JP6514849B2 - Method for obtaining a modified form of thermophilic bacterium-derived enzyme having improved enzyme activity at low temperature, and modified form of Thermus thermophilus-derived 3-isopropyl malate dehydrogenase having improved enzyme activity at low temperature - Google Patents

Description

  The present invention relates to a method for efficiently obtaining a modified thermophilic bacterium-derived enzyme with improved enzyme activity at low temperature. The present invention also relates to a modified version of Thermus thermophilus-derived 3-isopropylmalate dehydrogenase in which the enzyme activity at low temperature is improved.

  Generally, microorganisms having an optimum growth temperature of 45 ° C. or higher are classified as thermophilic bacteria. In particular, those having an optimum growth temperature of 80 ° C. or higher are also called hyperthermophilic bacteria. Enzymes collected from thermophilic bacteria are superior to thermophilic enzymes from the viewpoint of heat stability. On the other hand, since the optimum temperature for the enzyme activity is higher than normal temperature, the enzyme activity at normal temperature or lower is often significantly lower than the same enzyme derived from a cold bacterium. This point is a major issue in industrial use of thermophilic bacteria-derived enzymes.

  Therefore, in order to improve the enzyme activity of the thermophilic bacterium-derived enzyme in the normal temperature range, a modified form of the thermophilic bacterium-derived enzyme has been conventionally obtained. For example, Patent Document 1 discloses an example in which the enzyme activity at 10 ° C. or more and 37 ° C. or less of the hyperthermophilic bacterium Thermoproteus-derived glucose dehydrogenase is improved. Non-Patent Document 1 and Non-Patent Document 2 disclose examples in which the enzyme activity of the thermophilic bacterium Thermus thermophilus-derived 3-isopropylmalate dehydrogenase at 30 ° C. or less is improved.

  However, conventionally, in the method for obtaining the variant of the thermophilic bacterium-derived enzyme, a method combining random mutagenesis and screening is exclusively employed, and there is a disadvantage that the efficiency for obtaining the target variant is low. Furthermore, conventionally, when obtaining a modified form of a thermophilic bacterium-derived enzyme, information on which amino acid residue at which position can be changed to which amino acid residue can not be obtained, and information can not be obtained. At present, no method has been established to obtain a modified product with improved enzyme activity at low temperatures.

WO 2010/137489

Protein Eng. 14, p85, 2001 J. Biochem. 129, p 477, 2001

  The present invention has been made in view of the current state of the prior art, and an object thereof is to provide a modified technique for simply improving the enzyme activity at normal temperature or lower of an enzyme collected from a thermophilic bacterium. Another object of the present invention is to provide a modified version of Thermus thermophilus-derived 3-isopropylmalate dehydrogenase in which the enzyme activity at low temperature is improved.

As a result of repeated studies to solve the above problems, the present inventors focused attention on amino acid residues present at a distance of 8 Å from the substrate binding site and / or the coenzyme binding site in an enzyme derived from thermophilic bacteria. By introducing amino acid mutations, it has been found that variants with improved enzyme activity at low temperatures can be efficiently obtained. More specifically, it has been found that, by carrying out the following steps 1 to 4, it is possible to efficiently obtain a modified product in which the enzyme activity at low temperature is improved.
Step 1: analyzing the three-dimensional structure of the thermophilic bacterium-derived modified enzyme to identify an amino acid sequence site located at a distance of 8 Å from the substrate binding site and / or the coenzyme binding site of the modified enzyme
Step 2: The amino acid sequence of the mutant enzyme and the amino acid sequence of a reference enzyme of the same species as the mutant enzyme and derived from a thermophilic bacterium are analyzed by pairwise alignment, and the amino acid sequence site identified in step 1; Comparing corresponding amino acid sequence sites on the pairwise alignment in the amino acid sequence of the enzyme, and identifying an amino acid sequence site where the amino acid residue is not identical between the two;
Step 3: Substituting, substituting or deleting an amino acid in at least one of the amino acid sequence sites specified in Step 2 with respect to the mutant enzyme to obtain a mutant, and Step 4: Step A step of selecting a mutant having improved enzyme activity at a lower temperature than that of the mutant enzyme among the mutants obtained in 3.

  In this way, mutation is limited to a specific amino acid residue located within 8 Å from the substrate binding site and / or coenzyme binding site, and the enzyme activity at low temperature is improved regardless of random mutagenesis and screening. It is a finding first revealed by the present inventors that it is possible to easily and efficiently obtain the modified product.

  Furthermore, according to the method described above, the present inventors mutated a specific amino acid residue located within 8 Å from the substrate binding site and / or the coenzyme binding site in the Thermus thermophilus-derived 3-isopropylmalate dehydrogenase By applying, it was possible to obtain a modified product with improved enzyme activity at low temperatures.

The present invention has been completed by further investigation based on these findings. That is, the present invention provides the invention of the aspects listed below.
Item 1. A method for obtaining a modified product having improved enzyme activity at low temperature by mutating a thermophilic bacterium-derived modified enzyme,
Step 1: analyzing the three-dimensional structure of the thermophilic bacterium-derived modified enzyme to identify an amino acid sequence site located at a distance of 8 Å from the substrate binding site and / or the coenzyme binding site of the modified enzyme
Step 2: The amino acid sequence of the mutant enzyme and the amino acid sequence of a reference enzyme of the same species as the mutant enzyme and derived from a thermophilic bacterium are analyzed by pairwise alignment, and the amino acid sequence site identified in step 1; Comparing corresponding amino acid sequence sites on the pairwise alignment in the amino acid sequence of the enzyme, and identifying an amino acid sequence site where the amino acid residue is not identical between the two;
Step 3: Substituting, substituting or deleting an amino acid in at least one of the amino acid sequence sites specified in Step 2 with respect to the mutant enzyme to obtain a mutant, and Step 4: Step Selecting a mutant having improved enzyme activity at a lower temperature than the mutant enzyme among the mutants obtained in 3;
A method of obtaining a variant, comprising:
Item 2. Item 3. The method for obtaining a variant according to Item 1, wherein the amino acid substitution, deletion or insertion in Step 3 is performed so as to be identical to the amino acid residue of the corresponding site in the amino acid sequence of the reference enzyme.
Item 3. Item 3. A method for obtaining a variant according to item 1 or 2, wherein the thermophilic bacterium-derived modified enzyme is 3-isopropyl malate dehydrogenase.
Item 4. The polypeptide shown in any one of the following (A) to (O):
(A) a polypeptide comprising an amino acid sequence in which the 78th position in the amino acid sequence shown in SEQ ID NO: 1 is replaced with glutamic acid,
(B) in the amino acid sequence shown in SEQ ID NO: 1 at position 214, substituted with methionine, glycine or alanine, at position 216 for isoleucine, leucine or valine, at position 218 for asparagine, serine or glutamine, at position 219 for alanine, methionine Or a glycine, a 222nd amino acid substitution with glutamine, asparagine or serine, a 225nd amino acid substitution with lysine or histidine, and a 229th amino acid substitution with glutamine, asparagine or serine,
(C) a polypeptide comprising an amino acid sequence in which the 272nd position in the amino acid sequence shown in SEQ ID NO: 1 is substituted with alanine, methionine or glycine and the 273rd position is substituted with glycine, alanine or methionine,
(D) In the amino acid sequence shown in SEQ ID NO: 1, position 324 is substituted with arginine, lysine or histidine, position 325 is substituted with threonine, glutamine or asparagine, and glycine, alanine or methionine is inserted between positions 325 and 326 A polypeptide comprising the amino acid sequence
(E) In the amino acid sequence shown in SEQ ID NO: 1, position 272 is substituted with alanine, methionine or glycine, position 273 is substituted with glycine, alanine or methionine, position 324 is substituted with arginine, lysine or histidine, position 325 is threonine, glutamine Or a polypeptide comprising an amino acid sequence which is substituted with asparagine and glycine, alanine or methionine is inserted between the 325th and the 326th,
(F) In the amino acid sequence in which amino acid position 78 in the amino acid sequence shown in SEQ ID NO: 1 is substituted with glutamic acid, one or several amino acids other than amino acid position 78 are substituted, added, inserted or deleted, and -The 3-isopropylmalate dehydrogenase activity at low temperature is improved as compared to the 3-isopropylmalate dehydrogenase having the isopropylmalate dehydrogenase activity and comprising the amino acid sequence shown in SEQ ID NO: 1 Polypeptide,
(G) in the amino acid sequence shown in SEQ ID NO: 1 at position 214 substituted with methionine, glycine or alanine, at position 216 for isoleucine, leucine or valine, at position 218 for asparagine, serine or glutamine, at position 219 for alanine, methionine Or at glycine 222, at position 222 for glutamine, asparagine or serine, at position 225 for lysine or histidine, and at position 229 for glutamine, asparagine or serine at position 214, 216, 218 And one or several amino acids other than 219, 222, 225 and 229 are substituted, added, inserted or deleted and has 3-isopropylmalate dehydrogenase activity, It consists of the amino acid sequence shown to sequence number 1 - a polypeptide as compared to the isopropyl malate dehydrogenase 3-isopropyl malate dehydrogenase activity at low temperatures is improved,
(H) One of the amino acids other than 272 and 273 in the amino acid sequence in which the 272nd position in the amino acid sequence shown in SEQ ID NO: 1 is substituted with alanine, methionine or glycine and the 273rd position is substituted with glycine, alanine or methionine Or a few are substituted, added, inserted or deleted, and have 3-isopropylmalate dehydrogenase activity and are compared with the 3-isopropylmalate dehydrogenase consisting of the amino acid sequence shown in SEQ ID NO: 1 A polypeptide with improved 3-isopropylmalate dehydrogenase activity at low temperatures,
(I) In the amino acid sequence shown in SEQ ID NO: 1, position 324 is substituted with arginine, lysine or histidine, position 325 is substituted with threonine, glutamine or asparagine, and glycine, alanine or methionine is inserted between positions 325 and 326 And one or more amino acids other than the amino acids at positions 324 and 325 and the amino acid inserted between positions 325 and 326 are substituted, added, inserted or deleted, and Compared with 3-isopropyl malate dehydrogenase having 3-isopropyl malate dehydrogenase activity and comprising the amino acid sequence shown in SEQ ID NO: 1, the 3-isopropyl malate dehydrogenase activity at low temperature is improved Polypeptides,
(J) In the amino acid sequence shown in SEQ ID NO: 1, position 272 is substituted with alanine, methionine or glycine, position 273 is substituted with glycine, alanine or methionine, position 324 is substituted with arginine, lysine or histidine, position 325 is threonine, glutamine Or in the amino acid sequence formed by substitution of asparagine and glycine, alanine or methionine between 325 and 326, amino acids 272, 273, 324 and 325 and between 325 and 326 One or several amino acids other than the amino acid to be inserted are substituted, added, inserted or deleted, and have 3-isopropylmalate dehydrogenase activity and consist of the amino acid sequence shown in SEQ ID NO: 1 Low temperature compared to 3-isopropyl malate dehydrogenase Polypeptide is of 3-isopropyl malate dehydrogenase activity is enhanced,
(K) In an amino acid sequence in which amino acid 78 in the amino acid sequence shown in SEQ ID NO: 1 is substituted with glutamic acid, sequence identity excluding amino acid 78 is 80% or more, and 3-isopropylmalate dehydrogenase A polypeptide having activity and having improved low-temperature 3-isopropylmalate dehydrogenase activity as compared to 3-isopropylmalate dehydrogenase consisting of the amino acid sequence shown in SEQ ID NO: 1,
(L) in the amino acid sequence shown in SEQ ID NO: 1 at position 214 substituted with methionine, glycine or alanine, at position 216 for isoleucine, leucine or valine, at position 218 for asparagine, serine or glutamine, at position 219 for alanine, methionine Or at glycine 222, at position 222 for glutamine, asparagine or serine, at position 225 for lysine or histidine, and at position 229 for glutamine, asparagine or serine at position 214, 216, 218 The sequence identity excluding amino acids 219, 222, 225, and 229 is 80% or more, and has 3-isopropylmalate dehydrogenase activity, and the amino acid sequence shown in SEQ ID NO: 1 3-Isopropylate Polypeptide at low temperature 3-isopropyl malate dehydrogenase activity is enhanced as compared to the Gore-dehydrogenase,
(M) A sequence obtained by replacing amino acid 272 and 273 in an amino acid sequence in which amino acid 272 in the amino acid sequence shown in SEQ ID NO: 1 is substituted with alanine, methionine or glycine and amino acid 273 is substituted with glycine, alanine or methionine It has an identity of 80% or more, has 3-isopropylmalate dehydrogenase activity, and has a 3-isopropylmalate dehydrogenase having the amino acid sequence shown in SEQ ID NO: 1 at a low temperature as compared to 3-isopropylmalate dehydrogenase A polypeptide having improved isopropylmalate dehydrogenase activity,
(N) In the amino acid sequence shown in SEQ ID NO: 1, arginine, lysine or histidine substitutes for position 324, threonine, glutamine or asparagine for position 325, and glycine, alanine or methionine is inserted between positions 325 and 326 80% or more in sequence identity excluding amino acids 324 and 325 and amino acids inserted between 325 and 326, and 3-isopropylmalate dehydrogenase activity A polypeptide having improved 3-isopropylmalate dehydrogenase activity at low temperature as compared to 3-isopropylmalate dehydrogenase comprising the amino acid sequence shown in SEQ ID NO: 1,
(O) In the amino acid sequence shown in SEQ ID NO: 1, position 272 is substituted with alanine, methionine or glycine, position 273 is substituted with glycine, alanine or methionine, position 324 is substituted with arginine, lysine or histidine, position 325 is threonine, glutamine Or in the amino acid sequence formed by substitution of asparagine and glycine, alanine or methionine between 325 and 326, amino acids 272, 273, 324 and 325 and between 325 and 326 And 3-isopropylmalate dehydrogenase having an amino acid sequence shown in SEQ ID NO: 1 and having a sequence identity of 80% or more excluding the amino acid to be inserted, and having a 3-isopropylmalate dehydrogenase activity. 3-isopropyl malic acid at low temperature in comparison Polypeptide dehydrogenase activity is enhanced.
Item 5. Item 2. A DNA encoding the polypeptide according to Item 1.
Item 6. 6. A recombinant vector comprising the DNA according to item 5.
Item 7. Item 7. A transformant obtained by transforming a host with the recombinant vector according to Item 6.
Item 8. Item 8. A method for producing the polypeptide according to Item 1, comprising the step of culturing the transformant according to Item 7.

  The method for obtaining the variant of the present invention does not require random mutagenesis and screening, and can easily and efficiently obtain a variant in which the enzyme activity of the thermophilic bacterium-derived enzyme at low temperature is improved. Therefore, according to the present invention, a thermophilic bacterium-derived enzyme which is high in heat resistance and superior in storage stability can be modified to exhibit high activity at low temperature, and can be used, for example, for in vitro diagnostic agent applications.

  Furthermore, in the polypeptide of the present invention, the enzyme activity at low temperature is improved by applying an amino acid mutation to a predetermined site of Thermus thermophilus-derived 3-isopropylmalate dehydrogenase, and the utility is enhanced. There is.

The pairwise alignment figure of the Thermus thermophilus origin IPMDH (TthIPMDH) and E. coli origin IPMDH (EcoIPMDH) is shown. The underlined part shown in TthIPMDH indicates an amino acid residue present within 8 Å from the amino acid residue located at the coenzyme binding site and the substrate binding site. The schematic of SOE (Splicing by overlap extension) -PCR method is shown. The figure which compared the specific activity in 25 degreeC of various modified bodies is shown. In this figure, the relative ratio when the value of the specific activity of TthIPMDH (wild type) is 1.00 is graphed. The figure which compared the specific activity in 25 degreeC of various modified bodies is shown. In this figure, the relative ratio when the value of the specific activity of TthIPMDH (wild type) is 1.00 is graphed.

  Hereinafter, the present invention will be described in detail. In addition to the sequence listing, 20 types of amino acid residues in the amino acid sequence are expressed by one-letter abbreviations. That is, glycine (Gly) is G, alanine (Ala) is A, valine (Val) is V, leucine (Leu) is L, isoleucine (Ile) is I, phenylalanine (Phe) is F, and tyrosine (Tyr) is Y , Tryptophan (Trp) is W, serine (Ser) is S, threonine (Thr) is T, cysteine (Cys) is C, methionine (Met) is M, aspartic acid (Asp) is D, glutamic acid (Glu) is E Asparagine (Asn) is N, glutamine (Gln) is Q, lysine (Lys) is K, arginine (Arg) is R, histidine (His) is H, proline (Pro) is P.

  Expressions such as "F45V" in the present specification are notations for amino acid substitution. For example, “F45V” means that the 45th amino acid F from the N-terminal side in a specific amino acid sequence is substituted with the amino acid V. Moreover, expressions such as "V272A / H273G" in the present specification mean multiple mutations. For example, "V272A / H273G" means that amino acid substitutions of V272A and H273G are introduced at the same time. Furthermore, expressions such as "326G (INSERTION)" in the present specification mean that the amino acid G is inserted between the 325th and the 326th amino acid residues from the N-terminal side in the specific amino acid sequence.

  Also, as used herein, "nonpolar amino acids" include alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and triptyphan. Also, "non-charged amino acids" include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Also, "acidic amino acid" includes aspartic acid and glutamic acid. Also, "basic amino acids" include lysine, arginine and histidine.

1. Method for Obtaining Variant The method for obtaining the variant of the present invention is a method for obtaining a variant in which the enzyme activity at low temperature is improved by mutating the thermophilic bacterium-derived enzyme (modified enzyme). Hereinafter, in the method for obtaining the variant of the present invention, the modified enzyme to be introduced with a mutation, the reference enzyme referred to for specifying a mutation introduction site, and each step 1 to 4 will be described in detail.

Modified Enzyme In the method for obtaining the variant of the present invention, the modified enzyme is an enzyme to which a mutation is to be introduced, and a thermophilic bacterium-derived enzyme is used.

  The method for obtaining the variant of the present invention can be applied to obtain any variant of the enzyme regardless of the type of the modified enzyme, so that the improvement of the activity at low temperature is required for the type of the modified enzyme. The limit is not particularly limited. In the method for obtaining a variant according to the present invention, an enzyme requiring nicotinamide adenine dinucleotide (hereinafter sometimes referred to as NAD) as a coenzyme as a preferable example of the modified enzyme, preferably NAD as a coenzyme The required dehydrogenase is mentioned. Specifically as a dehydrogenase which requires NAD as a coenzyme, specifically, 3-isopropyl malate dehydrogenase, isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase, glucose dehydrogenase Enzymes, alcohol dehydrogenase, lactate dehydrogenase, glutamate dehydrogenase and the like can be mentioned. Among these, 3-isopropylmalate dehydrogenase is preferably used as a modified enzyme in the present invention because it is possible to more efficiently obtain a modified product with improved enzyme activity at low temperatures. Be done.

  The origin of the modified enzyme is not particularly limited as a thermophilic bacterium, and may be appropriately selected according to the type of the modified enzyme. In the present specification, the “thermophilic bacterium” is a microorganism having an optimum growth temperature of 45 ° C. or higher and is a microorganism belonging to archaea, gram positive eubacteria, or gram negative eubacteria.

  Specific examples of the thermophilic bacterium from which the modified enzyme is derived include Actinomadura, Actinopolyspora, Alicyclobacillus, Amycolatopsis, Anaerolinea, Aneurinibacillus, Archaeoglobus, Bacillus, Caldanaerobacter, Calderihabitans, Caldimonas, Caldisericum, Carboxydothermus, Chlorobaculum, Clostridium, Deferrobacter, Deinococcus, Desulfotomaculum, Exilispira, Fervidobacterium, Geobac Illus, Georgenia, Halobaculum, Hydronobacter, Hydrogenogenus, Hydrogenophilus, Ignavibacterium, Kosmotoga, Kyrpidia, Laceyella, Lacyella, Lutispora, Meriothermus, Melghirimyces, Metallosisphaella, Methanetechococcus , Methanothermobacter, Natrialba, Oceanithermus, Petrotoga, Picrophilus, Polycladomyces, Pseudonocardia, P Eudoxanthomonas, Pyrobaculum, Pyrococcus, Pyrococcus, Pyredictium, Roseiflexus, Rubrobacter, Saccharomonospora, Saccharopolyspora, Saccharopolyspora, Streptomyces, Sulfolibus, Sulfuritisphaera, , Thermobispora, Thermococcus, Thermocrispum, Thermodesulfobium, Ther Thermophilic bacteria such as moflavimicrobium, Thermomonospora, Thermoplasma, Thermoproteus, Thermosiphos, Thermosiphos, Thermosulfidibacter, Thermotoga, Thermus, Vulcanisaeta, Ureibacillus and the like can be mentioned. Among these thermophilic bacteria, from the viewpoint of obtaining a modified product in which the enzyme activity at low temperature is improved more efficiently, the genus is preferably Thermus, more preferably Thermus thermophilus (Thermus thermophilus).

  The modified enzyme used in the present invention may be a wild-type enzyme derived from a thermophilic bacterium, or a mutant-type enzyme in which a wild-type enzyme derived from a thermophilic bacterium is mutated .

Reference Enzyme In the method for obtaining the variant of the present invention, the reference enzyme is an enzyme referred to in determining the site for introducing a mutation into the modified enzyme, the type of mutation to be introduced, and the like.

  In the present invention, the same reference enzyme as the above-mentioned modified enzyme is used as the reference enzyme. Here, “homologous to the aforementioned modified enzyme” means that the reference enzyme has the same enzyme activity as the modified enzyme and belongs to the same family as the modified enzyme in the enzyme classification. . For example, if 3-isopropylmalate dehydrogenase is employed as the modified enzyme, 3-isopropylmalate dehydrogenase is also used as the reference enzyme.

  In the reference enzyme, the identity of the amino acid sequence to the modified enzyme to be used is not particularly limited as long as it is the same type as the modified enzyme, but for example, 20% or more, preferably 50% or more, further Preferably 70% or more, particularly preferably 80% or more can be mentioned. Here, “identity of amino acid sequence” means bl2seq program (Tatiana A. Tatsusova, Thomas L) of BLAST PACKAGE [sgi 32 bit edition, Version 2.0. 12; available from the National Center for Biotechnology Information [NCBI]]. Madden, FEMS Microbiol. Lett., Vol. 174, 247-250, 1999) shows the value of identity. The parameters may be set to Gap insertion Cost value: 11 and Gap extension Cost value: 1.

  Moreover, in the present invention, the reference enzyme used is that derived from a thermophilic bacterium. As used herein, a "cold bacteria" is a microorganism having an optimum growth temperature of less than 45 ° C, and belongs to archaea, gram positive eubacteria, gram negative eubacteria, or eukaryotes including yeast and mold. It is a microbe.

  In addition, the origin of the reference enzyme is not particularly limited as a limit to be a cold bacterium, and may be appropriately selected according to the type of the reference enzyme and the like. Specific examples of the origin of the reference enzyme include thermophilic bacteria such as Aspergillus, Bacillus, Escherichia, Mucor, Penicillium, Pseudomonas, Streptomyces and the like. Among these thermophilic bacteria, from the viewpoint of obtaining a modified product in which the enzyme activity at low temperature is further improved more efficiently, Escherichia coli is preferably mentioned, and more preferably Escherichia coli (E. coli).

  The reference enzyme used in the present invention may be a wild-type enzyme derived from a thermophilic bacterium, or may be a mutant-type enzyme obtained by introducing a mutation into a wild-type enzyme derived from a thermophilic bacterium.

Step 1
In step 1, the three-dimensional structure of the thermophilic bacterium-derived modified enzyme is analyzed, and an amino acid sequence site located at a distance of 8 Å from the substrate binding site and / or the coenzyme binding site of the modified enzyme is identified.

  The substrate binding site and / or the coenzyme binding site of the modified enzyme is known for each type of modified enzyme. For example, if it is a 3-isopropyl malate dehydrogenase derived from Thermus thermophilus (a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; hereinafter sometimes referred to as TthIPMDH), a substrate binding site and a coenzyme binding site Is the 7th to 12th, 15th, 41 to 42nd, 69th to 78th, 85th to 92nd, 94th, 102th, 104th, 132th to 134th, 139th parts in the amino acid sequence shown in SEQ ID NO: 1 -140th, 184th-188th, 196th, 213th-219th, 221st-222nd, 225th-226th, 229th, 237-238th, 240th-242nd, 244th-245 Th, 248th, 253rd-257th, 259th, 261st, 270th 280 th, 290 th 284 th, it is known to be located 324 th -327 th region.

  Also known is a method of acquiring the three-dimensional structure information from the amino acid sequence of a protein, visualizing the three-dimensional structure, and specifying an amino acid residue present at a specified distance from an arbitrary region in the protein. Therefore, in step 1, based on the amino acid sequence of the modified enzyme, the three-dimensional structure information of the modified enzyme is obtained and the three-dimensional structure of the modified enzyme is visualized according to a known method. The amino acid sequence site existing at a distance of 8 Å from the site may be specified.

  Specifically, based on the amino acid sequence of the modified enzyme, Protein Data Bank (http://www.rcsb.org/pdb/home/home.do) is used to obtain its conformational information. You just need to get the PBD file. For example, TthIPMDH is tagged with ID: 4F7I in Protein Data Bank, and by searching this value against the database, it is possible to obtain TthIPMDH three-dimensional structure information in the PDB file format.

  Also, the three-dimensional structure information (PBD file) obtained above is output by software such as Swiss-Pdb Viewer Version 4.1 (http://spdbv.vital-it.ch/) to obtain the three-dimensional structure of the enzyme. Can be visualized. In addition, by using the software, it is possible to specify an amino acid residue present at a distance of 8 Å from the substrate binding site and / or the coenzyme binding site in the three-dimensional structure of the modified enzyme.

  In the present specification, the amino acid sequence site existing at a distance of 8 Å from the substrate binding site and / or the coenzyme binding site is at least a substrate binding site and / or a coenzyme binding site in the three-dimensional structure of the modified enzyme. Starting from the amide bond part (one of two amide bond parts) of one amino acid residue, the part constituting the main chain (-NH-C-CO-; excluding the side chain of the amino acid residue The whole of “portion” means a sequence site of amino acid residue included within a distance of 8 Å or less.

  When the modified enzyme does not require a coenzyme at the time of the enzyme reaction, an amino acid sequence site present at a distance of 8 Å from the substrate binding site of the modified enzyme may be specified in this step 1. In addition, when the modified enzyme requires a coenzyme at the time of the enzyme reaction, an amino acid sequence site existing at a distance of 8 Å from at least one of the substrate binding site or the coenzyme binding site of the modified enzyme is identified. However, from the viewpoint of obtaining a variant with improved enzyme activity at low temperature more efficiently, an amino acid sequence site existing at a distance of 8 Å from both the substrate binding site and the coenzyme binding site is specified It is preferable to do.

Step 2
In step 2, the amino acid sequence of the mutant enzyme and the amino acid sequence of a reference enzyme derived from a thermophilic bacterium which is the same type as the mutant enzyme are analyzed by pairwise alignment, and the amino acid sequence site identified in step 1; In the amino acid sequence of the reference enzyme, corresponding amino acid sequence sites are compared on a pairwise alignment to identify an amino acid sequence site where the amino acid residue is not identical between them.

  Methods for making pairwise alignments of the amino acid sequences of two proteins are known. In this step 2, a pairwise alignment may be created between the amino acid sequence of the modified enzyme and the amino acid sequence of the reference enzyme according to a known method. Specifically, ClustalW Version 2.1 (http://clustalw.ddbj.nig.ac.jp/) on the DNA Data Bank of Japan website may be used to output the pairwise alignment.

  In this step 2, after creating a pairwise alignment, the amino acid sequence site specified in the step 1 in the amino acid sequence of the modified enzyme (hereinafter sometimes referred to as "modified enzyme 8 Å region site"); The corresponding sites (hereinafter referred to as "reference enzyme corresponding site") on the pairwise alignment in the amino acid sequence of the reference enzyme are compared, and an amino acid sequence site in which the amino acid residue is not identical between them is specified.

  Specifically, (1) when the amino acid residue at the 8 Å region of the modified enzyme is different from the amino acid residue at the corresponding site of the reference enzyme, (2) the amino acid residue at the 8 Å region of the modified enzyme is referred to When deleted at the enzyme corresponding site, and (3) when the amino acid residue at the reference enzyme corresponding site is deleted at the 8 Å region site of the modified enzyme, the 8 Å region site of the modified enzyme corresponds to the reference enzyme Amino acid residues are identified as inconsistent amino acid sequence sites between the sites.

  The amino acid site identified in step 2 is identified as a site that is likely to improve the enzyme activity at low temperature by mutating the modified enzyme. Hereinafter, the amino acid sequence site identified in step 2 may be referred to as a “mutation candidate site”.

Step 3
In step 3, amino acid substitution, deletion, or insertion is performed on at least one of mutation candidate sites in the enzyme to be modified to obtain a variant.

  In this step 3, when there are two or more mutation candidate sites, one mutation enzyme site may be selected to perform single mutation, or two or more mutation enzyme sites may be selected to perform multiple mutation. . Specifically, when there are two or more mutation candidate sites, the number of mutations to be introduced varies depending on the number of mutation candidate sites, the type of mutant enzyme, etc., but can not be generally described. 1 to 10, more preferably 1 to 5, still more preferably 1 to 4, particularly preferably 1 to 3 can be mentioned.

  In this step 3, the mutation to be made to the mutation candidate site of the modified enzyme may be any of amino acid substitution, deletion or insertion, but the modification in which the enzyme activity at low temperature is improved From the viewpoint of enhancing the acquisition efficiency of the body, it is preferable to introduce a mutation so as to be identical to the amino acid residue at the corresponding site of the reference enzyme. Specifically, if the amino acid residue at the mutation candidate site is different from the amino acid residue at the site corresponding to the reference enzyme as a preferable mutation introduction mode, refer to the amino acid residue at the mutation candidate site. (2) If the amino acid residue at the mutation candidate site is deleted at the reference enzyme corresponding site, the amino acid residue at the mutation candidate site is deleted; (3) If the amino acid residue at the site corresponding to the reference enzyme is deleted at the mutation candidate site, the amino acid residue at the site corresponding to the reference enzyme may be inserted into the mutation candidate site.

  In addition, a method for substituting, deleting or inserting an amino acid at a specific site of a protein is known, and in this step 3, substitution, deletion or insertion of an amino acid at a mutation candidate site should be carried out according to known methods. Can.

Step 4
In the step 4, among the variants obtained in the step 3, a variant having improved enzyme activity at a lower temperature than the modified enzyme is selected.

  In the present specification, the phrase "enzymatic activity at a lower temperature than the modified enzyme is improved" means 37 ° C or less, preferably 37 ° C or less, more preferably 30 ° C or less, still more preferably 10 to 25 ° C. It means that the enzyme activity in the temperature range is higher than that of the modified enzyme.

  Specifically, in this step 4, the specific activity (enzyme activity per enzyme protein weight) of the enzyme is measured at low temperature for the modified product obtained in step 3 and the modified enzyme, and the temperature is lower than that of the modified enzyme It is sufficient to select a variant in which the specific activity of the enzyme at. The method for determining the specific activity can not be generalized and described here because it varies depending on the type of enzyme, but for example, in the case of 3-isopropylmalate dehydrogenase, the item of [Measurement of IPMDH activity] described later, And [measurement of IPMDH specific activity].

  Thus, among the variants obtained in the step 3, by selecting a variant having improved enzyme activity at a lower temperature than the modified enzyme, the enzyme activity is effectively expressed even in a low temperature region. It is possible to obtain a variant of the enzyme that can

2. Polypeptide The present invention provides a polypeptide obtained by the method for obtaining the variant. The polypeptide of the present invention is a modified form of TthIPMDH, and is a 3-isopropyl malate dehydrogenase in which 3-isopropyl malate dehydrogenase activity at low temperature is improved over TthIPMDH. 3-isopropylmalate dehydrogenase is an oxidoreductase whose coenzyme is nicotinamide adenine dinucleotide (hereinafter also described as NAD ⁺ ), and 3-isopropylmalate and NAD ⁺ as a substrate, It is an enzyme that catalyzes a reaction that produces isopropyl-3 oxosuccinic acid and NADH, and H ⁺ , and the EC number is classified into 1.1.1.85.

Specifically, one aspect of the polypeptide of the present invention includes the woven peptides shown in the following (A) to (D).
(A) a polypeptide comprising an amino acid sequence in which the 78th position in the amino acid sequence shown in SEQ ID NO: 1 is replaced with glutamic acid,
(B) in the amino acid sequence shown in SEQ ID NO: 1 at position 214, substituted with methionine, glycine or alanine, at position 216 for isoleucine, leucine or valine, at position 218 for asparagine, serine or glutamine, at position 219 for alanine, methionine Or a glycine, a 222nd amino acid substitution with glutamine, asparagine or serine, a 225nd amino acid substitution with lysine or histidine, and a 229th amino acid substitution with glutamine, asparagine or serine,
(C) a polypeptide comprising an amino acid sequence in which the 272nd position in the amino acid sequence shown in SEQ ID NO: 1 is substituted with alanine, methionine or glycine and the 273rd position is substituted with glycine, alanine or methionine,
(D) In the amino acid sequence shown in SEQ ID NO: 1, position 324 is substituted with arginine, lysine or histidine, position 325 is substituted with threonine, glutamine or asparagine, and glycine, alanine or methionine is inserted between positions 325 and 326 A polypeptide comprising the amino acid sequence
(E) In the amino acid sequence shown in SEQ ID NO: 1, position 272 is substituted with alanine, methionine or glycine, position 273 is substituted with glycine, alanine or methionine, position 324 is substituted with arginine, lysine or histidine, position 325 is threonine, glutamine Or a polypeptide comprising an amino acid sequence which is substituted with asparagine and glycine, alanine or methionine is inserted between the 325th and the 326th.

  In the polypeptide (B), from the viewpoint of further improving 3-isopropylmalate dehydrogenase activity at low temperature, preferably, the 214th position in the amino acid sequence shown in SEQ ID NO: 1 is substituted with methionine, the 216th position Is isoleucine, 218 is substituted by asparagine, 219 is substituted by alanine, 222 is substituted by glutamine, 225 is substituted by lysine, and the 229 is substituted by glutamine. Be

  In the polypeptide of (C), from the viewpoint of further improving 3-isopropylmalate dehydrogenase activity at low temperature, preferably, the 272nd position in the amino acid sequence shown in SEQ ID NO: 1 is substituted with alanine, and 273 The polypeptide which consists of the amino acid sequence by which the th was substituted by glycine is mentioned.

  In the polypeptide of (D), from the viewpoint of further improving 3-isopropylmalate dehydrogenase activity at low temperature, preferably, the 324th position in the amino acid sequence shown in SEQ ID NO: 1 is substituted with arginine, 325th position And a threonine, and a polypeptide consisting of an amino acid sequence in which glycine is inserted between the 325th and the 326th.

  In the polypeptide of the above (E), from the viewpoint of further improving 3-isopropylmalate dehydrogenase activity at low temperature, preferably, at position 272 in the amino acid sequence shown in SEQ ID NO: 1 alanine, A polypeptide comprising an amino acid sequence comprising a glycine substitution, a 324th substitution with arginine, a 325th substitution with threonine, and a glycine inserted between 325th and 326th positions.

Moreover, the polypeptide shown to the following (F)-(O) as another aspect of the polypeptide of this invention is mentioned.
(F) In the amino acid sequence in which amino acid position 78 in the amino acid sequence shown in SEQ ID NO: 1 is substituted with glutamic acid, one or several amino acids other than amino acid position 78 are substituted, added, inserted or deleted, and A polypeptide having isopropylmalate dehydrogenase activity and having improved 3-isopropylmalate dehydrogenase activity at low temperature as compared to TthIPMDH,
(G) in the amino acid sequence shown in SEQ ID NO: 1 at position 214 substituted with methionine, glycine or alanine, at position 216 for isoleucine, leucine or valine, at position 218 for asparagine, serine or glutamine, at position 219 for alanine, methionine Or at glycine 222, at position 222 for glutamine, asparagine or serine, at position 225 for lysine or histidine, and at position 229 for glutamine, asparagine or serine at position 214, 216, 218 And one or several amino acids other than 219, 222, 225 and 229 are substituted, added, inserted or deleted and has 3-isopropylmalate dehydrogenase activity, 3 at low temperature compared to TthIPMDH Polypeptide isopropyl malate dehydrogenase activity is improved,
(H) One of the amino acids other than 272 and 273 in the amino acid sequence in which the 272nd position in the amino acid sequence shown in SEQ ID NO: 1 is substituted with alanine, methionine or glycine and the 273rd position is substituted with glycine, alanine or methionine Or several are substituted, added, inserted or deleted, and have 3-isopropylmalate dehydrogenase activity, and improve 3-isopropylmalate dehydrogenase activity at low temperature compared to TthIPMDH The polypeptide being
(I) In the amino acid sequence shown in SEQ ID NO: 1, position 324 is substituted with arginine, lysine or histidine, position 325 is substituted with threonine, glutamine or asparagine, and glycine, alanine or methionine is inserted between positions 325 and 326 And one or more amino acids other than the amino acids at positions 324 and 325 and the amino acid inserted between positions 325 and 326 are substituted, added, inserted or deleted, and A polypeptide having 3-isopropylmalate dehydrogenase activity and having improved low-temperature 3-isopropylmalate dehydrogenase activity as compared to TthIPMDH,
(J) In the amino acid sequence shown in SEQ ID NO: 1, position 272 is substituted with alanine, methionine or glycine, position 273 is substituted with glycine, alanine or methionine, position 324 is substituted with arginine, lysine or histidine, position 325 is threonine, glutamine Or in the amino acid sequence formed by substitution of asparagine and glycine, alanine or methionine between 325 and 326, amino acids 272, 273, 324 and 325 and between 325 and 326 One or several amino acids other than the amino acid to be inserted are substituted, added, inserted or deleted, and have 3-isopropylmalate dehydrogenase activity, and 3 at low temperature as compared to Tth IP MDH -The isopropyl malate dehydrogenase activity is improved Polypeptide,
(K) The sequence identity excluding amino acid 78 in the amino acid sequence shown in SEQ ID NO: 1 is 80% or more, and has 3-isopropylmalate dehydrogenase activity, and at low temperature as compared to TthIPMDH Polypeptide having improved 3-isopropylmalate dehydrogenase activity of
(L) in the amino acid sequence shown in SEQ ID NO: 1 at position 214 substituted with methionine, glycine or alanine, at position 216 for isoleucine, leucine or valine, at position 218 for asparagine, serine or glutamine, at position 219 for alanine, methionine Or at glycine 222, at position 222 for glutamine, asparagine or serine, at position 225 for lysine or histidine, and at position 229 for glutamine, asparagine or serine at position 214, 216, 218 The sequence identity excluding amino acids 219, 222, 225, and 229 is 80% or more, and has 3-isopropylmalate dehydrogenase activity, and at low temperature as compared to TthIPMDH 3-isopropyl phosphorus Polypeptide dehydrogenase activity is improved,
(M) A sequence obtained by replacing amino acid 272 and 273 in an amino acid sequence in which amino acid 272 in the amino acid sequence shown in SEQ ID NO: 1 is substituted with alanine, methionine or glycine and amino acid 273 is substituted with glycine, alanine or methionine A polypeptide having an identity of 80% or more, and having 3-isopropylmalate dehydrogenase activity and having improved low-temperature 3-isopropylmalate dehydrogenase activity at low temperature as compared to TthIPMDH,
(N) In the amino acid sequence shown in SEQ ID NO: 1, arginine, lysine or histidine substitutes for position 324, threonine, glutamine or asparagine for position 325, and glycine, alanine or methionine is inserted between positions 325 and 326 80% or more in sequence identity excluding amino acids 324 and 325 and amino acids inserted between 325 and 326, and 3-isopropylmalate dehydrogenase activity And a polypeptide having improved 3-isopropylmalate dehydrogenase activity at low temperature compared to TthIPMDH,
(O) In the amino acid sequence shown in SEQ ID NO: 1, position 272 is substituted with alanine, methionine or glycine, position 273 is substituted with glycine, alanine or methionine, position 324 is substituted with arginine, lysine or histidine, position 325 is threonine, glutamine Or in the amino acid sequence formed by substitution of asparagine and glycine, alanine or methionine between 325 and 326, amino acids 272, 273, 324 and 325 and between 325 and 326 The sequence identity excluding the inserted amino acid is 80% or more, and has 3-isopropylmalate dehydrogenase activity, and 3-isopropylmalate dehydrogenase activity at low temperature compared to TthIPMDH Improved polypeptides.

  Hereinafter, in the above-mentioned polypeptides (F) and (K), an amino acid site other than the 78th position in the amino acid sequence shown in SEQ ID NO: 1 may be referred to as an “optionally modified site”. In the above-mentioned polypeptides (G) and (L), amino acid sites other than 214, 216, 218, 219, 222, 225 and 229 in the amino acid sequence shown in SEQ ID NO: 1 are It may be described as "optionally modified site". Furthermore, in the polypeptides (H) and (M), amino acid sites other than the 272nd and 273rd amino acids in the amino acid sequence shown in SEQ ID NO: 1 may be referred to as “arbitrary modification sites”. Furthermore, in the above-mentioned polypeptides (I) and (N), “optionally modified except for the 324th and 325th amino acid sites in the amino acid sequence shown in SEQ ID NO: 1 and the amino acid site inserted between 325th and 326th It may be written as "part". In the above-mentioned polypeptides (J) and (O), amino acid positions 272, 273, 324 and 325 in the amino acid sequence shown in SEQ ID NO: 1 and amino acids inserted between 325 and 326 Other than the site may be described as "arbitrary alteration site".

  The amino acid modifications introduced into the arbitrary modification sites of the polypeptides of (F) to (J) above include only one type of modification (for example, substitution) among substitution, addition, insertion and deletion. And may include two or more modifications (eg, substitution and insertion). In the above-mentioned (F) to (J) polypeptides, one or more or several amino acids may be substituted, added, inserted or deleted at any modification site, for example, 1 to 10, preferably Is 1 to 7, more preferably 1 to 5, more preferably 1 to 3, particularly preferably 1 or 2 or 1.

  Further, in the polypeptides (K) to (O), the sequence identity excluding the specific amino acid in the amino acid sequence shown in SEQ ID NO: 1 may be 80% or more, preferably 90% or more, More preferably, it is 95% or more, particularly preferably 99% or more.

  Here, in the polypeptides (K) to (O) above, the sequence identity excluding the specific amino acid in the amino acid sequence shown in SEQ ID NO: 1 means only the above-mentioned arbitrary modification site from the amino acid sequence shown in SEQ ID NO: 1 The sequence identity is calculated by comparing only the arbitrary altered site. Furthermore, “sequence identity” means bl2seq program (Tatiana A. Tatsusova, Thomas L. Madden, FEMS) of BLAST PACKAGE [sgi 32 bit edition, Version 2.0. 12; available from National Center for Biotechnology Information (NCBI)]. The amino acid sequence identity value obtained by Microbiol.Lett., Vol. 174, p247-250, 1999) is shown. The parameters may be set to Gap insertion Cost value: 11 and Gap extension Cost value: 1.

  Moreover, it is preferable that the amino acid substitution introduce | transduced into the arbitrary modification site | part of the polypeptide of said (F)-(O) is a conservative substitution. That is, as the substitution at the arbitrary modification site, for example, if the amino acid before substitution is a nonpolar amino acid, substitution with another nonpolar amino acid, if the amino acid before substitution is a noncharged amino acid, other noncharged Substitutions with amino acids, substitution with other acidic amino acids if the amino acid before substitution is an acidic amino acid, and substitution with other basic amino acids with a basic amino acid before substitution are mentioned.

  In the above-mentioned polypeptides (F) to (O), “the 3-isopropylmalate dehydrogenase activity at low temperature is improved as compared to TthIPMDH” is 3-isopropyl under the temperature condition of 25 ° C. When the specific activity of malate dehydrogenase is measured, it means that the specific activity is higher than that of TthIPMDH. More specifically, when the specific activity of 3-isopropylmalate dehydrogenase is measured under a temperature condition of 25 ° C., the specific activity is 3 times or more, preferably 5 times or more, more preferably 10 as compared to TthIPMDH. Point out that it has improved more than twice. In addition, since the specific activity of TthIPMDH at 25 ° C. is 3.1 U / mg, 3-isopropyl malic acid at 25 ° C. as a preferred example of the polypeptides of (F) to (O). The specific activity of the dehydrogenase activity is 9.3 U / mg or more, preferably 10 U / mg or more, and more preferably 30 U / mg or more. In the present invention, 3-isopropylmalate dehydrogenase activity is specifically measured under the following conditions.

[Measurement of 3-isopropylmalate dehydrogenase activity]
The 3-isopropylmalate dehydrogenase activity is measured using the following reagent under the following measurement conditions.

(reagent)
Aqueous solution containing 50 mM HEPES-KOH buffer (pH 8.0), 5 mM magnesium chloride, 100 mM potassium chloride, 400 μM 3-isopropyl malic acid, 5 mM NAD ⁺

(Measuring method)
Place 0.9 mL of reagent in the spectrophotometer cell and preincubate at 25 ° C. for 5 minutes more. After adding 0.005 mL of an enzyme solution in which the polypeptide to be measured is diluted to a predetermined concentration is added and mixed well, the change in absorbance at 340 nm is recorded for 60 seconds with a spectrophotometer, and the change in absorbance (ΔOD) is measured. The blank is mixed with water in the reagent instead of the enzyme solution and the change in absorbance (ΔOD blank) is measured as described above. From these values, the activity value is determined according to the following formula.

In the above formula, 905 is the volume of the reagent and enzyme solution, 6.22 is the molecular absorption coefficient of NAD ⁺ (cm ² / micromole), 5 is the volume of the enzyme solution, 1.0 is the light path of the spectroscopic cell Indicates the length (cm).

[Determination of specific activity of 3-isopropyl malate dehydrogenase]
The specific activity of 3-isopropyl malate dehydrogenase is measured under the following measurement conditions. The activity (U / mL) of the enzyme solution is determined according to the above method, and the polypeptide concentration (mg / ml) of the enzyme solution used for the determination is further determined. From these values, the specific activity is determined according to the following formula.

3. DNA encoding the polypeptide
The DNA encoding the polypeptide of the present invention (hereinafter sometimes referred to as “the DNA of the present invention”) is, for example, the above amino acid mutation introduced into the DNA encoding TthIPMDH (SEQ ID NO: 1). It can be obtained by Moreover, the DNA of the present invention can also be artificially synthesized by a total gene synthesis method.

  Here, the DNA encoding TthIPMDH is known, for example, as the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing, and is determined by PCR from genomic DNA of Thermus thermophilus HB8 strain. It can be isolated.

  Methods for introducing specific mutations at specific sites of a base sequence are known, and for example, site-directed mutagenesis of DNA can be used. As a specific method for converting a base in DNA, for example, utilization of a commercially available kit (QuickChange Lightning Site-Directed Mutagenesis kit: manufactured by Stratagene, KOD-Plus-Mutagenesis kit: manufactured by Toyobo, etc.) may be mentioned.

  The DNA in which mutations have been introduced into the base sequence in this manner can be confirmed using the DNA sequencer. For the obtained base sequence, for example, analysis is performed using base sequence analysis software such as DNASIS (made by Hitachi Software Engineering) or GENETIX (made by software development) to identify the coding region of the ACS gene in DNA. be able to.

  The DNA sequence thus obtained can be confirmed by using a DNA sequencer. About the obtained base sequence, the said peptide (3-isopropyl malate dehydrogenation in DNA is carried out by analyzing with base sequence analysis software, such as DNASIS (made by Hitachi Soft Engineering Co., Ltd.) and GENETIX (made by software development company) etc. The coding region of the enzyme) can be identified.

  Once the base sequence is determined, the DNA encoding the polypeptide is then obtained by chemical synthesis, PCR using a cloned probe as a template, or hybridization using a DNA fragment having the base sequence as a probe. be able to.

  Furthermore, it is possible to synthesize a mutant of the DNA encoding the above-mentioned polypeptide by site-directed mutagenesis and the like, which has the same function as that before mutation. In addition, in order to introduce a mutation into the DNA encoding the above-mentioned peptide, known methods such as the Kunkel method, Gapped duplex method, megaprimer PCR method or the like, can be adopted.

  When producing a DNA encoding a fusion protein in which a DNA encoding the polypeptide and a DNA encoding a foreign protein or peptide are linked, the DNA encoding the polypeptide is a DNA encoding the foreign protein or peptide. It suffices to connect. Examples of the foreign protein or peptide include proteins or peptides used for protein purification (glutathione S-transferase, maltose binding protein, thioredoxin, cellulose binding domain, streptavidin binding peptide, histidine tag, etc.). The position where the foreign protein or peptide is linked to the polypeptide can be appropriately selected so that the polypeptide and the foreign protein or peptide have their respective functions or activities. In the method of ligating a DNA encoding a foreign protein or peptide to the DNA encoding the polypeptide, the purified DNA and the DNA encoding a foreign protein or peptide are digested with an appropriate restriction enzyme, respectively. And a method of linking. In addition, by providing regions homologous to each of the DNA encoding the polypeptide and the DNA encoding the foreign protein or peptide, the in vitro method using PCR etc. or the in vivo method using yeast etc. It may be a method of connecting both.

  Further, the DNA of the present invention encodes a polypeptide having 3-isopropylmalate dehydrogenase activity and having improved 3-isopropylmalate dehydrogenase activity at low temperature as compared to TthIPMDH, Included are DNAs that hybridize under stringent conditions with DNA comprising a base sequence complementary to the DNA encoding the polypeptide of (A) to (D) above.

  Here, "under stringent conditions" means 0.5% SDS, 5 × Denhartz's, 0.1% bovine serum albumin (BSA), 0.1% polyvinyl pyrrolidone, 0.1% Ficoll 4) to overnight at 50 ° C. to 65 ° C. in 6 × SSC (1 × SSC, 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) containing 400] and 100 μg / ml salmon sperm DNA It says the condition to keep warm.

Specifically, hybridization under stringent conditions is performed by the following procedure. That is, a nylon membrane on which a DNA library or a cDNA library is immobilized is prepared, and it is prepared at 65 ° C. in a prehybridization solution containing 6 × SSC, 0.5% SDS, 5 × denhaltz, 100 μg / ml salmon sperm DNA. Block the nylon membrane. Thereafter, each probe labeled with ³² P is added and incubated overnight at 65 ° C. This nylon membrane is incubated in 6 × SSC for 10 minutes at room temperature, in 2 × SSC containing 0.1% SDS for 10 minutes at room temperature, in 0.2 × SSC containing 0.1% SDS for 30 minutes at 45 ° C. After washing, autoradiography can be performed to detect DNA specifically hybridized with the probe.

  Furthermore, the DNA of the present invention encodes a polypeptide having 3-isopropylmalate dehydrogenase activity and having improved low-temperature 3-isopropylmalate dehydrogenase activity as compared to TthIPMDH, Also included are DNAs having 80% or more homology to the DNA encoding the polypeptides of (A) to (D) above. The homology is preferably 90% or more, more preferably 95% or more, and particularly preferably 98% or more.

  Here, the “homology” of DNA means the bl2seq program (Tatiana A. Tatsusova, Thomas L., BLAST PACKAGE [sgi 32 bit edition, Version 2.0. 12; available from the National Center for Biotechnology Information (NCBI)]). Madden, FEMS Microbiol. Lett., Vol. 174, 247-250, 1999) shows the value of identity obtained. The parameters may be set to Gap insertion Cost value: 11 and Gap extension Cost value: 1.

  The DNA of the present invention is preferably one in which codon usage frequency is optimized for the host, and more preferably DNA in which codon usage frequency is optimized for E. coli.

A total of host optimum codon usage frequency of each codon may be adopted as an index indicating the codon usage frequency. The optimal codon is defined as the codon most frequently used among the codons corresponding to the same amino acid. The codon usage frequency is not particularly limited as long as it is optimized for the host, and for example, the following may be mentioned as an example of the optimal codon of E. coli.
F: phenylalanine (ttt), L: leucine (ctg), I: isoleucine (att), M: methionine (atg), V: valine (gtg), Y: tyrosine (tat), stop codon (taa), H: Histidine (cat), Q: Glutamine (cag), N: Asparagine (aat), K: Lysine (aaa), D: Aspartate (gat), E: Glutamate (gaa), S: Serine (agc), P: Proline (ccg), T: threonine (acc), A: alanine (gcg), C: cysteine (tgc), W: tryptophan (tgg), R: arginine (cgc), G: glycine (ggc).

4. Recombinant vector A recombinant vector (hereinafter sometimes referred to as "the recombinant vector of the present invention") containing a DNA encoding the peptide of the present invention is obtained by inserting the DNA of the present invention into an expression vector Can.

  The recombinant vector of the present invention includes control elements such as a promoter operably linked to the DNA of the present invention. The control element typically includes a promoter, but may further contain a transcription element such as an enhancer, a CCAAT box, a TATA box, or an SPI site, if necessary. Further, operably linked means that the DNA of the present invention is operably linked in a host with various regulatory elements such as promoters, enhancers and the like that regulate the DNA of the present invention.

  As an expression vector, a vector constructed for genetic recombination from a phage or plasmid capable of autonomously propagating in a host is suitable.

  Examples of the phage include Lambda gt10, Lambda gt11 and the like when using Escherichia coli described later as a host.

  Examples of the plasmid include pBR322, pUC18, pUC118, pUC19, pUC119, pTrc99A, pBluescript, pET21-c, and Cosmid Super Cos I etc. when E. coli is used as the host.

  When Pseudomonas is used as a host, RSF1010, pBBR122, and pCN51, which are broad host range vectors for gram-negative bacteria, can be mentioned. In addition, animal viruses such as retrovirus and vaccinia virus, and insect virus vectors such as baculovirus can also be used.

5. Transformant A transformant (hereinafter sometimes referred to as "the transformant of the present invention") is obtained by transforming a host using the recombinant vector of the present invention.

  The host used for producing the transformant is not particularly limited as long as the recombinant vector is stable and capable of autonomous growth and capable of expressing a foreign gene trait, but, for example, Escherichia coli etc. Bacteria belonging to the genus Escherichia, bacteria belonging to the genus Bacillus such as Bacillus subtilis, and bacteria belonging to the genus Pseudomonas such as Pseudomonas putida; yeast; animal cells such as COS cells; insect cells such as Sf 9; Whole plants, plant organs (eg, leaves, petals, stems, roots and seeds), plant tissues (eg, epidermis, phloem, parenchyma, xylem and vascular bundles etc.), plant cultured cells, etc. Be Among these, E. coli is preferable, and E. coli DH5α, E. coli BL21, E. coli BL21 (DE3), E. coli Rosetta2, E. coli Rosetta2 (DE3), and E. coli JM109 are more preferable.

  The transformant of the present invention can be obtained by introducing the recombinant vector of the present invention into a host, and the conditions for introducing the recombinant vector into the host may be appropriately set according to the type of host and the like. When the host is a bacterium, for example, methods using competent cells by calcium ion treatment, electroporation methods and the like can be mentioned. When the host is yeast, for example, electroporation (electroporation), spheroplast method, lithium acetate method and the like can be mentioned. When the host is an animal cell, for example, electroporation, calcium phosphate method, lipofection method and the like can be mentioned. When the host is an insect cell, for example, calcium phosphate method, lipofection method, electroporation method and the like can be mentioned. When the host is a plant vesicle, for example, electroporation, Agrobacterium method, particle gun method, PEG method and the like can be mentioned.

  Whether or not the recombinant vector of the present invention has been incorporated into the host can be confirmed by PCR, Southern hybridization, Northern hybridization and the like.

  When confirming whether the recombinant vector of the present invention has been incorporated into the host by PCR, for example, the recombinant vector may be separated and purified from the transformant.

  For example, when the host is a bacterium, separation and purification of the recombinant vector are performed based on a lysate obtained by lysing the bacterium. The method of lysis is, for example, treated with a lysis enzyme such as lysozyme, and if necessary, a protease and other enzymes and a surfactant such as sodium lauryl sulfate (SDS) are used in combination.

  Furthermore, physical disruption methods such as freeze-thaw and French pressing may be combined. Separation and purification of DNA from lysates can be performed, for example, by appropriately combining dephenolization treatment with phenol treatment and protease treatment, ribonuclease treatment, alcohol precipitation treatment, and a commercially available kit.

  Cleavage of DNA can be performed according to a conventional method, for example, using restriction enzyme treatment. As a restriction enzyme, for example, a type II restriction enzyme that acts on a specific nucleotide sequence is used. The ligation of the DNA and the expression vector is performed using, for example, a DNA ligase.

  Thereafter, using the separated and purified DNA as a template, a primer specific to the DNA of the present invention is designed and PCR is performed. The amplified product obtained by PCR is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis and the like, stained with ethidium bromide and SYBR Green solution, and the like, and the amplified product is detected as a band to give a trait. It can be confirmed that it has been converted.

  Alternatively, amplification products can be detected by conducting PCR using primers previously labeled with a fluorescent dye or the like. Furthermore, a method may also be employed in which the amplification product is bound to a solid phase such as a microplate and the amplification product is confirmed by fluorescence, an enzyme reaction or the like.

6. Production of Polypeptide The polypeptide of the present invention can be produced by culturing the transformant of the present invention.

  The culture form of the transformant of the present invention may be selected from culture conditions in consideration of the nutritional and physiological properties of the host, but preferably includes liquid culture. Industrially, it is advantageous to carry out aeration and agitation culture.

  As a nutrient source of the medium, those required for growth of transformants can be used. The carbon source may be any assimilable carbon compound, and examples thereof include glucose, sucrose, lactose, maltose, molasses, pyruvic acid and the like.

  The nitrogen source may be any assimilable nitrogen compound, and examples thereof include peptone, meat extract, yeast extract, casein hydrolyzate, and soybean meal alkaline extract.

  In addition to carbon sources and nitrogen sources, for example, phosphates, carbonates, sulfates, salts such as magnesium, calcium, potassium, iron, manganese and zinc, specific amino acids and specific vitamins, etc., as required You may

  The culture temperature can be set as appropriate as long as the transformant of the present invention can grow and the transformant of the present invention can produce the polypeptide of the present invention, but is preferably about 15 to 37 ° C. Culturing may be completed at an appropriate time in anticipation of reaching the maximum yield of the polypeptide of the present invention, and the culturing time is usually about 12 to 48 hours.

The pH of the culture medium is such that the host develops and the host is mutated ACS
The pH may be suitably changed within the range of producing, but preferably in the range of about pH 5.0 to 9.0.

  The transformant of the present invention is cultured, and the culture supernatant is collected by a method such as centrifugation to recover the culture supernatant or cells, and the cells are treated with a mechanical method such as ultrasound and French press or a lytic enzyme such as lysozyme A water-soluble fraction containing the polypeptide of the present invention can be obtained by applying and using an enzyme such as protease or a surfactant such as sodium lauryl sulfate (SDS) if necessary.

  Alternatively, the expressed polypeptide of the present invention can be secreted into the culture solution by selecting an appropriate expression vector and host.

  The water-soluble fraction containing the polypeptide of the present invention obtained as described above may be subjected to purification treatment as it is, but after concentration of the polypeptide of the present invention in the water-soluble fraction, purification treatment may be carried out You may use it.

  The concentration can be carried out, for example, by vacuum concentration, membrane concentration, salting-out, fractional precipitation with a hydrophilic organic solvent (eg, methanol, ethanol and acetone), or the like.

  The purification process of the polypeptide of the present invention can be performed, for example, by appropriately combining methods such as gel filtration, adsorption chromatography, ion exchange chromatography, affinity chromatography and the like.

  The purification process is already known and can be carried out by referring to appropriate documents, magazines, textbooks and the like. The polypeptide of the present invention thus purified can, if necessary, be powdered by lyophilization, vacuum drying, spray drying or the like and distributed in the market.

  Next, the present invention will be specifically described, but the present invention is not limited to the following.

1. Identification of Modified Enzyme and Reference Enzyme In this example, TthIPMD was used as the modified enzyme, and IPMDH derived from E. coli (hereinafter also referred to as EcoIPMDH) was used as the reference enzyme.

  The amino acid sequence of TthIPMDH and the base sequence of DNA encoding TthIPMDH are already known, and the sequence information can be obtained from the NCBI website (http://www.ncbi.nlm.nih.gov/). The amino acid sequence of TthIPMDH is shown in SEQ ID NO: 1 of the Sequence Listing, and the base sequence of DNA encoding the amino acid sequence is shown in SEQ ID NO: 2 of the Sequence Listing. In addition, the amino acid sequence of EcoIPMDH is also known, and as described above, its sequence information can be obtained. The amino acid sequence of EcoIPMDH is shown in SEQ ID NO: 3 of the sequence listing.

2. Isolation of the gene of the modified enzyme (TthIPMDH) The wild-type TthIPMDH DNA was PCR-PCR using the oligonucleotides of SEQ ID NO: 4 and SEQ ID NO: 5 with the genomic DNA extracted by the standard method by culturing Thermus thermophilus strain HB8 as a template. It was amplified. The DNA purified by ethanol precipitation and the plasmid pET21c were digested with restriction enzymes NdeI and HindIII, respectively, the digested DNA fragments were subjected to agarose gel electrophoresis, and the DNA was purified from gel. The purified TthIPMDH DNA fragment and the pET21c fragment are mixed and ligated using Ligation High (Toyobo Co., Ltd.), E. coli JM109 is transformed and cultured, the plasmid is purified from a single colony, and the sequence is determined by DNA sequencing. It was confirmed that a DNA consisting of the nucleotide sequence shown in No. 2 was cloned. From the base sequence, it was also confirmed that it encoded TthIPMDH consisting of the amino acid sequence shown in SEQ ID NO: 1.

3. Determination of the site of mutation introduction into the modified enzyme (TthIPMDH) The tertiary structure information (PDB ID: 4F7I) of TthIPMDH complexed with the coenzyme NAD ⁺ and substrate 3-isopropylmalate was obtained from the PDB home page, and the structure Were visualized by Swiss-PdbViewer. Amino acid residues present within 8 Å were extracted from amino acid residues that bind to coenzyme NAD ⁺ and substrate 3-isopropylmalate. At the same time, a pairwise alignment was made with ClustalW using the amino acid sequence of TthIPMDH shown in SEQ ID NO: 1 and the amino acid sequence of EcoIPMDH shown in SEQ ID NO: 3, and the amino acid residues present within 8 Å were marked. The results are shown in FIG. In FIG. 1, the amino acid residues present within 8 Å in the amino acid sequence of TthIPMDH are underlined. Among these, the portions different in amino acid residue between TthIPMDH and EcoIPMDH were marked by open squares.

  For example, in FIG. 1, the 7th to 12th residues ("PGDGIG") from the N-terminus in the amino acid sequence of TthIPMDH are present within 8 Å. On the other hand, in the amino acid sequence of EcoIPMDH, the corresponding site is the same as TthIPMDH ("PGDGIG"), and no mutation site was designed in this region.

  Further, in FIG. 1, residues 41 to 42 ("FG") from the N-terminus in the amino acid sequence of TthIPMDH are present within 8 Å. On the other hand, in the amino acid sequence of EcoIPMDH, the corresponding site was "VG", and the 41st residue was different. Therefore, it was decided to introduce a mutation (F41V) that changes the 41st phenylalanine (F41) to valine (V).

  Furthermore, in FIG. 1, residues 324 to 327 ("PP-DL") from the N-terminus in the amino acid sequence of TthIPMDH are present within 8 Å. On the other hand, in the amino acid sequence of EcoIPMDH, the corresponding site was "RTGDL", which was different in the form in which the 324th and 325th amino acid residues and the 325th and 326th insertions were inserted. Therefore, mutations (P324R / P325T / 326G (INSERTION)) that insert proline (P) at position 324 arginine (R), proline 325 at position threonine (T), and positions 325 and 326 glycine (G) I decided to introduce it.

  The other regions were also examined in the same manner to determine the mutation site.

  As a result of the above examination, it was decided to introduce the mutations shown in Table 1 respectively.

4. Site-Directed Mutagenesis of the Modified Enzyme (TthIPMDH) into a Gene Site-directed mutagenesis of the TthIPMDH gene was carried out by using oligonucleotides for mutation shown in SEQ ID NOS: 6 to 27 listed in Table 1 and SEQ ID NO: 28 and The oligonucleotide for amplification shown to sequence number 29 and pET21c vector (Hereinafter, it describes also as pET21c-TthIPMDH) which cloned the said TthIPMDH gene were utilized.

  Mutagenesis was performed by SOE (Splicing by overlap extension) -PCR method. The outline of the method is shown in FIG.

  That is, as shown in FIG. 2, the synthesis of mut1 gene in Table 1 was carried out by the following procedures (1) to (3).

(1) PCR # 1
Primer a in FIG. 2 is a T7 promoter primer designed on pET21c sequence, and is an oligonucleotide shown in SEQ ID NO: 28. Primer d is an oligonucleotide shown in SEQ ID NO: 7 in the sequence listing. The oligonucleotide is designed to introduce a mutation of F41V. The PCR reaction solutions shown in Table 2 were prepared, and 25 cycles of 98 ° C .: 30 seconds, 53 ° C .: 30 seconds, 72 ° C .: 120 seconds were performed. The DNA fragment between the amplified primers a to d was subjected to agarose gel electrophoresis, and the DNA fragment was purified from the gel.

(2) PCR # 2
Primer b in FIG. 2 is a T7 terminator primer designed on pET21c sequence, and is an oligonucleotide shown in SEQ ID NO: 29. Primer c is the oligonucleotide shown in SEQ ID NO: 6. The oligonucleotide is designed to introduce a mutation of F41V. The PCR reaction solutions shown in Table 3 were prepared, and 25 cycles of 98 ° C .: 30 seconds, 53 ° C .: 30 seconds, 72 ° C .: 120 seconds were performed. The DNA fragment between the amplified primers c and b was subjected to agarose gel electrophoresis, and the DNA fragment was purified from the gel.

(3) PCR # 3
Prepare PCR reaction solutions shown in Table 4 using the DNA fragments obtained by PCR # 1 and PCR # 2 as templates and primers a and b, 98 ° C: 30 seconds, 53 ° C: 30 seconds, 72 ° C : 120 cycles of reaction were carried out for 25 cycles. The DNA fragment between the amplified primers c and b was subjected to agarose gel electrophoresis, and the DNA fragment was purified from the gel.

  The TthIPMDH subjected to site-directed mutagenesis of mut2 to mut11 was also synthesized according to the procedure of (1) to (3) above using a predetermined oligonucleotide for mutation shown in Table 1.

The obtained mut1 to mut11 genes were each treated with a restriction enzyme with a solution having the composition shown in Table 5, subjected to agarose gel electrophoresis, and purified from the gel.

  The mut1 to mut11 genes digested with NdeI and HindIII are ligated with a solution having the composition shown in Table 6, respectively, and the reaction solution is added to E. coli JM109 competent cells for transformation, and the cells are transformed onto LB agar medium (1. The transformant colonies appeared by culturing at 37 ° C. for 18 hours with 0% tryptone, 0.5% yeast extract, 1.0% sodium chloride, 1.5% agar, 0.1 g / ml ampicillin) . Inoculate a single colony into LB medium, culture overnight, purify the plasmid by alkaline-SDS method, introduce the desired mutation by sequencing, and do not introduce unexpected mutation by PCR reaction It was confirmed.

5. Recombinant Expression and Purification of TthIPMDH and its Modified Genes The plasmids of wild type TthIPMDH and mut1 to mut11 obtained in Example 1 were used to transform E. coli Rosetta2 (DE3), and the cells were plated on LB agar (1.0% The transformant colonies were appeared by culturing in tryptone, 0.5% yeast extract, 1.0% sodium chloride, 1.5% agar, 0.1 g / ml ampicillin) at 37 ° C. for 16 hours.

  Single colonies were inoculated in 100 ml of LB medium and cultured at 37 ° C. for 24 hours to express TthIPMDH and variants of TthIPMDH. The transformant was harvested by centrifugation, frozen once at -80 ° C, and then suspended in 20 mM phosphate buffer (pH 7.6) containing 0.5 mM EDTA. The suspended transformant is disrupted by an ultrasonic crusher, centrifuged (30,000 rpm, 20 minutes) to recover the supernatant of the disrupted solution, and subjected to heat treatment at 70 ° C. for 15 minutes to obtain E. coli-derived protein The solution was heat denatured and further centrifuged to recover the denatured protein-removed supernatant.

  The supernatant is applied to a HiTrap Q (manufactured by GE Healthcare) column previously equilibrated with 20 mM phosphate buffer (pH 7.6) containing 0.5 mM EDTA to adsorb TthIPMDH or a variant thereof, TthIPMDH or its variant was eluted with a potassium chloride concentration gradient. The eluted fraction of TthIPMDH or its variant was collected, ammonium sulfate was added to 20% saturation, and it was previously equilibrated with 20 mM phosphate buffer (pH 7.6) containing 20% saturated ammonium sulfate and 0.5 mM EDTA. The IPMDH was adsorbed onto a Butyl-toyopearl column, and purified TthIPMDH or its modified product was eluted with a concentration gradient of 20% saturated ammonium sulfate to 0% ammonium sulfate.

  The purified TthIPMDH and its modified product were dialyzed against 20 mM phosphate buffer (pH 8.0) containing 0.5 mM EDTA, respectively, to obtain a purified preparation to be used for the following activity measurement.

5. Comparison of the activity at 25 ° C. of each Tth IPMDH variant In order to compare the low temperature activity of the variant of Tth IP MDH with Tth IP MDH not introduced with mutation, the activity measurement at 25 ° C. was performed. The measurement conditions are as described above.

  The obtained result is shown in FIG. Eight variants except mut2, mut6 and mut7 showed specific activity as high as or higher than that of TthIPMDH (wild-type) at 25 ° C. In particular, relative to TthIPMDH (wild-type), mut3 showed a specific activity 3.40 times, mut5 3.16 times, mut9 10.86 times, and mut11 7.20 times higher.

  As a result of a model experiment using TthIPMDH as the modified enzyme, as many as four variants from 11 variants, that is, variants having an extremely improved enzyme activity at low temperature with an efficiency as high as 36% were successfully obtained. A widely used method combining random mutagenesis and screening requires searching for potential variants from 1,000 to 30,000 variants, so that such potential variants can be made with such high efficiency. What can be obtained is not assumed from the prior art, and it can be concluded that the present invention is extremely excellent as a method for modifying industrial enzymes.

6. Further improvement of the activity at 25 ° C. by the combination of mutations The mut9 / 11 variant enzyme combining mutations of mut9 and mut11 whose activity at 25 ° C. was greatly improved was prepared by the method described in Examples 1-2, The specific activity at 25 ° C. was compared to TthIPMDH (wild-type), mut9 and mut11 as in Example 3.

  The obtained result is shown in FIG. The specific activity of mut9 / 11 was improved 12.0 times at 25 ° C. as compared to TthIPMDH (wild-type), and the specific activity was also improved as compared to mut9 and mut11.

  As described above, in the modified enzyme (TthIPMDH), paying attention to the substrate binding site and / or the amino acid residue present at a distance of 8 Å from the coenzyme binding site, the amino acid sequence of the corresponding site of the reference enzyme (EcoIPMDH) As a reference, by introducing an amino acid mutation, it was possible to efficiently obtain a variant having improved enzyme activity at low temperature. The above test shows an example using TthIPMDH as the modified enzyme, but for the reason described below, the substrate binding site and the enzyme other than 3-isopropylmalate dehydrogenase are also similar to TthIPMDH. By introducing a mutation focusing on amino acid residues present at a distance of 8 Å from the coenzyme binding site, it is considered possible to efficiently obtain a variant with improved enzyme activity at low temperatures. .

  Thermophilic bacteria-derived enzymes have heat resistance, and in order to stabilize the structure, hydrophobic interactions and ionic bonds in the molecule are stronger than enzymes derived from thermophilic bacteria, and this tendency is The same applies to the formation of the enzyme / substrate complex. Near room temperature, this robust enzyme / coenzyme / substrate complex reduces turnover (the rate at which one molecule of enzyme catalyzes the substrate in one second). Therefore, the thermophilic bacterium-derived enzyme has a lower specific activity at around normal temperature than the enzyme derived from the thermophilic bacterium. On the other hand, in the thermophilic bacterium-derived enzyme, the above-mentioned enzyme / supplement at around normal temperature is substituted by substituting the amino acid residue derived from a thermophilic bacterium with the enzyme and coenzyme and the substrate binding site (and its surrounding 8 Å). It is believed that destabilization and destabilization of the enzyme / substrate complex contributes to the increase in turnover. This is analogized to the mechanism by which the mutation method activates the thermophilic bacterium-derived enzyme at a low temperature. Therefore, focusing on amino acid residues present at a distance of 8 Å from the substrate binding site and / or the coenzyme binding site, the method of introducing a mutation is a thermophilic bacterium-derived enzyme, preferably a coenzyme (preferably It is considered that the specific activity at low temperature can be improved regardless of the type of the enzyme if it requires NAD), the three-dimensional structure is clear, and the sequences of enzymes from cold bacteria can be compared. Of course, the present invention should not be construed as being limited to the mechanism and the like described herein.

Claims (5)

The polypeptide shown in any one of the following (A) to (O):
(A) a polypeptide comprising an amino acid sequence in which the 78th position in the amino acid sequence shown in SEQ ID NO: 1 is replaced with glutamic acid,
(B) in the amino acid sequence shown in SEQ ID NO: 1 at position 214, substituted with methionine, glycine or alanine, at position 216 for isoleucine, leucine or valine, at position 218 for asparagine, serine or glutamine, at position 219 for alanine, methionine Or a glycine, a 222nd amino acid substitution with glutamine, asparagine or serine, a 225nd amino acid substitution with lysine or histidine, and a 229th amino acid substitution with glutamine, asparagine or serine,
(C) a polypeptide comprising an amino acid sequence in which the 272nd position in the amino acid sequence shown in SEQ ID NO: 1 is substituted with alanine, methionine or glycine and the 273rd position is substituted with glycine, alanine or methionine,
(D) In the amino acid sequence shown in SEQ ID NO: 1, position 324 is substituted with arginine, lysine or histidine, position 325 is substituted with threonine, glutamine or asparagine, and glycine, alanine or methionine is inserted between positions 325 and 326 A polypeptide comprising the amino acid sequence
(E) In the amino acid sequence shown in SEQ ID NO: 1, position 272 is substituted with alanine, methionine or glycine, position 273 is substituted with glycine, alanine or methionine, position 324 is substituted with arginine, lysine or histidine, position 325 is threonine, glutamine Or a polypeptide comprising an amino acid sequence which is substituted with asparagine and glycine, alanine or methionine is inserted between the 325th and the 326th,
(F) In the amino acid sequence in which amino acid position 78 in the amino acid sequence shown in SEQ ID NO: 1 is substituted with glutamic acid, one or several amino acids other than amino acid position 78 are substituted, added, inserted or deleted, and The 3-isopropylmalate dehydrogenase activity at 25 ° C. is improved as compared to the 3-isopropylmalate dehydrogenase having the amino acid sequence shown in SEQ ID NO: 1 and having the isopropylmalate dehydrogenase activity. Polypeptides,
(G) in the amino acid sequence shown in SEQ ID NO: 1 at position 214 substituted with methionine, glycine or alanine, at position 216 for isoleucine, leucine or valine, at position 218 for asparagine, serine or glutamine, at position 219 for alanine, methionine Or at glycine 222, at position 222 for glutamine, asparagine or serine, at position 225 for lysine or histidine, and at position 229 for glutamine, asparagine or serine at position 214, 216, 218 And one or several amino acids other than 219, 222, 225 and 229 are substituted, added, inserted or deleted and has 3-isopropylmalate dehydrogenase activity, It consists of the amino acid sequence shown to sequence number 1 - a polypeptide as compared to the isopropyl malate dehydrogenase 3-isopropyl malate dehydrogenase activity at 25 ° C. is improved,
(H) One of the amino acids other than 272 and 273 in the amino acid sequence in which the 272nd position in the amino acid sequence shown in SEQ ID NO: 1 is substituted with alanine, methionine or glycine and the 273rd position is substituted with glycine, alanine or methionine Or a few are substituted, added, inserted or deleted, and have 3-isopropylmalate dehydrogenase activity and are compared with the 3-isopropylmalate dehydrogenase consisting of the amino acid sequence shown in SEQ ID NO: 1 A polypeptide with improved 3-isopropylmalate dehydrogenase activity at 25 ° C.,
(I) In the amino acid sequence shown in SEQ ID NO: 1, position 324 is substituted with arginine, lysine or histidine, position 325 is substituted with threonine, glutamine or asparagine, and glycine, alanine or methionine is inserted between positions 325 and 326 And one or more amino acids other than the amino acids at positions 324 and 325 and the amino acid inserted between positions 325 and 326 are substituted, added, inserted or deleted, and The 3-isopropylmalate dehydrogenase activity at 25 ° C. is improved as compared to the 3-isopropylmalate dehydrogenase having the 3-isopropylmalate dehydrogenase activity and comprising the amino acid sequence shown in SEQ ID NO: 1 Polypeptide,
(J) In the amino acid sequence shown in SEQ ID NO: 1, position 272 is substituted with alanine, methionine or glycine, position 273 is substituted with glycine, alanine or methionine, position 324 is substituted with arginine, lysine or histidine, position 325 is threonine, glutamine Or in the amino acid sequence formed by substitution of asparagine and glycine, alanine or methionine between 325 and 326, amino acids 272, 273, 324 and 325 and between 325 and 326 One or several amino acids other than the amino acid to be inserted are substituted, added, inserted or deleted, and have 3-isopropylmalate dehydrogenase activity and consist of the amino acid sequence shown in SEQ ID NO: 1 25 compared to 3-isopropyl malate dehydrogenase Polypeptide of 3-isopropyl malate dehydrogenase activity is enhanced by,
(K) In an amino acid sequence in which amino acid 78 in the amino acid sequence shown in SEQ ID NO: 1 is substituted with glutamic acid, sequence identity excluding amino acid 78 is 90% or more, and 3-isopropyl malate dehydrogenase A polypeptide having activity and having improved 3-isopropylmalate dehydrogenase activity at 25 ° C. as compared to 3-isopropylmalate dehydrogenase consisting of the amino acid sequence shown in SEQ ID NO: 1,
(L) in the amino acid sequence shown in SEQ ID NO: 1 at position 214 substituted with methionine, glycine or alanine, at position 216 for isoleucine, leucine or valine, at position 218 for asparagine, serine or glutamine, at position 219 for alanine, methionine Or at glycine 222, at position 222 for glutamine, asparagine or serine, at position 225 for lysine or histidine, and at position 229 for glutamine, asparagine or serine at position 214, 216, 218 Sequence identity excluding amino acids 219, 222, 225, and 229 is 90% or more, and has 3-isopropylmalate dehydrogenase activity, and the amino acid sequence shown in SEQ ID NO: 1 3-Isopropylate Polypeptide compared to the Gore-acid dehydrogenase 3-isopropyl malate dehydrogenase activity at 25 ° C. is improved,
(M) A sequence obtained by replacing amino acid 272 and 273 in an amino acid sequence in which amino acid 272 in the amino acid sequence shown in SEQ ID NO: 1 is substituted with alanine, methionine or glycine and amino acid 273 is substituted with glycine, alanine or methionine 3 at 25 ° C. in comparison with 3-isopropylmalate dehydrogenase having an identity of 90% or more and having 3-isopropylmalate dehydrogenase activity and consisting of the amino acid sequence shown in SEQ ID NO: 1 A polypeptide with improved isopropylmalate dehydrogenase activity,
(N) In the amino acid sequence shown in SEQ ID NO: 1, arginine, lysine or histidine substitutes for position 324, threonine, glutamine or asparagine for position 325, and glycine, alanine or methionine is inserted between positions 325 and 326 Amino acid sequence having at least 90% sequence identity excluding amino acids 324 and 325 and an amino acid inserted between 325 and 326, and 3-isopropylmalate dehydrogenase activity A polypeptide having improved 3-isopropylmalate dehydrogenase activity at 25 ° C. as compared to 3-isopropylmalate dehydrogenase consisting of the amino acid sequence shown in SEQ ID NO: 1,
(O) In the amino acid sequence shown in SEQ ID NO: 1, position 272 is substituted with alanine, methionine or glycine, position 273 is substituted with glycine, alanine or methionine, position 324 is substituted with arginine, lysine or histidine, position 325 is threonine, glutamine Or in the amino acid sequence formed by substitution of asparagine and glycine, alanine or methionine between 325 and 326, amino acids 272, 273, 324 and 325 and between 325 and 326 3-isopropylmalate dehydrogenase having a sequence identity of 90% or more excluding the amino acid to be inserted, and having 3-isopropylmalate dehydrogenase activity, and consisting of the amino acid sequence shown in SEQ ID NO: 1 3-isopropyl apple at 25 ° C in comparison Polypeptide dehydrogenase activity is enhanced. A DNA encoding the polypeptide according to claim 1 . A recombinant vector comprising the DNA of claim 2 . A transformant obtained by transforming a host with the recombinant vector according to claim 3 . A method for producing the polypeptide according to claim 1, comprising the step of culturing the transformant according to claim 4 .