reduced coenzyme I. The present invention further provides an application in improving the catalytic activity of a catalyst. [Effects of the Invention]

[0004] The present invention has the following beneficial effects. 1. The present invention provides various formate dehydrogenase mutants that can efficiently catalyze NADH regeneration. This mutant formate dehydrogenase has higher catalytic activity than the original formate dehydrogenase, As a result, NAD + The catalytic effect of reducing ATP to NADH is improved. Improved production efficiency of high-value-added chemicals that rely on the formate dehydrogenase regeneration circulation system, reducing production costs is reduced. 2. The mutants provided by the present invention effectively improve the regeneration efficiency of the reduced coenzyme NADH. The catalytic efficiency of the mutant N24D / K184S / M334I / V377T can be increased. is 1.5 times the catalytic efficiency of wild-type formate dehydrogenase. 3. The formate dehydrogenase mutant provided by the present invention has a mild reaction condition and a low reaction pH value. The reaction temperature range is 15-40℃, and the reaction pH value is 4-10. is improved compared to wild-type formate dehydrogenase. [Brief explanation of the drawings]

[0005] [Figure 1] FIG. 1 is a reaction diagram of the FDH-catalyzed reduction of NAD+ to NADH provided by an example of the present application. [Figure 2] FIG. 1 is a diagram showing the effect of the relative activity of NADH regenerated by wild-type formate dehydrogenase and mutant formate dehydrogenase catalysts provided by the examples of the present application. [Figure 3] FIG. 1 is a diagram showing the effect of the relative activity of wild-type formate dehydrogenase and NADH regenerated by mutant formate dehydrogenase catalysts under different temperatures provided by the examples of the present application. [Figure 4] FIG. 1 is a diagram showing the effect of the relative activity of wild-type formate dehydrogenase and NADH regenerated by mutant formate dehydrogenase catalysts under different pH values ​​provided by the examples of the present application. DETAILED DESCRIPTION OF THE INVENTION

[0006] The formate dehydrogenase mutants provided in the examples of the present application and their uses are In the field of biology, the formate dehydrogenase mutant catalyzed the synthesis of oxidized coenzyme I (Oxidized Coenzyme I) form of nicotinamide adenine dinucleoti de, NAD + ) into reduced form of coenzyme I (reduced form of nicotin amide adenine dinucleotide (NADH) can. For existing formate dehydrogenase (FDH) interventions As a result, high-value-added chemicals that depend on the NADH regeneration cycle are not available. In order to solve the problem of the background art that the production efficiency of the product is low and the production cost is high, The examples of the present application provide formate dehydrogenase mutants and uses thereof, and the formate dehydrogenase mutants The body uses formate and NAD as highly catalytically active enzymes. + It efficiently catalyzes reactions using It can generate carbon dioxide and NADH, and has high activity depending on the NADH regeneration cycle. The decrease in production efficiency of value-added chemicals can be improved, and the production costs can be reduced. In the examples of this application, the term "wild-type" refers to a gene isolated from a naturally occurring source. A wild-type gene is the gene that is most commonly observed in a population. refers to a child, and is therefore arbitrarily designed to be the "normal" or "wild-type" form of a gene. Conversely, the terms "modified," "mutant," or "variant" refer to a variant of a wild-type gene. sequence modifications (e.g., substitutions, truncations, or insertions), translational Genes or gene products that exhibit post-modification and / or functional properties (e.g., altered properties) It should also be noted that naturally occurring variants may be isolated, and their The fact that a mutant has altered properties compared to the wild-type gene or gene product The introduction or substitution of naturally occurring or non-naturally occurring amino acids is performed. Methods are well known in the art. In the examples of the present application, the relevant information of such common amino acids is shown in Table 1. The examples of the present application do not provide relevant information for all amino acids, and other amino acids other than those in Table 1 may be used. If an amino acid is present, other existing products can be referenced to obtain related information on other amino acids. Please understand that. Table 1: Common amino acid abbreviations and corresponding one-letter codes JPEG2025171967000001.jpg182129 Example 1: The examples of the present application provide formate dehydrogenase mutants and the architecture of the formate dehydrogenase mutants. The amino acid sequence is derived from the amino acid sequence of wild-type formate dehydrogenase shown in SEQ ID NO:1. It is obtained by at least one of the following mutations: N24D, K185S, M334I, and V377T. can be done. SEQ ID NO:1 is shown below. MAKVLCVLYDDPIDGYPTTYARDNLPKIDHYPGGQTLPTP KAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALT AGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVKHSYDLEAMSVGTVAA GRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYI VNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHMSGTSLTAQTRYAAGTREILECFFEGR PIRDEYLIVQGGNLAGVGAHSYSKGNATGGSEEAGKFKKA G Here, N24D, K185S, M334I and V377T are all standard amino acids. It employs standard substitution notation for typical single-character codes. The above-mentioned N24D has the amino acid sequence of the formate dehydrogenase mutant shown in SEQ ID NO: 1. The asparagine (N) at position 24 of the N-terminus is replaced with aspartic acid (D). . The above K185S is the amino acid sequence of the formate dehydrogenase mutant shown in SEQ ID NO: 1 The lysine (K) at position 185 of the N-terminus is replaced with serine (S). The above M334I has the amino acid sequence of the formate dehydrogenase mutant shown in SEQ ID NO: 1 The methionine (M) at position 334 of the N-terminus is replaced with isoleucine (I). . The above V377T is the amino acid sequence of the formate dehydrogenase mutant shown in SEQ ID NO: 1 The valine (V) at position 377 of the N-terminus is replaced with threonine (T). In an embodiment of the first aspect, at least one of N24D, K185S, M334I, V377T The formate dehydrogenase mutant obtained by mutation with one of: 1. Formate dehydrogenase mutant, amino acid sequence of formate dehydrogenase mutant, SEQ ID NO. :1 is obtained by N24D mutation from the amino acid sequence of wild-type formate dehydrogenase shown in The amino acid sequence of the formate dehydrogenase mutant (also called the N24D mutant) is shown in SEQ ID NO: Shown in NO:2. SEQ ID NO:2 MAKVLCVLYDDPIDGYPTTYARDDLPKIDHYPGGQTLPTP KAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALT AGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVKHSYDLEAMSVGTVAA GRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYI VNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHMSGTSLTAQTRYAAGTREILECFFEGR PIRDEYLIVQGGNLAGVGAHSYSKGNATGGSEEAGKFKKA G Second formate dehydrogenase mutant, amino acid sequence of formate dehydrogenase mutant, SEQ ID NO. :1 is obtained by the K185S mutation from the wild-type formate dehydrogenase amino acid sequence shown in The amino acid sequence of the formate dehydrogenase mutant (also called the K185S mutant) is SEQ ID NO: 1 Shown in D NO:3. SEQ ID NO:3 MAKVLCVLYDDPIDGYPTTYARDNLPKIDHYPGGQTLPTP KAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALT AGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVSHSYDLEAMSVGTVAA GRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYI VNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHMSGTSLTAQTRYAAGTREILECFFEGR PIRDEYLIVQGGNLAGVGAHSYSKGNATGGSEEAGKFKKA G 3. Formate dehydrogenase mutant, amino acid sequence of formate dehydrogenase mutant, SEQ ID NO. : The wild-type formate dehydrogenase shown in 1 is obtained by the M334I mutation, The amino acid sequence of the 3 formate dehydrogenase mutant (also called the M334I mutant) is SEQ ID NO: 1 Shown in D NO:4. SEQ ID NO:4 MAKVLCVLYDDPIDGYPTTYARDNLPKIDHYPGGQTLPTP KAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALT AGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVKHSYDLEAMSVGTVAA GRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYI VNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHISGTSLTAQTRYAAGTREILECFFEGR PIRDEYLIVQGGNLAGVGAHSYSKGNATGGSEEAGKFKKA G 4. Formate dehydrogenase mutant, amino acid sequence of formate dehydrogenase mutant, SEQ ID NO. : The wild-type formate dehydrogenase shown in 1 is obtained by the V377T mutation, The amino acid sequence of the 4 formate dehydrogenase mutant (also called the V377T mutant) is SEQ ID NO: 1 Shown in D NO:5. SEQ ID NO:5 MAKVLCVLYDDPIDGYPTTYARDNLPKIDHYPGGQTLPTP KAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALT AGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVKHSYDLEAMSVGTVAA GRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYI VNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHMSGTSLTAQTRYAAGTREILECFFEGR PIRDEYLIVQGGNLAGTGAHSYSKGNATGGSEEAGKFKKA G 5 formate dehydrogenase mutant, amino acid sequence of formate dehydrogenase mutant, SEQ ID NO : The amino acid sequence of wild-type formate dehydrogenase shown in 1 is N24D mutation and K185S mutation. and the fifth formate dehydrogenase mutant (also called the N24D / K185S mutant) The amino acid sequence of ) is shown in SEQ ID NO:6. SEQ ID NO:6 MAKVLCVLYDDPIDGYPTTYARDDLPKIDHYPGGQTLPTP KAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALT AGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVSHSYDLEAMSVGTVAA GRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYI VNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHMSGTSLTAQTRYAAGTREILECFFEGR PIRDEYLIVQGGNLAGVGAHSYSKGNATGGSEEAGKFKKA G 6 formate dehydrogenase mutant, amino acid sequence of formate dehydrogenase mutant, SEQ ID NO : The amino acid sequence of wild-type formate dehydrogenase shown in 1 is N24D mutation and M334I mutation. and the sixth formate dehydrogenase mutant (also called the N24D / M334I mutant) The amino acid sequence of ) is shown in SEQ ID NO:7. SEQ ID NO:7 MAKVLCVLYDDPIDGYPTTYARDDLPKIDHYPGGQTLPTP KAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALT AGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVKHSYDLEAMSVGTVAA GRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYI VNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHISGTSLTAQTRYAAGTREILECFFEGR PIRDEYLIVQGGNLAGVGAHSYSKGNATGGSEEAGKFKKA G Formate dehydrogenase mutant 7, amino acid sequence of formate dehydrogenase mutant, SEQ ID NO : The amino acid sequence of wild-type formate dehydrogenase shown in 1 is N24D mutation and V377T mutation. and the seventh formate dehydrogenase mutant (also called the N24D / V377T mutant) The amino acid sequence of ) is shown in SEQ ID NO:8. SEQ ID NO:8 MAKVLCVLYDDPIDGYPTTYARDDLPKIDHYPGGQTLPTP KAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALT AGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVKHSYDLEAMSVGTVAA GRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYI VNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHMSGTSLTAQTRYAAGTREILECFFEGR PIRDEYLIVQGGNLAGTGAHSYSKGNATGGSEEAGKFKKA G Formate dehydrogenase mutant 8, amino acid sequence of formate dehydrogenase mutant, SEQ ID NO The amino acid sequence of wild-type formate dehydrogenase shown in 1 was analyzed using the K185S and M334I mutations. The eighth formate dehydrogenase mutant (also called the K185S / M334I mutant) was obtained by The amino acid sequence of ATP (pronounced ATP) is shown in SEQ ID NO:9. SEQ ID NO:9 MAKVLCVLYDDPIDGYPTTYARDNLPKIDHYPGGQTLPTP KAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALT AGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVSHSYDLEAMSVGTVAA GRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYI VNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHISGTSLTAQTRYAAGTREILECFFEGR PIRDEYLIVQGGNLAGVGAHSYSKGNATGGSEEAGKFKKA G 9 formate dehydrogenase mutant, amino acid sequence of formate dehydrogenase mutant, SEQ ID NO The amino acid sequence of wild-type formate dehydrogenase shown in 1 was analyzed using the K185S and V377T mutations. The 9th formate dehydrogenase mutant (also called the K185S / V377T mutant) was obtained by The amino acid sequence of ATP (pronounced ATP) is shown in SEQ ID NO:10. SEQ ID NO:10 MAKVLCVLYDDPIDGYPTTYARDNLPKIDHYPGGQTLPTP KAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALT AGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVSHSYDLEAMSVGTVAA GRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYI VNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHMSGTSLTAQTRYAAGTREILECFFEGR PIRDEYLIVQGGNLAGTGAHSYSKGNATGGSEEAGKFKKA G 10th formate dehydrogenase mutant, amino acid sequence of formate dehydrogenase mutant, SEQ ID N The amino acid sequence of wild-type formate dehydrogenase shown in O:1 contains the M334I mutation and V377T The 10th formate dehydrogenase mutant (also known as the M334I / V377T mutant) was obtained by mutation. The amino acid sequence of ATP-1, ATP-2, ATP-3, ATP-4, ATP-5, ATP-6, ATP-7, ATP-8, ATP-9, ATP-10, ATP-11, ATP-12, ATP-13, ATP-14, ATP-15, ATP-16, ATP-17, ATP-18, ATP-19, ATP-20, ATP-21, ATP-22 SEQ ID NO:11 MAKVLCVLYDDPIDGYPTTYARDNLPKIDHYPGGQTLPTP KAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALT AGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVKHSYDLEAMSVGTVAA GRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYI VNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHISGTSLTAQTRYAAGTREILECFFEGR PIRDEYLIVQGGNLAGTGAHSYSKGNATGGSEEAGKFKKA G Formate dehydrogenase mutant 11, amino acid sequence of formate dehydrogenase mutant, SEQ ID N The amino acid sequence of wild-type formate dehydrogenase shown in O:1 contains the N24D mutation, K185S mutation, and and M334I mutations, and the 11th formate dehydrogenase mutant (N24D / K185 The amino acid sequence of the S / M334I mutant is shown in SEQ ID NO: 12. can be. SEQ ID NO:12 MAKVLCVLYDDPIDGYPTTYARDDLPKIDHYPGGQTLPTP KAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALT AGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVSHSYDLEAMSVGTVAA GRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYI VNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHISGTSLTAQTRYAAGTREILECFFEGR PIRDEYLIVQGGNLAGVGAHSYSKGNATGGSEEAGKFKKA G 12th formate dehydrogenase mutant, amino acid sequence of formate dehydrogenase mutant, SEQ ID N The amino acid sequence of wild-type formate dehydrogenase shown in O:1 contains the N24D mutation, K185S mutation, and and V377T mutations, and the 12th formate dehydrogenase mutant (N24D / K185 The amino acid sequence of the S / V377T variant is shown in SEQ ID NO: 13. can be. SEQ ID NO:13 MAKVLCVLYDDPIDGYPTTYARDDLPKIDHYPGGQTLPTP KAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALT AGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVSHSYDLEAMSVGTVAA GRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYI VNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHMSGTSLTAQTRYAAGTREILECFFEGR PIRDEYLIVQGGNLAGTGAHSYSKGNATGGSEEAGKFKKA G 13th formate dehydrogenase mutant, amino acid sequence of formate dehydrogenase mutant, SEQ ID N O: The amino acid sequence of wild-type formate dehydrogenase shown in 1 is similar to that of the K185S and M334I mutations. and V377T mutations, resulting in the 13th formate dehydrogenase mutant (K185S / M3 The amino acid sequence of the 34I / V377T variant is set forth in SEQ ID NO:14. is shown. SEQ ID NO:14 MAKVLCVLYDDPIDGYPTTYARDNLPKIDHYPGGQTLPTP KAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALT AGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVSHSYDLEAMSVGTVAA GRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYI VNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHISGTSLTAQTRYAAGTREILECFFEGR PIRDEYLIVQGGNLAGTGAHSYSKGNATGGSEEAGKFKKA G 14th formate dehydrogenase mutant, amino acid sequence of formate dehydrogenase mutant, SEQ ID N From the amino acid sequence of wild-type formate dehydrogenase shown in O:1, the N24D mutation, M334I mutation, and and V377T mutations, and the 14th formate dehydrogenase mutant (N24D / M334 The amino acid sequence of the IL-1 / V377T mutant is shown in SEQ ID NO: 15. can be. SEQ ID NO:15 MAKVLCVLYDDPIDGYPTTYARDDLPKIDHYPGGQTLPTP KAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALT AGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVKHSYDLEAMSVGTVAA GRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYI VNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHISGTSLTAQTRYAAGTREILECFFEGR PIRDEYLIVQGGNLAGTGAHSYSKGNATGGSEEAGKFKKA G 15th formate dehydrogenase mutant, amino acid sequence of formate dehydrogenase mutant, SEQ ID N The amino acid sequence of wild-type formate dehydrogenase shown in O:1 is N24D mutation, K185S mutation, The 15th formate dehydrogenase mutant (N The amino acid sequence of the mutant (also called the 24D / K185S / M334I / V377T mutant) is S Shown in EQ ID NO:16. SEQ ID NO:16 MAKVLCVLYDDPIDGYPTTYARDDLPKIDHYPGGQTLPTP KAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALT AGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVSHSYDLEAMSVGTVAA GRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYI VNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHISGTSLTAQTRYAAGTREILECFFEGR PIRDEYLIVQGGNLAGTGAHSYSKGNATGGSEEAGKFKKA G Example 2: The examples of the present application relate to nucleic acids encoding the formate dehydrogenase mutants in Example 1, An expression vector encoding the nucleic acid, and a method for transforming or transferring the expression vector. A host cell that is transfectant is provided. The examples of the present application utilize the sequence and structural information of formate dehydrogenase that has already been reported. By using this method and performing a non-redundant search in databases such as NCBI, protein structures can be identified. Based on principles such as similarity, conserved site analysis and diversity of host sources, several potential enzymes were identified. These genes are functionally expressed in an E. coli expression system, and then The formate dehydrogenase gene was then purified to obtain a purified formate dehydrogenase mutant. Transformed by directed evolution using rational design to produce formate and NAD + is used as a substrate for catalytic reaction We have developed a highly catalytically active formate dehydrogenase mutant that produces carbon dioxide and NADH in the reaction. The above reaction process is shown in FIG. In the examples of the present application, the nucleic acid, an expression vector encoding the nucleic acid, and The process for constructing a host cell to transform or transfect the vector is as follows: do. Step 1: A nucleic acid encoding the formate dehydrogenase mutant in Example 1 and the nucleic acid encoding the mutant An expression vector for this gene is constructed. In the examples of this application, the whole-plasmid PCR method was used to identify Ancylobacter aquaticus. The formate dehydrogenase wild-type gene of Ancylobacter aquaticus (Kin The target mutant gene (i.e., (i.e., the nucleic acid encoding the formate dehydrogenase mutant in Example 1) was obtained, and the pET22b plasmid (i.e., an expression vector encoding the nucleic acid). Ancylobacter aquaticus formate dehydrogenase The gene is shown in SEQ ID NO:17. SEQ ID NO:17 ATGGCGAAAGTTCTGTGCGTTCTGTACGATGATCCGATCG ATGGTTACCCGACCACCTACGCGCGTGACAACCTGCCGAA AATCGACCACTATCCGGGTGGTCAGACCCTGCCGACCCCG AAAGCGATCGATTTCACTCCGGGCACCATGCTGGGTTCTG TTTCTGGTGAACTGGGCCTGCGTAAATACCTGGAAAGCAA CGGTCACACCCTGGTTGTTACCTCTGATAAAGATGGTCCG GATTCTGTTTTCGAAAAAGAATTGGTTGATGCGGATATTG TTATCAGCCAGCCGTTCTGGCCGGCTTACCTGACCCCGGA ACGCTTCGCTAAAGCTAAAAACCTGAAACTGGCTCTGACC GCGGGCATCGGCTCTGATCACGTTGACCTGCAAAGCGCAA TTGATCGTGGTGTTACCGTTGCGGAAGTGACCTACTGCAA CTCTATCAGCGTGGCGGAACACGTTGTGATGATGATCCTG GGCCTGGTTCGTAACTACCTGCCGGCGCACGACTGGGCGC GTAAAGGTGGCTGGAACATCGCAGATTGCGTGAAACACTC TTACGATCTGGAAGCGATGTCTGTTGGTACTGTGGCGGCG GGCCGCATCGGTCTGGCGGTGCTGCGCCGCCTGGCTCCGT TCGACGTGAAATTACACTATACCGACCGTCACCGCCTGCC GGAAAGCGTTGAAAAAGAACTGAACCTGACCTGGCACGCT TCTCCGACCGATATGTACCCGCACTGCGACGTGGTTACCC TGAACTGCCCGCTGCACCCGGAAACCGAACACATGGTTAA CGAAGAAACCCTGAAACTGTTCAAACGTGGTGCGTACATC GTTAACACCGCGCGTGGTAAACTGTGCGACCGTGATGCGA TCGCGCGCGCGCTGGAAAACGGCACCCTGGCCGGTTATGC GGGCGATGTTTGGTTCCCGCAGCCGGCGCCGGCTGATCAC CCGTGGCGTACTATGGCATGGAACGGCATGACCCCGCACA TGAGCGGCACTAGCCTGACCGCGCAGACCCGTTATGCTGC GGGCACCCGTGAAATCCTGGAATGCTTCTTTGAAGGCCGT CCGATCCGTGATGAATACCTGATCGTTCAGGGCGGTAACC TGGCGGGTGTTGGCGCACACAGCTACTCTAAAGGCAACGC TACCGGTGGTTCTGAAGAAGCGGGTAAATTTAAAAAAGCG GGCTAATCTCTC The above Ancylobacter aquaticus s) In the formate dehydrogenase gene, A stands for adenine and T stands for thymine. Thymine, C stands for cytosine, and G stands for guanine. uanine), The primers for the mutation sites N24D, K185S, M334I, and V377T are shown in Table 2. can be. Table 2: Primer list for N24D, K185S, M334I and V377T JPEG2025171967000002.jpg182129 The primers for the target mutant gene encoding the formate dehydrogenase mutant in Example 1 are explained below. do. (1) Mutations due to any one of N24D, K185S, M334I, and V377T The primers for the thus obtained formate dehydrogenase mutants are as follows: The targeted mutation gene encoding the first formate dehydrogenase mutant (i.e., encoding the first formate dehydrogenase For the nucleic acid of the enzyme mutant, the primer was a primer corresponding to N24D (SEQ ID NO:18 and SEQ ID NO:19). The targeted mutation gene encoding the second formate dehydrogenase mutant (i.e., encoding the second formate dehydrogenase) For the nucleic acid of the enzyme mutant, the primer was the primer corresponding to K185S (SE SEQ ID NO:20 and SEQ ID NO:21). The targeted mutation gene encoding the third formate dehydrogenase mutant (i.e., encoding the third formate dehydrogenase) For the nucleic acid of the enzyme mutant, the primer was a primer corresponding to M334I (SE SEQ ID NO:22 and SEQ ID NO:23). The targeted mutation gene encoding the 4th formate dehydrogenase mutant (i.e., encoding the 4th formate dehydrogenase For the nucleic acid of the enzyme mutant, the primer was a primer corresponding to V377T (SE SEQ ID NO:24 and SEQ ID NO:25). (2) mutations due to any two of N24D, K185S, M334I, and V377T The primers for the thus obtained formate dehydrogenase mutants are as follows: The targeted mutation gene encoding the 5th formate dehydrogenase mutant (i.e., encoding 5th formate dehydrogenase) For the nucleic acid of the enzyme mutant, the primers were the primers corresponding to N24D and K This is a primer corresponding to 185S. The targeted mutation gene encoding the 6th formate dehydrogenase mutant (i.e., encoding the 6th formate dehydrogenase For the nucleic acid of the enzyme mutant, the primers were the primers corresponding to N24D and M This is a primer corresponding to 334I. The targeted mutation gene encoding the 7th formate dehydrogenase mutant (i.e., encoding 7th formate dehydrogenase) For the nucleic acid of the enzyme mutant, the primers were the primers corresponding to N24D and V This is a primer corresponding to 377T. The targeted mutation gene encoding the 8th formate dehydrogenase mutant (i.e., encoding the 8th formate dehydrogenase For the nucleic acid of the enzyme mutant, the primers were the primers corresponding to K185S and This is a primer corresponding to M334I. The targeted mutation gene encoding the 9th formate dehydrogenase mutant (i.e., encoding 9th formate dehydrogenase) For the nucleic acid of the enzyme mutant, the primers were the primers corresponding to K185S and This is a primer corresponding to V377T. The targeted mutation gene encoding the 10th formate dehydrogenase mutant (i.e., the coding region for the 10th formate dehydrogenase) For the nucleic acid of the hydrogenase mutant, the primers were the primers corresponding to M334I and and V377T. (3) mutations due to any three of N24D, K185S, M334I, and V377T The primers for the thus obtained formate dehydrogenase mutants are as follows: The targeted mutation gene encoding the 11th formate dehydrogenase mutant (i.e., the coding region for the 11th formate dehydrogenase) For the nucleic acid of the hydrogenase mutant, the primers are the primers corresponding to N24D, K A primer corresponding to 185S and a primer corresponding to M334I. The targeted mutation gene encoding the 12th formate dehydrogenase mutant (i.e., the coding region for the 12th formate dehydrogenase) For the nucleic acid of the hydrogenase mutant, the primers are the primers corresponding to N24D, K A primer corresponding to 185S and a primer corresponding to V377T. The targeted mutation gene encoding the 13th formate dehydrogenase mutant (i.e., the coding region for the 13th formate dehydrogenase) For the nucleic acid of the hydrogenase mutant, the primers were: A primer corresponding to M334I and a primer corresponding to V377T. The targeted mutation gene encoding the 14th formate dehydrogenase mutant (i.e., the 14th formate dehydrogenase For the nucleic acid of the hydrogenase mutant, the primers were the primers corresponding to N24D, M The primers are those corresponding to 334I and V377T. (4) A total of four mutations, N24D, K185S, M334I, and V377T, were obtained. Primers for selected formate dehydrogenase mutants. The targeted mutation gene encoding the 15th formate dehydrogenase mutant (i.e., the 15th formate dehydrogenase For the nucleic acid of the hydrogenase mutant, the primers are the primers corresponding to N24D, K The primers for 185S, M334I, and V377T were used. It is a primer that responds to The PCR reaction system for the above whole-plasmid PCR is shown in Table 3. Table 3: PCR reaction system JPEG2025171967000003.jpg182129 The PCR reaction sequence for the whole plasmid PCR is shown in Table 4. Table 4: PCR reaction schedule JPEG2025171967000004.jpg182129 After PCR amplification of the target fragment, the amplified product was detected by 0.9% agarose gel electrophoresis. The amplification products are each a single band of approximately 6000 bp in size. The amplified product was purified and recovered using a kit manufactured by the company. Finally, the pET22b plasmids for the 1st and 15th formate dehydrogenase mutants were used. The pET22b plasmid for the first formate dehydrogenase mutant was obtained. Expression vector for the second formate dehydrogenase mutant, expression vector for the third formate dehydrogenase mutant Expression vector for the fourth formate dehydrogenase mutant, expression vector for the fifth formate dehydrogenase mutant Expression vector for the 6th formate dehydrogenase mutant, and expression vector for the 7th formate dehydrogenase mutant. Expression vector for the 8th formate dehydrogenase mutant, and expression vector for the 9th formate dehydrogenase mutant. Expression vector, expression vector for formate dehydrogenase mutant 10, and formate dehydrogenase mutant 11 expression vector for the 12th formate dehydrogenase mutant, expression vector for the 13th formate dehydrogenase mutant Expression vectors for variants, expression vectors for variants of formate dehydrogenase XIV and variants of formate dehydrogenase XV An expression vector for the mutant enzyme is obtained. Step 2: Constructing host cells that are transformed or transfected with the expression vector. Build. In the examples of the present application, the E. coli BL21(DE3) strain (hereinafter referred to as E. coli BL21 (DE3)) as an expression host (i.e., host cell), and we successfully analyzed the base sequence. The pET22b plasmid was transfected into E. coli BL21(DE3). The recombinant mutant expression strain E. coli BL21(DE3) / pET22b-AqFDH Build. Specifically, the purified gene fragment was digested with EasyCut endonuclease-DpnI. The template was removed by PCR, and then the recombinant product was recombined with recombinase. H5α receptor cells were transformed with 100 μg / mL ampicillin ), and cultured at 37°C for 14 hours. Single colonies were collected from the LB liquid medium. The culture was then incubated at 37°C for 16 hours with shaking at 220 rpm. Sterile glycerol was added to a final glycerol concentration of 25%, and then the numbered 8 Store at 0°C and prepare the recombinant mutant plasmid clone strain E. coli DH5α / p ET22b-AqFDH was obtained, and a portion of the bacterial solution was centrifuged at 8,000 rpm for 3 minutes to separate the cells. and extracted into E. coli DH5α / pET2 using a high-purity plasmid mini extraction kit. Extract the plasmid from 2b-AqFDH and confirm the accuracy of the mutation site by sequencing. did. Finally, the recombinant mutant plasmid clone strains of the first formate dehydrogenase mutant to the second formate dehydrogenase mutant were prepared. Recombinant mutant plasmid clone strains of 15 formate dehydrogenase mutants and the corresponding recombinant mutants Obtaining the plasmids, i.e., transfectants carrying the first formate dehydrogenase mutants, respectively The host cells were transfected with an expression vector expressing the second formate dehydrogenase mutant. The host cells of the present vector, the expression vector transfectant for the third formate dehydrogenase mutant, The host cells were transfected with the fourth formate dehydrogenase mutant. Host cells, transfectants of expression vectors expressing the fifth formate dehydrogenase mutant host cells transfected with an expression vector encoding the sixth formate dehydrogenase mutant, 7. Transfectant host cells containing expression vectors expressing formate dehydrogenase mutants. Transfectant host cells expressing the 9th formate dehydrogenase mutant Transfectant host cells with expression vectors expressing mutant formate dehydrogenase 10 Transfectant host cells with expression vectors expressing mutant formate dehydrogenase 11 Transfectant host cells with expression vectors containing the 12th formate dehydrogenase mutant Transfectant host cells expressing the 13th formate dehydrogenase mutant. The host cells were transfected with the 14th formate dehydrogenase mutant. Transfectant expression vectors in host cells and transfectant 15 formate dehydrogenase mutant Obtain host cells for the fectant expression vector. The examples of the present application show the use of an expression vector transfectant containing the 15th formate dehydrogenase mutant. Host cells for the vector (hereafter referred to as Escherichia coli NJUXR-FX-1) The above Escherichia coli (E. coli) NJUXR-FX-1 is a typical Chinese culture. It is stored in the Conservation Center at Wuhan University, Wuhan, China, and the storage date is 2025. The date is January 13th, and the storage number given by the storage unit is CCTCC NO:M 20 25101, and the above-mentioned Escherichia coli (E. coli) NJUXR-FX-1 (biological materials and The taxonomic name of the strain (also called Escherichia coli) is NJUXR-FX-1. The Latin name of this taxonomic name is Escherichia coli NJUXR- It's the FX-1. Example 3: The method for preparing the formate dehydrogenase mutant described in this example involves the use of recombinant mutant proteins. The method includes constructing a protein-expressing strain and obtaining a culture containing the formate dehydrogenase mutant. In the examples of this application, the pET22b plasmid, whose base sequence was successfully analyzed, was used in E. coli. i BL21(DE3) was transfected as an expression host (i.e., host cell). , recombinant mutant protein expression strain E. coli BL21(DE3) / pET22b-A qFDH was constructed. The above preparation method will be described in detail below. The successfully constructed recombinant mutant plasmids were cultured in E. coli BL21(DE3) recipient cells. The transformants were then plated onto ampicillin plates with a final concentration of 100 μg / mL, and single clones were then cultured. The cells were then excised and placed in a culture tube containing 5 mL of LB liquid medium containing 100 μg / mL ampicillin. The mixture was inoculated and cultured at 37°C and 220 rpm for 16 hours with shaking to express the recombinant mutant protein. The strain E. coli BL21(DE3) / pET22b-AqFDH was obtained. Transfer the seed volume to 500 mL of antibiotic LB medium containing 100 μg / mL ampicillin, and measure the OD 60 When the pH reached approximately 0.6, add 1 mL of 0.5 M IPTG (i.e., isopropyl-β- D-thiogalactopyranoside) was added to the medium to make the final concentration of IPTG 0.5 mM, and the mixture was incubated at 18°C. The culture was then induced for approximately 16 hours under these conditions to obtain a culture containing the formate dehydrogenase mutant. Finally, the cultures containing the 1st to 15th formate dehydrogenase mutants were prepared. A culture containing After induction, the cells were collected by centrifugation, resuspended in buffer, and then sonicated (5 min) in an ice bath. 2 seconds operation every 30 minutes, 4°C, 12,000 rpm / min, 20 The supernatant was collected and filtered through a 0.22 μm aqueous filter tip. The enzyme was purified by passing it through a nickel column. Calculate the molar extinction coefficient of the protein and use the A 280 Protein by method By measuring the absorbance of the protein (wild-type formate dehydrogenase and mutant formate dehydrogenase), The concentration of the heterogeneous substance (a general term for heterogeneous substances) can be calculated. Example 4: The examples of the present application are based on the formate dehydrogenase mutants provided in Example 1, Nucleic acids of formate dehydrogenase mutants provided by Example 2, nucleic acids provided by Example 2 the expression vector of Example 2 or the host cell of Example 3 The catalytic activity of the culture prepared by the method for reducing oxidized coenzyme I to reduced coenzyme I is It provides applications in sexual enhancement. In one embodiment, the culture containing the first to fifteenth formate dehydrogenase mutants obtained in Example 3 is From the culture containing the acid dehydrogenase mutant, NAD + To obtain a catalyst that catalyzes the reduction of The catalyst may be a whole cell or a pure enzyme solution. In one application scenario, the third formate dehydrogenase mutant (also called the M334I mutant), 8 formate dehydrogenase mutant (also called K185S / M334I mutant), 6 formate dehydration Formate dehydrogenase mutant (also called N24D / M334I mutant), formate dehydrogenase 12 mutant (also called the N24D / K185S / V377T mutant) and formate dehydrogenase XV For example, let's take the example of the N24D / K185S / M334I / V377T mutant. If you take it, NAD + The reaction system that catalyzes the reduction of to NADH is as follows: 0.5mg / ml formate dehydrogenase mutant or OD 600 = 0.1 of recombinant mutant expression strains Whole cell bacterial solution, 1mM NAD + , 5 mM HCOONa, reaction buffer 100 mM, pH = 7 potassium dihydrogen phosphate / dipotassium hydrogen phosphate buffer solution. After 10 minutes of reaction, The change in absorbance of the product NADH at 340 nm was measured using an enzyme marker, and the relative activity was compared. The results of the comparison are shown in Figure 2, and are compared with wild-type formate dehydrogenase (aqFDH (WT) in Figure 2). In comparison with the 3rd formate dehydrogenase mutant, the 8th formate dehydrogenase mutant, and the 6th formate dehydrogenase mutant, In the hydrogenase mutant, the formate dehydrogenase 12 mutant, and the formate dehydrogenase 15 mutant, NAD + The efficiency of catalytic reduction of β-amycin to NADH was significantly improved. Here, the results of catalytic reactions using whole cell bacterial suspension are shown in Table 5. Formate dehydrogenase The results of catalysis carried out via pure enzyme solutions of the mutants are shown in Table 6. Table 5: Catalytic reaction results for whole cell bacterial suspension JPEG2025171967000005.jpg182129 Table 6: Catalytic reaction results of formate dehydrogenase mutants JPEG2025171967000006.jpg182129 As can be seen from Tables 5 and 6, the catalytic conversion rates of the mutants were all higher than those of the wild-type formate dehydrogenase. In particular, the formate dehydrogenase 15 mutant (N24D / K184S / M3 The catalytic efficiency of the mutant (also called the 34I / V377T mutant) is significantly higher than that of wild-type formate dehydrogenase. From the above, it can be seen that the formate dehydrogenase mutant obtained in the examples of the present application The variant is NAD + to NADH is higher than that of wild-type formate dehydrogenase. , improve the production efficiency of high-value-added chemicals that depend on the NADH regeneration circulation system, and This can reduce the production costs of valuable chemicals. Furthermore, the catalytic conditions for the above catalytic reactions are discussed. (1) Formate dehydrogenase mutants are NAD + Determine the preferred temperature for catalyzing the reduction of NADH. Determine. Using the whole cells or a pure enzyme solution of the formate dehydrogenase mutant obtained in Example 2 as a catalyst, Formate dehydrogenase mutants NAD + To determine the favorable temperature for the catalytic reduction of β-glucan to NADH, Sticky. Taking the 15th formate dehydrogenase mutant as an example, the reaction system is as follows: final concentration 0.0 0.5mg / ml protein or OD 600 =0.1 whole cell suspension, 1mM NAD + , 5 mM HCOONa, reaction buffer was 100 mM potassium dihydrogen phosphate / phosphate buffer, pH 7 The reaction temperature was adjusted to 20℃, 25℃, 30℃, and 35℃ using a water bath. The temperature was controlled at 40℃, 45℃, 50℃, and 55℃, and the enzyme marker was used to measure the product NADH at 340℃. The absorbance values ​​at 50°C were detected and the relative activity was compared. The results are shown in Figure 3. A significant catalytic effect was observed, and the enzyme activity was good in the range of 25 to 55°C. According to the study, temperature differences significantly affected the catalytic activity of different mutants, and their reaction activity It was shown that the temperature distribution tends to be biased towards a normal distribution. A significant effect was observed, and outside this temperature the enzyme activity was relatively low. (1) Formate dehydrogenase mutants are NAD + The preferred pH value for catalytic reduction of to NADH Make a decision. Using the whole cells or a pure enzyme solution of the formate dehydrogenase mutant obtained in Example 2 as a catalyst, Formate dehydrogenase mutants NAD + The preferred pH value for the catalytic reduction of NADH I looked it up. Specifically, taking the 15th formate dehydrogenase mutant as an example, the reaction system of the above catalytic reaction is as follows: Final concentration OD 600 =0.1 whole cell suspension, 1mM NAD + , 5mM HC OONa, reaction buffer is 100 mM, pH = 7 potassium dihydrogen phosphate / dibasic potassium phosphate The reaction buffer solutions are buffers of different pH, i.e., pH is 100 mM potassium dihydrogen phosphate / dihydrogen phosphate, which are 6, 6.5, 7, 7.5, and 8, respectively. Potassium buffer (KPi) with pH values ​​of 8, 8.5, 9, and 9.5, respectively, 100 mM Tris-HCl buffer, pH 10.25, glycine-sodium hydroxide ( The reaction temperature was controlled at 30°C, and the product N was measured using an enzyme marker. The absorbance value of ADH at 340 nm was detected, and the results of comparing the relative activity are shown in Figure 4. The effect of different pH levels on the catalytic activity of formate dehydrogenase is small. , the optimum catalytic effect was observed. Experiments have shown that different buffer solutions have different effects on enzyme activity, and potassium dihydrogen phosphate When dipotassium hydrogen phosphate buffer is used as the reaction system, the enzyme activity is maintained at a high level. When Tris-HCl buffer was used as the reaction system, the wild-type enzyme and the mutant enzyme were activated at pH 9.5. The sex is at its maximum. (2) Formate dehydrogenase mutants are NAD + Favorable kinetics in the catalytic reduction of NADH to Determine the parameters. Based on the reaction system of the above catalytic reaction, the NADH concentration of wild-type formate dehydrogenase and its mutants was calculated as follows: The reaction rate is calculated by the double inverse graph method from the reaction rate and the reciprocal of the substrate concentration. The theoretical parameters were calculated and the results are shown in Table 7. Compared with wild-type formate dehydrogenase, 15 Formate dehydrogenase mutants showed a decreased Km and an increased Kcat, and the mutant catalytic activity was significantly increased. It was shown to have risen. Table 7: Kinetic parameters table JPEG2025171967000007.jpg182129 Note: Km is the Michaelis constant and r is the reciprocal is the reaction rate, and Kcat is the catalytic constant. <Copy of receipt of deposit of microorganism> JPEG2025171967000008.jpg181129[Sequence table] <st26sequencelisting originalfreetextlanguagecode="ja" dtdversion="V1_3" filenam e="ギ酸脱水素酵素変異体およびその用途.xml" softwarename="WIPO Sequence" software version="2.3.0" productiondate="2025-03-31"> <applicationidentification> <ipofficecode> JP< / ipofficecode> <applicationnumbertext / > <filingdate / > < / applicationidentification> <applicantfilereference> 10284< / applicantfilereference> <earliestpriorityapplicationidentification> <ipofficecode> CN< / ipofficecode> <applicationnumbertext> 202410562403.9< / applicationnumbertext> <filingdate> 2024-05-08< / filingdate> < / earliestpriorityapplicationidentification> <applicantname languagecode="ja"> Nanjing University< / applicantname> <applicantnamelatin> Nanjing 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DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALTAGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVKHSYDLEAMSVGTVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYIVNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHMSGTSLTAQTRYAAGTREILECFFEGRPIRDEYLIVQGGNLAGVGAHSYSKGNATGGSEEAGKFKKA G < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="2"> <insdseq> <INSDSeq_length>401< / INSDSeq_length> <INSDSeq_moltype>AA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..401< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q4"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> MAKVLCVLYDDPIDGYPTTYARDDLPKIDHYPGGQTLPTPKAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALTAGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVKHSYDLEAMSVGTVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYIVNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHMSGTSLTAQTRYAAGTREILECFFEGRPIRDEYLIVQGGNLAGVGAHSYSKGNATGGSEEAGKFKKA G < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="3"> <insdseq> <INSDSeq_length>401< / INSDSeq_length> <INSDSeq_moltype>AA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..401< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q6"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> MAKVLCVLYDDPIDGYPTTYARDNLPKIDHYPGGQTLPTPKAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALTAGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVSHSYDLEAMSVGTVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYIVNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHMSGTSLTAQTRYAAGTREILECFFEGRPIRDEYLIVQGGNLAGVGAHSYSKGNATGGSEEAGKFKKA G < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="4"> <insdseq> <INSDSeq_length>401< / INSDSeq_length> <INSDSeq_moltype>AA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..401< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q8"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> MAKVLCVLYDDPIDGYPTTYARDNLPKIDHYPGGQTLPTPKAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALTAGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVKHSYDLEAMSVGTVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYIVNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHISGTSLTAQTRYAAGTREILECFFEGRPIRDEYLIVQGGNLAGVGAHSYSKGNATGGSEEAGKFKKA G < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="5"> <insdseq> <INSDSeq_length>401< / INSDSeq_length> <INSDSeq_moltype>AA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..401< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q10"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> MAKVLCVLYDDPIDGYPTTYARDNLPKIDHYPGGQTLPTPKAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALTAGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVKHSYDLEAMSVGTVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYIVNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHMSGTSLTAQTRYAAGTREILECFFEGRPIRDEYLIVQGGNLAGTGAHSYSKGNATGGSEEAGKFKKA G < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="6"> <insdseq> <INSDSeq_length>401< / INSDSeq_length> <INSDSeq_moltype>AA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..401< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q12"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> MAKVLCVLYDDPIDGYPTTYARDDLPKIDHYPGGQTLPTPKAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALTAGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVSHSYDLEAMSVGTVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYIVNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHMSGTSLTAQTRYAAGTREILECFFEGRPIRDEYLIVQGGNLAGVGAHSYSKGNATGGSEEAGKFKKA G < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="7"> <insdseq> <INSDSeq_length>401< / INSDSeq_length> <INSDSeq_moltype>AA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..401< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q14"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> MAKVLCVLYDDPIDGYPTTYARDDLPKIDHYPGGQTLPTPKAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALTAGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVKHSYDLEAMSVGTVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYIVNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHISGTSLTAQTRYAAGTREILECFFEGRPIRDEYLIVQGGNLAGVGAHSYSKGNATGGSEEAGKFKKA G < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="8"> <insdseq> <INSDSeq_length>401< / INSDSeq_length> <INSDSeq_moltype>AA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..401< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q16"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> MAKVLCVLYDDPIDGYPTTYARDDLPKIDHYPGGQTLPTPKAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALTAGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVKHSYDLEAMSVGTVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYIVNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHMSGTSLTAQTRYAAGTREILECFFEGRPIRDEYLIVQGGNLAGTGAHSYSKGNATGGSEEAGKFKKA G < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="9"> <insdseq> <INSDSeq_length>401< / INSDSeq_length> <INSDSeq_moltype>AA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..401< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q18"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> MAKVLCVLYDDPIDGYPTTYARDNLPKIDHYPGGQTLPTPKAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALTAGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVSHSYDLEAMSVGTVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYIVNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHISGTSLTAQTRYAAGTREILECFFEGRPIRDEYLIVQGGNLAGVGAHSYSKGNATGGSEEAGKFKKA G < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="10"> <insdseq> <INSDSeq_length>401< / INSDSeq_length> <INSDSeq_moltype>AA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..401< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q20"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> MAKVLCVLYDDPIDGYPTTYARDNLPKIDHYPGGQTLPTPKAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALTAGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVSHSYDLEAMSVGTVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYIVNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHMSGTSLTAQTRYAAGTREILECFFEGRPIRDEYLIVQGGNLAGTGAHSYSKGNATGGSEEAGKFKKA G < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="11"> <insdseq> <INSDSeq_length>401< / INSDSeq_length> <INSDSeq_moltype>AA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..401< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q22"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> MAKVLCVLYDDPIDGYPTTYARDNLPKIDHYPGGQTLPTPKAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALTAGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVKHSYDLEAMSVGTVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYIVNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHISGTSLTAQTRYAAGTREILECFFEGRPIRDEYLIVQGGNLAGTGAHSYSKGNATGGSEEAGKFKKA G < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="12"> <insdseq> <INSDSeq_length>401< / INSDSeq_length> <INSDSeq_moltype>AA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..401< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q24"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> MAKVLCVLYDDPIDGYPTTYARDDLPKIDHYPGGQTLPTPKAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALTAGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVSHSYDLEAMSVGTVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYIVNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHISGTSLTAQTRYAAGTREILECFFEGRPIRDEYLIVQGGNLAGVGAHSYSKGNATGGSEEAGKFKKA G < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="13"> <insdseq> <INSDSeq_length>401< / INSDSeq_length> <INSDSeq_moltype>AA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..401< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q26"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> MAKVLCVLYDDPIDGYPTTYARDDLPKIDHYPGGQTLPTPKAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALTAGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVSHSYDLEAMSVGTVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYIVNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHMSGTSLTAQTRYAAGTREILECFFEGRPIRDEYLIVQGGNLAGTGAHSYSKGNATGGSEEAGKFKKA G < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="14"> <insdseq> <INSDSeq_length>401< / INSDSeq_length> <INSDSeq_moltype>AA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..401< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q28"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> MAKVLCVLYDDPIDGYPTTYARDNLPKIDHYPGGQTLPTPKAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALTAGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVSHSYDLEAMSVGTVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYIVNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHISGTSLTAQTRYAAGTREILECFFEGRPIRDEYLIVQGGNLAGTGAHSYSKGNATGGSEEAGKFKKA G < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="15"> <insdseq> <INSDSeq_length>401< / INSDSeq_length> <INSDSeq_moltype>AA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..401< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q30"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> MAKVLCVLYDDPIDGYPTTYARDDLPKIDHYPGGQTLPTPKAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALTAGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVKHSYDLEAMSVGTVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYIVNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHISGTSLTAQTRYAAGTREILECFFEGRPIRDEYLIVQGGNLAGTGAHSYSKGNATGGSEEAGKFKKA G < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="16"> <insdseq> <INSDSeq_length>401< / INSDSeq_length> <INSDSeq_moltype>AA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..401< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q32"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> MAKVLCVLYDDPIDGYPTTYARDDLPKIDHYPGGQTLPTPKAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALTAGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVSHSYDLEAMSVGTVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYIVNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHISGTSLTAQTRYAAGTREILECFFEGRPIRDEYLIVQGGNLAGTGAHSYSKGNATGGSEEAGKFKKA G < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="17"> <insdseq> <INSDSeq_length>1212< / INSDSeq_length> <INSDSeq_moltype>DNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..1212< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other DNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q34"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> atggcgaaagttctgtgcgttctgtacgatgatccgatcgatggttacccgaccacctacgcgcgtgacaacctgccgaa aatcgaccactatccgggtggtcagaccctgccgaccccgaaagcgatcgatttcactccgggcaccatgctgggttctg tttctggtgaactgggcctgcgtaaatacctggaaagcaacggtcacaccctggttgttacctctgataaagatggtccg gattctgttttcgaaaaagaattggttgatgcggatattgttatcagccagccgttctggccggcttacctgaccccgga acgcttcgctaaagctaaaaacctgaaactggctctgaccgcgggcatcggctctgatcacgttgacctgcaaagcgcaa ttgatcgtggtgttaccgttgcggaagtgacctactgcaactctatcagcgtggcggaacacgttgtgatgatgatcctg ggcctggttcgtaactacctgccggcgcacgactgggcgcgtaaaggtggctggaacatcgcagattgcgtgaaacactc ttacgatctggaagcgatgtctgttggtactgtggcggcgggccgcatcggtctggcggtgctgcgccgcctggctccgt tcgacgtgaaattacactataccgaccgtcaccgcctgccggaaagcgttgaaaaagaactgaacctgacctggcacgct tctccgaccgatatgtacccgcactgcgacgtggttaccctgaactgcccgctgcacccggaaaccgaacacatggttaa cgaagaaaccctgaaactgttcaaacgtggtgcgtacatcgttaacaccgcgcgtggtaaactgtgcgaccgtgatgcga tcgcgcgcgcgctggaaaacggcaccctggccggttatgcgggcgatgtttggttcccgcagccggcgccggctgatcac ccgtggcgtactatggcatggaacggcatgaccccgcacatgagcggcactagcctgaccgcgcagacccgttatgctgc gggcacccgtgaaatcctggaatgcttctttgaaggccgtccgatccgtgatgaatacctgatcgttcagggcggtaacc tggcgggtgttggcgcacacagctactctaaaggcaacgctaccggtggttctgaagaagcgggtaaatttaaaaaagcg ggctaatctctc < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="18"> <insdseq> <INSDSeq_length> 28< / INSDSeq_length> <INSDSeq_moltype> DNA< / INSDSeq_moltype> <INSDSeq_division> PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..28< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other DNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q36"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> gcgcgtgacgacctgccgaaaatcgacc< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="19"> <insdseq> <INSDSeq_length>28< / INSDSeq_length> <INSDSeq_moltype>DNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..28< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other DNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q38"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>ggtcgattttcggcaggtcgtcacgcgc< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="20"> <insdseq> <INSDSeq_length>28< / INSDSeq_length> <INSDSeq_moltype>DNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..28< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other DNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q40"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>gcagattgcgtgagccactcttacgatc< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="21"> <insdseq> <INSDSeq_length>28< / INSDSeq_length> <INSDSeq_moltype>DNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..28< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other DNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q42"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>gatcgtaagagtggctcacgcaatctgc< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="22"> <insdseq> <INSDSeq_length>26< / INSDSeq_length> <INSDSeq_moltype>DNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..26< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other DNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q44"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>gaccccgcacatcagcggcactagcc< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="23"> <insdseq> <INSDSeq_length>26< / INSDSeq_length> <INSDSeq_moltype>DNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..26< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other DNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q46"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>ggctagtgccgctgatgtgcggggtc< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="24"> <insdseq> <INSDSeq_length>24< / INSDSeq_length> <INSDSeq_moltype>DNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..24< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other DNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q48"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>ctggcgggtactggcgcacacagc< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="25"> <insdseq> <INSDSeq_length>25< / INSDSeq_length> <INSDSeq_moltype>DNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..25< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other DNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q50"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>gctgtgtgcgccagtacccgccagg< / INSDSeq_sequence> < / insdseq> < / sequencedata> < / inventiontitle> < / st26sequencelisting>

[0007]

Claims

1. A formate dehydrogenase mutant, The amino acid sequence of the formate dehydrogenase mutant is the same as that of the wild-type formate dehydrogenase shown in SEQ ID NO:

1. The amino acid sequence of the dehydrogenase revealed at least N24D, K185S, M334I, and V377T. A formate dehydrogenase mutant characterized in that it is obtained by mutation with either one of the following:

2. A nucleic acid encoding the formate dehydrogenase mutant of claim 1.

3. An expression vector comprising the nucleic acid of claim 2.

4. A host cell transformed or transfected with the expression vector of claim 3.

5. A method for preparing the formate dehydrogenase mutant of claim 1, comprising: and obtaining a culture containing the formate dehydrogenase mutant. Method for preparing formate dehydrogenase mutants.

6. The formate dehydrogenase mutant according to claim 1, the nucleic acid according to claim 2, and the expression method according to claim 3. A vector, a host cell according to claim 4, or a preparation method according to claim 5. Use of a culture in improving the catalytic activity of reducing oxidized coenzyme I to reduced coenzyme I.

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