Luciferase mutants

Abstract

In one embodiment, an object of the present disclosure is to provide a firefly luciferase having improved thermal stability. In one embodiment, the present disclosure relates to: a mutant of a firefly luciferase comprising an amino acid sequence in which an amino acid residue at a position corresponding to position such as position 252 of SEQ ID NO: 1 is substituted; a luciferase mutant having improved thermal stability; a polynucleotide encoding the luciferase mutant; and a method for producing the luciferase mutant.

Description

Technical Field

[0001] The present disclosure relates to a luciferase mutant with improved thermal stability, a polynucleotide encoding the luciferase mutant, a method for producing the luciferase mutant, a kit for detecting at least one of ATP, ADP and AMP comprising the luciferase mutant, and a method for detecting at least one of ATP, ADP and AMP comprising using the luciferase mutant. Background Art

[0002] Firefly luciferase is an enzyme that converts adenosine triphosphate (ATP), D-luciferin, and enzymes into adenosine monophosphate (AMP), oxyluciferin, and carbon dioxide in the presence of magnesium ions and oxygen, and generates light. Applying the luminescence principle of firefly luciferase, it is possible to measure trace amounts of enzyme reaction substrates with extremely high sensitivity. Therefore, firefly luciferase can be widely used, for example, in microbial detection in beverages and foods using ATP as an indicator, determination of food residues and stains attached to fingers and utensils, or high-sensitivity assays using various antibody technologies and gene amplification technologies.

[0003] However, beetle luciferases, such as firefly luciferase, are generally unstable to heat and therefore easily inactivated when stored as reagents. Therefore, various attempts have been made to overcome this shortcoming and obtain luciferases with good thermal stability.

[0004] One attempt has been to improve the reagent composition by adding salts to the assay reagent to ensure a certain degree of stability. However, this approach is limited in terms of reagent composition and cannot be widely applied. Furthermore, the addition of salts often has the disadvantage of causing disruptions to the luciferase reaction.

[0005] In addition to working on the reagent components, another different approach being tried is to explore mutant luciferases with suitable properties. For example, non-patent document 1 reports on the acquisition of a North American firefly luciferase in which the 342nd amino acid is mutated to alanine, and the luminescence persistence of the firefly luciferase is improved. In addition, patent document 1 discloses that the 217th amino acid of the firefly luciferase of Genji or the firefly luciferase of the family Heike is substituted with a hydrophobic amino acid and has heat resistance. Patent document 2 discloses that the thermal stability of the firefly luciferase having an amino acid sequence equivalent to the 287th amino acid of the firefly luciferase is mutated to alanine, or the amino acid equivalent to the 392nd amino acid is mutated to isoleucine is improved.

[0006] Patent Document 3 reports on a luciferase mutant in which position 393 of Heike luciferase (SEQ ID NO: 1) is substituted.

[0007] A luciferase with high thermostability is desired.

[0008] Prior art literature

[0009] Patent Literature

[0010] Patent Document 1: Japanese Patent Application Laid-Open No. 5-244942

[0011] Patent Document 2: Japanese Patent Application Laid-Open No. 2011-120559

[0012] Patent Document 3: International Publication No. 2020 / 009215

[0013] Patent Document 4: Japanese Patent Application Laid-Open No. 2018-153133

[0014] Patent Document 5: International Publication No. 1999 / 014336

[0015] Patent Document 6: International Publication No. 2001 / 020002

[0016] Patent Document 7: International Publication No. 2016 / 070788

[0017] Patent Document 8: International Publication No. 2001 / 31028

[0018] Patent Document 9: International Publication No. 2018 / 071807

[0019] Patent Document 10: International Publication No. 2019 / 236731

[0020] Patent Document 11: Japanese Patent Application Laid-Open No. 2011-188787

[0021] Patent Document 12: International Publication No. 2019 / 193391

[0022] Patent Document 13: International Publication No. 2007 / 017684

[0023] Non-patent literature

[0024] Non-patent literature 1: Biochemistry 2003, Vol. 42, pp. 10429-10436 Summary of the Invention

[0025] Problems to be solved by the invention

[0026] In a specific embodiment, the present disclosure aims to provide firefly luciferase with improved thermostability. In addition, in a specific embodiment, the present disclosure aims to provide firefly luciferase with significantly improved thermostability.

[0027] Technical solutions to solve problems

[0028] The present inventors discovered that firefly luciferase mutants with specific amino acid substitutions exhibit improved thermostability, and thus incorporated this as an embodiment into the present invention, completing the present invention. The present inventors also discovered that firefly luciferase mutants with more than one specific amino acid substitution exhibit significantly improved thermostability, and thus incorporated this as an embodiment into the present invention, completing the present invention.

[0029] Therefore, the present disclosure includes the following embodiments.

[0030] [1] A luciferase mutant, wherein the luciferase before amino acid substitution has at least 70%, 80% or 90% amino acid sequence identity with SEQ ID NO: 10, and the luciferase after amino acid substitution is substituted with leucine at the position corresponding to position 252 of SEQ ID NO: 1, and the thermal stability of the luciferase after amino acid substitution is improved compared with the luciferase before amino acid substitution.

[0031] [2] According to the luciferase mutant described in embodiment 1, further, the position corresponding to position 262 of SEQ ID NO: 1 is substituted by tyrosine, and / or the position corresponding to position 294 of SEQ ID NO: 1 is substituted by leucine.

[0032] [3] The luciferase mutant according to embodiment 1 or 2, wherein the amino acid residue corresponding to position 90, 371, 41, 427, 53, 306 or 312 of SEQ ID NO: 1 is substituted.

[0033] [4] The luciferase mutant according to embodiment 3, wherein

[0034] (a) the amino acid residue at position 90 of SEQ ID NO: 1 is substituted, where the amino acid residue at position 90 of SEQ ID NO: 1 is an amino acid residue selected from the group consisting of tyrosine, leucine, isoleucine, cysteine, serine, threonine, aspartic acid, glutamic acid, proline, valine, tryptophan, methionine, alanine, glycine, glutamine, asparagine, lysine, histidine, and arginine;

[0035] (b) the amino acid residue at position 371 of SEQ ID NO: 1 is substituted with an amino acid residue selected from the group consisting of leucine, tyrosine, cysteine, threonine, aspartic acid, glutamic acid, isoleucine, proline, valine, tryptophan, methionine, alanine, glycine, glutamine, asparagine, lysine, histidine, and arginine;

[0036] (c) the amino acid residue at position 41 of SEQ ID NO: 1 is substituted, where the amino acid residue at position 41 of SEQ ID NO: 1 is an amino acid residue selected from the group consisting of valine, leucine, isoleucine, methionine, tyrosine, cysteine, serine, threonine, aspartic acid, glutamic acid, tryptophan, phenylalanine, proline, glycine, glutamine, asparagine, lysine, histidine, and arginine;

[0037] (d) the amino acid residue at position 427 of SEQ ID NO: 1 is substituted with an amino acid residue selected from the group consisting of phenylalanine, leucine, isoleucine, cysteine, serine, threonine, aspartic acid, glutamic acid, tryptophan, methionine, valine, alanine, proline, glycine, glutamine, asparagine, lysine, histidine, and arginine;

[0038] (e) the amino acid residue at position 53 of SEQ ID NO: 1 is substituted with an amino acid residue selected from the group consisting of leucine, isoleucine, cysteine, serine, threonine, aspartic acid, glutamic acid, tryptophan, methionine, valine, phenylalanine, alanine, proline, glycine, glutamine, asparagine, lysine, histidine, and arginine;

[0039] (f) the amino acid residue at position 306 of SEQ ID NO: 1 is substituted, where the amino acid residue at position 306 of SEQ ID NO: 1 is selected from the group consisting of phenylalanine, leucine, isoleucine, cysteine, serine, threonine, aspartic acid, glutamic acid, tryptophan, methionine, valine, alanine, proline, glycine, glutamine, asparagine, lysine, histidine, and arginine; or

[0040] (g) the amino acid residue at position 312 of SEQ ID NO:1 is substituted, and the amino acid residue at position 312 of SEQ ID NO:1 is an amino acid residue selected from the group consisting of tyrosine, phenylalanine, leucine, isoleucine, cysteine, serine, threonine, aspartic acid, glutamic acid, tryptophan, methionine, alanine, proline, glycine, glutamine, asparagine, lysine, histidine and arginine.

[0041] [5] The luciferase mutant according to embodiment 4, wherein

[0042] (a) the amino acid residue at position 90 of SEQ ID NO: 1 is substituted, and the amino acid residue at position 90 of SEQ ID NO: 1 is tyrosine;

[0043] (b) the amino acid residue at position 371 of SEQ ID NO: 1 is substituted with leucine;

[0044] (c) the amino acid residue at position 41 of SEQ ID NO: 1 is substituted, and the amino acid residue at position 41 of SEQ ID NO: 1 is valine;

[0045] (d) the amino acid residue at position 427 of SEQ ID NO: 1 is substituted with phenylalanine;

[0046] (e) the amino acid residue at position 53 of SEQ ID NO: 1 is substituted with leucine;

[0047] (f) the amino acid residue at position 306 of SEQ ID NO: 1 is substituted with phenylalanine; or

[0048] (g) The amino acid residue at position 312 of SEQ ID NO: 1 is substituted, and the amino acid residue at position 312 of SEQ ID NO: 1 is glutamic acid, lysine or arginine.

[0049] [6] The luciferase mutant according to any one of embodiments 1 to 5, further comprising an amino acid sequence in which the amino acid residue corresponding to position 221, 17, 18, 75, 110, 223, 224, 257, 229 or 158 of SEQ ID NO: 1 is substituted.

[0050] [7] The luciferase mutant according to embodiment 6, wherein

[0051] (i) the amino acid residue at position 221 of SEQ ID NO: 1 is substituted, where the amino acid residue at position 221 of SEQ ID NO: 1 is an amino acid residue selected from the group consisting of leucine, tyrosine, cysteine, serine, threonine, aspartic acid, glutamic acid, isoleucine, proline, valine, tryptophan, methionine, alanine, glycine, glutamine, asparagine, lysine, histidine, and arginine; or

[0052] (ii) the amino acid residue at position 17 of SEQ ID NO: 1 is substituted, where the amino acid residue at position 17 of SEQ ID NO: 1 is selected from the group consisting of aspartic acid, glutamic acid, lysine, histidine, arginine, tyrosine, cysteine, serine, threonine, tryptophan, methionine, leucine, isoleucine, valine, alanine, proline, glycine, glutamine, and asparagine;

[0053] (iii) the amino acid residue at position 18 of SEQ ID NO: 1 is substituted, where the amino acid residue at position 18 of SEQ ID NO: 1 is selected from the group consisting of leucine, isoleucine, cysteine, serine, threonine, aspartic acid, glutamic acid, tryptophan, methionine, valine, phenylalanine, alanine, proline, glycine, glutamine, asparagine, lysine, histidine, and arginine;

[0054] (iv) the amino acid residue at position 75 of SEQ ID NO: 1 is substituted, where the amino acid residue at position 75 of SEQ ID NO: 1 is selected from the group consisting of lysine, histidine, arginine, aspartic acid, glutamic acid, tyrosine, cysteine, serine, threonine, tryptophan, methionine, leucine, isoleucine, phenylalanine, alanine, proline, glycine, glutamine, and asparagine;

[0055] (v) the amino acid residue at position 110 of SEQ ID NO: 1 is substituted, where the amino acid residue at position 110 of SEQ ID NO: 1 is selected from the group consisting of aspartic acid, glutamic acid, lysine, arginine, histidine, tyrosine, cysteine, serine, threonine, tryptophan, methionine, leucine, valine, phenylalanine, alanine, proline, glycine, glutamine, and asparagine;

[0056] (vi) the amino acid residue at position 223 of SEQ ID NO: 1 is substituted, wherein the amino acid residue at position 223 of SEQ ID NO: 1 is an amino acid residue selected from the group consisting of valine, isoleucine, leucine, tyrosine, cysteine, serine, threonine, aspartic acid, glutamic acid, tryptophan, methionine, phenylalanine, alanine, proline, glycine, glutamine, asparagine, lysine, and arginine;

[0057] (vii) the amino acid residue at position 224 of SEQ ID NO: 1 is substituted, wherein the amino acid residue at position 224 of SEQ ID NO: 1 is an amino acid residue selected from the group consisting of isoleucine, leucine, valine, methionine, cysteine, tyrosine, serine, threonine, aspartic acid, glutamic acid, tryptophan, phenylalanine, proline, glycine, glutamine, asparagine, lysine, histidine, and arginine;

[0058] (viii) the amino acid residue at position 257 of SEQ ID NO: 1 is substituted with an amino acid residue selected from the group consisting of phenylalanine, leucine, isoleucine, cysteine, serine, threonine, aspartic acid, glutamic acid, tryptophan, methionine, valine, alanine, proline, glycine, glutamine, asparagine, lysine, histidine, and arginine;

[0059] (ix) the amino acid residue at position 229 of SEQ ID NO: 1 is substituted, where the amino acid residue at position 229 of SEQ ID NO: 1 is selected from the group consisting of phenylalanine, leucine, isoleucine, cysteine, serine, threonine, aspartic acid, glutamic acid, tryptophan, methionine, valine, alanine, proline, glycine, glutamine, asparagine, lysine, histidine, and arginine; or

[0060] (x) the amino acid residue at position 158 of SEQ ID NO:1 is substituted, and the amino acid residue at position 158 of SEQ ID NO:1 is an amino acid residue selected from the group consisting of aspartic acid, glutamic acid, lysine, arginine, histidine, tyrosine, cysteine, serine, threonine, tryptophan, methionine, leucine, isoleucine, phenylalanine, alanine, proline, glycine, glutamine and asparagine.

[0061] [8] The luciferase mutant according to embodiment 7, wherein

[0062] (i) the amino acid residue at position 221 of SEQ ID NO: 1 is substituted with leucine;

[0063] (ii) the amino acid residue at position 17 of SEQ ID NO: 1 is substituted, and the amino acid residue at position 17 of SEQ ID NO: 1 is aspartic acid, glutamic acid, or lysine;

[0064] (iii) the amino acid residue at position 18 of SEQ ID NO: 1 is substituted, and the amino acid residue at position 18 of SEQ ID NO: 1 is leucine;

[0065] (iv) the amino acid residue at position 75 of SEQ ID NO: 1 is substituted, and the amino acid residue at position 75 of SEQ ID NO: 1 is lysine;

[0066] (v) the amino acid residue at position 110 of SEQ ID NO: 1 is substituted, and the amino acid residue at position 110 of SEQ ID NO: 1 is aspartic acid, glutamic acid, lysine, or arginine;

[0067] (vi) the amino acid residue at position 223 of SEQ ID NO: 1 is substituted, and the amino acid residue at position 223 of SEQ ID NO: 1 is valine, isoleucine, or leucine;

[0068] (vii) the amino acid residue at position 224 of SEQ ID NO: 1 is substituted with isoleucine;

[0069] (viii) the amino acid residue at position 257 of SEQ ID NO: 1 is substituted with phenylalanine;

[0070] (ix) the amino acid residue at position 229 of SEQ ID NO: 1 is substituted, and the amino acid residue at position 229 of SEQ ID NO: 1 is phenylalanine or leucine; or

[0071] (x) The amino acid residue at position 158 of SEQ ID NO: 1 is substituted, and the amino acid residue at position 158 of SEQ ID NO: 1 is aspartic acid or glutamic acid.

[0072] [9] The luciferase mutant according to any one of embodiments 1 to 8, wherein

[0073] The luciferase before amino acid substitution comprises an amino acid sequence selected from the group consisting of the following (A) to (F):

[0074] (A) an amino acid sequence having at least 70%, 80% or 90% amino acid sequence identity to SEQ ID NO: 10;

[0075] (B) Regarding the amino acid sequence of (A), further, an amino acid sequence having a sequence identity of 90% or more between the homology region of SEQ ID NO: 1 and the homology region of luciferase before the amino acid substitution;

[0076] (C) an amino acid sequence in which, in the amino acid sequence of SEQ ID NO: 10, one or more amino acids are substituted, deleted, or added at positions other than the position corresponding to position 252 of SEQ ID NO: 1;

[0077] (D) Regarding the amino acid sequence of (C), further, in any one of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 10, SEQ ID NO: 69, SEQ ID NO: 71 or SEQ ID NO: 73, one or more amino acids are substituted, deleted or added at positions other than the positions corresponding to positions 90, 262, 371, 41, 427, 53, 306, 312, 294, 221, 17, 18, 75, 110, 223, 224, 257, 229 or 158 of SEQ ID NO: 1;

[0078] (E) the amino acid sequence of SEQ ID NO: 10; and

[0079] (F) Amino acid sequence of Heike luciferase.

[0080]

[10] The luciferase mutant according to embodiment 9, wherein

[0081] The luciferase before amino acid substitution comprises an amino acid sequence selected from the group consisting of the following (a) to (d):

[0082] (a) an amino acid sequence having an amino acid sequence identity of 90% or more to SEQ ID NO: 10;

[0083] (b) an amino acid sequence in which one or more amino acids are substituted, deleted, or added at positions other than the position corresponding to position 252 of SEQ ID NO: 1 in the amino acid sequence of SEQ ID NO: 1;

[0084] (c) With respect to the amino acid sequence of (b), further, in the amino acid sequence of SEQ ID NO: 1, one or more amino acids are substituted, deleted or added at positions other than the positions corresponding to positions 262, 371, 41, 427, 53, 306, 312, 252, 294, 221, 17, 18, 75, 110, 223, 224, 257, 229 or 158 of SEQ ID NO: 1; and

[0085] (d) The amino acid sequence of SEQ ID NO: 10.

[0086]

[11] A polynucleotide encoding the luciferase mutant according to any one of embodiments 1 to 10.

[0087]

[12] A vector comprising the polynucleotide according to embodiment 11.

[0088]

[13] A host cell comprising the polynucleotide of embodiment 11 or the vector of embodiment 12.

[0089]

[14] A method for producing a luciferase mutant with improved thermal stability, the method comprising the step of culturing the host cell described in embodiment 13.

[0090]

[15] A kit for detecting at least one of ATP, ADP, and AMP, comprising the luciferase mutant according to any one of embodiments 1 to 10.

[0091]

[16] A method for detecting at least one of ATP, ADP, and AMP, comprising using the luciferase mutant according to any one of embodiments 1 to 10.

[0092]

[17] A luciferase mutant comprising an amino acid sequence in which one or more amino acid residues corresponding to positions 252, 262, 294, 90, 371, 41, 427, 53, 306, 312, 221, 17, 18, 75, 110, 223, 224, 257, 229 and 158 of SEQ ID NO: 1 are substituted, and the thermal stability of the luciferase after the amino acid substitution is improved compared to the luciferase before the amino acid substitution.

[0093]

[18] The luciferase mutant according to embodiment 17, wherein

[0094] The position corresponding to position 252 of SEQ ID NO: 1 is substituted with leucine;

[0095] The position corresponding to position 262 of SEQ ID NO: 1 is substituted with tyrosine;

[0096] The position corresponding to position 294 of SEQ ID NO: 1 is substituted with leucine;

[0097] The position corresponding to position 90 of SEQ ID NO: 1 is substituted with tyrosine;

[0098] The position corresponding to position 371 of SEQ ID NO: 1 is substituted with leucine;

[0099] The position corresponding to position 41 of SEQ ID NO: 1 is substituted with valine;

[0100] The position corresponding to position 427 of SEQ ID NO: 1 is substituted with phenylalanine;

[0101] The position corresponding to position 53 of SEQ ID NO: 1 is substituted with leucine;

[0102] The position corresponding to position 306 of SEQ ID NO: 1 is substituted with phenylalanine;

[0103] The position corresponding to position 312 of SEQ ID NO: 1 is substituted with glutamic acid, lysine or arginine;

[0104] The position corresponding to position 221 of SEQ ID NO: 1 is substituted with leucine;

[0105] The position corresponding to position 17 of SEQ ID NO: 1 is substituted with aspartic acid, glutamic acid or lysine;

[0106] The position corresponding to position 18 of SEQ ID NO: 1 is substituted with leucine;

[0107] The position corresponding to position 75 of SEQ ID NO: 1 is substituted with lysine;

[0108] The position corresponding to position 110 of SEQ ID NO: 1 is substituted with aspartic acid, glutamic acid, lysine or arginine;

[0109] The position corresponding to position 223 of SEQ ID NO: 1 is substituted with valine, isoleucine or leucine;

[0110] The position corresponding to position 224 of SEQ ID NO: 1 is substituted with isoleucine;

[0111] The position corresponding to position 257 of SEQ ID NO: 1 is substituted with phenylalanine;

[0112] The position corresponding to position 229 of SEQ ID NO: 1 is substituted with phenylalanine or leucine; or

[0113] The position corresponding to position 158 of SEQ ID NO: 1 is substituted with aspartic acid or glutamic acid.

[0114] This specification incorporates the disclosure of Japanese Patent Application No. 2023-010710, upon which the present application claims priority.

[0115] Effects of the Invention

[0116] According to the present disclosure, firefly luciferase with improved thermostability is provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0117] Figure 1 The results of an alignment of wild-type luciferases from Luciola lateralis, Luciola cruciata, Photinus pyralis, and Photuris pennsylvanica are shown. In the figure, identical amino acid residues in the four amino acid sequences are enclosed in boxes.

[0118] Figure 2 yes Figure 1 Continuation of . DETAILED DESCRIPTION

[0119] (Benchmark sequence)

[0120] Unless otherwise specified, this specification uses SEQ ID NO:1 as the reference sequence to define sequence positions. SEQ ID NO:1 is the amino acid sequence of luciferase from Firefly. SEQ ID NO:10 is a mutant generated based on SEQ ID NO:1. Compared to SEQ ID NO:1, SEQ ID NO:10 has amino acid substitutions A217L, E490K, C393L, G326S, V392I, and F467I.

[0121] (Luciferase mutant)

[0122] In one embodiment, the present disclosure relates to a firefly luciferase mutant comprising an amino acid sequence in which the amino acid residues corresponding to positions 252, 262, 294, 90, 371, 41, 427, 53, 306, and / or 312 of SEQ ID NO: 1 are substituted (in this specification, these positions may sometimes be referred to as position 252, etc.). In one embodiment, the present disclosure also relates to a firefly luciferase mutant comprising an amino acid sequence in which the amino acid residues corresponding to positions 221, 17, 18, 75, 110, 223, 224, 257, 229, and / or 158 of SEQ ID NO: 1 are substituted. In one embodiment, these positions may be substituted with any of 19 amino acids other than the amino acid residues in the wild-type sequence. In one embodiment, the present disclosure relates to a firefly luciferase mutant having one or more amino acid substitutions corresponding to F252L, F262Y, F294L, F90Y, F371L, A41V, Y427F, Y53L, Y306F, F221L, F17 (D, E, K), Y18L, V75K, I110 (D, E, K, R), H223 (V, I, L), A224I, and Y257F, Y229 (F, L), and V158 (D, E) with respect to SEQ ID NO: 1. In one embodiment, the luciferase mutant may have improved thermal stability compared to the luciferase before the amino acid substitution.

[0123] In this specification, "wild type" refers to the most common trait in nature within the same species population.

[0124] Firefly luciferase can be used from any firefly. For example, Luciola lateralis, Luciola cruciata, Photinus pyralis, Photuris pennsylvanica, Lampyris noctiluca, Pyrocoelia miyako, Pyrophorus plagiophthalamus, or Luciola mingrelica can be used, with firefly luciferase from Luciola lateralis, Luciola cruciata, Photinus pyralis, or Photuris pennsylvanica being preferred. Alternatively, chimeric proteins prepared from luciferase genes derived from various fireflies can also be used.

[0125] In this specification, the corresponding relationship of amino acid positions can be easily determined by comparing the amino acid sequences of various firefly luciferases using existing amino acid homology analysis software such as GENETYX (manufactured by GENETYX). For example, the amino acid position of luciferase corresponding to position X of the amino acid sequence of SEQ ID NO: 1 can be determined by aligning the amino acid sequence of the luciferase with the amino acid sequence of SEQ ID NO: 1. For example, "the position corresponding to position 262 of SEQ ID NO: 1" is position 262 of the amino acid sequence of SEQ ID NO: 69, position 260 of SEQ ID NO: 71, and position 259 of SEQ ID NO: 73. Figure 1 This table shows the alignment of Heike, Genji, P. firefly, and P. pennsylvanicum luciferases. By referring to this alignment, corresponding positions in each amino acid sequence can be determined.

[0126] In one embodiment, the amino acid at position 252 of SEQ ID NO: 1 in the luciferase mutant can be a non-acidic amino acid other than phenylalanine (e.g., leucine, tyrosine, isoleucine, cysteine, serine, threonine, aspartic acid, glutamic acid, tryptophan, methionine, valine, alanine, proline, glycine, glutamine, asparagine, lysine, histidine, or arginine). The substitution (mutation) of phenylalanine at position 252 of SEQ ID NO: 1 with leucine is sometimes referred to as F252L in this specification. However, the target of introduction of F252L is not limited to SEQ ID NO: 1, and it can be introduced into the position corresponding to position 252 of SEQ ID NO: 1 in any luciferase. In addition, depending on the luciferase to be introduced, the position corresponding to position 252 of SEQ ID NO: 1 before substitution may not be F. That is, F252L is based on SEQ ID NO: 1 for convenience of description. As long as L is introduced into the position corresponding to position 252 of SEQ ID NO: 1, such amino acid substitution belongs to F252L. However, the situation where the position corresponding to position 252 of SEQ ID NO: 1 in the wild-type luciferase is originally L (L252L) is not within the scope of mutations disclosed in this disclosure. In other words, this disclosure excludes wild-type sequences and also excludes natural products. Substitution of other amino acids at other positions is also recorded as "amino acid before substitution-position-amino acid after substitution". In one embodiment, the luciferase mutant can be a mutant comprising an amino acid sequence having a high sequence identity with the amino acid sequence of SEQ ID NO: 10, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity. In one embodiment, the luciferase before amino acid substitution may be a luciferase derived from Fluorophore heike or a luciferase derived from Fluorophore genji.

[0127] In one embodiment, the mutation at position 252 or the like of SEQ ID NO: 1 is an artificially introduced mutation, which can be achieved by artificially introducing a mutation into the gene sequence encoding luciferase.

[0128] In one embodiment, the firefly luciferase mutant may further include mutations other than mutations at positions corresponding to position 252 of SEQ ID NO: 1. The aforementioned further mutations may be artificially introduced to achieve certain specific effects, or may be randomly or non-artificially introduced. Mutations introduced to achieve specific effects, such as additions, deletions, and modifications to sequences for increasing the expression level of firefly luciferase and additions, deletions, and modifications to sequences for improving the purification efficiency of firefly luciferase, may also include various mutations that confer practically preferred properties to firefly luciferase. Examples of such known mutations include: a mutation for improving the duration of luminescence as described in Japanese Patent Application Laid-Open No. 2000-197484; a mutation for changing the wavelength of luminescence as described in Japanese Patent Application Laid-Open No. 3-285683 or Japanese Patent Table No. 2003-512071; a mutation for improving surfactant resistance as described in Japanese Patent Application Laid-Open No. 11-239493; a mutation for improving the resistance to surfactant as described in International Publication No. 99 / 02697, Japanese Patent Table No. 10-512750 or Japanese Patent Table No. 2 Mutations that alter substrate affinity as described in Japanese Patent Application Laid-Open No. 5-244942, Japanese Patent Application Laid-Open No. 2011-120559, Japanese Patent Application Laid-Open No. 2000-197487, Japanese Patent Translation Laid-Open No. 9-510610, or Japanese Patent Translation Laid-Open No. 2003-518912, or mutations that improve luminescence duration, stability, and luminescence intensity as described in Japanese Patent Application Laid-Open No. 2011-188787, etc. For example, Japanese Patent Application Laid-Open No. 2011-120559 discloses improved thermal stability of firefly luciferase having an amino acid sequence in which the amino acid at position 287 of the Heike luciferase is mutated to alanine, or the amino acid at position 392 of the Heike luciferase is mutated to isoleucine. Japanese Patent Application Laid-Open No. 2011-120559 describes that by combining these mutations with a serine substitution at amino acid position 326 and / or an isoleucine substitution at amino acid position 467, firefly luciferase with further improved stability can be obtained. For example, International Publication No. 2020 / 009215 describes a thermostable luciferase mutant with a substitution at position 393 of SEQ ID NO: 1.

[0129] In one embodiment, in the luciferase mutant disclosed herein,

[0130] (a) The amino acid residue at position 252 of SEQ ID NO: 1 is an amino acid residue selected from the group consisting of leucine, tyrosine, cysteine, serine, threonine, aspartic acid, glutamic acid, isoleucine, proline, valine, tryptophan, alanine, glycine, glutamine, asparagine, lysine, histidine, arginine and methionine; for example, leucine.

[0131] In one embodiment, in the luciferase mutant disclosed herein,

[0132] (b) the amino acid residue at position 262 of SEQ ID NO: 1 is substituted, and the amino acid residue at position 262 of SEQ ID NO: 1 is an amino acid residue selected from the group consisting of tyrosine, leucine, isoleucine, cysteine, serine, threonine, aspartic acid, glutamic acid, tryptophan, methionine, valine, alanine, proline, glycine, glutamine, asparagine, lysine, histidine and arginine; for example, tyrosine.

[0133] In one embodiment, in the luciferase mutant disclosed herein,

[0134] (c) The amino acid residue at position 294 of SEQ ID NO: 1 is an amino acid residue selected from the group consisting of leucine, tyrosine, cysteine, serine, threonine, aspartic acid, glutamic acid, isoleucine, proline, valine, tryptophan, methionine, alanine, glycine, glutamine, asparagine, lysine, histidine and arginine; for example, leucine.

[0135] In one embodiment, in the luciferase mutant disclosed herein, further,

[0136] (a) the amino acid residue at position 90 of SEQ ID NO: 1 is substituted, wherein the amino acid residue at position 90 of SEQ ID NO: 1 is an amino acid residue selected from the group consisting of tyrosine, leucine, isoleucine, cysteine, serine, threonine, aspartic acid, glutamic acid, proline, valine, tryptophan, methionine, alanine, glycine, glutamine, asparagine, lysine, histidine, and arginine; for example, tyrosine;

[0137] (b) the amino acid residue at position 371 of SEQ ID NO: 1 is substituted, wherein the amino acid residue at position 371 of SEQ ID NO: 1 is an amino acid residue selected from the group consisting of leucine, tyrosine, cysteine, threonine, aspartic acid, glutamic acid, isoleucine, proline, valine, tryptophan, methionine, alanine, glycine, glutamine, asparagine, lysine, histidine, and arginine; for example, leucine;

[0138] (c) the amino acid residue at position 41 of SEQ ID NO: 1 is substituted, wherein the amino acid residue at position 41 of SEQ ID NO: 1 is an amino acid residue selected from the group consisting of valine, leucine, isoleucine, methionine, tyrosine, cysteine, serine, threonine, aspartic acid, glutamic acid, tryptophan, phenylalanine, proline, glycine, glutamine, asparagine, lysine, histidine, and arginine; for example, valine;

[0139] (d) the amino acid residue at position 427 of SEQ ID NO: 1 is substituted, wherein the amino acid residue at position 427 of SEQ ID NO: 1 is an amino acid residue selected from the group consisting of phenylalanine, leucine, isoleucine, cysteine, serine, threonine, aspartic acid, glutamic acid, tryptophan, methionine, valine, alanine, proline, glycine, glutamine, asparagine, lysine, histidine, and arginine; for example, phenylalanine;

[0140] (e) the amino acid residue at position 53 of SEQ ID NO: 1 is substituted, wherein the amino acid residue at position 53 of SEQ ID NO: 1 is an amino acid residue selected from the group consisting of leucine, isoleucine, cysteine, serine, threonine, aspartic acid, glutamic acid, tryptophan, methionine, valine, phenylalanine, alanine, proline, glycine, glutamine, asparagine, lysine, histidine and arginine; for example, leucine; or,

[0141] (f) the amino acid residue at position 306 of SEQ ID NO: 1 is substituted, wherein the amino acid residue at position 306 of SEQ ID NO: 1 is an amino acid residue selected from the group consisting of phenylalanine, leucine, isoleucine, cysteine, serine, threonine, aspartic acid, glutamic acid, tryptophan, methionine, valine, alanine, proline, glycine, glutamine, asparagine, lysine, histidine, and arginine; for example, phenylalanine; or,

[0142] (g) the amino acid residue at position 312 of SEQ ID NO:1 is substituted, and the amino acid residue at position 312 of SEQ ID NO:1 is an amino acid residue selected from the group consisting of tyrosine, phenylalanine, leucine, isoleucine, cysteine, serine, threonine, aspartic acid, glutamic acid, tryptophan, methionine, alanine, proline, glycine, glutamine, asparagine, lysine, histidine and arginine.

[0143] (Further amino acid substitutions)

[0144] In one embodiment, in the luciferase mutant disclosed herein, further, the amino acid residues corresponding to positions 221, 17, 18, 75, 110, 223, 224, 257, 229 and / or 158 of SEQ ID NO: 1 are substituted, and the thermal stability of the luciferase after the amino acid substitution can be improved compared to the luciferase before the amino acid substitution.

[0145] In one embodiment, the luciferase mutant having further amino acid substitutions may have one or more of the following amino acid substitutions:

[0146] (i) the amino acid residue at position 221 of SEQ ID NO: 1 is substituted, wherein the amino acid residue at position 221 of SEQ ID NO: 1 is an amino acid residue selected from the group consisting of leucine, tyrosine, cysteine, serine, threonine, aspartic acid, glutamic acid, isoleucine, proline, valine, tryptophan, methionine, alanine, glycine, glutamine, asparagine, lysine, histidine, and arginine; for example, leucine;

[0147] (ii) the amino acid residue at position 17 of SEQ ID NO: 1 is substituted, wherein the amino acid residue at position 17 of SEQ ID NO: 1 is an amino acid residue selected from the group consisting of aspartic acid, glutamic acid, lysine, histidine, arginine, tyrosine, cysteine, serine, threonine, tryptophan, methionine, leucine, isoleucine, valine, alanine, proline, glycine, glutamine, and asparagine; for example, aspartic acid, glutamic acid, or lysine;

[0148] (iii) the amino acid residue at position 18 of SEQ ID NO: 1 is substituted, wherein the amino acid residue at position 18 of SEQ ID NO: 1 is an amino acid residue selected from the group consisting of leucine, isoleucine, cysteine, serine, threonine, aspartic acid, glutamic acid, tryptophan, methionine, valine, phenylalanine, alanine, proline, glycine, glutamine, asparagine, lysine, histidine, and arginine; for example, leucine;

[0149] (iv) the amino acid residue at position 75 of SEQ ID NO: 1 is substituted, wherein the amino acid residue at position 75 of SEQ ID NO: 1 is an amino acid residue selected from the group consisting of lysine, histidine, arginine, aspartic acid, glutamic acid, tyrosine, cysteine, serine, threonine, tryptophan, methionine, leucine, isoleucine, phenylalanine, alanine, proline, glycine, glutamine, and asparagine; for example, lysine;

[0150] (v) the amino acid residue at position 110 of SEQ ID NO: 1 is substituted, wherein the amino acid residue at position 110 of SEQ ID NO: 1 is an amino acid residue selected from the group consisting of aspartic acid, glutamic acid, lysine, arginine, histidine, tyrosine, cysteine, serine, threonine, tryptophan, methionine, leucine, valine, phenylalanine, alanine, proline, glycine, glutamine, and asparagine; for example, aspartic acid, glutamic acid, lysine, or arginine;

[0151] (vi) the amino acid residue at position 223 of SEQ ID NO: 1 is substituted, wherein the amino acid residue at position 223 of SEQ ID NO: 1 is an amino acid residue selected from the group consisting of valine, isoleucine, leucine, tyrosine, cysteine, serine, threonine, aspartic acid, glutamic acid, tryptophan, methionine, phenylalanine, alanine, proline, glycine, glutamine, asparagine, lysine, and arginine; for example, valine, isoleucine, or leucine;

[0152] (vii) the amino acid residue at position 224 of SEQ ID NO: 1 is substituted, wherein the amino acid residue at position 224 of SEQ ID NO: 1 is an amino acid residue selected from the group consisting of isoleucine, leucine, valine, methionine, cysteine, tyrosine, serine, threonine, aspartic acid, glutamic acid, tryptophan, phenylalanine, proline, glycine, glutamine, asparagine, lysine, histidine, and arginine; for example, isoleucine;

[0153] (viii) the amino acid residue at position 257 of SEQ ID NO: 1 is substituted, wherein the amino acid residue at position 257 of SEQ ID NO: 1 is an amino acid residue selected from the group consisting of phenylalanine, leucine, isoleucine, cysteine, serine, threonine, aspartic acid, glutamic acid, tryptophan, methionine, valine, alanine, proline, glycine, glutamine, asparagine, lysine, histidine, and arginine; for example, phenylalanine;

[0154] (ix) the amino acid residue at position 229 of SEQ ID NO: 1 is substituted, wherein the amino acid residue at position 229 of SEQ ID NO: 1 is an amino acid residue selected from the group consisting of phenylalanine, leucine, isoleucine, cysteine, serine, threonine, aspartic acid, glutamic acid, tryptophan, methionine, valine, alanine, proline, glycine, glutamine, asparagine, lysine, histidine, and arginine; for example, phenylalanine or leucine; and / or,

[0155] (x) the amino acid residue at position 158 of SEQ ID NO:1 is substituted, and the amino acid residue at position 158 of SEQ ID NO:1 is an amino acid residue selected from the group consisting of aspartic acid, glutamic acid, lysine, arginine, histidine, tyrosine, cysteine, serine, threonine, tryptophan, methionine, leucine, isoleucine, phenylalanine, alanine, proline, glycine, glutamine and asparagine; for example, aspartic acid or glutamic acid.

[0156] (Double mutant)

[0157] In one embodiment, the luciferase mutant disclosed herein may have a double mutation selected from the group consisting of F252L / F262Y, F262Y / F294L, and F252L / F294L based on SEQ ID NO: 1.

[0158] (Triple Mutant)

[0159] In one embodiment, the luciferase mutant disclosed herein may have a triple mutation consisting of F252L / F262Y / F294L based on SEQ ID NO: 1. One or more amino acid substitutions disclosed herein may also be introduced into the triple mutant. The triple mutant may have an amino acid sequence identity of 90% or more to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 10. In one embodiment, when the triple mutant is heat-treated at 60° C. for 5 minutes, the residual activity after heat treatment is increased by 2-fold, 3-fold, 4-fold, or 5-fold or more compared to the luciferase before the introduction of the three amino acid substitutions. In one embodiment, when the triple mutant is heat-treated at 60° C. for 5 minutes, the residual activity after heat treatment is increased by 100% or more, 200% or more, 300% or more, or 400% or more compared to the luciferase before the introduction of the three amino acid substitutions. In this specification, a firefly luciferase with significantly improved thermal stability refers to a firefly luciferase whose residual activity after heat treatment at 60°C for 5 minutes is increased by 100% or more, 200% or more, 300% or more, or 400% or more compared to a firefly luciferase before mutation as a comparison.

[0160] In one embodiment, the luciferase before amino acid substitution may have an amino acid sequence selected from the group consisting of the following (A) to (F):

[0161] (A) an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 10;

[0162] (B) With respect to the amino acid sequence of (A), further, an amino acid sequence having a sequence identity of at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% between the homology region of SEQ ID NO: 1 and the homology region of luciferase before the amino acid substitution;

[0163] (C) an amino acid sequence in which, in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 10, one or more amino acids are substituted, deleted, or added at positions other than the position corresponding to position 252 of SEQ ID NO: 1;

[0164] (D) Regarding the amino acid sequence of (C), further, in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 10, one or more amino acids are substituted, deleted or added at positions other than the positions corresponding to position 90, 262, 371, 41, 427, 53, 306, 312, 294, 221, 17, 18, 75, 110, 223, 224, 257, 229 or 158 of SEQ ID NO: 1;

[0165] (E) the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 10; and

[0166] (F) Amino acid sequence of Heike luciferase and amino acid sequence of Genji luciferase.

[0167] Regions of homology can be identified as Figure 1The regions of the same amino acid residues are conserved in the four firefly luciferases shown (Firefly taurus, Firefly genji, Firefly pyralis, and Firefly pulsatilla).In this specification, the homology region of SEQ ID NO: 1 is the region represented by SEQ ID The region consisting of all amino acid sequences at the following positions of NO: 1: positions 4-5, 9-10, 13-14, 16-17, 19, 23, 25-26, 28, 35-37, 40, 42-43, 45, 47, 55, 57, 62, 65, 72-74, 80, 83-86, 90-91, 93, 98, 101, 105-106, 111, 114-116, 119, 122, 125, 129, 131-132, 137, 141 151st, 153rd, 155th, 159th, 162nd, 164th, 169th, 183rd, 190th, 195th-196th, 198th, 200th-202nd, 204th-205th, 208th-210th, 212th, 214th, 220th-223rd, 226th-227th, 230th-231st, 235th, 237th-238th, 240th, 242nd, 244th-251st, 253rd-257th, 260th-263rd, 270th, 272nd, 275th-276th, 279th-283rd, 286th, 289-293rd, 300th, 302nd, 305-309th, 311th, 313-324th, 326-327th, 329th, 332-333rd, 335th, 339-350th, 353-355th, 358th, 361-362nd, 365-366th, 368-369th, 374-375th, 377th, 380th, 382nd, 384-385th, 387th, 390-391st, 396th, 398th, 400th, 403rd, 406th, 408-410th, 414th, 418-420th 20th, 423rd-425th, 427th, 429th, 433rd-434th, 436th-451st, 453rd-455th, 457th, 460th-464th, 466th, 468th-471st, 473rd, 475th-476th, 479th-483rd, 485th, 487th-488th, 492nd-493rd, 497th, 504th, 506th-508th, 511th-512th, 514th-518th, 520th, 522nd-523rd, 525th-527th, 529th-531st, 533rd, 538th, 543rd, and 547th. In addition,Unless otherwise specified, the homology region of firefly luciferase used as the subject in this specification is the region corresponding to the homology region of SEQ ID NO: 1. When the homology regions are compared with each other, the respective positions correspond. For example, when comparing the homology region of sequence A and the homology region of sequence B, the position corresponding to the 4th position of SEQ ID NO: 1 in sequence A and the position corresponding to the 4th position of SEQ ID NO: 1 in sequence B are compared as corresponding positions. It should be noted that the homology region is defined for the sequence before amino acid substitution and does not hinder the introduction of mutations for luciferase into the homology region. For example, position 262 of SEQ ID NO: 1 is contained in the homology region of SEQ ID NO: 1, and mutations can be introduced into this position. The same applies to other mutation positions recorded in this specification.

[0168] In this specification, the consistency of amino acid sequence and gene sequence can be calculated by programs such as maximum match and homology search of GENETYX (GENETYX company production), or multiple sequence alignment of CLUSTAL W, double sequence alignment using BLAST, etc. In order to calculate amino acid sequence consistency, when comparing more than 2 luciferases, the identical amino acid position in the more than 2 luciferases can be studied. According to such information, the identical region in the amino acid sequence can be determined. Wherein, in more than 2 amino acid sequences, consistency % refers to when utilizing BLAST (BLASTP) etc. that utilize amino acid sequence as object to compare more than 2 amino acid sequences, using the total number of amino acids in the region that can be compared as denominator, using the number of positions occupied by the identical amino acid as molecule when percentage. Therefore, generally in more than 2 amino acid sequences, when there is a region in which consistency cannot be observed completely, for example, when the amino acid sequence in which the added sequence that consistency cannot be observed completely at the C-terminus is located on one side, the region without consistency cannot be compared, and therefore cannot be used to calculate consistency %.

[0169] In addition, similar amino acid positions in more than two luciferases can also be studied. For example, using CLUSTALW can align more than one amino acid sequence. In this case, the algorithm uses Blosum62. When aligning more than one amino acid sequence, amino acids that are sometimes judged to be similar are referred to as similar amino acids. In the mutants disclosed herein, amino acid substitutions can be mutants formed by substitutions between such similar amino acids. Through such an alignment, for more than one amino acid sequence, regions with identical amino acid sequences and positions occupied by similar amino acids can be studied. Based on such information, regions of homology (also referred to as highly conserved regions) in the amino acid sequence can be determined.

[0170] (High Throughput Screening)

[0171] In order to obtain functional luciferase mutants, luciferase can also be used for high-throughput screening. For example, a library of transformed or transduced strains with introduced mutations in the luciferase gene can be prepared and used for high-throughput screening based on microtiter plates, or it can be used for ultra-high-throughput screening based on droplet-type microfluidics. As an example, a combinatorial library of mutant genes encoding mutations can be constructed, and then a large population of mutant luciferases can be screened using phage display technology (e.g., Chem. Rev. 105(11):4056-72, 2005), yeast display technology (e.g., Comb Chem High Throughput Screen. 2008; 11(2):127-34), bacterial display technology (e.g., Curr Opin Struct Biol 17:474-80, 2007), etc. can be used. In addition, refer to Agresti et al, "Ultrahigh-throughput screening indrop-based microfluidics for directed evolution" Proceedings of the National Academy of Sciences 107(9):4004-4009 (Mar, 2010). The description of the ultrahigh-throughput screening method that can be used for luciferase mutation screening is incorporated into this specification by reference. For example, a library can be constructed by mismatch PCR. In addition, saturation mutagenesis can be used to introduce mutations into the region and position described in this specification or the region and position corresponding thereto to construct a library. The library is transformed into appropriate cells such as electroporated EBY-100 cells to obtain about 10 7 (10 million) mutants. Yeast cells transformed with this library can then be used for cell sorting. Polydimethylsiloxane (PDMS) microfluidic devices made using standard soft lithography can also be used. A flow focusing device can be used to form monodisperse droplets. The droplets formed containing individual mutants are used in an appropriate sorting device. The presence or absence of luciferase activity can be used when sorting cells. For example, a reaction solution with a component that emits light when the above-mentioned luciferase is activated can be used. For example, 96-well plates, 192-well plates, 384-well plates, 9600-well plates, etc. and a plate reader can be used to measure luminescence. Mutation introduction and sorting can be repeated multiple times. The mutations mentioned here include amino acid substitutions, insertions, deletions and / or additions.

[0172] For example, 1 to 10 mutations can be introduced into luciferase to confirm luciferase activity. Then, starting with the luciferase mutant confirmed to have activity, another 1 to 10 mutations can be introduced to confirm activity. A series of high-throughput screening (e.g., obtaining about 107 The above method for screening mutants can be repeated for more than 2 rounds, more than 5 rounds, more than 10 rounds, more than 15 rounds, for example, more than 20 rounds. A high-throughput screening in which one or more mutations, five or more mutations, for example, more than 10 mutations, are introduced into each round can be repeated for 10 rounds, thereby introducing more than 10 mutations, more than 50 mutations, for example, more than 100 mutations into the starting luciferase, and rapidly obtaining active mutants. In addition, by repeating 20 rounds, more than 20 mutations, more than 100 mutations, for example, more than 200 mutations can be introduced into the starting luciferase, and rapidly obtaining active mutants. This operation can be performed by an automatic device or by repeating conventional procedures.

[0173] Mutations can be introduced into any one or more positions between the first and last amino acids in the full-length luciferase amino acid sequence. However, important regions related to enzyme function, such as the active center, substrate recognition site, ATP recognition site, and their vicinity, are excluded. Luciferase is widely used in industry, and those skilled in the art are familiar with important regions related to enzyme function, including its active center, substrate recognition site, and ATP recognition site. In a specific embodiment, for example, within the full-length luciferase sequence, one or more mutations can be first introduced at positions 1 to 10. Then, starting with a confirmed active luciferase mutant, one or more mutations can be introduced at positions 11 to 20 to confirm activity. This procedure can be repeated n times (n ≤ 55). For example, one or more mutations can be introduced at positions 541 to 548 on the 55th pass. During the process, important regions related to enzyme function and regions not intended for modification can be skipped as appropriate. For example, position 208 of SEQ ID NO: 1 can be skipped. Thus, in addition to important regions related to enzyme function, any mutation can be introduced into any position in the full-length sequence. In addition, for example, luciferase mutants having 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 110 or more, 120 or more, 130 or more, 140 or more, 150 or more, 160 or more, 170 or more, 180 or more, 190 or more, for example, 200 or more mutations and having activity can be rapidly obtained. In one embodiment, mutations that abolish luciferase activity are not introduced. In one embodiment, mutations are not introduced at positions corresponding to positions 202, 208, 346, 424, 439, 448, and 457 of SEQ ID NO: 1.

[0174] Mutations can be introduced randomly or by rational design. In one embodiment, mutations introduced by rational design or randomly introduced mutations can be conservative amino acid substitutions. Conservative amino acid substitutions include amino acid substitutions in which the amino acid before and after substitution have similar chemical properties (e.g., Stryer et al., Biochemistry, 5th ed., 2002, pp. 44-49). For example, conservative amino acid substitutions can be selected from the group consisting of: (i) substitution of a basic amino acid with a different type of basic amino acid; (ii) substitution of an acidic amino acid with a different type of acidic amino acid; (iii) substitution of an aromatic amino acid with a different type of aromatic amino acid; (iv) substitution of a non-polar aliphatic amino acid with a different type of non-polar aliphatic amino acid; (v) substitution of a polar uncharged amino acid with a different type of polar uncharged amino acid. Basic amino acids can be, for example, selected from arginine, histidine, and lysine. Acidic amino acids can be, for example, aspartic acid or glutamic acid. Aromatic amino acids can be, for example, selected from phenylalanine, tyrosine, and tryptophan. Non-polar aliphatic amino acids can be selected from, for example, glycine, alanine, valine, leucine, methionine, proline, and isoleucine. Polar uncharged amino acids can be selected from, for example, serine, threonine, cysteine, asparagine, and glutamine. Because the chemical properties of the amino acid residues before and after the substitution are similar in conservative amino acid substitutions, and the positions of the conservative amino acid substitutions are present in the same position in the three-dimensional structure of the protein, variants having these conservative amino acid substitutions can maintain their three-dimensional structure and are highly likely to be active.

[0175] In one embodiment, the mutations introduced by rational design or random introduction include substitutions with functionally similar amino acids. Lists of functionally similar amino acids are well known in the art. In one embodiment, in substitutions with functionally similar amino acids, the amino acid before and after substitution can conform to any of the following specific amino acid categories:

[0176] 1) Glycine (G), Alanine (A);

[0177] 2) Aspartic acid (D), glutamic acid (E);

[0178] 3) Asparagine (N), glutamine (Q);

[0179] 4) Arginine (N), Lysine (K), Histidine (H);

[0180] 5) Isoleucine (I), Leucine (L), Valine (V), Proline (P);

[0181] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W);

[0182] 7) serine (S), threonine (T); and,

[0183] 8) Cysteine (C), methionine (M).

[0184] In contrast to conservative amino acid substitutions, non-conservative amino acid substitutions are substitutions of a certain amino acid with any amino acid that does not conform to the conservative substitutions (i) to (v) summarized above. In one embodiment, the amino acid substitution may be a non-conservative amino acid substitution. In this case, for example, before and after the introduction of the non-conservative amino acid substitution, it may be determined whether the dehydrogenase activity is maintained. If activity is confirmed, the non-conservative amino acid substitution may be adopted.

[0185] In one embodiment, the amino acid substitution may be a substitution by a similar amino acid (similarity substitution). In this specification, unless otherwise specified, substitution by a similar amino acid refers to substitution by an amino acid that is evaluated as a positive value or a neutral value (zero) in the amino acid substitution matrix used in ClustalW software and Blosum62 algorithm (for example, see S. Heinkoff and J. G. Henikoff, Proc. Natl. Acad. Sci. USA, Vol. 89, pp. 10915-10919, 1992, in particular Figure 2 ; and Thompson, Nucleic Acid Research, 1994, Vol. 22, No. 22, pp. 4673-4680). This matrix was generated based on approximately 2,000 aligned sequence fragments from over 500 related proteins. Furthermore, even starting with a single matrix, repeated application using subsets of the proteome ultimately yields a roughly similar set of scores. Therefore, this substitution matrix is universal. It is the most widely used method, leveraging the evolutionary relationship between homologous sequences. Therefore, variants of a luciferase enzyme that incorporate similar substitutions are highly likely to be active. For example, in this substitution matrix, a serine-to-threonine substitution is evaluated as a positive value of "1." Therefore, a serine-to-threonine substitution at position 24 in SEQ ID NO:1 qualifies as a similar amino acid substitution. Since this position is threonine in SEQ ID NO:71, and any enzyme is active, the S24T mutant of SEQ ID NO:1 is highly likely to be active. The same applies to other similar substitution variants.

[0186] In one embodiment, conservative amino acid substitutions or substitutions with functionally similar amino acids are not located in important regions related to enzyme function, such as the enzyme's active center, substrate recognition site, coenzyme recognition motif, or their vicinity, and therefore have no significant effect on enzyme activity. In another embodiment, conservative amino acid substitutions or substitutions with functionally similar amino acids are located in the enzyme's active center, substrate recognition site, coenzyme recognition motif, or their vicinity, but have no substantial effect on enzyme activity.

[0187] In this specification, "1 or more than 1" with respect to amino acid substitution, deletion or addition can be 1 to 10, for example, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, for example, 1 or 2.

[0188] The luciferase mutant may include an amino acid deletion compared to the sequence before the amino acid substitution. In a typical embodiment, the amino acid deletion is not in an important region related to the function of the enzyme and therefore has no significant effect on the activity of the enzyme. In one embodiment, the deletion may be a short deletion of 1 to 2 amino acids. In one embodiment, when the amino acid sequence of a luciferase is compared with the amino acid sequence of another luciferase, when an amino acid is deleted in one sequence, the deletion may be introduced into the other luciferase. Both luciferases exhibit activity, so there is a high possibility that the deletion has no significant effect on the activity of the enzyme.

[0189] The following are examples of amino acid deletions. For example, when SEQ ID NO: 73 is compared with SEQ ID NO: 1, the position corresponding to position 7 in SEQ ID NO: 1 is deleted in SEQ ID NO: 73. Therefore, even if position 7 is deleted from SEQ ID NO: 1, it is likely that the enzyme activity will not be significantly affected. Other deletions can be found in Figure 1 Sure.

[0190] Luciferase mutants may also include mutants in which additional amino acids are inserted compared to the sequence before mutation. In a typical embodiment, the amino acid insertion is not in the active center, substrate recognition site, coenzyme recognition motif, and other important areas related to the function of the enzyme near them, and therefore has no significant effect on the activity of the enzyme. In one embodiment, the insertion may be 1 to 4 amino acids. In one embodiment, when an amino acid is inserted into the sequence of one luciferase compared to the amino acid sequence of another luciferase, the inserted amino acid can be introduced into the other luciferase. Both luciferases exhibit activity, so there is a high possibility that the insertion has no significant effect on the activity of the enzyme.

[0191] The following are examples of inserted amino acids. When SEQ ID NO: 1 and SEQ ID NO: 73 are compared, a proline is inserted between positions 39 and 40 of SEQ ID NO: 1, corresponding to position 37 of SEQ ID NO: 1. Both luciferases show activity, so it is highly likely that the insertion has no significant effect on the activity of the enzyme. Therefore, an amino acid (e.g., proline) can be inserted between positions 39 and 40 of SEQ ID NO: 1. Other insertion positions can be selected from Figure 1 The inserted amino acid may be an amino acid inserted in another sequence, or an amino acid substituted with a conservative amino acid.

[0192] Luciferase mutants can also include mutants with additional amino acids added compared to the sequence before mutation. In a typical embodiment, the addition of amino acids is carried out at the N-terminus or C-terminus of the luciferase and has no obvious effect on the activity of the enzyme. In one embodiment, the addition can be 1 to 6 amino acids, 1 to 5 amino acids, for example, 1 to 4 amino acids. Examples of additions can include but are not limited to short segments of histidine residues (for example, 2 to 6 histidine residues) for assisting luciferase purification. In addition, examples of additions can include but are not limited to the addition of signal peptides for assisting luciferase expression. Signal peptides can include well-known signal sequences or functionally equivalent substances thereof.

[0193] The following are examples of added amino acids. When SEQ ID NO:1 and SEQ ID NO:71 are aligned, SEQ ID NO:1 has three additional amino acids further upstream from the N-terminus of SEQ ID NO:71. Since both luciferases exhibit activity, it is highly likely that these additions have no significant effect on the enzyme's activity. Therefore, one, two, or three amino acids may be added to the N-terminus of SEQ ID NO:71. The added amino acids may be corresponding amino acids in the other sequence, or they may be conservative amino acid substitutions with the corresponding amino acids.

[0194] Mutations introduced into luciferase can be performed without disrupting its secondary structure and structural motifs, such as the α-helix and β-sheet structures. Regions of secondary structure can be determined, for example, using secondary structure prediction algorithms. Examples of such prediction algorithms include, but are not limited to, NetSurfP-2.0. The same applies to other structural motifs, such as nests and niches.

[0195] In one embodiment, the amino acid residues or amino acid sequence motifs required for the activity of luciferase are not substituted. In another embodiment, these positions can be subjected to conservative amino acid substitutions or similarity substitutions, but the activity of the variant after the substitution must be confirmed. In one embodiment, no amino acid deletion or insertion is performed before or after the amino acid residues required for the activity of luciferase. The before and after positions of the amino acid residues required for the activity of luciferase refer to positions 1 or 2 N-terminal sides of the amino acid residues required for the activity, or positions 1 or 2 C-terminal sides. In another embodiment, amino acid deletion or insertion can be performed before or after the amino acid residues required for the activity of luciferase, but in this case, the activity of the variant after the substitution must be confirmed. Whether the variant has activity can be routinely confirmed, for example, by high-throughput screening.

[0196] In one embodiment, the luciferase mutant disclosed herein has luciferase activity. The presence or absence of luciferase activity can be measured, for example, using Lumitester C-110 (manufactured by Kikkoman Biochemifa Co., Ltd.) according to the method described in the Examples.

[0197] As used herein, "thermostability" can be evaluated, for example, by using as an indicator the residual activity of firefly luciferase after heat treatment at a predetermined temperature for a predetermined time. Specifically, the thermal stability of firefly luciferase can be evaluated by comparing the residual activity after heat treatment at a high temperature, for example, at a reaction temperature of typically 30-50°C, for example, 35-45°C or 35-40°C, for a predetermined time, typically 5-180 minutes or 10-180 minutes, for example, 60-180 minutes or approximately 90 minutes.

[0198] The residual activity of firefly luciferase is the activity after heat treatment, when the activity of firefly luciferase before the treatment under the aforementioned high temperature conditions is set to 1. For example, when the residual activity after heat treatment is halved, the residual activity after heat treatment becomes 0.5, relative to the activity before heat treatment of 1. The residual activity rate of firefly luciferase is calculated as the ratio of the activity after heat treatment to the activity of firefly luciferase before the treatment under the aforementioned high temperature conditions. As used herein, improved thermal stability refers to the situation where the residual activity rate of a firefly luciferase mutant when subjected to the aforementioned conditions is improved by 1.01-fold, 1.02-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, or 2-fold or more compared to the luciferase before introduction of the mutation disclosed herein (mutations at positions corresponding to position 252, etc., of SEQ ID NO: 1). In other words, in this specification, improved thermal stability means that the residual activity rate of the firefly luciferase mutant when subjected to the above-mentioned conditions shows an improvement of 1% or more, 2% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more relative to the residual activity rate of the luciferase before introduction of the mutation disclosed herein.

[0199] (polynucleotide)

[0200] In one embodiment, the present disclosure provides a polynucleotide encoding a luciferase mutant (hereinafter also referred to as a "luciferase gene"). The sequence of the polynucleotide can be easily determined based on the amino acid sequence of the firefly luciferase mutant. For example, as a polynucleotide encoding the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 10, the polynucleotide of SEQ ID NO: 3 or SEQ ID NO: 11 can be cited respectively. The polynucleotide encoding the luciferase before amino acid substitution can, for example, include the following nucleotide sequence:

[0201] (i) a nucleotide sequence that has a sequence identity of 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, more preferably 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more to the entire length of any one of SEQ ID NO: 3 and SEQ ID NO: 11, and encodes an active luciferase;

[0202] (ii) a nucleotide sequence encoding active luciferase in which one or more nucleotides are substituted, deleted or added in any one of the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 11; or

[0203] (iii) a nucleotide sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 11.

[0204] To obtain these luciferase-encoding nucleotides, commonly used gene cloning methods are generally used. For example, chromosomal DNA or mRNA can be extracted from firefly tissues or cells capable of producing luciferase using conventional methods, such as those described in Current Protocols in Molecular Biology (WILEY Interscience, 1989). mRNA can then be used as a template to synthesize cDNA. Chromosomal DNA or cDNA obtained in this way can be used to create a chromosomal DNA or cDNA library.

[0205] Next, based on the amino acid sequence of the luciferase, an appropriate probe DNA is synthesized, and the probe DNA is used to screen for a polynucleotide encoding luciferase from a chromosomal DNA or cDNA library; or, based on the amino acid sequence, appropriate primer DNA is prepared, and an appropriate polymerase chain reaction (PCR) such as a 5'RACE method or a 3'RACE method is used to amplify a DNA containing a target nucleotide fragment encoding the luciferase, and these DNA fragments are ligated to obtain a DNA containing the full-length nucleotide encoding the target luciferase.

[0206] If the nucleotide sequence encoding luciferase is known, it can also be artificially synthesized. Such artificial gene synthesis services are provided by, for example, IDT.

[0207] (Method for producing luciferase gene)

[0208] Mutation of the luciferase gene can be performed using any known method, depending on the desired mutation type. Specifically, methods such as contacting and allowing the luciferase gene or a recombinant DNA incorporating the gene to act with a mutagenic agent, ultraviolet irradiation, genetic engineering techniques, or protein engineering techniques can be widely employed.

[0209] Examples of the mutagen used in the above-mentioned mutation treatment include hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine, nitrous acid, sulfurous acid, hydrazine, formic acid, and 5-bromouracil.

[0210] The conditions for contact and action can be adapted to the type of agent used, and are not particularly limited as long as they induce the desired mutation in the luciferase gene. Typically, contact and action are preferably performed at a concentration of 0.5 to 12 M of the agent at a reaction temperature of 20 to 80°C for at least 10 minutes, preferably 10 to 180 minutes, to induce the desired mutation. Ultraviolet irradiation can also be performed according to conventional methods as described above (Modern Chemistry, 024-30, June 1989).

[0211] As a method utilizing protein engineering means, a method known as site-specific mutagenesis is generally used, for example, the Kramer method (Nucleic Acids Res., 12, 9441-9456 (1984)), the Eckstein method (Nucleic Acids Res., 13, 8749-8764 (1985); Nucleic Acids Res., 13, 8765 (1985); Nucleic Acids Res, 14, 9679 (1986)), and the Kunkel method (Proc. Natl. Acid. Sci. USA, 82, 488-492 (1985)).

[0212] In addition to the above-mentioned gene modification methods, the desired modified luciferase gene can also be directly synthesized by organic synthesis or enzymatic synthesis.

[0213] The base sequence of the luciferase gene can be confirmed using, for example, the multi-capillary DNA analysis system Applied Biosystems 3730x1 DNA Analyzer (manufactured by Thermo Fisher Scientific).

[0214] (vector, host cell)

[0215] In one embodiment, the present disclosure relates to vectors comprising the above-mentioned polynucleotides. These luciferase genes are preferably ligated to various vectors according to conventional methods. Examples of vectors include plasmids, and any vector known to those skilled in the art, such as phages and cosmids, can be used. The type of vector can be selected based on the host cell, and specifically, for example, pET16-b or pKK223-3 are preferred.

[0216] In one embodiment, the present disclosure relates to a host cell comprising the polynucleotide or vector. The host cell is not limited and can be bacteria such as Escherichia coli and Bacillus subtilis, yeast cells, insect cells, animal cells (e.g., mammalian cells), and plant cells, preferably bacterial cells such as Escherichia coli.

[0217] (Transformation and Transduction)

[0218] Can be by conventional method the luciferase gene obtained as mentioned above is integrated on the carriers such as the plasmid used in the transformation of phage, clay or prokaryotic cell or eukaryotic cell, then by conventional method the host corresponding to each carrier is transformed or transduced.For example, as host, can use the microorganism belonging to Escherichia, for example, use the recombinant DNA transformation or transduction such as intestinal bacteria K-12 strain obtained, or intestinal bacteria B strain, preferably intestinal bacteria JM109 strain, intestinal bacteria DH5α strain, intestinal bacteria BL21 strain, intestinal bacteria BL21 (DE3) strain (all manufactured by treasure biological company) etc., thereby obtain each bacterial strain.This method that recombinant vector is moved into host cell, for example, when host cell is the microorganism belonging to Escherichia coli, can adopt the method etc. of moving into of recombinant DNA in the presence of calcium ion, can also adopt electroporation.In addition, can also use commercially available competent cells (for example ECOS Competent Escherichia coli BL21 (DE3, Nippon Gene manufactures). It should be noted that the luciferase gene may be a gene that has been codon-optimized according to the expression host.

[0219] (Method for producing luciferase mutants)

[0220] In one embodiment, the present disclosure relates to a method for producing a luciferase mutant with improved thermal stability, comprising the step of culturing the host cell described above. The culturing can be performed using various known methods, such as solid culture, or preferably liquid culture.

[0221] The production method may include: culturing the host cell under conditions capable of expressing luciferase protein; and any step of isolating the luciferase from the culture medium or culture fluid. The conditions capable of expressing luciferase protein refer to transcribing and translating the luciferase gene to produce the polypeptide encoded by the gene.

[0222] In one embodiment, the production method includes: before the culturing step, artificially introducing a mutation at a position corresponding to position 252 of SEQ ID NO: 1 of the luciferase protein. This can be achieved by artificially introducing a mutation into the gene sequence encoding luciferase.

[0223] In addition, the culture medium for culturing the above-mentioned host cells can be, for example, a culture medium obtained by adding one or more inorganic salts such as sodium chloride, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, magnesium chloride, ferrous chloride, ferrous sulfate or manganese sulfate to one or more nitrogen sources such as yeast extract, tryptone, peptone, meat extract, corn steep liquor or soybean or wheat bran extract, and further adding carbohydrate raw materials, vitamins, etc. as needed.

[0224] The initial pH of the culture medium is preferably adjusted to pH 7 to 9. Cultivation can be carried out by deep aeration stirring culture, shaking culture, static culture, etc. at a culture temperature of 20 to 42°C, preferably about 25 to 37°C, for 4 to 24 hours, more preferably about 25 to 37°C for 8 to 16 hours.

[0225] After the culture is completed, luciferase can be obtained by collecting the culture using conventional enzyme collection methods. For example, the bacteria can be subjected to ultrasonic destruction treatment, grinding treatment, etc. by conventional methods, or the enzyme can be extracted using a lytic enzyme such as lysozyme, or shaken or placed in the presence of toluene to lyse the bacteria so that the enzyme is discharged from the bacteria. Then, the solution is filtered and centrifuged to remove the solid portion, and after removing the nucleic acid using streptomycin sulfate, protamine sulfate, or manganese sulfate as needed, ammonium sulfate, ethanol, acetone, etc. are added thereto and separated, and the precipitate is collected to obtain a crude luciferase enzyme.

[0226] In order to further obtain purified luciferase from the crude luciferase, for example, gel filtration using Sephadex, Superdex, or Ultrogel; adsorption elution using ion exchange carriers, hydrophobic carriers, or hydroxyapatite; electrophoresis using polyacrylamide gel; sedimentation methods such as sucrose density gradient centrifugation; affinity chromatography; separation methods using molecular sieve membranes or hollow fiber membranes, etc., or a combination of these methods can be appropriately selected to obtain purified luciferase. Thus, the desired luciferase can be obtained.

[0227] The produced luciferase can be used in the kit described in this specification, or in the method for detecting at least one of ATP, ADP, and AMP.

[0228] (Kit for detecting at least one of ATP, ADP and AMP)

[0229] In one embodiment, the present disclosure relates to a kit for detecting at least one of ATP, ADP, and AMP, comprising a luciferase mutant described herein. In addition to the luciferase mutant, the kit may also contain luciferin. In this case, the kit may also contain metal ions such as magnesium, manganese, and calcium. Those skilled in the art can determine the concentration of the metal ions based on the enzyme used. ATP, O2, and luciferin are converted into AMP, pyrophosphate, CO2, and oxyluciferin by luciferase, resulting in luminescence. The reactions that occur are as follows.

[0230] Luciferin + ATP + O2 → Oxyluciferin + adenosine monophosphate (AMP) + pyrophosphate (PPi) + CO2 + light

[0231] In one embodiment, the kit further comprises an enzyme that catalyzes the reaction of generating ATP from ADP. The enzyme that catalyzes the reaction of generating ATP from ADP can be selected from the group consisting of pyruvate kinase (PK), acetate kinase (AK), creatine kinase (CK), polyphosphate kinase (PPK), hexokinase, glucokinase, glycerokinase, fructokinase, phosphofructokinase, riboflavin kinase, and fructose bisphosphatase. In another embodiment, the kit further comprises pyruvate phosphate dikinase (PPDK), adenylate kinase (ADK), or pyruvate water dikinase (PWDK).

[0232] When a sample contains ATP, ATP is converted to AMP by luciferase, which then emits light. In a system where an enzyme catalyzes the reaction of generating ATP from ADP is present, when the sample contains ADP, the enzyme converts ADP into ATP, which is then used in the luminescence reaction. This allows the total amount of ATP and ADP present in the system to be measured. Furthermore, in a system where PPDK is present, when a sample contains AMP, AMP is converted to ATP by PPDK, PEP, and PPi. Alternatively, in a system where PWDK is present, when a sample contains AMP, AMP is converted to ATP by PWDK, PEP, and phosphate. The generated ATP is then again activated by luciferase to emit light. Luminescence is maintained steadily, and the amount of luminescence correlates with the total amount of ATP and AMP present in the system, allowing for quantification of ATP and AMP. When an enzyme catalyzing the reaction of generating ATP from ADP is present along with PPDK, ADK, or PWDK, the total amount of ATP, ADP, and AMP can be measured.

[0233] Regarding luciferin, there is no limitation as long as it is recognized as a substrate by the luciferase used, and both natural products and chemical syntheses are acceptable. In addition, any known luciferin derivative can also be used. The basic framework of luciferin is imidazopyrazinone, and there are many tautomers. Luciferin can include firefly luciferin. Firefly luciferin is a substrate for firefly luciferase (EC 1.13.12.7). Luciferin derivatives can be derivatives described in Japanese Patent Application Laid-Open No. 2007-91695, Japanese Patent Application No. 2010-523149 (International Publication No. 2008 / 127677), etc.

[0234] In addition to the above components, the kit may further include at least one of a stabilizer, a buffer, and instructions.

[0235] (Method for detecting at least one of ATP, ADP and AMP)

[0236] In one embodiment, the present disclosure relates to a method for detecting at least one of ATP, ADP, and AMP, comprising using a luciferase mutant described herein. The method may include the steps of catalyzing an oxidation reaction of luciferin using the luciferase mutant described herein, and measuring the luminescence generated by the oxidation reaction.

[0237] The catalysis of the oxidation reaction of luciferin by the luciferase mutant is consistent with that described in the "Kit for Detecting At Least One of ATP, ADP, and AMP." This can be performed by reacting the luciferase mutant described in this specification with luciferin and a sample. When the sample contains ATP, the ATP is converted to AMP by the action of luciferase, emitting light, allowing the measurement of ATP. In a system in which an enzyme catalyzes the reaction of generating ATP from ADP is present, the total amount of ATP and ADP present in the system can be measured. Furthermore, in a system in which PPDK or PWDK is present, ATP and AMP can be quantified. When an enzyme catalyzing the reaction of generating ATP from ADP is present along with PPDK, ADK, or PWDK, the total amount of ATP, ADP, and AMP can be measured.

[0238] The determination of the luminescence amount of luciferase can be carried out by a known method, and the relative luminescence intensity (RLU) obtained using an appropriate luminescence measuring device, such as an illuminometer (manufactured by Berthold, CentroLB960 or Lumat3 LB9508; manufactured by Kikkoman Biochemi Corporation, Lumitester C-110, Lumitester C-100, Lumitester PD-20, Lumitester PD-30, etc.) can be evaluated as an index. Generally, the luminescence generated when luciferin is converted to oxyluciferin is measured. The luminescence measuring device can also use a device with a photomultiplier tube (manufactured by 3M Company, etc.) and a device with a photodiode (manufactured by Hygiena Company, Neogen Company, etc.) that can perform high-sensitivity measurement.

[0239] In one embodiment, the luciferase mutant disclosed herein (e.g., a luciferase mutant having at least 70%, 75%, 80%, 85%, 90% or 95% amino acid sequence identity with SEQ ID NO: 10, wherein the position corresponding to position 252 of SEQ ID NO: 1 is leucine) has an isoleucine at position 393 of SEQ ID NO: 1, a serine at position 326 of SEQ ID NO: 1, an isoleucine at position 392 of SEQ ID NO: 1, and an isoleucine at position 467 of SEQ ID NO: 1. In one embodiment, the mutant has one or more amino acid substitutions corresponding to 252L, 262Y, 294L, 90Y, 371L, 41V, 427F, 53L, 306F, 221L, 17D, 17E, 17K, 18L, 75K, 110D, 110E, 110K, 110R, 223V, 223L, 223I, 224I, 257F, 229F, 229L, 158D or 158E based on SEQ ID NO:1.

[0240] The luciferase mutants disclosed herein exclude the wild-type sequence itself. In addition, the luciferase mutants disclosed herein exclude the natural product.

[0241] In one embodiment, the peroxisome targeting signal 1 of luciferase can be deleted. PTS1 is located at the 3 amino acids at the C-terminus, and its consensus sequence is "-(S / A / C)-(K / R / H)-(L / M)-COOH". For example, in one embodiment, 3, 4 or 5 amino acids on the C-terminal side of luciferase can be deleted, but the present disclosure is not limited thereto. For example, in one embodiment, 6, 7, 8, 9, 10, 11 or 12 amino acids on the C-terminal side of luciferase can be deleted, but the present disclosure is not limited thereto. Whether the deletion mutant has activity can be simply confirmed by routine operations.

[0242] The present invention will be described in more detail below with reference to the following embodiments. However, the technical scope of the present invention is not limited by these embodiments.

[0243] [Example]

[0244] The mutant luciferase disclosed herein is further illustrated by the following examples, which are for illustrative purposes only and are not intended to limit the present invention.

[0245] pET-16b-LlLuc-1M is a plasmid for expressing luciferase, and a gene (SEQ ID NO: 3) encoding mutant Heike luciferase (SEQ ID NO: 2) is inserted into the multiple cloning site of the pET-16b vector (refer to WO2020 / 009215 publication).

[0246] 1. Preparation of luciferase mutants derived from Heike Firefly (LlLuc)

[0247] Site-specific mutagenesis was performed by PCR using pET-16b-LlLuc-1M as a template. PCR reagents and reaction conditions were based on the KOD One PCR Master Mix (Toyobo Co., Ltd.) manual. The reaction was repeated 15 times in a cycle of 98°C for 10 seconds, 55°C for 5 seconds, and 68°C for 30 seconds.

[0248] To 20 μL of the PCR product, 1 μL of DpnI was added and the reaction was incubated at 37°C for 1 hour to degrade the template DNA. This reaction product was then used to transform Escherichia coli JM109. The resulting transformants were cultured in LB medium containing 50 μg / mL ampicillin, recovered by centrifugation, and plasmids carrying genes encoding LlLuc-1M mutants were extracted from the transformants using the FastGene Plasmid Miniprep Kit (manufactured by Nippon Genetics). Single mutation introduction was repeated to construct expression plasmids for multiple LlLuc mutants. For example, PCR was performed using pET-16b-LlLuc-1M as a template and primers of SEQ ID NO:4 and SEQ ID NO:5 to construct an expression plasmid for LlLuc-2M. The names of the generated mutants, the amino acid substitutions introduced into LlLuc-1M, and the template plasmids and primers used in PCR are shown in Table 1.

[0249] [Table 1]

[0250]

[0251] 2. Preparation of LlLuc-4M mutant

[0252] Mutants of L1Luc-4M (SEQ ID NO: 10) were prepared according to the method described in "1. Preparation of Luciferase (L1Luc) Mutants from Firefly Heike." The base sequence of the gene encoding L1Luc-4M is shown in SEQ ID NO: 11. Single mutation introduction was repeated to construct plasmids for expressing multiple L1Luc-4M mutants. For example, PCR was performed using pET-16b-L1Luc-5M as a template and primers of SEQ ID NO: 16 and SEQ ID NO: 17 to construct a plasmid for expressing L1Luc-6M. The names of the mutants prepared, the amino acid substitutions introduced into L1Luc-4M, and the template plasmids and primers used in PCR are shown in Table 2.

[0253] [Table 2]

[0254]

[0255] 3. Recombinant production of the LlLuc-4M mutant

[0256] Escherichia coli BL21(DE3) was transformed with a plasmid expressing LlLuc-4M or its mutant enzyme to obtain a strain producing LlLuc-4M or its mutant enzyme. These strains were inoculated into 2 mL of LB medium containing 50 μg / mL ampicillin and 0.1 mM IPTG and cultured at 180 rpm at 25°C or 30°C for 20 hours.

[0257] The culture was centrifuged at 15,000 rpm for 2 minutes, and the resulting pellet was resuspended in 1 mL of 230 mM Tricine buffer (pH 7.85). The bacterial suspension was disrupted by ultrasound and then centrifuged at 15,000 rpm for 15 minutes. The resulting supernatant was recovered as a crude enzyme solution of L1Luc-4M or its mutant enzyme.

[0258] 4. Evaluation of the heat resistance of the LlLuc-4M mutant

[0259] 50 μL of crude enzyme solution of LlLuc-4M or its mutant enzyme was mixed with 50 μL of a heat treatment reagent (19 mM disodium EDTA, 0.4 mM dithiothreitol, 74 mM magnesium acetate, 20% xylitol, 0.2% Tween 80) and heated at 50°C for 120 minutes. The residual enzyme activity was assessed (Table 3).

[0260] [Table 3]

[0261]

[0262] The residual activities of the LlLuc-4M mutants shown in Table 3 increased by 0.19 to 0.62 compared to LlLuc-4M, in other words, by 58% to 187%. This indicates that the thermal stability of the LlLuc-4M mutants was improved.

[0263] 5. Recombinant production of LlLuc-4M, 5M, 6M, and 7M mutants

[0264] Escherichia coli BL21(DE3) was transformed with LlLuc-4M, 5M, 6M, and 7M expression plasmids to generate LlLuc-4M or mutant enzyme production strains. These production strains were inoculated into 2.5 mL of LB medium containing 100 μg / mL ampicillin and precultured at 30°C and 160 rpm for 12 hours. One mL of the preculture was inoculated into 100 mL of 2×LB medium containing 50 μg / mL ampicillin and cultured at 30°C and 120 rpm for 21 hours. After this, 1 mL of 10 mM IPTG was added and cultured at 28°C and 110 rpm for 17 hours.

[0265] The culture was centrifuged at 10,000 rpm for 10 minutes, and the resulting pellet was resuspended in 40 mL of buffer A (5% glycerol, 1 mM EDTA-2Na, 2 mM 3-mercapto-1,2-propanediol, phosphoric acid, pH 7.1). The bacterial suspension was disrupted by ultrasound and then centrifuged at 10,000 rpm for 50 minutes. The pH of the resulting supernatant was adjusted to 7.1 with phosphoric acid and filtered through a 0.2 μm filter to recover the crude enzyme solution.

[0266] The crude enzyme solution was loaded onto a cation exchange chromatography column (HiScreen SP FF, manufactured by Cytiva) equilibrated with the aforementioned buffer A. The active fraction was recovered by gradient elution using buffer B (200 mM potassium phosphate, 5% glycerol, 1 mM EDTA-2Na, 2 mM 3-mercapto-1,2-propanediol, pH 7.1). The recovered fraction was concentrated approximately 30-fold using an Amicon Ultra-3K (manufactured by Merck) to obtain a purified enzyme solution.

[0267] 6. Evaluation of the heat resistance of LlLuc-4M, 5M, 6M, and 7M mutants

[0268] The purified enzyme solution was diluted 500-fold or more with a heat treatment buffer (0.3 M Tricine, 0.2% BSA, 5% glycerol), heated at 60°C for 5 minutes, and the residual enzyme activity was evaluated (Table 4).

[0269] [[Table 4]

[0270]

[0271] The residual activities of the LlLuc-5M, 6M, and 7M mutants shown in Table 4 increased by 0.07 to 0.52 compared to LlLuc-4M, or in other words, by 54% to 400%. Combined with the amino acid substitutions shown in Table 3, it can be said that the thermal stability of the LlLuc-4M mutant has been significantly improved.

[0272] 7. Production of LlLuc-7M mutant

[0273] Site-specific mutagenesis was performed by PCR using pET-16b-LlLuc-7M as a template and primers of SEQ ID NO:25 and SEQ ID NO:26. PCR reagents and reaction conditions were based on the instructions for KOD One PCR Master Mix (Toyobo Co., Ltd.). The reaction was repeated seven times in a cycle of "98°C for 10 seconds → 55°C for 5 seconds → 68°C for 30 seconds."

[0274] To 20 μL of the PCR product, 1 μL of DpnI was added, and the reaction was incubated at 37°C for 1 hour to degrade the template DNA. Next, to 2 μL of the reaction product, 5 μL of Ligation High Ver. 2 (Toyobo Co., Ltd.), 1 μL of T4 polynucleotide kinase (Toyobo Co., Ltd.), and 7 μL of ion-exchanged water were added, and the reaction was incubated at 16°C for 1 hour. This reaction solution was then used to transform Escherichia coli JM109. The resulting transformants were cultured in LB medium containing 50 μg / mL ampicillin, recovered by centrifugation, and a plasmid carrying the gene (SEQ ID NO: 28) encoding LlLuc-7Md (SEQ ID NO: 27) was extracted from the transformants using the FastGene Plasmid Miniprep Kit. It should be noted that LlLuc-7Md is a mutant lacking the five amino acids (P544, V545, A546, K547, and M548) at the C-terminus of LlLuc-7M, which contain the peroxisome targeting signal 1 (PTS1). PTS1 consists of three amino acids at the C-terminus, and its consensus sequence is "-(S / A / C)-(K / R / H)-(L / M)-COOH" (see, for example, FEBS J., 272, 2362 (2005), Plant Cell Physiol., 38, 759 (1997), and Eur. J. Cell Biol., 71, 248 (1996)).

[0275] Next, mutants of L1Luc-7Md were prepared according to the method described in "1. Preparation of Luciferase (L1Luc) Mutants from Firefly Heike." Single mutation introduction was repeated to construct plasmids for expressing multiple mutant L1Luc-7Md. For example, PCR was performed using pET-16b-L1Luc-8Md as a template and primers of SEQ ID NO: 66 and SEQ ID NO: 67 to construct a plasmid for expressing L1Luc-9Md. The names of the mutants prepared, the amino acid substitutions introduced into L1Luc-7Md, and the template plasmids and primers used in PCR are shown in Table 5.

[0276] [Table 5]

[0277]

[0278]

[0279] 8. Evaluation of the heat resistance of the LlLuc-7Md mutant

[0280] Crude enzyme solutions of LlLuc-7Md and its mutant enzymes were prepared according to the method described in "3. Recombinant production of LlLuc-4M mutants". The residual activity of LlLuc-7Md and its mutant enzymes after heating at 55°C for 60 minutes or 60°C for 30 minutes was evaluated according to the method described in "4. Evaluation of heat resistance of LlLuc-4M mutants" (Table 6).

[0281] [Table 6]

[0282]

[0283]

[0284] The residual activity of the LlLuc-7M mutants shown in Table 6 increased by 0.04 to 0.50 compared to LlLuc-7M, or in other words, by 13% to 161%. This indicates that the thermostability of the LlLuc-7M mutants has been improved. These mutations are likely to similarly improve thermostability when introducing luciferases from other sources.

[0285] The residual activities of LlLuc-7Md and its mutant enzymes after heating at 60°C for 30 minutes are shown in the table (Table 7).

[0286] [Table 7]

[0287]

[0288] The residual activities of the LlLuc-8Md to 16Md and 15Md-2 mutants shown in Table 7 increased by 0.02 to 1.01 compared to LlLuc-7Md, in other words, by 200% to 10100%. Combined with the amino acid substitutions shown in Table 6, it can be said that the thermal stability of the LlLuc-7Md mutant has been significantly improved.

[0289] Industrial applicability

[0290] According to the present disclosure, a luciferase with improved thermal stability is provided. In addition, according to the present disclosure, a luciferase with significantly improved thermal stability is provided. In addition, according to the present disclosure, a luciferase with improved affinity for a substrate is provided. These can be used for ATP determination, ATP detection, luminescence determination, etc.

[0291] This specification cites various documents, including patent applications and manufacturer's manuals. The disclosures of these documents are not considered relevant to the patentability of the present disclosure and are incorporated herein by reference in their entirety. More specifically, all cited documents are incorporated herein by reference, as if each document were specifically and individually indicated to be incorporated by reference.

[0292] Sequence Description

[0293] SEQ ID NO: 1: Luciferase from Luciola lateralis

[0294] SEQ ID NO: 2: Mutant luciferase from L. lateralis (also known as HLK-C393L, see WO2020 / 009215)

[0295] SEQ ID NO: 3: Base sequence of the gene encoding SEQ ID NO: 2

[0296] SEQ ID NO: 4 to SEQ ID NO: 9: Primer sequences for mutation introduction

[0297] SEQ ID NO: 10: Mutant luciferase from L. lateralis (LlLuc-4M)

[0298] SEQ ID NO: 11: Base sequence of the gene encoding SEQ ID NO: 10

[0299] SEQ ID NO: 12 to SEQ ID NO: 26: Primer sequences for mutation introduction

[0300] SEQ ID NO: 27: Mutant luciferase from L. lateralis (L1Luc-7Md)

[0301] SEQ ID NO: 28: Base sequence of the gene encoding SEQ ID NO: 27

[0302] SEQ ID NO: 29 to SEQ ID NO: 68: Primer sequences for mutation introduction

[0303] SEQ ID NO:69 Amino acid sequence of Lucidum luciferase

[0304] SEQ ID NO: 70 Base sequence of Lucidum luciferase

[0305] SEQ ID NO:71 Amino acid sequence of Photinia firefly luciferase

[0306] SEQ ID NO: 72 Base sequence of Firefly luciferase

[0307] SEQ ID NO:73 Amino acid sequence of Photinus pennsylvanicus luciferase

[0308] SEQ ID NO:74 Base sequence of Photinus pennsylvanicus luciferase

[0309] Sequences

[0310] Amino acid sequence of firefly luciferase from Luciola lateralis, SEQ ID NO:1

[0311] MENMENDENIVYGPEPFYPIEEGSAGAQLRKYMDRYAKLGAIAFTNALTG

[0312] VDYTYAEYLEKSCCLGEALKNYGLVVDGRIALCSENCEEFFIPVLAGLFI

[0313] GVGVAPTNEIYTLRELVHSLGISKPTIVFSSKKGLDKVITVQKTVTAIKT

[0314] IVILDSKVDYRGYQSMDNFIKKNTPQGFKGSSFKTVEVNRKEQVALIMNS

[0315] SGSTGLPKGVQLTHENAVTRFSHARDPIYGNQVSPGTAILTVVPFHHGFG

[0316] MFTTLGYLTCGFRIVMLTKFDEETFLKTLQDYKCSSVILVPTLFAILNRS

[0317] ELLDKYDLSNLVEIASGGAPLSKEIGEAVARRFNLPGVRQGYGLTETTSA

[0318] IIITPEGDDKPGASGKVVPLFKAKVIDLDTKKTLGPNRRGEVCVKGPMLM

[0319] KGYVDNPEATREIIDEEGWLHTGDIGYYDEEKHFFIVDRLKSLIKYKGYQ

[0320] VPPAELESVLLQHPNIFDAGVAGVPDPIAGELPGAVVVLEKGKSMTEKEV

[0321] MDYVASQVSNAKRLRGGVRFVDEVPKGLTGKIDGKAIREILKKPVAKM

[0322] SEQ ID NO: 2 Amino acid sequence of mutant Luciola lateralis-derived luciferase (also known as HLK-C393L)

[0323] MENMENDENIVYGPEPFYPIEEGSAGAQLRKYMDRYAKLGAIAFTNALTG

[0324] VDYTYAEYLEKSCCLGEALKNYGLVVDGRIALCSENCEEFFIPVLAGLFI

[0325] GVGVAPTNEIYTLRELVHSLGISKPTIVFSSKKGLDKVITVQKTVTAIKT

[0326] IVILDSKVDYRGYQSMDNFIKKNTPQGFKGSSFKTVEVNRKEQVALIMNS

[0327] SGSTGLPKGVQLTHENLVTRFSHARDPIYGNQVSPGTAILTVVPFHHGFG

[0328] MFTTLGYLTCGFRIVMLTKFDEETFLKTLQDYKCSSVILVPTLFAILNRS

[0329] ELLDKYDLSNLVEIASGGAPLSKEIGEAVARRFNLPGVRQGYGLTETTSA

[0330] IIITPEGDDKPGASGKVVPLFKAKVIDLDTKKTLGPNRRGEVLVKGPMLM

[0331] KGYVDNPEATREIIDEEGWLHTGDIGYYDEEKHFFIVDRLKSLIKYKGYQ

[0332] VPPAELESVLLQHPNIFDAGVAGVPDPIAGELPGAVVVLKKGKSMTEKEV

[0333] MDYVASQVSNAKRLRGGVRFVDEVPKGLTGKIDGKAIREILKKPVAKM

[0334] SEQ ID NO: 3 Base sequence of the mutant Luciola lateralis-derived luciferase gene (also known as HLK-C393L)

[0335] atggaaaacatggagaacgatgaaaatattgtgtatggtcctgaaccattttaccctatt

[0336] gaagagggatctgctggagcacaattgcgcaagtatatggatcgatatgcaaaacttgga

[0337] gcaattgcttttactaacgcacttaccggtgtcgattatacgtacgccgaatacttagaa

[0338] aaatcatgctgtctaggagaggctttaaagaattatggtttggttgttgatggaagaatt

[0339] gcgttatgcagtgaaaactgtgaagagttcttttcctgtattagccggtttatttata

[0340] ggtgtcggtgtggctccaactaatgagatttacactctacgtgaattggttcacagttta

[0341] ggcatctctaagccaacaattgtatttagttctaaaaaaggattagataaagttataact

[0342] gtacaaaaaacggtaactgctattaaaaccattgttatattggacagcaaagtggattat

[0343] agaggttatcaatccatggacaactttattaaaaaaaacactccacaaggtttcaaagga

[0344] tcaagttttaaaactgtagaagttaaccgcaaagaacaagttgctcttataatgaactct

[0345] tcgggttcaaccggtttgccaaaaggtgtgcaacttactcatgaaaatttggtcacgcgt

[0346] ttttctcacgtagagatccaatttatggaaaccaagtttcaccaggcacggctatttta

[0347] actgtagtaccattccatcatggttttggtatgtttactactttaggctatctaacttgt

[0348] ggttttcgtattgtcatgttaacgaaatttgacgaagagacttttttaaaaaacactgcaa

[0349] gattacaaatgttcaagcgttatcttgtaccgactttgtttgcaattcttaatagaagat

[0350] gaattactcgataaatatgatttatcaaatttagttgaaattgcatctggcggagcacct

[0351] ttatctaaagaaattggtgaagctgttgctagacgttttaatttaccgggtgttcgtcaa

[0352] ggctatggtttaacagaaacaacctctgcaattattatcacaccggaaggcgatgataaa

[0353] ccaggtgcttctggcaaagttgtgccattatttaaagcaaaagttatcgatcttgatact

[0354] aaaaaaaactttgggcccgaacagacgtggagaagttctggtaaagggtcctatgcttatg

[0355] aaaggttatgtagataatccagaagcaacaagagaaatcatagatgaagaaaggttggttg

[0356] cacacaggagagatattgggtattacgatgaagaaaaacatttctttatcgtggatcgtttg

[0357] aagtctttaatcaaatacaaaggatatcaagtaccacctgctgaattagaatctgttctt

[0358] ttgcaacatccaaatatttttgatgccggcgttgctggcgttccagatcctatagctggt

[0359] gagcttccgggagctgttgttgtacttaagaaaggaaaatctatgactgaaaaagaagta

[0360] atggattacgttgctagtcaagtttcaaatgcaaaacgtttgcgtggtggtgtccgtttt

[0361] gtggacgaagtacctaaaggtctcactggtaaaattgacggtaaagcaattagagaaata

[0362] ctgaagaaaccagttgctaagatgtaa

[0363] SEQ ID NO:4 G326S introduction primer

[0364] aatttctttagataaaggtgctccgccaga

[0365] SEQ ID NO:5 G326S introduction primer

[0366] tctaaagaaattagcgaagctgttgctaga

[0367] SEQ ID NO:6 V392I introduction primer

[0368] ttctccacgtctgttcgggcccaaagtttt

[0369] SEQ ID NO:7 V392I introduction primer

[0370] agacgtggagaaattctggtaaagggtcct

[0371] SEQ ID NO:8 Primer for F467I introduction

[0372] aatatttggatgttgcaaaagaacagattc

[0373] Primer for F467I introduction, SEQ ID NO:9

[0374] catccaaatattattgatgccggcgttgct

[0375] Amino acid sequence of mutant firefly luciferase (LlLuc-4M) derived from Luciola lateralis, SEQ ID NO:10

[0376] MENMENDENIVYGPEPFYPIEEGSAGAQLRKYMDRYAKLGAIAFTNALTG

[0377] VDYTYAEYLEKSCCLGEALKNYGLVVDGRIALCSENCEEFFIPVLAGLFI

[0378] GVGVAPTNEIYTLRELVHSLGISKPTIVFSSKKGLDKVITVQKTVTAIKT

[0379] IVILDSKVDYRGYQSMDNFIKKNTPQGFKGSSFKTVEVNRKEQVALIMNS

[0380] SGSTGLPKGVQLTHENLVTRFSHARDPIYGNQVSPGTAILTVVPFHHGFG

[0381] MFTTLGYLTCGFRIVMLTKFDEETFLKTLQDYKCSSVILVPTLFAILNRS

[0382] ELLDKYDLSNLVEIASGGAPLSKEISEAVARRFNLPGVRQGYGLTETTSA

[0383] IIITPEGDDKPGASGKVVPLFKAKVIDLDTKKTLGPNRRGEILVKGPMLM[[ID=3(1]]

[0384] KGYVDNPEATREIIDEEGWLHTGDIGYYDEEKHFFIVDRLKSLIKYKGYQ

[0385] VPPAELESVLLQHPNIIDAGVAGVPDPIAGELPGAVVVLKKGKSMTEKEV

[0386] MDYVASQVSNAKRLRGGVRFVDEVPKGLTGKIDGKAIREILKKPVAKM

[0387] Base sequence of the luciferase (LlLuc-4M) gene derived from the SEQ ID NO:11 mutant Luciola lateralis

[0388] atggaaaacatggagaacgatgaaaatattgtgtatggtcctgaaccattttaccctatt

[0389] gaagagggatctgctggagcacaattgcgcaagtatatggatcgatatgcaaaacttgga

[0390] gcaattgcttttactaacgcacttaccggtgtcgattatacgtacgccgaatacttagaa

[0391] aaatcatgctgtctaggagaggctttaaagaattatggtttggttgttgatggaagaatt

[0392] gcgttatgcagtgaaaactgtgaagagttctttattcctgtattagccggtttatttata

[0393] ggtgtcggtgtggctccaactaatgagatttacactctacgtgaattggttcacagttta

[0394] ggcatctctaagccaacaattgtatttagttctaaaaaaggattagataaagttataact [[ID=2)]

[0395] gtacaaaaaacggtaactgctattaaaaccattgttatattggacagcaaagtggattat

[0396] agaggttatcaatccatggacaactttattaaaaaaaacactccacaaggtttcaaagga

[0397] tcaagttttaaaactgtagaagttaaccgcaaagaacaagttgctcttataatgaactct

[0398] tcgggttcaaccggtttgccaaaaggtgtgcaacttactcatgaaaatttggtcacgcgt

[0399] ttttctcacgtagagatccaatttatggaaaccaagtttcaccaggcacggctatttta

[0400] actgtagtaccattccatcatggttttggtatgtttactactttaggctatctaacttgt

[0401] ggttttcgtattgtcatgttaacgaaatttgacgaagagacttttttaaaaaacactgcaa

[0402] gattacaaatgttcaagcgttatcttgtaccgactttgtttgcaattcttaatagaagat

[0403] gaattactcgataaatatgatttatcaaatttagttgaaattgcatctggcggagcacct

[0404] ttatctaaagaaattagcgaagctgttgctagacgttttaatttaccgggtgttcgtcaa

[0405] ggctatggtttaacagaaacaacctctgcaattattatcacaccggaaggcgatgataaa

[0406] ccaggtgcttctggcaaagttgtgccattatttaaagcaaaagttatcgatcttgatact

[0407] aaaaaaaactttgggcccgaacagacgtggagaaattctggtaaagggtcctatgcttatg

[0408] aaaggttatgtagataatccagaagcaacaagagaaatcatagatgaagaaaggttggttg

[0409] cacacaggagatattgggtattacgatgaagaaaaacatttctttatcgtggatcgtttg

[0410] aagtctttaatcaaatacaaaggatatcaagtaccacctgctgaattagaatctgttctt

[0411] ttgcaacatccaaatattattgatgccggcgttgctggcgttccagatcctatagctggt

[0412] gagcttccgggagctgttgttgtacttaagaaaggaaaatctatgactgaaaaagaagta

[0413] atggattacgttgctagtcaagtttcaaatgcaaaacgtttgcgtggtggtgtccgtttt

[0414] gtggacgaagtacctaaaggtctcactggtaaaattgacggtaaagcaattagagaaata

[0415] ctgaagaaaccagttgctaagatgtaa

[0416] Primer for F90Y introduction, SEQ ID NO:12

[0417] ctcttcacagttttcactgcataacgcaat

[0418] Primer for F90Y introduction, SEQ ID NO:13

[0419] aactgtgaagagtactttattcctgtatta

[0420] Primer for F221L introduction, SEQ ID NO:14

[0421] acgcgtgaccaaattctcgtgagtaagttg

[0422] Primer for F221L introduction, SEQ ID NO:15

[0423] ttggtcacgcgtttatctcacgccagagat

[0424] SEQ ID NO: 16 Primer for F252L introduction

[0425] cataccaaaaccatggtgaaatgggactac

[0426] SEQ ID NO: 17 Primer for F252L introduction

[0427] ggttttggtatgttaactactttaggctat

[0428] SEQ ID NO: 18 F262Y introduction primer

[0429] accacaagttagatagcctaaagtagtaaa

[0430] SEQ ID NO: 19 F262Y introduction primer

[0431] ctaacttgtggttatcgtattgtcatgtta

[0432] SEQ ID NO: 20 Primer for F371L introduction

[0433] taatggcacaaccttaccagaagcacctgg

[0434] SEQ ID NO:21 Primer for F371L introduction

[0435] gttgtgccattattgaaagcaaaagttatc

[0436] SEQ ID NO: 22 Primer for F294L introduction

[0437] caaagtcggtacaagaataacgcttgaaca

[0438] SEQ ID NO: 23 Primer for F294L introduction

[0439] gtaccgactttgctggcaattcttaataga

[0440] SEQ ID NO: 24 Primer for F252 / F262Y introduction

[0441] accacaagttagatagcctaaagtagttaa

[0442] SEQ ID NO:25 Primer for deletion of P544-M548

[0443] tttcttcagtatttctctaattgctttacc

[0444] Primers for P544 - M548 deletion, SEQ ID NO:26

[0445] taaggatccggctgctaacaaagcccgaaa

[0446] SEQ ID NO:27 Mutant firefly luciferase (LlLuc - 7Md) from Luciola lateralis

[0447] MENMENDENIVYGPEPFYPIEEGSAGAQLRKYMDRYAKLGAIAFTNALTG

[0448] VDYTYAEYLEKSCCLGEALKNYGLVVDGRIALCSENCEEFFIPVLAGLFI

[0449] GVGVAPTNEIYTLRELVHSLGISKPTIVFSSKKGLDKVITVQKTVTAIKT

[0450] IVILDSKVDYRGYQSMDNFIKKNTPQGFKGSSFKTVEVNRKEQVALIMNS

[0451] SGSTGLPKGVQLTHENLVTRFSHARDPIYGNQVSPGTAILTVVPFHHGFG

[0452] MLTTLGYLTCGYRIVMLTKFDEETFLKTLQDYKCSSVILVPTLLAILNRS

[0453] ELLDKYDLSNLVEIASGGAPLSKEIGEAVARRFNLPGVRQGYGLTETTSA

[0454] IIITPEGDDKPGASGKVVPLFKAKVIDLDTKKTLGPNRRGEVLVKGPMLM

[0455] KGYVDNPEATREIIDEEGWLHTGDIGYYDEEKHFFIVDRLKSLIKYKGYQ

[0456] VPPAELESVLLQHPNIFDAGVAGVPDPIAGELPGAVVVLKKGKSMTEKEV

[0457] MDYVASQVSNAKRLRGGVRFVDEVPKGLTGKIDGKAIREILKK

[0458] Base sequence of the gene of the luciferase (LlLuc-7Md) derived from the SEQ ID NO:28 mutant Luciola lateralis

[0459] atggaaaacatggagaacgatgaaaatattgtgtatggtcctgaaccattttaccctatt

[0460] gaagagggatctgctggagcacaattgcgcaagtatatggatcgatatgcaaaacttgga

[0461] gcaattgcttttactaacgcacttaccggtgtcgattatacgtacgccgaatacttagaa

[0462] aaatcatgctgtctaggagaggctttaaagaattatggtttggttgttgatggaagaatt

[0463] gcgttatgcagtgaaaactgtgaagagttctttattcctgtattagccggtttatttata

[0464] ggtgtcggtgtggctccaactaatgagatttacactctacgtgaattggttcacagttta

[0465] ggcatctctaagccaacaattgtatttagttctaaaaaaggattagataaagttataact

[0466] gtacaaaaaacggtaactgctattaaaaccattgttatattggacagcaaagtggattat

[0467] agaggttatcaatccatggacaactttattaaaaaaaacactccacaaggtttcaaagga

[0468] tcaagtttaaaactgtagaagttaaccgcaaagaacaagttgctcttataatgaactct

[0469] tcgggttcaaccggtttgccaaaaggtgtgcaacttactcatgaaaatttggtcacgcgt

[0470] ttttctcacgtagagatccaatttatggaaaccaagtttcaccaggcacggctatttta

[0471] actgtagtcccatttcaccatggttttggtatgttaactactttaggctatctaacttgt

[0472] ggttatcgtattgtcatgttaacgaaatttgacgaagagacttttttaaaaaacactgcaa

[0473] gattacaaatgttcaagcgttatcttgtaccgactttgctggcaattcttaatagaagat

[0474] gaattactcgataaatatgatttatcaaatttagttgaaattgcatctggcggagcacct

[0475] ttatctaaagaaattagcgaagctgttgctagacgttttaatttaccgggtgttcgtcaa

[0476] ggctatggtttaacagaaacaacctctgcaattattatcacaccggaaggcgatgataaa

[0477] ccaggtgcttctggcaaagttgtgccattatttaaagcaaaagttatcgatcttgatact

[0478] aaaaaaaactttgggcccgaacagacgtggagaaattctggtaaagggtcctatgcttatg

[0479] aaaggttatgtagataatccagaagcaacaagagaaatcatagatgaagaaggttggttg

[0480] cacacaggagatattgggtattacgatgaagaaaaacatttctttatcgtggatcgtttg

[0481] aagtctttaatcaaatacaaaggatatcaagtaccacctgctgaattagaatctgttctt

[0482] ttgcaacatccaaatattattgatgccggcgttgctggcgttccagatcctatagctggt

[0483] gagcttccgggagctgttgttgtacttaagaaaggaaaatctatgactgaaaaagaagta

[0484] atggattacgttgctagtcaagtttcaaatgcaaaacgtttgcgtggtggtgtccgtttt

[0485] gtggacgaagtacctaaaggtctcactggtaaaattgacggtaaagcaattagagaaata

[0486] ctgaagaaataa

[0487] SEQ ID NO:29 Primer for F17D / E / K introduction

[0488] tggttcaggaccatacacaatattttcatc

[0489] SEQ ID NO:30 Primer for F17D introduction

[0490] ggtcctgaaccagattaccctattgaagag

[0491] SEQ ID NO:31 Primer for F17E introduction

[0492] ggtcctgaaccagaataccctattgaagag

[0493] SEQ ID NO:32 Primer for F17K introduction

[0494] ggtcctgaaccaaagtaccctattgaagag

[0495] SEQ ID NO:33 Y18L introduction primer

[0496] aaatggttcaggaccatacacaatattttc

[0497] SEQ ID NO:34 Y18L introduction primer

[0498] cctgaaccatttttacctattgaagaggga

[0499] SEQ ID NO:35 A41V introduction primer

[0500] tccaagttttgcatatcgatccatatactt

[0501] SEQ ID NO:36 A41V introduction primer

[0502] gcaaaacttggagtaattgcttttaactaac

[0503] SEQ ID NO:37 Y53L introduction primer

[0504] atcgacaccggtaagtgcgttagtaaaagc

[0505] SEQ ID NO:38 Y53L introduction primer

[0506] accggtgtcgatttaacgtacgccgaatac

[0507] SEQ ID NO:39 I110D / E / K / R introduction primers

[0508] ctcattagttggagccacaccgacacctat

[0509] SEQ ID NO:40 I110D introduction primer

[0510] ccaactaatgaggattacactctacgtgaa

[0511] SEQ ID NO:41 Primer for I110E introduction

[0512] ccaactaatgaggaatacactctacgtgaa

[0513] SEQ ID NO:42 Primer for I110K introduction

[0514] ccaactaatgagaagtacactctacgtgaa

[0515] SEQ ID NO:43 Primer for I110R introduction

[0516] ccaactaatgagcgttacactctacgtgaa

[0517] SEQ ID NO:44 V158D / E introduction primer

[0518] tttgctgtccaatataacaatggttttaat

[0519] SEQ ID NO:45 V158D introduction primer

[0520] ttggacagcaaagatgattatagaggttat

[0521] SEQ ID NO:46 V158E introduction primer

[0522] ttggacagcaaagaggattatagaggttat

[0523] SEQ ID NO:47 Primers for H223V / I / L introduction

[0524] agaaaaacgcgtgaccaaattttcatgagt

[0525] SEQ ID NO:48 H223V introduction primer

[0526] acgcgtttttctgtcgctagagatccaatt

[0527] SEQ ID NO:49 H223I introduction primer

[0528] acgcgtttttctatcgctagagatccaatt

[0529] SEQ ID NO: 50 H223L introduction primer

[0530] acgcgtttttctctcgctagagatccaatt

[0531] SEQ ID NO:51 A224I introduction primer

[0532] gtgagaaaaacgcgtgaccaaattttcatg

[0533] SEQ ID NO:52 A224I introduction primer

[0534] cgtttttctcacattagagatccaatttat

[0535] SEQ ID NO:53 Primer for Y229F / L introduction

[0536] aattggatctctagcgtgagaaaaacgcgt

[0537] SEQ ID NO:54 Y229F introduction primer

[0538] agagatccaatttttggaaaccaagtttca

[0539] SEQ ID NO:55 Y229L introduction primer

[0540] agagatccaattttaggaaaccaagtttca

[0541] SEQ ID NO:56 Y257F introduction primer

[0542] gcctaaagtagttaacataccaaaaccatg

[0543] SEQ ID NO: 57 Y257F introduction primer

[0544] actactttaggctttctaacttgtggttat

[0545] SEQ ID NO:58 Y306F introduction primer

[0546] tttatcgagtaattcacttctattaagaat

[0547] SEQ ID NO: 59 Y306F introduction primer

[0548] ttactcgataaattcgatttatcaaattta

[0549] SEQ ID NO:60 V312D / E / K introduction primers

[0550] taaatttgataaatcatattttatcgagtaa

[0551] SEQ ID NO:61 V312R introduction primer

[0552] ttatcaaatttacgtgaaattgcatctggc

[0553] SEQ ID NO:62 V312E introduction primer

[0554] ttatcaaatttagaagaaattgcatctggc

[0555] SEQ ID NO:63 V312K introduction primer

[0556] ttatcaaatttaaaggaaattgcatctggc

[0557] SEQ ID NO:64 Y427F introduction primer

[0558] cccaatatctcctgtgtgcaaccaaccttc

[0559] SEQ ID NO:65 Y427F introduction primer

[0560] ggagatattgggttttacgatgaagaaaaa

[0561] SEQ ID NO:66 V75K introduction primer

[0562] caaaccataattctttaaagcctctcctag

[0563] SEQ ID NO:67 V75K introduction primer

[0564] aattatggtttgaaagttgatggaagaatt

[0565] SEQ ID NO:68 Primer for introducing L229Y (Y229L reverse mutation)

[0566] agagatccaatttatggaaaccaagtttca

[0567] SEQ ID NO:69 Amino acid sequence of Lucidum luciferase

[0568]

[0569]

[0570]

[0571] SEQ ID NO: 70 Base sequence of Lucidum luciferase

[0572]

[0573]

[0574] SEQ ID NO:71 Amino acid sequence of Photinia firefly luciferase

[0575]

[0576]

[0577] SEQ ID NO: 72 Base sequence of Firefly luciferase

[0578]

[0579]

[0580] SEQ ID NO:73 Amino acid sequence of Photinus pennsylvanicus luciferase

[0581]

[0582]

[0583] SEQ ID NO:74 Base sequence of Photinus pennsylvanicus luciferase

[0584]

[0585]

Claims

1. A luciferase mutant, wherein: The luciferase before the amino acid substitution has at least 70%, 80% or 90% amino acid sequence identity with SEQ ID NO:

10. The luciferase after the amino acid substitution is substituted with leucine at the position corresponding to position 252 of SEQ ID NO:

1. Compared with the luciferase before the amino acid substitution, the thermal stability of the luciferase after the amino acid substitution is improved.

2. The luciferase mutant according to claim 1, wherein the position corresponding to position 262 of SEQ ID NO: 1 is substituted by tyrosine, and / or the position corresponding to position 294 of SEQ ID NO: 1 is substituted by leucine.

3. The luciferase mutant according to claim 1 or 2, wherein the amino acid residue at position 90, 371, 41, 427, 53, 306 or 312 of SEQ ID NO: 1 is substituted.

4. The luciferase mutant according to claim 3, wherein (a) the amino acid residue at position 90 of SEQ ID NO: 1 is substituted, where the amino acid residue at position 90 of SEQ ID NO: 1 is an amino acid residue selected from the group consisting of tyrosine, leucine, isoleucine, cysteine, serine, threonine, aspartic acid, glutamic acid, proline, valine, tryptophan, methionine, alanine, glycine, glutamine, asparagine, lysine, histidine, and arginine; (b) the amino acid residue at position 371 of SEQ ID NO: 1 is substituted with an amino acid residue selected from the group consisting of leucine, tyrosine, cysteine, threonine, aspartic acid, glutamic acid, isoleucine, proline, valine, tryptophan, methionine, alanine, glycine, glutamine, asparagine, lysine, histidine, and arginine; (c) the amino acid residue at position 41 of SEQ ID NO: 1 is substituted, where the amino acid residue at position 41 of SEQ ID NO: 1 is an amino acid residue selected from the group consisting of valine, leucine, isoleucine, methionine, tyrosine, cysteine, serine, threonine, aspartic acid, glutamic acid, tryptophan, phenylalanine, proline, glycine, glutamine, asparagine, lysine, histidine, and arginine; (d) the amino acid residue at position 427 of SEQ ID NO: 1 is substituted with an amino acid residue selected from the group consisting of phenylalanine, leucine, isoleucine, cysteine, serine, threonine, aspartic acid, glutamic acid, tryptophan, methionine, valine, alanine, proline, glycine, glutamine, asparagine, lysine, histidine, and arginine; (e) the amino acid residue at position 53 of SEQ ID NO: 1 is substituted with an amino acid residue selected from the group consisting of leucine, isoleucine, cysteine, serine, threonine, aspartic acid, glutamic acid, tryptophan, methionine, valine, phenylalanine, alanine, proline, glycine, glutamine, asparagine, lysine, histidine, and arginine; (f) the amino acid residue at position 306 of SEQ ID NO: 1 is substituted, where the amino acid residue at position 306 of SEQ ID NO: 1 is selected from the group consisting of phenylalanine, leucine, isoleucine, cysteine, serine, threonine, aspartic acid, glutamic acid, tryptophan, methionine, valine, alanine, proline, glycine, glutamine, asparagine, lysine, histidine, and arginine; or (g) the amino acid residue at position 312 of SEQ ID NO:1 is substituted, and the amino acid residue at position 312 of SEQ ID NO:1 is an amino acid residue selected from the group consisting of tyrosine, phenylalanine, leucine, isoleucine, cysteine, serine, threonine, aspartic acid, glutamic acid, tryptophan, methionine, alanine, proline, glycine, glutamine, asparagine, lysine, histidine and arginine.

5. The luciferase mutant according to claim 4, wherein (a) the amino acid residue at position 90 of SEQ ID NO: 1 is substituted, and the amino acid residue at position 90 of SEQ ID NO: 1 is tyrosine; (b) the amino acid residue at position 371 of SEQ ID NO: 1 is substituted with leucine; (c) the amino acid residue at position 41 of SEQ ID NO: 1 is substituted, and the amino acid residue at position 41 of SEQ ID NO: 1 is valine; (d) the amino acid residue at position 427 of SEQ ID NO: 1 is substituted with phenylalanine; (e) the amino acid residue at position 53 of SEQ ID NO: 1 is substituted with leucine; (f) the amino acid residue at position 306 of SEQ ID NO: 1 is substituted with phenylalanine; or (g) The amino acid residue at position 312 of SEQ ID NO: 1 is substituted, and the amino acid residue at position 312 of SEQ ID NO: 1 is glutamic acid, lysine or arginine.

6. The luciferase mutant according to any one of claims 1 to 5, further comprising an amino acid sequence in which the amino acid residue corresponding to position 221, 17, 18, 75, 110, 223, 224, 257, 229 or 158 of SEQ ID NO: 1 is substituted.

7. The luciferase mutant according to claim 6, wherein (i) the amino acid residue at position 221 of SEQ ID NO: 1 is substituted, where the amino acid residue at position 221 of SEQ ID NO: 1 is an amino acid residue selected from the group consisting of leucine, tyrosine, cysteine, serine, threonine, aspartic acid, glutamic acid, isoleucine, proline, valine, tryptophan, methionine, alanine, glycine, glutamine, asparagine, lysine, histidine, and arginine; or (ii) the amino acid residue at position 17 of SEQ ID NO: 1 is substituted, where the amino acid residue at position 17 of SEQ ID NO: 1 is selected from the group consisting of aspartic acid, glutamic acid, lysine, histidine, arginine, tyrosine, cysteine, serine, threonine, tryptophan, methionine, leucine, isoleucine, valine, alanine, proline, glycine, glutamine, and asparagine; (iii) the amino acid residue at position 18 of SEQ ID NO: 1 is substituted, where the amino acid residue at position 18 of SEQ ID NO: 1 is selected from the group consisting of leucine, isoleucine, cysteine, serine, threonine, aspartic acid, glutamic acid, tryptophan, methionine, valine, phenylalanine, alanine, proline, glycine, glutamine, asparagine, lysine, histidine, and arginine; (iv) the amino acid residue at position 75 of SEQ ID NO: 1 is substituted, where the amino acid residue at position 75 of SEQ ID NO: 1 is selected from the group consisting of lysine, histidine, arginine, aspartic acid, glutamic acid, tyrosine, cysteine, serine, threonine, tryptophan, methionine, leucine, isoleucine, phenylalanine, alanine, proline, glycine, glutamine, and asparagine; (v) the amino acid residue at position 110 of SEQ ID NO: 1 is substituted, where the amino acid residue at position 110 of SEQ ID NO: 1 is selected from the group consisting of aspartic acid, glutamic acid, lysine, arginine, histidine, tyrosine, cysteine, serine, threonine, tryptophan, methionine, leucine, valine, phenylalanine, alanine, proline, glycine, glutamine, and asparagine; (vi) the amino acid residue at position 223 of SEQ ID NO: 1 is substituted, wherein the amino acid residue at position 223 of SEQ ID NO: 1 is an amino acid residue selected from the group consisting of valine, isoleucine, leucine, tyrosine, cysteine, serine, threonine, aspartic acid, glutamic acid, tryptophan, methionine, phenylalanine, alanine, proline, glycine, glutamine, asparagine, lysine, and arginine; (vii) the amino acid residue at position 224 of SEQ ID NO: 1 is substituted, wherein the amino acid residue at position 224 of SEQ ID NO: 1 is an amino acid residue selected from the group consisting of isoleucine, leucine, valine, methionine, cysteine, tyrosine, serine, threonine, aspartic acid, glutamic acid, tryptophan, phenylalanine, proline, glycine, glutamine, asparagine, lysine, histidine, and arginine; (viii) the amino acid residue at position 257 of SEQ ID NO: 1 is substituted with an amino acid residue selected from the group consisting of phenylalanine, leucine, isoleucine, cysteine, serine, threonine, aspartic acid, glutamic acid, tryptophan, methionine, valine, alanine, proline, glycine, glutamine, asparagine, lysine, histidine, and arginine; (ix) the amino acid residue at position 229 of SEQ ID NO: 1 is substituted, where the amino acid residue at position 229 of SEQ ID NO: 1 is selected from the group consisting of phenylalanine, leucine, isoleucine, cysteine, serine, threonine, aspartic acid, glutamic acid, tryptophan, methionine, valine, alanine, proline, glycine, glutamine, asparagine, lysine, histidine, and arginine; or (x) the amino acid residue at position 158 of SEQ ID NO:1 is substituted, and the amino acid residue at position 158 of SEQ ID NO:1 is an amino acid residue selected from the group consisting of aspartic acid, glutamic acid, lysine, arginine, histidine, tyrosine, cysteine, serine, threonine, tryptophan, methionine, leucine, isoleucine, phenylalanine, alanine, proline, glycine, glutamine and asparagine.

8. The luciferase mutant according to claim 7, wherein (i) the amino acid residue at position 221 of SEQ ID NO: 1 is substituted with leucine; (ii) the amino acid residue at position 17 of SEQ ID NO: 1 is substituted, and the amino acid residue at position 17 of SEQ ID NO: 1 is aspartic acid, glutamic acid, or lysine; (iii) the amino acid residue at position 18 of SEQ ID NO: 1 is substituted, and the amino acid residue at position 18 of SEQ ID NO: 1 is leucine; (iv) the amino acid residue at position 75 of SEQ ID NO: 1 is substituted, and the amino acid residue at position 75 of SEQ ID NO: 1 is lysine; (v) the amino acid residue at position 110 of SEQ ID NO: 1 is substituted, and the amino acid residue at position 110 of SEQ ID NO: 1 is aspartic acid, glutamic acid, lysine, or arginine; (vi) the amino acid residue at position 223 of SEQ ID NO: 1 is substituted, and the amino acid residue at position 223 of SEQ ID NO: 1 is valine, isoleucine, or leucine; (vii) the amino acid residue at position 224 of SEQ ID NO: 1 is substituted with isoleucine; (viii) the amino acid residue at position 257 of SEQ ID NO: 1 is substituted with phenylalanine; (ix) the amino acid residue at position 229 of SEQ ID NO: 1 is substituted, and the amino acid residue at position 229 of SEQ ID NO: 1 is phenylalanine or leucine; or (x) The amino acid residue at position 158 of SEQ ID NO: 1 is substituted, and the amino acid residue at position 158 of SEQ ID NO: 1 is aspartic acid or glutamic acid.

9. The luciferase mutant according to any one of claims 1 to 8, wherein The luciferase before amino acid substitution comprises an amino acid sequence selected from the group consisting of the following (A) to (F): (A) an amino acid sequence having at least 70%, 80% or 90% amino acid sequence identity to SEQ ID NO: 10; (B) Regarding the amino acid sequence of (A), further, an amino acid sequence having a sequence identity of 90% or more between the homology region of SEQ ID NO: 1 and the homology region of luciferase before the amino acid substitution; (C) an amino acid sequence in which, in the amino acid sequence of SEQ ID NO: 10, one or more amino acids are substituted, deleted, or added at positions other than the position corresponding to position 252 of SEQ ID NO: 1; (D) Regarding the amino acid sequence of (C), further, in any one of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 10, SEQ ID NO: 69, SEQ ID NO: 71 or SEQ ID NO: 73, one or more amino acids are substituted, deleted or added at positions other than the positions corresponding to positions 90, 262, 371, 41, 427, 53, 306, 312, 294, 221, 17, 18, 75, 110, 223, 224, 257, 229 or 158 of SEQ ID NO: 1; (E) the amino acid sequence of SEQ ID NO: 10; and (F) Amino acid sequence of Heike luciferase.

10. The luciferase mutant according to claim 9, wherein The luciferase before amino acid substitution comprises an amino acid sequence selected from the group consisting of the following (a) to (d): (a) an amino acid sequence having an amino acid sequence identity of 90% or more to SEQ ID NO: 10; (b) an amino acid sequence in which one or more amino acids are substituted, deleted, or added at positions other than the position corresponding to position 252 of SEQ ID NO: 1 in the amino acid sequence of SEQ ID NO: 1; (c) With respect to the amino acid sequence of (b), further, in the amino acid sequence of SEQ ID NO: 1, one or more amino acids are substituted, deleted or added at positions other than the positions corresponding to positions 262, 371, 41, 427, 53, 306, 312, 252, 294, 221, 17, 18, 75, 110, 223, 224, 257, 229 or 158 of SEQ ID NO: 1; and (d) The amino acid sequence of SEQ ID NO:

10. A polynucleotide encoding the luciferase mutant according to any one of claims 1 to 10. A vector comprising the polynucleotide according to claim 11 .

13. A host cell comprising the polynucleotide of claim 11 or the vector of claim 12. 14 . A method for producing a luciferase mutant with improved thermal stability, comprising the step of culturing the host cell according to claim 13 . 15 . A kit for detecting at least one of ATP, ADP and AMP, comprising the luciferase mutant according to claim 1 .

16. A method for detecting at least one of ATP, ADP and AMP, the method comprising using the luciferase mutant according to any one of claims 1 to 10.

17. A luciferase mutant comprising an amino acid sequence in which one or more amino acid residues corresponding to positions 252, 262, 294, 90, 371, 41, 427, 53, 306, 312, 221, 17, 18, 75, 110, 223, 224, 257, 229, and 158 of SEQ ID NO: 1 are substituted, wherein the thermal stability of the luciferase after the amino acid substitution is improved compared to the luciferase before the amino acid substitution.

18. The luciferase mutant according to claim 17, wherein The position corresponding to position 252 of SEQ ID NO: 1 is substituted with leucine; The position corresponding to position 262 of SEQ ID NO: 1 is substituted with tyrosine; The position corresponding to position 294 of SEQ ID NO: 1 is substituted with leucine; The position corresponding to position 90 of SEQ ID NO: 1 is substituted with tyrosine; The position corresponding to position 371 of SEQ ID NO: 1 is substituted with leucine; The position corresponding to position 41 of SEQ ID NO: 1 is substituted with valine; The position corresponding to position 427 of SEQ ID NO: 1 is substituted with phenylalanine; The position corresponding to position 53 of SEQ ID NO: 1 is substituted with leucine; The position corresponding to position 306 of SEQ ID NO: 1 is substituted with phenylalanine; The position corresponding to position 312 of SEQ ID NO: 1 is substituted with glutamic acid, lysine or arginine; The position corresponding to position 221 of SEQ ID NO: 1 is substituted with leucine; The position corresponding to position 17 of SEQ ID NO: 1 is substituted with aspartic acid, glutamic acid or lysine; The position corresponding to position 18 of SEQ ID NO: 1 is substituted with leucine; The position corresponding to position 75 of SEQ ID NO: 1 is substituted with lysine; The position corresponding to position 110 of SEQ ID NO: 1 is substituted with aspartic acid, glutamic acid, lysine or arginine; the position corresponding to position 223 of SEQ ID NO: 1 is substituted with valine, isoleucine or leucine; The position corresponding to position 224 of SEQ ID NO: 1 is substituted with isoleucine; The position corresponding to position 257 of SEQ ID NO: 1 is substituted with phenylalanine; The position corresponding to position 229 of SEQ ID NO: 1 is substituted with phenylalanine or leucine; or, the position corresponding to position 158 of SEQ ID NO: 1 is substituted with aspartic acid or glutamic acid.

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