Novel malate dehydrogenase

Abstract

To provide a novel malate dehydrogenase.SOLUTION: There is provided a malate dehydrogenase having an amino acid sequence X1X2RX3X4GX5X6RX7DLX8 in an active center loop (where, X1 is V, M, L, I or A; X2 is P or A; X3 is K or R; X4 is P, E, D or A; X5 is a hydrophobic amino acid other than methionine; X6 is T, E, D or S; X 7 is D, K, S or R; X8 is F, L or V), the malate dehydrogenase having a higher malate dehydrogenase activity compared with a malate dehydrogenase which has the same sequence except that the hydrophobic amino acid X5 contained in the active center loop is methionine, and the sequence identity with the amino acid sequence of SEQ ID NO:1 being 90% or less.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present application relates to a novel malate dehydrogenase. [Background technology]

[0002] Malate dehydrogenase (MDH) is a NAD + In the presence of NADH, it reversibly converts malate and oxaloacetate. + Since MDH and NADH have different wavelengths of UV absorption peaks, the enzyme reaction of MDH can be easily detected by a spectrophotometer. Therefore, MDH is used in tests for the amount of aspartate aminotransferase (AST) to check for heart and liver dysfunction. This is because NADH and NAD are converted when oxaloacetate produced by AST in the sample is converted to malate by MDH. + This detection method is called the MDH-UV method. Since the activity and stability of MDH contained in AST test reagents are related to the quality of the test reagents, research is being conducted on stable thermophilic bacterium-derived MDH and highly active mutants (Patent Document 1, Non-Patent Document 1).

[0003] MDH is known to function in the TCA cycle in eukaryotes and in the glyoxylate cycle in plants and yeast, and three isozymes (MDH1-3) have been reported in budding yeast. In budding yeast, MDH1 is localized in mitochondria, MDH2 in the cytosol, and MDH3 in glyoxysomes. MDH1 functions in the TCA cycle and MDH2 in the glyoxylate cycle. Meanwhile, MDH3 converts NADH, which is necessary for the β-oxidation of fatty acids, to NAD. +It is known that MDH plays a role in reoxidizing oxaloacetate and has a lower affinity for oxaloacetate than MDH1 and MDH2 (Non-Patent Document 2). Many complex structures with coenzymes and substrates have been determined for MDH localized in the cytosol or mitochondria, and their reaction mechanisms have been elucidated (Non-Patent Documents 3-5), whereas only the structures of the Apo type and citrate-bound type of MDH from Watermelon have been reported for MDH localized in glyoxysomes. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special table 2013-503631 [Non-patent literature]

[0005] [Non-Patent Document 1] Nishiyama M et al., Biochem Biophys Res Commun. 1996 Aug 23; 225(3); 844-848 [Non-Patent Document 2] Steffan JS, McAlister-Henn L. J Biol Chem. 1992 Dec 5(34): 267 [Non-Patent Document 3] P. Minarik, N. Tomaskova, Gen Physiol Biophys. 2002 Sep; 21(3): 257-65. [Non-Patent Document 4] Goward CR, Nicholls DJ. Protein Sci. 1994 Oct; 3(10): 1883-8. [Non-Patent Document 5] Bell JK et al., J Biol Chem. 2001 Aug 17; 276(33): 31156-62 [Non-Patent Document 6] Moriyama S, Nishio K and Mizushima T, Acta Cryst. 2018; F74: 617-624 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present application is to provide a malate dehydrogenase obtained by a method for highly activating malate dehydrogenase. [Means for solving the problem]

[0007] The present invention was completed by finding a method for highly activating malate dehydrogenase. The method provided in the present application is not limited to a specific malate dehydrogenase, and can be used to prepare highly active mutants of various malate dehydrogenases. In other words, the present application provides malate dehydrogenase obtained by the method for highly activating malate dehydrogenase.

[0008] It is known that budding yeast MDH3 has a lower affinity for oxaloacetate than budding yeast MDH1 and MDH2. The present inventors measured the malate dehydrogenase activity of budding yeast MDH2 and MDH3 and found that MDH3 has a much higher malate dehydrogenase activity than MDH2 (see Reference Example 1, FIG. 2).

[0009] In general, it has been reported that the active center loop of MDH changes from an open state to a closed state when oxaloacetate, malate, or a dicarboxylic acid molecule similar to these binds (Non-Patent Document 4). The active center loop of budding yeast MDH3 is composed of amino acid residues Val79-Phe90, and Arg86-Phe91 forms an α-helix structure. The present inventors have investigated the apo form of budding yeast MDH3 and NAD + The structures of the / OAA-bound MDH3 and the two were compared, but no structural change in the active center was observed, indicating that the active center loop in the budding yeast MDH3 remains open.

[0010] We compared the sequences of the active center loop of budding yeast MDH1 and 2 with those of budding yeast MDH3, and identified L84 (M110 in MDH2) in MDH3 as a site that is conserved in MDH1 and MDH2 but not in MDH3, and M92 (V118 in MDH2) in MDH3 as a site located at the hinge of the conformational change of the active center loop, with low amino acid homology, and with a side chain close to L84 and expected to come into contact with it. We created mutants in budding yeast MDH2 and MDH3 in which these sites were mutated to the corresponding amino acid residues of the other, and examined the effect on malate dehydrogenase activity.

[0011] The activities of the budding yeast MDH3 L84M mutant and the MDH3 L84M / M92V mutant were measured (see Reference Example 2, FIG. 3). The activities of the two MDH3 mutants were lower than that of wild-type MDH3 and similar to that of wild-type MDH2. Next, the activities of the budding yeast MDH2 M110L mutant and the MDH2 M110L / V118M mutant were measured (see Example 1, FIG. 4). The activities of the two MDH2 mutants were higher than that of wild-type MDH2 and similar to that of wild-type MDH3.

[0012] These results indicate that by mutually substituting the above two sites in budding yeast MDH2 and MDH3, malate dehydrogenase activity similar to that of the other is exhibited. The amino acid corresponding to M110 in MDH2 is conserved as methionine in a great many species except for budding yeast MDH3. Therefore, it was predicted that highly active MDHs could be produced by substituting the methionine in various malate dehydrogenases with hydrophobic amino acids such as leucine.

[0013] To clarify whether the properties of the two residues identified in budding yeast MDH are conserved in species other than budding yeast, we used Thermoplasma acidophilum (TA) MDH (TA-MDH; aa1-325), Geobacillus stearothermophilus (GS) MDH (GS-MDH; aa1-312), and Thermus thermophilus (TT) MDH (TT-MDH; aa1-327), which have low amino acid homologies with budding yeast MDH3, respectively, at about 25%, 29%, and 25%. First, we measured the activity of the TA-MDH M92L / K100M mutant, in which mutations were introduced into M92 and K100 of TA-MDH, which correspond to L84 and M92 of budding yeast MDH3 (see Example 2, Figure 5). As a result, the TA-MDH M92L / K100M mutant showed malate dehydrogenase activity more than twice as high as that of wild-type TA-MDH. Similarly, the activity of the GS-MDH M91L / T99M mutant, in which mutations were introduced into M91 and T99 of GS-MDH corresponding to L84 and M92 of budding yeast MDH3, was measured (see Example 3, FIG. 6). As a result, the GS-MDH M91L / T99M mutant showed malate dehydrogenase activity more than twice as high as that of wild-type GS-MDH. Furthermore, the activity of the TT-MDH M96L / V104M mutant, in which mutations were introduced into M96 and V104 of TT-MDH corresponding to L84 and M92 of budding yeast MDH3, was measured (see Example 4, FIG. 7). As a result, the TT-MDH M96L / V104M mutant showed malate dehydrogenase activity more than twice as high as that of wild-type TT-MDH. These results suggest that highly active MDH can be generated by mutating the conserved methionine in various MDHs to a hydrophobic amino acid such as leucine.

[0014] That is, the present application includes the following inventions. In one aspect, the present application provides: a malate dehydrogenase having an amino acid sequence of X1X2RX3X4GX5X6RX7DLX8 (wherein X1 is V, M, L, I or A, X2 is P or A, X3 is K or R, X4 is P, E, D or A, X5 is a hydrophobic amino acid other than methionine, X6 is T, E, D or S, X7 is D, K, S or R, and X8 is F, L or V) in an active center loop; It has a higher malate dehydrogenase activity than a malate dehydrogenase having the same sequence as that of the malate dehydrogenase having the same sequence except that the hydrophobic amino acid X5 contained in the active center loop is methionine, and The present invention provides a malate dehydrogenase having a sequence identity of 90% or less with the amino acid sequence of SEQ ID NO:1.

[0015] In another aspect, the present application provides: (1) an amino acid sequence of SEQ ID NO: 9, 10, 15, or 16, wherein Xaa is a hydrophobic amino acid other than methionine; (2) an amino acid sequence having malate dehydrogenase activity, in which 1 to 30 amino acids other than the amino acid sequence of the active center loop in the amino acid sequence of SEQ ID NO: 9, 10, 15, or 16 are deleted, added, and / or substituted; or (3) An amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 9, 10, 15 or 16, an amino acid sequence having the same amino acid sequence of the active center loop, and having malate dehydrogenase activity; The present invention provides a malate dehydrogenase having a higher malate dehydrogenase activity than a malate dehydrogenase having the same sequence except that Xaa is methionine. Effect of the Invention

[0016] The present application provides a malate dehydrogenase obtained by a method for highly activating malate dehydrogenase. [Brief description of the drawings]

[0017] [Figure 1]Comparison of amino acid sequences of the active center loop of various malate dehydrogenases (MDH). MDH3, Saccharomyces cerevisiae MDH3; Cl_gMDH, Citrullus lanatus glyoxysome-localized MDH; MDH1, Saccharomyces cerevisiae MDH1; Cl_mMDH, Citrullus lanatus mitochondrial-localized MDH; Hs_mMDH, human mitochondrial-localized MDH (human MDH2); Ss_mMDH, Sus scrofa mitochondrial-localized MDH (porcine MDH2); MDH2, Saccharomyces cerevisiae MDH2; Hs_cMDH, human cytosol-localized MDH (human MDH1); Ss_cMDH, Sus scrofa cytosol-localized MDH (porcine MDH1); Ec_MDH, Escherichia coli MDH. [Diagram 2] Results of activity measurement of wild-type MDH2 and MDH3 in budding yeast. The horizontal axis is the OAA concentration in the sample, and the vertical axis is ΔO.D. / min. Error bars indicate standard deviation (±SD). [Diagram 3] Results of activity measurement of budding yeast MDH3 mutants. The activities of the two MDH3 mutants are lower than that of wild-type MDH3 and similar to that of wild-type MDH2. The horizontal axis is the OAA concentration in the sample, and the vertical axis is ΔO.D. / min. Error bars indicate standard deviation (±SD). [Figure 4] Results of activity measurement of budding yeast MDH2 mutants. The activities of the two MDH2 mutants are higher than that of wild-type MDH2 and similar to that of wild-type MDH3. The horizontal axis is the OAA concentration in the sample, and the vertical axis is ΔO.D. / min. Error bars indicate standard deviation (±SD). [Diagram 5] Results of activity measurement of wild-type TA-MDH and TA-MDH mutant. The activity of the TA-MDH mutant is more than twice as high as that of wild-type MDH. The horizontal axis is the OAA concentration in the sample, and the vertical axis is ΔO.D. / min. Error bars indicate standard deviation (±SD). [Figure 6] Results of activity measurement of wild-type GS-MDH and GS-MDH mutant. Vmax (ΔO.D. / min) of GS-MDH mutant is 4 times higher than that of wild-type MDH. The horizontal axis is OAA concentration in the sample, and the vertical axis is ΔO.D. / min. Error bars indicate standard deviation (±SD). [Figure 7] Results of activity measurement of wild-type TT-MDH and TT-MDH mutant. Vmax (ΔO.D. / min) of TT-MDH mutant is more than four times higher than that of wild-type MDH. The horizontal axis is OAA concentration in the sample, and the vertical axis is ΔO.D. / min. Error bars indicate standard deviation (±SD). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] In this disclosure, when a numerical value is accompanied by the term "about," it is intended to include a range of ±10% of that value. For example, "about 20" is intended to include "18-22." A range of numerical values ​​includes all values ​​between and at the endpoints. "About" in reference to a range applies to both endpoints of the range. Thus, for example, "about 20-30" is intended to include "18-33."

[0019] In this disclosure, amino acid residues are represented by the following abbreviations: Ala or A: Alanine Arg or R: Arginine Asn or N: Asparagine Asp or D: Aspartic acid Cys or C: Cysteine Gln or Q: Glutamine Glu or E: glutamic acid Gly or G: glycine His or H: histidine Ile or I: Isoleucine Leu or L: leucine Lys or K: Lysine Met or M: methionine Phe or F: Phenylalanine Pro or P: Proline Ser or S: serine Thr or T: Threonine Trp or W: Tryptophan Tyr or Y: Tyrosine Val or V: Valine

[0020] In the present disclosure, "comprising" a certain amino acid sequence means that one or more amino acid residues may be added to the N-terminus and / or C-terminus of the amino acid sequence. For example, a "peptide comprising the amino acid sequence of SEQ ID NO:X" includes a peptide consisting of the amino acid sequence of SEQ ID NO:X and a peptide having one or more amino acid residues added to the N-terminus and / or C-terminus of the amino acid sequence of SEQ ID NO:X.

[0021] Malate dehydrogenase In the present application, malate dehydrogenase (MDH) is classified into EC 1.1.1.37 in the enzyme classification, and is an NAD + or NADH. The amino acid sequences of Saccharomyces cerevisiae MDH3 and Saccharomyces cerevisiae MDH2 are shown in SEQ ID NOs: 1 and 2, respectively. The amino acid sequences of Thermoplasma acidophilum (TA) MDH (TA-MDH; aa1-325), Geobacillus stearothermophilus (GS) MDH (GS-MDH; aa1-312), and Thermus thermophilus (TT) MDH (TT-MDH; aa1-327) are shown in SEQ ID NOs: 3, 11, and 12, respectively.

[0022] Examples of MDHs having an amino acid sequence X1X2RX3X4GMX6RX7DLX8 (wherein X1 is V, M, L, I, or A, X2 is P or A, X3 is K or R, X4 is P, E, D, or A, X6 is T, E, D, or S, X7 is D, K, S, or R, and X8 is F, L, or V) in the active center loop, or a sequence similar thereto, include those described under the following Uniprot accession numbers: P58407, B7VID0, B2SMP6, Q5WU94, P17783, Q1QQR2, Q82WB9, B4F2A1, Q1Q932, Q8ZVB2, and A1S. RP8, Q0BAF9, B5ZSS0, Q9EYJ6, A1KI28, A3MBR2, Q0S365, A3P7Q9, C1B155, Q2 J3G8, B9KNB4, A1W9K7, Q42972, B5R0N2, A4FFX3, B5BGR3, Q0HZ38, Q46YU4, B8 CSY7, Q8CQ25, C1CY73, A0A0S3QTC6, A1TP96, B1JMK1, B2IG85, B1Y8A3, Q6ZZC6, Q3AS98, B3PHI3, B0T6C1, Q8TSH7, Q9LKA3, Q5NVR2, Q9SML8, Q92IA0, B4TJ T3, B2FQL8, A8GRV2, O67581, Q67LB8, P61891, P10584, A7MWD3, C5BF98, Q5N YA9, Q74D53, Q64P62, Q3SVK5, B3Q761, B7UJW8, Q6F7X1, B3CM44, Q7CWK7, B9J CF5, Q87E35, B4U831, A9VJQ4, Q6MAA3, A4SWW0, Q32BA3, Q5SKV7, Q5HBC0, A8ILC6, Q72ZE5, Q4QL89, A6TEQ3, Q6FYD0, A1WV94, Q11CV9, P80039, B3QPY9, Q8 EPE2, A1R2B5, P37226, Q3IZ83, Q11QQ3, A9KFT9, Q9FSF0, B7M0U8, A4WF48, B 2K2N5, Q8PC25, Q7VFV4, Q9K849, Q2L068, P61975, Q0I491, Q13DR6, B7NKU9, Q 5U907, P61889, Q8YJE7, Q6D9D1, A4WNM7, A5UPY6, B7N0M1, Q7MP97, Q73G44, Q1R6A3, A4SIV0, B2SH29, P25077, A4J5N8, Q9ZP05, A7IBL9, Q5WEG2, Q8A0W0,<h2 style=";text-align:left;direction:ltr">Q7VW97, Q65G89, A0L113, Q59202, Q5R030, A8FG47, C0RFH2, A0PVV1, B3EEE5, B5REV7, Q07YA5, A8LJK6, Q14IT0, B0TCL9, Q89X59, Q97VN4, B8EB55, A9NDV1 Q8PVJ7, Q47C34, Q9K3J3, Q704B2, B7MBZ7, B7LHU4, P61892, Q0BXA0, B5XSQ7, B0BXA8, Q68WZ8, A5G320, B1XHK9, Q0HEW2, P61890, A5FHP3, A4TRK3, A9R58 4, Q5KWB7, C6E487, Q7VP41, A8G8Y7, Q47VL0, P11386, Q0AFK6, A1SMP3, Q3J7E7, A3PN14, Q6A6Z5, Q3ZZJ7, Q6G1M0, Q051U6, Q255I4, B9MBP0, Q87SU7, B7ND L4, Q7M9A7, Q5PAV3, Q5H496, Q1CEJ3, A1UQV8, Q57AX1, B8GVT2, Q2T7J2, P19446, B3R570, Q83Q04, B1I3T1, Q5X2T6, B0SN74, Q84FY8, Q9PK18, C3M9U0, Q9P E17, P61973, B1ZG93, A9M8R3, Q3B2R4, B3EP06, P50917, B2HRH5, Q3JKE9, A3NM97, Q169U9, Q8Z3E0, A5FS18, A3QB91, Q3Z9A4, O02640, B0CIT1, B2JQD2, P9 3819、O88989、A6WXE7、Q7MYW9、Q3IFH4、A0QCI6、B1K394、B3PQ91、A1RS58、B 2U705、Q393V1、A9AMD5、A2S105、Q62AG8、A1UZ10、P80536、Q13S42、Q7X3X5、Q 6L0C3、Q15YH0、A3MWU9、A0AZ43、O33525、C1AMN4、Q7UNC6、B6IYP5、Q2YAQ4、 B2T9P8、Q8XXW5、A5WGM2、Q5LXE1、Q8YP78、Q9CN86、Q6AW21、A1VRQ1、Q126N9、 A9MNX5, C0PZQ4, Q83C87, Q1LKG0, B1W3N4, C5BU70, Q30RQ5, B8GPC3, B9KZS7, Q2GCH6, Q21K60, Q07UX5, B2J5F8, A7HT37, Q3A5S0, A7NG29, C6DKH1, A8GWI0<h2 style=";text-align:left;direction:ltr">A1S3C4, Q7WS85, Q0T052, A0KG16, A9IZV5, ​​B0B7U5, P40926, P40925, Q6PAB3, Q0K8F5, B1KGG7, Q0TCN0, A0Q6K3, B7HFA6, B2GKC8, Q65T37, Q5YTI1, C3PN92 A0LFF8, Q979N9, C5CSI5, A8A545, Q8DEC2, Q2P736, C0R4Y0, B0TZT2, P0C7R5, Q3BWU8, C1EUT6, A0RJJ0, A9W386, B1LZN1, B2S881, P9WK12, A6Q388, A8GN8 2, B0TUH8, A8FRU0, Q47TT4, A7NC13, Q0VQ52, Q2GK85, O08349, A1K5Q9, A4YAE8, A7MNR3, B2UKY5, B7LRL0, C3L8X1, B7GGT8, B5EIU8, B9J092, Q54D04, P082 49、B9LLP6、Q9HJL5、B5YSW2、Q9X4K8、A4IRP9、A0L5T9、Q60B71、Q0AKT3、A8M AC1、Q7YRU4、B4TWK9、Q2S289、B0VQX5、B1IQP3、B1LGK2、Q5GT41、Q2GGI2、Q0R E66, A4G5Z9, P80458, B3QSH8, Q9Y7R8, P0C890, P48364, Q1BM38, Q4FQU7, B4EFB0, Q2K3E9, Q98EC4, P0A5J7, A3D075, Q31WA4, P17505, A3M928, A3N1U6, B9 JTS9、Q25QU7、P44427、B0BC10、Q5ZME2、Q2IK16、Q43744、A1RFX8、B2VGW7、B 9KIH0、A7FMU2、A8EUE8、A5G3L2、B0UUR6、Q817F9、Q5L5E3、B8GDA2、B7KVX2、A 8EZ58, Q2RV34, C4K2E2, A9L340, B2U1U9, Q5HR46, Q1GQY3, Q9ZF99, A7GTN7, A5IEF4, Q04RS5, A5E935, Q1AWH4, Q82HS2, A9BVK0, Q0ABE6, Q3MDN9, B7JRW5 P49814, Q46BQ2, A9N855, Q6AQI3, O82399, B0BQN0, A1AGC9, B6I1V4, Q8PNP8, Q2A3K7, Q8GNM0, Q39VY0, Q28U77, A9IIS3, Q57JA9, B4SLI5, B9DK67, A8H0U0Q1IWC9, B7GW58, A1WR02, C4ZSX4, B3H269, Q6HCU0, C1DB66, Q2G946, A9A450, A6L903, Q4FP28, P46488, Q7NZ60, P14152, Q9ZDF3, Q2T4T8, K 0J107, A6WSM1, Q3YX11, P82177, Q49VN8, B0V6R7, P61977, B2HZ52, Q9KUT3, B7IJZ0, B7HRN2, Q9Z6N1, O84381, P57106, A5VSQ4, Q8FYF4, Q6 AW23, B4SFQ8, B1XV63, B4S9F1, Q5E875, B2I8M1, B0SF41, Q2W064, Q7WD94, A2SHT9, Q2YLR9, B8EM38, P46487, B5FGF5, B0U5Q1, B6EL39, B0R U49, B3E9R5, A7Z7J8, A6SY47, Q633K5, A8AQC8, P83778, Q4R568, Q7W5Q8, B8IJB4, B0UCF0, Q9A2B1, A1T9L9, Q822E9, P61976, Q2IIC2, Q54VM 2, Q7XDC8, Q9SN86, A1JIV0, Q5L8Z8, B8F5K4, A4YKC5, Q1RIT9, B5FIT7, B5F7L9, B4T769, Q12R11, A5V5U9, A6UDP3, Q4URH2, Q1CBY7, A5UCQ1 , C3PAI1, P9WK13, O67655, A4SFT4, P83373, Q9RXI8, Q55383, A7ZSD0, B7I9D2, Q5FGT9, Q3YS64, Q2J7E7, A4IY35, C5D654, Q6HSF4, B9M1D2, Q7NHJ3, A5UIX3, Q5ZT13, Q8F4A2, A5U1T8, P80040, O24047, Q21XH1, Q21CW7, P37228, P00346, A1BHN9, Q9ZP06, F1C7I4, Q04820, Q43743, Q42686, O48905, Q9FJU0, P11708, P04636, Q6DIY9, P22133, Q08062, B6J7Q0, B6IZN7, Q3T145, A4JKE6, Q9XFW3, A4JM71, Q32LG3, and Q54GE6.

[0023] Malate dehydrogenase of the present invention The present application relates to a malate dehydrogenase having an amino acid sequence of X1X2RX3X4GX5X6RX7DLX8 (wherein X1 is V, M, L, I or A, X2 is P or A, X3 is K or R, X4 is P, E, D or A, X5 is a hydrophobic amino acid other than methionine, X6 is T, E, D or S, X7 is D, K, S or R, and X8 is F, L or V) in an active center loop, It has a higher malate dehydrogenase activity than a malate dehydrogenase having the same sequence as that of the malate dehydrogenase having the same sequence except that the hydrophobic amino acid X5 contained in the active center loop is methionine, and The present invention provides a malate dehydrogenase having a sequence identity of 90% or less with the amino acid sequence of SEQ ID NO:1.

[0024] The MDH of the present application has the amino acid sequence X1X2RX3X4GX5X6RX7DLX8 in the active center loop. The active center loop means a loop structure present in the active site of MDH, and is the amino acid sequence corresponding to positions 104 to 116 of SEQ ID NO: 2. That is, the MDH of the present application has the amino acid sequence X1X2RX3X4GX5X6RX7DLX8 in the amino acid sequence corresponding to positions 104 to 116 of SEQ ID NO: 2.

[0025] In this specification, the term "position corresponding to" a given position in an amino acid sequence means the position of another amino acid sequence that corresponds to a given position in the given amino acid sequence in an alignment of the other amino acid sequence. The alignment can be performed, for example, using known gene analysis software. Specific examples of gene analysis software include DNASIS manufactured by Hitachi Solutions, GENETYX manufactured by Genetyx, and FASTA, BLAST, and ClustalW published by DDBJ.

[0026] The amino acid sequence X1X2RX3X4GX5X6RX7DLX8 is not particularly limited, as long as X1 is V, M, L, I or A, X2 is P or A, X3 is K or R, X4 is P, E, D or A, X5 is a hydrophobic amino acid other than methionine, X6 is T, E, D or S, X7 is D, K, S or R, and X8 is F, L or V. "Hydrophobic amino acid" refers to an amino acid having a hydrophobic side chain, and includes, for example, valine, leucine, isoleucine, and methionine. In some embodiments, the hydrophobic amino acid other than methionine can be valine, leucine, or isoleucine, for example, leucine.

[0027] The MDH of the present application can be obtained by substituting the methionine residue corresponding to position 110 of SEQ ID NO: 2 in the MDH indicated by the above Uniprot registration number with a hydrophobic amino acid other than methionine, and, if necessary, substituting other amino acid residues with amino acid residues within the range specified in the present application. Preferably, the MDH has the amino acid sequence X1X2RX3X4GMX6RX7DLX8 in the active center loop, and the methionine residue between G and X6 is substituted with a hydrophobic amino acid other than methionine. More preferably, the MDH may have VPRKPGXTRDDLF (SEQ ID NO: 4), MPRREGXERKDLL (SEQ ID NO: 5), MPRRDGXERKDLL (SEQ ID NO: 6), VARKPGXDRSDLF (SEQ ID NO: 7), LARKPGXSRDDLF (SEQ ID NO: 8), IARKPGXSRDDLV (SEQ ID NO: 13), or APRKAGXERRDLL (SEQ ID NO: 14) (wherein X in each sequence is a hydrophobic amino acid other than methionine) in the active center loop, for example, VPRKPGXTRDDLF (SEQ ID NO: 4), MPRRDGXERKDLL (SEQ ID NO: 6), LARKPGXSRDDLF (SEQ ID NO: 8), IARKPGXSRDDLV (SEQ ID NO: 13), or APRKAGXERRDLL (SEQ ID NO: 14). Here, SEQ ID NO: 4 shows the sequence in which methionine is replaced with a hydrophobic amino acid (X) other than methionine in the active center loops of budding yeast MDH1, budding yeast MDH2, human MDH2, and porcine MDH2. SEQ ID NO: 5 shows a sequence in which methionine in the active center loop of human MDH1 is replaced with a hydrophobic amino acid (X) other than methionine. SEQ ID NO: 6 shows a sequence in which methionine in the active center loop of porcine MDH1 is replaced with a hydrophobic amino acid (X) other than methionine. SEQ ID NO: 7 shows a sequence in which methionine in the active center loop of Escherichia coli MDH is replaced with a hydrophobic amino acid (X) other than methionine. SEQ ID NO: 8 shows a sequence in which methionine in the active center loop of thermophilic bacterium Thermoplasma acidophilum MDH (TA-MDH) is replaced with a hydrophobic amino acid (X) other than methionine. SEQ ID NO: 13 shows a sequence in which methionine in the active center loop of Geobacillus stearothermophilus MDH (GS-MDH) is replaced with a hydrophobic amino acid (X) other than methionine.SEQ ID NO: 14 shows the sequence in which methionine in the active center loop of Thermus thermophilus MDH (TT-MDH) is replaced with a hydrophobic amino acid (X) other than methionine.

[0028] In certain embodiments, the MDH of the present application may have a methionine as the amino acid at the position corresponding to position 118 of the amino acid sequence of SEQ ID NO:2.

[0029] Moreover, the MDH of the present application has a higher malate dehydrogenase activity than MDH having the same sequence except that the hydrophobic amino acid X5 or X contained in the active center loop is methionine. The malate dehydrogenase activity is defined, for example, by the specific activity (unit / mg) (unit=μmol / min) indicating how many μmoles of oxaloacetic acid (OAA) or malic acid are produced or consumed per minute per 1 mg of enzyme. For example, the MDH of the present application has a specific malate dehydrogenase activity that is about 1.2 times or more, about 1.5 times or more, about 2 times or more, about 3 times or more, or about 4 times or more higher than that of MDH having the same sequence except that the hydrophobic amino acid X5 or X contained in the active center loop is methionine. The malate dehydrogenase activity is also defined, for example, by the unit, where the amount of enzyme that catalyzes the conversion of 1 μmol of substrate per minute under a specified condition is one unit. For example, the MDH of the present application has about 1.2 times or more, about 1.5 times or more, about 2 times or more, about 3 times or more, or about 4 times or more more malate dehydrogenase units than an MDH having the same sequence except that the hydrophobic amino acid X5 or X contained in the active center loop is methionine.

[0030] A known method can be used to measure malate dehydrogenase activity. For example, NADH may be added to a solution containing MDH and the substrate oxaloacetate, and the enzyme activity may be measured by measuring the rate of decrease of NADH. The rate of decrease of NADH can be measured, for example, by monitoring the absorbance at 340 nm, which is the absorption wavelength of NADH, using a spectrophotometer or the like. The rate of decrease of NADH can also be measured by the ratio of NADH and NAD +It can also be measured by a fluorescence method (for example, excitation wavelength 340 nm, fluorescence wavelength 460 nm) based on the difference in fluorescence.

[0031] Further, the MDH of the present application may have a sequence identity to the amino acid sequence of SEQ ID NO:1 of 98% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, or 55% or less, for example, 90% or less.

[0032] As used herein, "sequence identity" with respect to a nucleic acid sequence or an amino acid sequence means the percentage of matching bases or amino acid residues between two sequences aligned optimally (maximally identical) across the entire region of the sequences to be compared. Here, the sequences to be compared may have additions or deletions (e.g., gaps, etc.) in the optimal alignment of the two sequences. Sequence identity can be calculated using programs such as FASTA, BLAST, and CLUSTAL W provided in public databases (e.g., DDBJ (http: / / www.ddbj.nig.ac.jp)). Alternatively, it can be determined using commercially available sequence analysis software (e.g., Vector NTI (registered trademark) software, GENETYX (registered trademark) ver. 12).

[0033] As a specific example of malate dehydrogenase, the present application (1) an amino acid sequence of SEQ ID NO: 9, 10, 15, or 16, wherein Xaa is a hydrophobic amino acid other than methionine; (2) an amino acid sequence having malate dehydrogenase activity, in which 1 to 30 amino acids other than the amino acid sequence of the active center loop in the amino acid sequence of SEQ ID NO: 9, 10, 15, or 16 are deleted, added, and / or substituted; or (3) An amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 9, 10, 15 or 16, an amino acid sequence having the same amino acid sequence of the active center loop, and having malate dehydrogenase activity; The present invention provides a malate dehydrogenase having a higher malate dehydrogenase activity than a malate dehydrogenase having the same sequence except that Xaa is methionine.

[0034] SEQ ID NO: 9 shows the amino acid sequence of budding yeast MDH2 in which the methionine residue in the active center loop has been replaced with a hydrophobic amino acid (Xaa) other than methionine. SEQ ID NO: 10 shows the amino acid sequence of TA-MDH in which the methionine residue in the active center loop has been replaced with a hydrophobic amino acid (Xaa) other than methionine. SEQ ID NO: 15 shows the amino acid sequence of GS-MDH in which the methionine residue in the active center loop has been replaced with a hydrophobic amino acid (Xaa) other than methionine. SEQ ID NO: 16 shows the amino acid sequence of TT-MDH in which the methionine residue in the active center loop has been replaced with a hydrophobic amino acid (Xaa) other than methionine.

[0035] In some embodiments, the MDH of the present application can comprise (1) the amino acid sequence of SEQ ID NO: 9, 10, 15, or 16, wherein Xaa is a hydrophobic amino acid other than methionine.

[0036] In another aspect, the MDH of the present application may comprise an amino acid sequence having malate dehydrogenase activity, in which (2) 1 to 10, 1 to 15, 1 to 20, 1 to 25, 1 to 30, 1 to 40, or 1 to 50, for example 1 to 30, amino acids other than the amino acid sequence of the active center loop in the amino acid sequence of SEQ ID NO: 9, 10, 15, or 16 are deleted, added, and / or substituted.

[0037] In yet another embodiment, the MDH of the present application may comprise an amino acid sequence that has (3) at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, e.g., at least 90%, sequence identity to the amino acid sequence of SEQ ID NO: 9, 10, 15, or 16, has the same amino acid sequence of the active center loop, and has malate dehydrogenase activity.

[0038] The MDH of these embodiments has a higher malate dehydrogenase activity than MDH having the same sequence except that the amino acid (Xaa) corresponding to position 110 of SEQ ID NO: 9 is methionine. For example, compared to MDH having the same sequence except that Xaa is methionine, the malate dehydrogenase activity is about 1.2 times or more, about 1.5 times or more, about 2 times or more, about 3 times or more, or about 4 times or more higher. The method for measuring malate dehydrogenase activity is as described above.

[0039] Method for Producing Malate Dehydrogenase of the Present Application The MDH of the present application can be produced, for example, by preparing DNA encoding the amino acid sequence of the MDH of the present application, ligating it to an expression vector, transforming a host with the expression vector, producing it as a heterologous or homologous protein, and isolating and purifying it.

[0040] A method for preparing DNA encoding the MDH of the present application can be a conventionally known method. For example, a method for totally synthesizing DNA encoding a target amino acid sequence by gene synthesis, a method for introducing a mutation into a specific site by site-directed mutagenesis into DNA encoding wild-type MDH derived from the above-mentioned various species, etc. can be mentioned. A conventionally known method can be used as the site-directed mutagenesis method, for example, the inverse PCR method can be used. When the inverse PCR method is used, a commercially available kit can be used, for example, the KOD-Plus-Mutagenesis Kit, the Q5 Site-Directed Mutagenesis Kit (New England BioLabs), and the QuickChange Site-Directed Mutagenesis Kit (Agilent), etc. can be used.

[0041] An expression vector containing the DNA can be prepared by ligating the DNA encoding the MDH of the present application downstream of a promoter in an appropriate expression vector using a restriction enzyme and DNA ligase.

[0042] Examples of expression vectors include bacterial plasmids, yeast plasmids, phage DNA (lambda phage, etc.), viral DNA such as retrovirus, baculovirus, vaccinia virus, adenovirus, and derivatives of SV40, but any other vector can be used as long as it is replicable and viable in the host cell. For example, when the host is E. coli, examples include pET, pCold, pUC, and pBAD.

[0043] The promoter may be any promoter appropriate for the host used to express the gene, for example, when the host is Escherichia coli, the promoter may be the lac promoter, T7 promoter, Trp promoter, PL promoter, or PR promoter.

[0044] In the present specification, the host is not particularly limited as long as it can express the desired MDH by introducing the expression vector, and examples of the host include bacteria such as Escherichia coli and Bacillus subtilis, fungi such as yeast, animal cells, insect cells, and plant cells.

[0045] When a bacterium is used as a host, the host is, for example, Escherichia coli. Examples of Escherichia coli include E. coli K12 strain and E. coli B strain, and their wild-derived derivatives such as BL21 strain, JM109 strain, and XL1-Blue strain. These strains are easily available from, for example, the American Type Culture Collection (ATCC). The method of introducing a recombinant vector into a bacterium is not particularly limited as long as it is a method for introducing DNA into a bacterium. Examples include a method using calcium ions and electroporation.

[0046] When yeast is used as a host, the type of yeast used is not particularly limited, but the genera Saccharomyces, Debaryomyces, Schizosaccharomyces, Candida, Yarrowia, Rhodotorula, Lipomyces, Kluyveromyces, Rhodosporidium, Trichoderma, Torulopsis, and Pichia can be used. Examples include Saccharomyces cerevisiae, Saccharomyces bayanus, and Schizosaccharomyces pombe. The method of introducing a recombinant vector into yeast is not particularly limited as long as it is a method for introducing DNA into yeast, and examples include electroporation, spheroplast, and lithium acetate methods.

[0047] When the target malate dehydrogenase is secreted outside the cells, it is purified directly from the medium, and when it is present inside the cells, it is purified after disrupting the cells by physical means such as ultrasonic disruption or mechanical disruption or chemical means such as cell lysing agents. Specifically, malate dehydrogenase can be partially or completely purified from the medium of recombinant cells by a combination of techniques such as ammonium sulfate, polyethylene glycol (PEG) or ethanol precipitation, acid extraction, anion or cation exchange chromatography, reversed-phase high performance liquid chromatography, affinity chromatography, gel filtration chromatography, and electrophoresis.

[0048] Composition for measuring aspartate aminotransferase The present application also provides a composition for measuring aspartate aminotransferase (AST), comprising the malate dehydrogenase of the present application.

[0049] AST catalyzes the transfer reaction between the α-keto group of α-ketoglutaric acid and the amino group of L-aspartic acid to produce oxaloacetic acid and glutamic acid. In conjunction with this reaction, MDH converts NADH to NAD in the presence of the generated oxaloacetic acid. AST activity can be calculated by measuring the rate at which NADH decreases. The method for measuring the rate at which NADH decreases is as described above.

[0050] The composition for AST measurement of the present application is a composition containing the MDH of the present application and used to measure the amount of AST in a measurement sample. The measurement sample is not particularly limited as long as it contains AST, and is, for example, a biological sample such as plasma or serum.

[0051] In the composition for measuring AST of the present application, the concentration of MDH of the present application is not particularly limited as long as it enables measurement of AST, and is, for example, 0.1 U / mL to 100 U / mL, 1 U / mL to 10 U / mL, or 1 U / mL to 5 U / mL during measurement.

[0052] The composition for measuring AST of the present application may contain, in addition to MDH of the present application, α-ketoglutaric acid, aspartic acid and NADH as reaction substrates. The concentration of each substrate is not particularly limited as long as AST can be measured.

[0053] The composition for AST measurement of the present application may further contain a buffering agent, a preservative, a pH adjusting agent, and / or an antioxidant, etc. As the buffering agent, known buffering agents can be used, for example, carbonate buffer, Tris buffer, 2-amino-2-hydroxymethyl-1,3-propanediol buffer, phosphate buffer, borate buffer, glycine buffer, diethanolamine buffer, etc. EXAMPLES

[0054] The present invention will be described in more detail below by showing examples, but the present invention is not limited to these examples in any way.

[0055] Materials and Methods Generation of wild-type MDH3 and MDH3 mutants His-SUMO1-MDH3 cDNA, His-SUMO1-MDH3 L84M cDNA, or His-SUMO1-MDH3 L84M / M92V cDNA was cloned into pCold vector to prepare recombinant vectors. E. coli was transformed with the recombinant vectors. Culture was performed at 37°C, and when the absorbance at 600 nm (OD600) reached 0.5-0.6, isopropyl β-d-1 thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM to induce expression of the MDH3 gene. The culture was then incubated at 16°C for a further 24 hours.

[0056] Cells were harvested by centrifugation at 5053 g for 15 min, resuspended in lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl) and lysed by sonication. 2+ The bound protein was loaded onto a Sepharose column (GE Healthcare) and eluted from the column with elution buffer (300 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl pH 7.5). The tag was cleaved by adding ubiquitin-like specific protease (Ulp1) and incubating overnight at 4°C. Further purification was performed using a HiLoad Superdex 75 column (GE Healthcare) filled with gel filtration buffer (25 mM Tris-HCl pH 7.5, 10 mM β-mercaptoethanol, 150 mM NaCl).

[0057] Generation of wild-type MDH2 GST-MDH2 cDNA was cloned into the pet28a vector to prepare a recombinant vector. E. coli was transformed with the recombinant vector. Culture, centrifugation, and cell disruption were performed in the same manner as for the purification of MDH3.

[0058] The cell lysate was applied to a Glutathione Sepharose 4B column (GE Healthcare), and bound proteins were eluted from the column with elution buffer (20 mM glutathione, 50 mM Tris-HCl pH 7.5). The GST tag was cleaved by adding 3C protease and incubating overnight at 4°C. The sample was further purified by adsorbing it again to the Glutathione Sepharose column and collecting the unadsorbed fraction.

[0059] Generation of MDH2 mutants His-SUMO1-MDH2 M110L cDNA or His-SUMO1-MDH2 M110L / V1184M cDNA was cloned into pCold vector to prepare recombinant vector. E. coli was transformed with the recombinant vector. After culturing, centrifugation, and cell disruption in the same manner as for MDH3 purification, gel filtration chromatography using a HiLoad Superdex 75 column was performed to purify the MDH2 mutant.

[0060] The sample was then further purified by anion exchange chromatography using a HiTrap QHP (GE Healthcare). Elution was performed using a linear gradient of buffer A (20 mM Tris-HCl pH 7.5, 10 mM β-mercaptoethanol) and buffer B (20 mM Tris-HCl pH 7.5, 10 mM β-mercaptoethanol, 2 M NaCl).

[0061] Generation of wild-type TA-MDH and TA-MDH mutants His-SUMO1-TA-MDH cDNA or His-SUMO1-TA-MDH M92L / K100M cDNA was cloned into the pet28a vector to prepare a recombinant vector. E. coli was transformed with the recombinant vector. After culturing, centrifugation, and cell disruption in the same manner as for the purification of MDH3, the cells were incubated at 4°C for 1 h and then incubated for 1 h. 2+The sample was purified by affinity chromatography using a Sepharose column. The tag was cleaved by adding ubiquitin-like specific protease (Ulp1) and incubating overnight at 4°C. The sample was then further purified by anion exchange chromatography using a HiTrap QHP (GE Healthcare). Elution was performed using a linear gradient of buffer A (20 mM Tris-HCl pH 7.5, 10 mM β-mercaptoethanol) and buffer B (20 mM Tris-HCl pH 7.5, 10 mM β-mercaptoethanol, 2 M NaCl).

[0062] Generation of wild-type GS-MDH and GS-MDH mutants GS-MDH cDNA or GS-MDH M91L / T99M cDNA was cloned into the pet21a vector to prepare a recombinant vector. E. coli was transformed with the recombinant vector. After culturing, centrifugation, and cell disruption were performed in the same manner as for the purification of MDH3, the cells were heat-treated at 70°C for approximately 10 minutes. The cells were then further purified using a HiLoad Superdex 75 column (GE Healthcare) filled with gel filtration buffer (25 mM Tris-HCl pH 7.5, 10 mM β-mercaptoethanol, 150 mM NaCl).

[0063] Generation of wild-type TT-MDH and TT-MDH mutants TT-MDH cDNA or TT-MDH M96L / V104M cDNA was cloned into the pet21a vector to prepare a recombinant vector. E. coli was transformed with the recombinant vector. After culturing, centrifugation, and cell disruption in the same manner as for the purification of MDH3, the cells were heat-treated at 70°C for approximately 10 minutes. The cells were then further purified using a HiLoad Superdex 75 column (GE Healthcare) filled with gel filtration buffer (25 mM Tris-HCl pH 7.5, 10 mM β-mercaptoethanol, 150 mM NaCl).

[0064] Activity measurement

[0065] NADH was added to the sample (45 mM potassium phosphate buffer (KPB), oxaloacetic acid (OAA), each MDH), and the absorbance at 340 nm, which is the absorption wavelength of NADH, was measured using a spectrophotometer (Smart Spec Plus, Bio-Rad) to obtain ΔO.D. / min. In the activity measurement of MDH2, MDH3, and TA-MDH, the concentration range of OAA was 0.01 to 4 mM, and the concentration of each MDH was 0.02 μg / ml. In the activity measurement of GS-MDH and TT-MDH, the concentration range of OAA was 0.002 to 0.3 mM, and the concentration of each MDH was 0.002 μg / ml. ΔO.D. / min was obtained for each concentration of OAA. Measurements were performed three times for each condition.

[0066] Based on the ΔO.D. / min at each concentration of OAA, the dissociation constant K between MDH and OAA was calculated using the Excel add-in program Solver so that the error of the Michaelis-Menten equation was minimized. m (mM) value and maximum reaction rate V max (μM / min) was calculated. The molar extinction coefficient of NADH at 340 nm was 6.3 × 10 3 From these values, the unit (μmol / min) is the amount of enzyme that catalyzes the conversion of 1 μmol of substrate per minute under the specified conditions, the specific activity (unit / mg) is the enzyme activity per mg of enzyme protein, and the K cat , a parameter used to compare the catalytic efficiency of different enzymes, K cat / K m was calculated.

[0067] [result] Reference Example 1: Measurement of wild-type MDH2 and wild-type MDH3 activity in budding yeast The results of measuring the activity of wild-type MDH2 and wild-type MDH3 in budding yeast are shown in Figure 2. From the graph in Figure 2, the K m Values ​​(mM) and V max From these values, unit (μmol / min), specific activity (unit / mg), and Kcat , K cat / K m was calculated (Table 1). Table 1. Various parameters of wild-type MDH2 and wild-type MDH3 in budding yeast [Table 1]

[0068] The measurement results showed that the specific activity of MDH3 was 639 units / mg, which was more than four times higher than the specific activity of MDH2 (151 units / mg). m The value is 0.138 mM, and the K m This was more than three times higher than the V value of MDH3 (0.040 mM). max is 0.080 (ΔO.D. / min), and the V of MDH2 max This was more than four times higher than the value (0.019ΔO.D. / min) of MDH3, demonstrating that MDH3 is an enzyme with much higher activity than MDH2.

[0069] Reference Example 2: Activity measurement of MDH3 mutants of budding yeast The results of activity measurement of the budding yeast MDH3 mutants are shown in Figure 3. Various parameters were calculated in the same manner as in the activity measurement of wild-type MDH2 and MDH3 (Table 2). Table 2. Various parameters of MDH3 mutants of budding yeast [Table 2]

[0070] The specific activities of the MDH3 L84M and L84M / M92V mutants were 284 and 193 units / mg, respectively, which were much lower than the specific activity of wild-type MDH3 (639 units / mg) and close to the specific activity of MDH2 (151 units / mg). m The values ​​were 0.026 mM and 0.023 mM, respectively, which is the K mThis is a much lower value than the K value of MDH2 (0.138 mM). m The V of the MDH3 L84M and L84M / M92V mutants was close to the value of 0.04 mM. max were 0.035 (ΔO.D. / min) and 0.024 (ΔO.D. / min), respectively, which is higher than the V of wild-type MDH3. max (0.080ΔO.D. / min), which is lower than the V max The value was close to (0.019ΔO.D. / min).

[0071] Example 1: Activity measurement of MDH2 mutants of budding yeast The results of activity measurement of the budding yeast MDH2 mutants are shown in Figure 4. Various parameters were calculated in the same manner as above (Table 3). Table 3. Various parameters of MDH2 mutants of budding yeast [Table 3]

[0072] The specific activities of the MDH2 M110L mutant and the MDH2 M110L / V118M mutant were 664 unit / mg and 653 unit / mg, respectively, which were higher than the specific activity of wild-type MDH2 (151 unit / mg) and close to the specific activity of MDH3 (639 unit / mg). m The K values ​​were 0.156 mM and 0.329 mM, respectively, which is the K m The K value of MDH3 was higher than that of m The V value of the MDH2 M110L mutant and the MDH2 M110L / V118M mutant was close to the value of 0.138 mM. max were 0.083 (ΔO.D. / min) and 0.081 (ΔO.D. / min), respectively, which was higher than the V of wild-type MDH2. max (0.019ΔO.D. / min), and V max The value was close to (0.080ΔO.D. / min).

[0073] Example 2: Activity measurement of wild-type TA-MDH and TA-MDH mutants The results of measuring the activity of wild-type TA-MDH and the TA-MDH mutant are shown in Figure 5. Various parameters were calculated in the same manner as above (Table 4). Table 4. Various parameters of wild-type TA-MDH and TA-MDH mutants [Table 4]

[0074] The specific activities of wild-type TA-MDH and the TA-MDH M92L / K100M mutant were 152 and 339 units / mg, respectively, and the mutants showed specific activities more than twice as high as the wild-type. m The values ​​were 0.007 mM and 0.018 mM, respectively, and the K m Furthermore, V max were 0.019 (ΔO.D. / min) and 0.042 (ΔO.D. / min), respectively, and the mutant showed activity more than twice as high as the wild type.

[0075] Example 3: Activity measurement of wild-type GS-MDH and GS-MDH mutants The results of measuring the activity of wild-type GS-MDH and GS-MDH mutants are shown in Figure 6. Various parameters were calculated in the same manner as above (Table 5). Table 5. Various parameters of wild-type GS-MDH and GS-MDH mutants [Table 5]

[0076] The specific activities of wild-type GS-MDH and the GS-MDH M91L / T99M mutant were 863 and 3516 units / mg, respectively, and the mutant showed a specific activity four times higher than that of the wild-type. m The K values ​​were 0.001 mM and 0.017 mM, respectively, and the mutant had a K value more than 15-fold higher than that of the wild type. mFurthermore, V max were 0.011 (ΔO.D. / min) and 0.044 (ΔO.D. / min), respectively, and the mutant showed four-fold higher activity than the wild type.

[0077] Example 4: Activity measurement of wild-type TT-MDH and TT-MDH mutants The results of measuring the activity of wild-type TT-MDH and TT-MDH mutants are shown in Figure 7. Various parameters were calculated in the same manner as above (Table 6). Table 6. Various parameters of wild-type TT-MDH and TT-MDH mutants [Table 6]

[0078] The specific activities of wild-type TT-MDH and the TT-MDH M96L / V104M mutant were 519 unit / mg and 2441 unit / mg, respectively, and the mutant showed a specific activity more than four times higher than that of the wild-type. m The K values ​​were 0.003 mM and 0.016 mM, respectively, and the mutant had a K value more than 5-fold higher than that of the wild type. m Furthermore, V max were 0.034 (ΔO.D. / min) and 0.158 (ΔO.D. / min), respectively, and the mutant showed activity more than four times higher than that of the wild type.

Claims

1. A sequence having a sequence identity of 90% or less with the amino acid sequence of SEQ ID NO:1, and any one of the following amino acid sequences (1) to (3): (1) an amino acid sequence of SEQ ID NO: 10, 15, or 16, wherein Xaa is V, L, or I; (2) In the amino acid sequence of SEQ ID NO: 10, 15 or 16 (wherein Xaa is V, L or I), 1 to 30 amino acids other than the amino acid sequence of the active center loop, which is the amino acid sequence LARKPGXSRDDLF (SEQ ID NO: 8), IARKPGXSRDDLV (SEQ ID NO: 13), or APRKAGXERRDLL (SEQ ID NO: 14) (wherein X is V, K or I), are deleted, added and / or substituted; (3) having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 10, 15 or 16 (wherein Xaa is V, L or I), and having an active center loop of the amino acid sequence LARKPGXSRDDLF (SEQ ID NO: 8), IARKPGXSRDDLV (SEQ ID NO: 13), or APRKAGXERRDLL (SEQ ID NO: 14) (wherein X is V, K or I); A malate dehydrogenase having the formula: A malate dehydrogenase having a higher malate dehydrogenase activity than a malate dehydrogenase having the same sequence except that Xaa is methionine.

2. A sequence having a sequence identity of 90% or less with the amino acid sequence of SEQ ID NO:1, and any one of the following amino acid sequences (1) to (3): (1) an amino acid sequence of SEQ ID NO:9, wherein Xaa is V or L; (2) In the amino acid sequence of SEQ ID NO:9 (wherein Xaa is V or L), 1 to 30 amino acids other than the amino acid sequence of the active center loop, which is the amino acid sequence VPRKPGXTRDDLF (SEQ ID NO:4) (wherein X is V or L), are deleted, added and / or substituted; (3) having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:9 and having an active center loop of the amino acid sequence VPRKPGXTRDDLF (SEQ ID NO:4) (wherein X is V or L); A malate dehydrogenase having the formula: A malate dehydrogenase having a higher malate dehydrogenase activity than a malate dehydrogenase having the same sequence except that Xaa is methionine.

3. The malate dehydrogenase according to claim 1 or 2, wherein X or Xaa is leucine.

4. The malate dehydrogenase according to any one of claims 1 to 3, wherein the amino acid at the position corresponding to position 118 in the amino acid sequence of SEQ ID NO:2 is methionine.

5. A DNA encoding the malate dehydrogenase according to any one of claims 1 to 4.

6. An expression vector comprising the DNA of claim 5.

7. A composition for measuring aspartate aminotransferase, comprising the malate dehydrogenase described in any one of claims 1 to 4.

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