Proteins with laccase activity

Abstract

Provided is a novel CueO mutant having improved activity compared to a wild type. A protein according to one or more embodiments of the present invention comprises an amino acid sequence [1] or [2]: [1] an amino acid sequence comprising at least the 29th to 516th regions in the amino acid sequence obtained by introducing mutations (a) and (b) in the amino acid sequence represented by SEQ ID NO: 14: (a) D360 is replaced with an amino acid other than D; (b) one or more elements selected from the group consisting of substitution of G304 with an amino acid other than G, substitution of D373 with an amino acid other than D, and substitution of Q374 with an amino acid other than Q; [2] An amino acid sequence having 90% or more sequence identity with the amino acid sequence [1] and having amino acid residues corresponding to positions 360, 304, 373, and 374 in the amino acid sequence of SEQ ID NO: 14 identical to the amino acid sequence [1], and a protein having laccase activity.

Description

Technical Field

[0001] One or more embodiments of the present invention relate to a protein having laccase activity, a polynucleotide comprising a base sequence encoding the protein, a vector or a DNA fragment comprising the polynucleotide, and a transformed cell harboring the vector or the DNA fragment.

[0002] One or more embodiments of the present invention also relate to methods of oxidizing laccase substrate compounds using the proteins.

[0003] One or more embodiments of the present invention also relate to a biofuel cell comprising the protein. Background Art

[0004] CueO (Copper efflux oxidase) from Escherichia coli is a multi-copper oxidase with laccase activity.

[0005] The multi-copper oxidase with laccase activity is used in various chemical reactions and industrial fields. For example, it is expected to be used in the removal of lignin in the drainage treatment, pulp manufacturing treatment, etc., including phenolic compounds and aromatic amines, electrode catalysts, the manufacture of artificial lacquers, the synthesis of organic compounds, the browning treatment of tea, the bitterness reduction of food and beverages, the manufacture of cosmetic raw materials such as gelling agents and hair dyes for food, as clinical test reagents, bleaching agents, etc., and in multiple industrial fields such as the utilization. In addition, it is known that the electrode is used as an electron donor, and by combining with appropriate electrode substrates such as carbon substrates, it is expected to be used as an electrode catalyst for the cathode (oxygen reduction pole) of a biofuel cell, and an electrode catalyst for a biosensor (e.g., patent document 1).

[0006] CueO contains multiple copper ions coordinated to the active center.

[0007] Non-Patent Document 1 describes that the laccase activity of CueO (yacK) decreases over time when copper ions are not added, whereas the activity is sustained when copper ions are added.

[0008] In non-patent document 2, it is recorded that the fifth copper ion plays an important role in the electron transfer activity of CueO. In non-patent document 2, the following is recorded: the electron transfer activity of mutants with amino acid substitution introduced into one of the four amino acid residues M335, D360, D439 and M441 that coordinate the fifth copper ion in the amino acid sequence of wild-type CueO was studied, and 11 CueO mutants such as D360K had significantly improved electron transfer activity compared with the wild type.

[0009] The following is recorded in non-patent document 3: G304 (G276 excluding the signal peptide consisting of 28 amino acids at the N-terminus) of the amino acid sequence of wild-type CueO plays an important role in the laccase activity of CueO. The CueO mutants in which this residue is replaced with K, R, N and Y have significantly improved laccase activity compared to the wild-type CueO, especially the G304K mutant has 2.7 times the laccase activity of the wild-type.

[0010] Non-patent document 4 is a paper by the same group of researchers as non-patent document 3. Non-patent document 4 states that in the G304K mutant having high laccase activity, in the presence of copper ions, the substrate is easily accessible to type 1 copper ions due to conformational changes in the MR (Met-rich) helix and the regulatory loop (R-loop), and that the triple mutant in which the mutations D373A and Q374A are further introduced into the G304K mutant has higher laccase activity than the G304K mutant.

[0011] Prior art literature

[0012] Patent Literature

[0013] Patent Document 1: Japanese Patent Application Publication No. 2014-3970

[0014] Non-patent literature

[0015] Non-patent document 1: Kim, C. et al., Journal of bacteriology, 183, 4866-4875 (2001)

[0016] Non-patent document 2: Zhang, L. et al., Chem. Eur. J. 26, 4974-4979 (2020)

[0017] Non-patent document 3: Yue, Q. et al., Bioresour. Bioprocess, 4, 48 (2017)

[0018] Non-patent document 4: Wang, H. et al., Sci. Rep. 8, 14252 (2018) Summary of the invention

[0019] Technical problem to be solved by the invention

[0020] Non-Patent Document 1 suggests that copper ions coordinated to CueO are easily detached.

[0021] As described in Non-Patent Documents 2, 3, and 4, there has long been a demand for improving the activity of CueO.

[0022] Therefore, an object of the present invention is to provide a novel CueO mutant having improved activity compared to the wild type.

[0023] Technical solutions for solving technical problems

[0024] In this specification, the following embodiments are disclosed as one or more embodiments of the present invention.

[0025] <1> A protein comprising the amino acid sequence of [1] or [2]:

[0026] [1] An amino acid sequence comprising at least the region from positions 29 to 516 in the amino acid sequence of SEQ ID NO: 14 into which the mutations (a) and (b) are introduced (an amino acid sequence represented by any one of SEQ ID NOs: 17, 18, and 19):

[0027] (a) D360 is replaced by an amino acid other than D;

[0028] (b) one or more selected from the group consisting of G304 being replaced by an amino acid other than G, D373 being replaced by an amino acid other than D, and Q374 being replaced by an amino acid other than Q,

[0029] [2] an amino acid sequence having a sequence identity of 90% or more to the amino acid sequence of [1], and having the same amino acid residues at positions 360, 304, 373 and 374 as those of the amino acid sequence of [1],

[0030] The protein has laccase activity.

[0031] <2> according to <1> The protein, wherein

[0032] The mutation of (a) is that D360 is replaced by K or S,

[0033] The mutation of (b) includes replacement of G304 by K, N, R, V, L, S, Y, C, D, F, P, T, A or H.

[0034] <3> according to <2> The protein, wherein

[0035] The mutation of (a) is that D360 is replaced by K,

[0036] The mutation of (b) includes replacement of G304 by K, N, R, V, L, S, Y, C, D or F.

[0037] <4> according to <3> The protein, wherein

[0038] The mutation of (b) includes replacement of G304 by K, N or R.

[0039] <5> according to <2> The protein, wherein

[0040] The mutation of (a) is that D360 is replaced by S,

[0041] The mutation of (b) includes replacement of G304 by V, P, T, N, R, H, A, L, F, C or D.

[0042] <6> according to <5> The protein, wherein

[0043] The mutation of (b) includes replacement of G304 by V, P, T, N, R or H.

[0044] <7> according to <2> ~ <6> The protein described in any one of the preceding claims, wherein

[0045] The mutation of (b) further includes substitution of D373 by A and substitution of Q374 by A.

[0046] <8> according to <1> ~ <7> The protein described in any one of the preceding claims, wherein

[0047] The amino acid sequence of [2] contains the same region as the region corresponding to positions 299 to 398 of the amino acid sequence of SEQ ID NO: 14 in the amino acid sequence of [1].

[0048] <9> according to <1> ~ <8> The protein described in any one of the preceding claims, wherein

[0049] The protein comprises the amino acid sequence of [1].

[0050] <10> according to <9> The protein, wherein

[0051] The mutation of (a) is that D360 is replaced by K or S,

[0052] The mutation of (b) includes replacement of G304 by K, N, R, V, L, S, Y, C, D, F, P, T, A or H.

[0053] <11> according to <10> The protein, wherein

[0054] The mutation of (a) is that D360 is replaced by K,

[0055] The mutation of (b) includes replacement of G304 by K, N, R, V, L, S, Y, C, D or F.

[0056] <12> according to <11> The protein, wherein

[0057] The mutation of (b) includes replacement of G304 by K, N or R.

[0058] <13> according to <10> The protein, wherein

[0059] The mutation of (a) is that D360 is replaced by S,

[0060] The mutation of (b) includes replacement of G304 by V, P, T, N, R, H, A, L, F, C or D.

[0061] <14> according to <13> The protein, wherein

[0062] The mutation of (b) includes replacement of G304 by V, P, T, N, R or H.

[0063] <15> according to <10> ~ <14> The protein described in any one of the preceding claims, wherein

[0064] The mutation of (b) further includes substitution of D373 by A and substitution of Q374 by A.

[0065] <16> A polynucleotide comprising <1> ~ <15> The base sequence of the amino acid sequence of any one of the proteins described above.

[0066] <17> A vector or DNA fragment containing <16> The polynucleotide.

[0067] <18> A transformed cell that maintains <17> The vector or DNA fragment.

[0068] <19> A method for oxidizing a laccase substrate compound comprising <1> ~ <15> The laccase substrate compound is oxidized in a reaction solution of any one of the proteins and the laccase substrate compound.

[0069] <20> according to <19> The method, wherein

[0070] The method does not include adding copper ions to the reaction solution.

[0071] <21> A biofuel cell comprising <1> ~ <15> A cathode side electrode of a protein as described in any one of the above.

[0072] <22> according to <21> The biofuel cell, wherein

[0073] The biofuel cell further comprises:

[0074] an anode-side electrode; and

[0075] An electrolyte layer to which copper ions are not added is disposed between the cathode-side electrode and the anode-side electrode.

[0076] This specification includes the disclosure of Japanese Patent Application No. 2022-132106 which serves as the basis for the priority of the present application.

[0077] Furthermore, all publications, patents, and patent applications cited in this specification are incorporated herein by direct reference.

[0078] Effects of the Invention

[0079] The protein according to one or more embodiments of the present invention has higher laccase activity than wild-type CueO, and can maintain the laccase activity even without the addition of copper ions.

[0080] The polynucleotide, vector, DNA fragment, and transformed cell according to one or more embodiments of the present invention are useful for producing the above-mentioned protein.

[0081] According to the method for oxidizing a laccase substrate compound according to one or more embodiments of the present invention, the laccase substrate compound can be oxidized using the protein.

[0082] The biofuel cell according to one or more embodiments of the present invention can be used in various applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 This is a diagram illustrating primers used to prepare mut1 and mut2 in Example 1 and DNA fragments amplified by PCR using the primers.

[0084] Figure 2 This is a diagram illustrating the primers used to prepare mut3 in Example 1 and the DNA fragment amplified by PCR using the primers.

[0085] Figure 3 The photographs show the results of SDS-PAGE of the culture supernatant (S), filtrate (F), ammonium sulfate fractionated enzyme solution (A) and purified enzyme solution (D) of the enzymes WT, mut1, mut2 and mut3 respectively produced by the recombinant Escherichia coli BL21(pKL-CueO), BL21(pKL-CueO1), BL21(pKL-CueO2) and BL21(pKL-CueO3) prepared in Example 1.

[0086] Figure 4A The graph shows the residual activity of ABTS oxidation activity of WT, mut1, mut2, and mut3 stored at 60°C.

[0087] Figure 4B This is a graph showing the residual activity of ABTS oxidation activity of WT, mut1, mut2, and mut3 stored at 70°C.

[0088] Figure 4C This is a graph showing the residual activity of ABTS oxidation activity of WT, mut1, mut2, and mut3 stored at 80°C.

[0089] Figure 5A It is a graph showing the residual activity of ABTS oxidation activity of WT after storage at 4°C, 25°C, and 40°C for 7 days.

[0090] Figure 5B This is a graph showing the residual activity of ABTS oxidation activity of mut1 after storage at 4°C, 25°C, and 40°C for 7 days.

[0091] Figure 5C It is a graph showing the residual activity of ABTS oxidation activity of mut2 after storage at 4°C, 25°C, and 40°C for 7 days.

[0092] Figure 5D It is a graph showing the residual activity of ABTS oxidation activity of mut3 after storage at 4°C, 25°C, and 40°C for 7 days.

[0093] Figure 6 A preferred example of a biofuel cell including a cathode-side electrode containing a mutant protein according to one embodiment of the present invention is shown. DETAILED DESCRIPTION

[0094] <Protein>

[0095] The protein according to one or more embodiments of the present invention comprises the amino acid sequence of [1] or [2] above and has laccase (EC: 1.10.3.2) activity.

[0096] (Laccase activity)

[0097] Laccase activity refers to substrate oxidation activity, and refers to the activity of oxidizing a laccase substrate compound, which is selected from 2,2'-azino-bis-(3-ethylbenzothiazoline)-6-sulfonic acid (hereinafter referred to as "ABTS"), 2,6-dimethoxyphenol, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (hereinafter referred to as "TOOS"), dimethylaniline, diethylaniline, N,N-dimethyl-p-phenylenediamine, catechol, resorcinol, hydroquinone, phenol, guaiacol, pyrogallol, p-hydroxybenzoic acid, caffeic acid, hydrocaffeic acid, o-cresol, p-toluidine, o-chlorophenol, m-chlorophenol, p-chlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, 2,4,6-trichlorophenol, p-phenylenediamine, propyl gallate, etc., and especially refers to the activity of oxidizing ABTS.

[0098] The magnitude of the laccase activity of the protein involved in the present embodiment is not particularly limited, and preferably has a laccase activity higher than the laccase activity of the protein consisting of positions 29 to 516 in the amino acid sequence shown in SEQ ID NO: 14 (wild-type CueO). The laccase activity compared to the wild-type CueO is preferably the ABTS oxidation activity (specific activity) per 1 mg of protein measured by the measurement method described in Experiment 5 of Example 1 in which copper ions are not added to the reaction system during the measurement. The specific activity measured by the measurement method of the protein involved in the present embodiment is, for example, 5.0 times or more, preferably 7.0 times or more, more preferably 10.0 times or more, and most preferably 13 times or more of the wild-type CueO measured under the same conditions, and the upper limit is not particularly limited, for example, 50 times or less. That is, the specific activity measured by the measurement method of the protein involved in the present embodiment may be 5.0 times or more and 50 times or less of the specific activity of the wild-type CueO measured under the same conditions, and may preferably be within a more specific range determined by the above-mentioned lower limit value and / or upper limit value. The specific value of the specific activity measured by the determination method of the protein involved in the present embodiment is, for example, 5,000 U / mg or more, preferably 7,000 U / mg or more, more preferably 10,000 U / mg or more, and most preferably 13,000 U / mg or more, and the upper limit is not particularly limited, for example, 50,000 U / mg or less. That is, the specific activity of the protein involved in the present embodiment can be 5,000 U / mg or more and 50,000 U / mg or less, and can preferably be a more specific range determined by the above lower limit and / or upper limit. Here, the unit of activity (U) is the amount of enzyme that oxidizes 1 μmol of ABTS in 1 minute.

[0099] The wild-type CueO has the problem that copper ions are easily separated from it, and the laccase activity is reduced under the condition that copper ions are not present in the surroundings. In contrast, the protein involved in the present embodiment has the advantageous effect that copper ions are not easily separated from it, and the activity is maintained even in an environment where copper ions are not present in the surroundings. For example, the transformed cells described later are cultured in a culture medium containing a sufficient amount of copper ions (for example, as CuSO4), and the protein involved in the present embodiment purified and recovered from the culture has high activity even in an environment where copper ions are not present in the surroundings. In addition, unless otherwise specified, the protein involved in the present embodiment having laccase activity refers to a protein (holoenzyme) that retains copper ions. The protein involved in the present embodiment may also be expressed as a protein (apoenzyme) that does not contain copper ions, and the protein retained by culturing it in a buffer containing copper ions is obtained.

[0100] The protein according to the present embodiment is preferably a purified or roughly purified protein that retains copper ions.

[0101] (Amino acid sequence of [1])

[0102] The amino acid sequence of [1] is an amino acid sequence containing at least the region from positions 29 to 516 in the amino acid sequence of SEQ ID NO: 14 of wild-type CueO from Escherichia coli (an amino acid sequence represented by any one of SEQ ID NOs: 17, 18, and 19) into which the mutations (a) and (b) are introduced:

[0103] (a) D360 is replaced by an amino acid other than D;

[0104] (b) at least one selected from the group consisting of substitution of G304 with an amino acid other than G, substitution of D373 with an amino acid other than D, and substitution of Q374 with an amino acid other than Q.

[0105] Amino acid sequence of SEQ ID NO: 14:

[0106] MQRRDFLKYSVALGVASALPLWSRAVFAAERPTLPIPDLLTTDARNRIQLTIGAGQSTFGGKTATTWGYNGNLLGPAVKLQRGKAVTVDIYNQLTEETTLHWHGLEVPGEVDGGPQGIIPPGGKRSVTL NVDQPAATCWFHPHQHGKTGRQVAMGLAGLVVIEDDEILKLMLPKQWGIDDVPVIVQDKKFSADGQIDYQLDVMTAAVGWFGDTLLTNGAIYPQHAAPRGWLRLRLLNGCNARSLNFATSDNRPLYVIA SDGGLLPEPVKVSELPVLMGERFEVLVEVNDNKPFDLVTLPVSQMGMAIAAPFDKPHPVMRIQPIAISASGALPDTLSSLPALPSLEGLTVRKLQLSMDPMLDMMGMQMLMEKYGDQAMAGMDHSQMMGH MGHGNMNHMNHGGKFDFHHANKINGQAFDMNKPMFAAAKGQYERWVISGVGDMMLHPFHIHGTQFRILSENGKPPAAHRAGWKDTVKVEGNVSEVLVKFNHDAPKEHAYMAHCHLLEHEDTGMMLGFTV.

[0107] The 1st to 28th positions of the amino acid sequence of SEQ ID NO. 14 are signal sequences and therefore do not directly participate in the laccase activity of the protein. Therefore, the amino acid sequence of [1] may be the entire amino acid sequence (an amino acid sequence represented by any one of SEQ ID NOs. 17, 18 and 19) into which the mutations (a) and (b) are introduced into the amino acid sequence of SEQ ID NO. 14, or may be an amino acid sequence consisting of only the region from positions 29 to 516 in the amino acid sequence into which the mutations (a) and (b) are introduced into the amino acid sequence of SEQ ID NO. 14, or may be an amino acid sequence consisting of the region from positions N (N is an integer of 2 to 28) to 516 in the amino acid sequence into which the mutations (a) and (b) are introduced into the amino acid sequence of SEQ ID NO. 14.

[0108] In the present specification, "amino acid" means L-amino acid unless otherwise specified.

[0109] In the Examples, it was confirmed that the CueO mutant introduced with both the mutation (a) and the mutation (b) had significantly higher laccase activity than the wild-type CueO under the condition without adding copper ions.

[0110] In (a), "amino acid other than D" is preferably K or S, more preferably K. That is, the mutation in (a) is preferably D360K (D360 is replaced by K) or D360S (D360 is replaced by S), more preferably D360K. In the following, amino acid substitution mutations may be shown in the same manner.

[0111] The mutation of (b) can be selected from one or more of "G304 is replaced by an amino acid other than G", "D373 is replaced by an amino acid other than D" and "Q374 is replaced by an amino acid other than Q", and can be any one of one to three combinations. The mutation of (b) is preferably composed of only "G304 is replaced by an amino acid other than G", or is selected from a combination of one or more of "G304 is replaced by an amino acid other than G", "D373 is replaced by an amino acid other than D" and "Q374 is replaced by an amino acid other than Q", and more preferably is a combination of "G304 is replaced by an amino acid other than G", "D373 is replaced by an amino acid other than D" and "Q374 is replaced by an amino acid other than Q".

[0112] The mutation of (b) preferably includes at least "G304 is replaced by an amino acid other than G", and more preferably includes G304 being replaced by K, N, R, V, L, S, Y, C, D, F, P, T, A or H. In these cases, the mutation of (a) is particularly preferably D360K or D360S.

[0113] In the case where the mutation in (a) is D360K, the mutation in (b) preferably includes at least "G304 is replaced by an amino acid other than G", more preferably includes G304 being replaced by K, N, R, V, L, S, Y, C or D, and most preferably includes G304 being replaced by K, N or R.

[0114] When the mutation in (a) is D360S, the mutation in (b) preferably includes at least "G304 is replaced by an amino acid other than G", more preferably includes G304 being replaced by V, P, T, N, R, H, A, L, F, C or D, and most preferably includes G304 being replaced by V, P, T, N, R or H.

[0115] In the mutation of (b) above, "D373 is replaced by an amino acid other than D" is preferably D373A, and "Q374 is replaced by an amino acid other than Q" is preferably Q374A.

[0116] Particularly preferred combinations of the mutation (a) and the mutation (b) include one or more selected from the group consisting of combination numbers 1 to 112 shown in the following table. From the viewpoint of the strength of laccase activity, the combination of the mutation (a) and the mutation (b) is particularly preferably selected from one or more of combination numbers 1 to 10, 16 to 19, 22 to 28, 29 to 38, 44 to 47, 50 to 56, 57 to 66, 72 to 75, 78 to 84, 85 to 94, 100 to 103, and 106 to 112, more preferably selected from one or more of combination numbers 1 to 3, 16 to 18, 25, 26, 28, 29 to 31, 44 to 46, 53, 54, 56, 57 to 59, 72 to 74, 81, 82, 84, 85 to 87, 100 to 102, 109, 110, and 112, further preferably selected from one or more of combination numbers 1 to 3, 16 to 18, 25, 26, 28, and 85, and most preferably combination numbers 1 to 3 and 85. SEQ ID NO: 20 represents an amino acid sequence including a signal peptide, in which any one of the mutations in combination numbers 1 to 112 is introduced into the amino acid sequence represented by SEQ ID NO: 14.

[0117]

Table 1

[0118]

[0119]

[0120]

[0121] (Amino acid sequence of [2])

[0122] The "sequence identity" in the amino acid sequence of [2] is preferably 92% or more, more preferably 94% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, and more preferably 99% or more. However, the "sequence identity" in the amino acid sequence of [2] is less than 100%.

[0123] The "sequence identity" in the amino acid sequence of [2] refers to the ratio (%) of the number of identical amino acid residues relative to the total number of amino acid residues in the amino acid sequence of [1] when the amino acid sequence of [2] is aligned (aligned) with the amino acid sequence of [1] and gaps are introduced as necessary so that the amino acids of the two have the highest degree of identity. Sequence identity can be calculated using a protein search system based on BLAST or FASTA (Karlin, S. et al., 1993, Proc. Natl. Acad. Sci. USA, 90: 5873-5877; Altschul, SF et al., 1990, J. Mol. Biol., 215: 403-410; Pearson, WR et al., 1988, Proc. Natl. Acad. Sci. USA, 85: 2444-2448).

[0124] The “amino acid residues corresponding to positions 360, 304, 373 and 374 of the amino acid sequence of SEQ ID NO: 14” in the amino acid sequence of [2] refer to the amino acid residues in the amino acid sequence of [2] when the amino acid sequence of [2] is aligned with the amino acid sequence of [1], and the amino acid residues in the amino acid sequence of [2] correspond to the amino acid residues in the amino acid sequence of [1] corresponding to positions 360, 304, 373 and 374 of the amino acid sequence of SEQ ID NO: 14. That is, the amino acid residues in the amino acid sequence of [1] corresponding to positions 360, 304, 373 and 374 of the amino acid sequence of SEQ ID NO: 14 are also conserved in the amino acid sequence of [2].

[0125] The amino acid sequence of [2] comprises a region identical to the region corresponding to preferably positions 299 to 398, more preferably positions 250 to 450, more preferably positions 250 to 500, more preferably positions 250 to 510, more preferably positions 100 to 510, and more preferably positions 30 to 510 of the amino acid sequence of SEQ ID NO: 14 in the amino acid sequence of [1]. When these regions are identical, the amino acid sequence of [2] is particularly likely to have the same activity as the amino acid sequence of [1], and is therefore preferred.

[0126] <Polynucleotide comprising a base sequence encoding an amino acid sequence of a mutant protein>

[0127] One or more embodiments of the present invention also relate to polynucleotides comprising a base sequence encoding the amino acid sequence of the protein involved in the above-mentioned embodiment (hereinafter sometimes referred to as "mutant protein of the present disclosure"). The mutant protein of the present disclosure can be produced using various mutation introduction techniques known in the art. For example, it can be produced by mutating a polynucleotide encoding the amino acid residues of the modified object in the wild-type CueO gene encoding the reference amino acid sequence into a polynucleotide encoding the modified amino acid residues, and then expressing the protein from the mutant gene.

[0128] The polynucleotide involved in the present embodiment can include single-stranded or double-stranded DNA, cDNA, RNA or other artificial nucleic acids. The DNA, cDNA and RNA can also be chemically synthesized. In addition, the polynucleotide can also include the nucleotide sequence of the untranslated region (UTR) in addition to the open reading frame (ORF). In addition, the polynucleotide involved in the present embodiment can also be coordinated with the biological species of the transformed cell that keeps the vector or DNA fragment containing the polynucleotide to carry out codon optimization. The information of the codon used by various organisms can be obtained from the Codon Usage Database ([www.kazusa.or.jp / codon / ]).

[0129] As a means of mutating amino acid residues in the amino acid sequence of wild-type CueO, various mutation introduction techniques known in the art can be used. For example, in a polynucleotide comprising a base sequence encoding the amino acid sequence of wild-type CueO (hereinafter also referred to as a parent gene), by mutating the base sequence encoding the amino acid residue to be mutated to a base sequence encoding the mutated amino acid residue, a polynucleotide comprising a base sequence encoding the amino acid sequence of the mutant protein of the present invention can be obtained.

[0130] The target mutation can be introduced into the parental gene using various site-specific mutation introduction methods known to those skilled in the art. The site-specific mutation introduction method can be carried out by any method such as an inverse PCR method or an annealing method. It is also possible to use a commercially available site-specific mutation introduction kit (such as Stratagene's QuickChange II Site-Directed Mutagenesis Kit, QuickChange Multi Site-Directed Mutagenesis Kit, etc.).

[0131] The most common method for site-specific mutation introduction into a parental gene is to use a mutation primer containing a nucleotide mutation to be introduced. The mutation primer is designed to anneal to a region containing a base sequence encoding an amino acid residue to be mutated in the parental gene, and has a base sequence containing a base sequence (codon) encoding the amino acid residue after the mutation to replace the base sequence (codon) encoding the amino acid residue to be mutated. As long as it is a person skilled in the art, the base sequence (codon) encoding the amino acid residue before and after the mutation can be appropriately identified and selected based on common textbooks, etc. Alternatively, site-specific mutation introduction can also be used by the following method: DNA fragments on the upstream and downstream sides of the mutation site are respectively amplified using two complementary primers containing the nucleotide mutation to be introduced, and connected into one by SOE (splicing by overlap extension)-PCR (Gene, 1989, 77 (1): p61-68).

[0132] Template DNA containing the parent gene can be prepared by extracting genomic DNA from a microorganism that produces CueO using a conventional method. Alternatively, the corresponding base sequence can be chemically synthesized based on the amino acid sequence of wild-type CueO and used as template DNA.

[0133] Primers for mutation can be prepared by known oligonucleotide synthesis methods such as the phosphoramidite method (Nucleic Acids Research, 1989, 17: 7059-7071). Such primer synthesis can also be implemented using a commercially available oligonucleotide synthesizer (manufactured by ABI, etc.). Using a primer set including the primer for mutation, the polynucleotide involved in the present embodiment having the target mutation can be obtained by using the parental gene as the template DNA for site-specific mutation introduction as described above.

[0134] <Vector or DNA fragment>

[0135] One or more embodiments of the present invention also relate to a vector or a DNA fragment containing a polynucleotide comprising a base sequence encoding the amino acid sequence of the mutant protein disclosed herein.

[0136] The type of the vector involved in the present embodiment is not particularly limited, and may be any vector such as a plasmid, a phage, a phagemid, a cosmid, a virus, a YAC vector, a shuttle vector, etc. In addition, the vector involved in the present embodiment is not limited, and is preferably a vector that can be amplified in bacteria, preferably in Escherichia coli, and more preferably an expression vector that can induce the expression of the introduced gene in Escherichia coli. The vector involved in the present embodiment may also be a polynucleotide that has been integrated with a base sequence comprising an amino acid sequence encoding a mutant protein disclosed herein in a plasmid vector from Escherichia coli (e.g., pET22b (+), pBR 322, pBR325, pUC57, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.).

[0137] The vector or DNA fragment involved in the present embodiment can include the replication start region of DNA or the DNA region including the replication start point. Alternatively, in the vector or DNA fragment involved in the present embodiment, the promoter region, terminator region or the control sequence such as the secretion signal region for making the transcription of the gene start or the secretion of the expressed protein into the cell can also be operably connected with the polynucleotide (i.e. mutant gene) of the base sequence comprising the amino acid sequence of the mutant protein encoding the present disclosure. In addition, gene and control sequence "operably connected" refers to that gene and control region are configured to be able to express under the control of the control region with the gene.

[0138] The types of control sequences such as the promoter region, terminator region, and secretion signal region are not particularly limited, and commonly used promoter sequences, terminator sequences, and secretion signal sequences can be appropriately selected and used depending on the host to be introduced.

[0139] Alternatively, a marker gene (e.g., drug resistance genes such as ampicillin, neomycin, kanamycin, chloramphenicol, etc.) for selecting a host into which the vector is appropriately introduced may also be further integrated into the vector or DNA fragment involved in the present embodiment. Alternatively, in the case of using an auxotrophic strain in the host, a gene encoding a synthetase of the desired nutrition may also be integrated into the vector as a marker gene. Alternatively, in the case of using a selective culture medium that must be subjected to a specific metabolism for growth, an associated gene of the metabolism may also be integrated into the vector as a marker gene.

[0140] The polynucleotide containing the base sequence encoding the amino acid sequence of the mutant protein of the present disclosure can be linked to the control sequence and the marker gene by a method known in the art such as SOE-PCR. The step of introducing the linked fragment into a vector is known in the art.

[0141] <Transformed Cells>

[0142] One or more embodiments of the present invention also relate to a transformed cell that retains a vector or DNA fragment containing a polynucleotide containing a base sequence encoding an amino acid sequence of a mutant protein disclosed herein. The transformed cell involved in this embodiment can be obtained by introducing the vector into a host cell, or introducing the DNA fragment into the genome of a host cell. The transformed cell retains the vector or DNA fragment so that the mutant protein disclosed herein can be expressed.

[0143] As host cells, microorganisms such as bacteria, filamentous fungi, yeasts can be enumerated. As examples of bacteria, bacteria belonging to Escherichia coli, Staphylococcus, Enterococcus, Bacillus, etc. can be enumerated, wherein, preferably Escherichia coli. As examples of filamentous fungi, Trichoderma, Aspergillus, Rhizopus, etc. can be enumerated. As examples of yeast, Pichia, Saccharomyces, Schizosaccharomyces, etc. can be enumerated.

[0144] As the method for introducing the vector into the host cell, the methods commonly used in the field such as protoplast method and electroporation can be used. By selecting the strain appropriately introduced with the expression of the marker gene, auxotrophy, etc. as an index, the target transformant introduced with the vector can be obtained.

[0145] Alternatively, the transformed cells involved in the present embodiment can be obtained by directly introducing a DNA fragment containing a polynucleotide containing a base sequence encoding an amino acid sequence of a mutant protein disclosed herein into the genome of a host cell. For example, a DNA fragment having a base sequence complementary to the genome of a host cell attached to both ends of a fragment connecting the polynucleotide, a control sequence, and a marker gene sequence is constructed by the SOE-PCR method, etc., and introduced into a host cell, so that homologous recombination occurs between the genome of the host cell and the DNA fragment, thereby introducing the DNA fragment containing the polynucleotide into the genome of the host cell.

[0146] If the transformed cell involved in the present embodiment into which the vector or DNA fragment containing the polynucleotide containing the base sequence encoding the amino acid sequence of the mutant protein disclosed in the present invention is introduced in this way is cultured in an appropriate culture medium, the mutant gene on the vector or DNA fragment is expressed to produce the mutant protein disclosed in the present invention. The culture medium used in the culture of the transformed cell involved in the present embodiment can be appropriately selected by those skilled in the art in accordance with the type of the microorganism of the transformant.

[0147] Alternatively, the mutant protein of the present invention can also be expressed by a polynucleotide comprising a base sequence encoding the amino acid sequence of the mutant protein of the present invention or its transcription product using a cell-free translation system. "Cell-free translation system" refers to a suspension obtained by mechanically disrupting cells that serve as a host and adding reagents such as amino acids required for protein translation to form an in vitro transcription-translation system or an in vitro translation system.

[0148] The culture of the transformed cell involved in the present embodiment or the mutant protein of the present invention generated in the above-mentioned cell-free translation system can be separated or purified by using the general method used in protein purification, such as centrifugation, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., alone or in appropriate combination. At this time, on the vector or DNA fragment in the transformed cell, the gene encoding the amino acid sequence of the mutant protein of the present invention is operably connected to the secretion signal sequence, and the generated protein is secreted outside the cell, so it can be more easily recovered from the culture. The protein recovered from the culture can also be further purified by known means.

[0149] The mutant protein of the present disclosure as an expression product can be obtained by culturing the transformed cells involved in the present embodiment and collecting from the culture. "Culture" refers to any one of the culture supernatant, cultured cells or cultured cell fragments. The method of culturing transformed cells can be carried out according to the usual method used in the culture of host cells.

[0150] As a culture medium for culturing the transformed cells involved in the present embodiment, a culture medium containing copper ions is preferably used. Copper ions can be added to the culture medium as CuSO4 or its hydrate, for example. The mutant protein of the present disclosure obtained from the culture of the transformed cells involved in the present embodiment cultured in a culture medium containing copper ions maintains the copper ions required for activity in a stable state.

[0151] The culture medium for culturing the transformed cells involved in the present embodiment may also contain carbon sources, nitrogen sources, inorganic salts, etc. that can be assimilated by the host cells. As a culture medium, any one of natural culture medium and synthetic culture medium may also be used. As a carbon source, carbohydrates such as glucose, galactose, fructose, xylose, sucrose, raffinose, starch, organic acids such as acetic acid and propionic acid, alcohols such as ethanol and propanol may be cited. As a nitrogen source, inorganic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate or ammonium salts of organic acids or other nitrogen-containing compounds may be cited. In addition, peptone, meat extract, corn steep liquor, various amino acids, etc. may also be used. As an inorganic substance, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, etc. may be cited. In addition, defoamers such as vegetable oil, surfactant, silicon, etc. may also be added as needed.

[0152] The culture conditions can be appropriately selected according to the type of culture medium, the culture method, etc., and are not particularly limited as long as they are conditions under which the host cells proliferate and can produce the mutant protein disclosed herein. In addition, the culture method is also not particularly limited, and from the perspective of mass culture, liquid culture medium can be preferably used.

[0153] When the mutant protein of the present disclosure accumulates in the transformed cells, after the culture is completed, the transformed cells are recovered by centrifugation, the transformed cells are disrupted by ultrasonic treatment, etc., and then a cell-free extract is obtained by centrifugation, etc. This can be used as a starting material and purified by general protein purification methods such as salting-out method, ion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, affinity chromatography and various other chromatography methods. When the expressed mutant protein of the present disclosure is secreted outside the transformed cells, it can be purified from the culture supernatant in the same manner.

[0154] <Method for Oxidizing Laccase Substrate Compound>

[0155] One or more embodiments of the present invention relate to a method of oxidizing a laccase substrate compound.

[0156] The method of oxidizing a laccase substrate compound comprises oxidizing the laccase substrate compound in a reaction solution comprising a mutant protein of the present disclosure and a laccase substrate compound.

[0157] Here, examples of the laccase substrate compound include the above-mentioned compounds, and ABTS is particularly preferred.

[0158] The reaction solution may contain water as a solvent, and preferably may contain a buffer solution whose pH is adjusted to an appropriate range, for example, a range of 4.0 to 10.0.

[0159] The reaction of oxidizing the laccase substrate compound by the mutant protein of the present disclosure can be carried out at a temperature of 10 to 40°C, more preferably 25 to 35°C, for example.

[0160] The method involved in this embodiment preferably does not include adding copper ions to the reaction solution. The inventors surprisingly found that the mutant protein disclosed herein maintains laccase activity even without adding copper ions, and the laccase activity is improved when copper ions are not added. The mutant protein disclosed herein retains copper ions in advance, and it is believed that copper ions can be stably retained even in a reaction solution to which copper ions are not added.

[0161] <Biofuel Cell>

[0162] One or more embodiments of the present invention relate to a biofuel cell.

[0163] The biofuel cell includes a cathode-side electrode comprising the mutant protein of the present disclosure.

[0164] Figure 6 An example of a biofuel cell according to this embodiment is shown. Figure 6 The biofuel cell 1 shown includes a cathode electrode 11 including the mutant protein of the present disclosure, an anode electrode 12, and an electrolyte layer 13 disposed between the cathode electrode 11 and the anode electrode 12. The cathode electrode 11 and the anode electrode 12 are electrically connected via an external circuit 14, and a load 15 is disposed on the external circuit 14. In the anode electrode 12, the reduced form Red of the substrate (fuel) is oxidized to an oxidized product Ox, and the electrons generated by the reaction reach the cathode electrode 11 via the external circuit 14 including the load 15 to generate current, and the protons generated by the reaction reach the cathode electrode 11 via the electrolyte layer 13. In the cathode electrode 11, due to the activity of the mutant protein of the present disclosure, the electrons, protons and oxygen in the air react to generate water. In the biofuel cell 1 involved in this embodiment, the electrolyte layer 13 is preferably an electrolyte layer to which copper ions are not added. As described above, the mutant protein of the present disclosure maintains laccase activity even without the addition of copper ions, and the laccase activity is high when copper ions are not added. The biofuel cell according to the present embodiment can achieve higher performance by using an electrolyte layer to which copper ions are not added.

[0165] In the biofuel cell 1 involved in this embodiment, the cathode side electrode 11 is preferably an electrode for immobilizing the mutant protein of the present invention on a conductive substrate (e.g., a porous carbon electrode) connected to an external circuit 14. In addition, the mutant protein of the present invention can also be supplied to the conductive substrate in the form of being dissolved in an appropriate buffer. When immobilized, the mutant protein of the present invention is preferably immobilized in the state of a holoenzyme containing copper ions. However, the mutant protein of the present invention can also be immobilized in the form of an apoenzyme, and supplied in the form of copper ions as another layer or dissolved in an appropriate buffer.

[0166] The anode side electrode 12 may be an electrode in which an enzyme having an activity of oxidizing the reduced product Red of the substrate (fuel) into the oxidized product Ox is immobilized on a conductive substrate (e.g., a porous carbon electrode). As the reduced product Red of the substrate (fuel), for example, a reducing substance that can be used as an energy source by organisms such as sugar, alcohol, organic acid, amine, hydrogen, and other inorganic compounds can be used.

[0167] As the electrolyte layer 13, as long as it has ion conductivity that can permeate protons, etc., and has the property of not permeating the anode side constituent components, cathode side constituent components and electrons other than proton ions, then its raw materials and shape are not limited. For example, a solid electrolyte membrane can be used as a diaphragm. As a solid electrolyte membrane, examples include solid membranes with ion exchange functions such as organic polymers with strong acid groups such as sulfonic acid groups, phosphoric acid groups, phosphonic acid groups and phosphinic acid groups, weak acid groups such as carboxyl groups, and polar groups. In addition, the substance required for the expression of the catalytic activity of the enzyme can be supplied as another layer or dissolved in an appropriate buffer.

[0168] In the cathode electrode 11 of the biofuel cell 1 according to the present embodiment, the mutant protein disclosed herein catalyzes a reaction in which electrons are received from the anode electrode and oxygen is reduced to water. The mutant protein disclosed herein has excellent catalytic ability such as high laccase activity, so the efficiency of the redox reaction on the cathode electrode 11 of the biofuel cell 1 according to the present embodiment having the mutant protein is high. As a result, the obtained electric energy increases, and high-output and stable power generation can be performed.

[0169] <Other Uses>

[0170] The mutant protein disclosed in the present invention has excellent catalytic ability such as improved laccase activity (specific activity). Therefore, the mutant protein disclosed in the present invention can be applied to technologies requiring redox reactions of phenolic compounds in various industrial fields such as medicine, food, and environment, in addition to the electrochemical field.

[0171] Example

[0172] <Example 1>

[0173] (Experiment 1: Construction of plasmid pKL223)

[0174] Plasmid pTrcHis (Invitrogen, trade name) was used as a template and PCR was performed using primers Bsa-lacI-forward (SEQ ID NO: 1) and Bam-lacI-reverse (SEQ ID NO: 2) to amplify the lacI-containing q Gene and its promoter-controlling region. PCR was performed using KOD-plus- (trade name, manufactured by Toyobo Co., Ltd.) under the following reaction conditions. Step 1: 94°C, 2 minutes; Step 2: 94°C, 15 seconds; Step 3: 62°C, 30 seconds; Step 4: 68°C, 1 minute; Step 5: 4°C, ∞; Steps 2 to 4 were repeated for 30 cycles. The obtained amplified fragment of about 1400 bp was double-digested with BsaAI and BamHI, and inserted into the BsaAI-BamHI gap upstream of the tac promoter of plasmid pKK223-3 (trade name, manufactured by GE Healthcare Japan) to obtain plasmid pKL223. The sequences of the primers used are shown below.

[0175] Bsa-lacI-forward: 5'-TATCGCTACGTGACTGGGTCATGGCT-3' (SEQ ID NO: 1)

[0176] Bam-lacI-reverse: 5'-CGGGATCCTCGCGCTAACTCACATTAATTGC GTTGC-3' (SEQ ID NO: 2)

[0177] (Experiment 2: Construction of plasmid for CueO expression)

[0178] Based on the amino acid sequence of CueO from Escherichia coli (UniProtKB / Swiss-Prot Accession No. P36649, sequence number 14), the corresponding DNA sequence registered in the database (CueO gene, GenBank Accession No. AP009048.1: 137083-138633, sequence number 3) was searched. PCR primers EcoRI-CueO-F (sequence number 5) and HindIII-CueO-R (sequence number 6) were designed to attach a WSD sequence (AGGAGGTATTAT, sequence number 4) immediately upstream of the start codon. Using the two designed primers, the CueO gene was amplified by PCR using chromosomal DNA of Escherichia coli W3110 (NBRC12713) strain prepared using QIAGEN Genomic-tip and Genomic DNA Buffer Set (manufactured by Qiagen, trade name) as a template. PCR was performed using PrimeSTAR (registered trademark) GXL DNA Polymerase (trade name, manufactured by Takara Bio) under the following reaction conditions. Step 1: 98°C, 2 minutes; Step 2: 98°C, 10 seconds; Step 3: 68°C, 1 minute 30 seconds; Step 4: 12°C, ∞; Steps 2 to 3 were repeated for 30 cycles. The obtained DNA fragment was digested with EcoRI and HindIII and inserted into the EcoRI-HindIII gap located downstream of the tac promoter of plasmid pKL223 to construct plasmid pKL-CueO. Next, plasmid pKL-CueO was introduced into Escherichia coli BL21 strain to obtain transformed Escherichia coli (sometimes referred to as "recombinant Escherichia coli") BL21 (pKL-CueO). The base sequences of the primers used are shown below.

[0179] EcoRI-CueO-F (serial number 5):

[0180] 5'-AAAAGTTGAATTCAGGAGGTATTATATGCAACGTCGTGATTTC TTAAAATATTCCG-3'

[0181] HindIII-CueO-R (SEQ ID NO: 6):

[0182] 5'-CCCAAGCTTTTATACCGTAAACCCTAACATCATCCCGTATCTT C-3'

[0183] Cultivation of recombinant E. coli BL21 (pKL-CueO)

[0184] The recombinant Escherichia coli BL21 (pKL-CueO) was cultured in 5 ml of LBMiller medium (manufactured by Nacalai Tesque, trade name) supplemented with ampicillin at a final concentration of 50 μg / ml, while being heated to 37°C and shaken at 200 rpm overnight to prepare a seed culture solution. Next, 50 μL of the seed culture solution was added to 2×LB Lennox medium (manufactured by Nacalai Tesque, trade name) containing 50 ml of CuSO4·5H2O (5 mM) and lactose (2%) using a 500-ml Erlenmeyer flask with baffles (manufactured by PYREX), and culture was started at 37°C and 160 rpm. After 24 or 48 hours, the bacterial cells were collected from the culture solution by centrifugation (10,000×g, 15 minutes, 4°C).

[0185] (Experiment 3: Preparation of CueO mutants)

[0186] Construction of plasmid for expressing D360K mutant (mut1) (refer to Figure 1 upper paragraph)

[0187] Design PCR primers ClaI-mCueO-F (sequence number 7), mCueO-R1 (sequence number 8), mCueO-F1 (sequence number 9), and HindIII-CueO-R (sequence number 6). Use the designed primers and pKL-CueO as a template to amplify the two fragments by PCR. Next, use ClaI-mCueO-F (sequence number 7) and HindIII-CueO-R (sequence number 6) to perform PCR using the mixture of the two amplified fragments as a template to produce a DNA fragment of the mutant CueO-D360K mutation introduction sequence. PCR uses the same conditions as Experiment 2. The obtained DNA fragment is digested with ClaI and HindIII, and inserted into the ClaI-HindIII gap of the plasmid pKL-CueO, thereby constructing the plasmid pKL-mCueO1. The base sequences of the primers used are shown below.

[0188] ClaI-mCueO-F (SEQ ID NO: 7):

[0189] 5'-TGGGGTATCGATGATGTTCCGGTGATCG-3'

[0190] mCueO-R1 (SEQ ID NO: 8): The underline indicates the site of mutation introduction

[0191] 5'-GCATCCCCATCAT T T TGAGCATCGGGTCC-3'

[0192] mCueO-F1 (SEQ ID NO: 9): The underline indicates the site of mutation introduction

[0193] 5'-GGACCCGATGCTC A A A ATGATGGGGATGC-3'

[0194] HindIII-CueO-R (SEQ ID NO: 6):

[0195] 5'-CCCAAGCTTTTATACCGTAAACCCTAACATCATCCCGTATCTT C-3'

[0196] Construction of plasmid for expressing G304K / D373A / Q374A mutant (mut2) (refer to Figure 1 (lower paragraph)

[0197] Design PCR primers ClaI-mCueO-F (sequence number 7) and mCueO-R2 (sequence number 10), mCueO-F2 (sequence number 11) and mCueO-R3 (sequence number 12), mCueO-F3 (sequence number 13) and HindIII-CueO-R (sequence number 6). Use the designed primers and pKL-CueO as a template to amplify the three fragments by PCR. Next, use ClaI-mCueO-F (sequence number 7) and HindIII-CueO-R (sequence number 6) to perform PCR using the mixture of the three amplified fragments as a template to produce a DNA fragment of the mutant CueO-G304K / D373A / Q374A mutation introduction sequence. PCR uses the same conditions as Experiment 2. The obtained DNA fragment was digested with ClaI and HindIII and inserted into the ClaI-HindIII gap of plasmid pKL-CueO to construct plasmid pKL-mCueO2. The base sequences of the primers used are shown below.

[0198] ClaI-mCueO-F (SEQ ID NO: 7):

[0199] 5'-TGGGGTATCGATGATGTTCCGGTGATCG-3'

[0200] mCueO-R2 (SEQ ID NO: 10): The underline indicates the site of mutation introduction

[0201] 5'-GCAATCGCCATC TT CATCTGGCTGACC-3'

[0202] mCueO-F2 (serial number 11): The underline indicates the changed insertion position

[0203] 5'-GGTCAGCCAGATG AA GATGGCGATTGC-3'

[0204] mCueO-R3 (SEQ ID NO: 12): The underline indicates the site of mutation introduction

[0205] 5'-GGCCATCGCC GC A G CGCCATATTTC-3'

[0206] mCueO-F3 (SEQ ID NO: 13): The underline indicates the site of mutation introduction

[0207] 5'-GAAATATGGCG C T GC GGCGATGGCC-3'

[0208] HindIII-CueO-R (SEQ ID NO: 6):

[0209] 5'-CCCAAGCTTTTATACCGTAAACCCTAACATCATCCCGTATCTT C-3'

[0210] Construction of plasmids for expressing G360K and G304K / D373A / Q374A mutants (mut3) (refer to Figure 2 )

[0211] Using primers designed for the construction of mutant mut1, pKL-CueO2 was used as a template to amplify two fragments by PCR. Next, ClaI-mCueO-F (sequence number 7) and HindIII-CueO-R (sequence number 6) were used to perform PCR using the mixture of the two amplified fragments as a template to produce DNA fragments of mutant CueO-G360K and G304K / D373A / Q374A mutation introduction sequences. PCR used the same conditions as Experiment 2. The resulting DNA fragment was digested with ClaI and HindIII and inserted into the ClaI-HindIII gap of plasmid pKL-CueO, thereby constructing plasmid pKL-mCueO3. The base sequences of the primers used are shown below. Sequence number 21 represents the amino acid sequence of mut3 containing the signal peptide.

[0212] ClaI-mCueO-F (SEQ ID NO: 7):

[0213] 5'-TGGGGTATCGATGATGTTCCGGTGATCG-3'

[0214] mCueO-R1 (SEQ ID NO: 8): The underline indicates the site of mutation introduction

[0215] 5'-GCATCCCCATCAT T T T GAGCATCGGGTCC-3'

[0216] mCueO-F1 (SEQ ID NO: 9): The underline indicates the site of mutation introduction

[0217] 5'-GGACCCGATGCTC A A A ATGATGGGGATGC-3'

[0218] HindIII-CueO-R (SEQ ID NO: 6):

[0219] 5'-CCCAAGCTTTTATACCGTAAACCCTAACATCATCCCGTATCTT C-3'

[0220] (CueO gene (SEQ ID NO: 3)

[0221]

[0222] (Experiment 4: Preparation of CueO protein)

[0223] The plasmids pKL-mCueO1, pKL-mCueO2 and pKL-mCueO3 constructed in Experiment 3 were respectively introduced into Escherichia coli BL21 strain to obtain recombinant Escherichia coli BL21 (pKL-CueO1), BL21 (pKL-CueO2) and BL21 (pKL-CueO3).

[0224] Recombinant Escherichia coli BL21(pKL-CueO1), BL21(pKL-CueO2), BL21(pKL-CueO3) and BL21(pKL-CueO) prepared in Experiment 2 were cultured with stirring at 30°C for 48 hours in 2×LB Lennox medium (manufactured by Nacalai Tesque, trade name) containing 5 mM CuSO4·5H2O and 2% lactose.

[0225] The culture solution was heat-treated at 62°C for 2 hours and then ultrasonically disrupted to collect the supernatant (S).

[0226] Calcium chloride was added to the supernatant (S), and after the aggregates were removed by centrifugation (10,000×g, 30 minutes, 4° C.), the supernatant was filtered through a 0.2 μm filter to recover the filtrate (F).

[0227] Ammonium sulfate was added to the filtrate (F) to prepare a 55% ammonium sulfate solution, and salting out was performed. The precipitate was redissolved in deionized water to prepare an ammonium sulfate fractionated enzyme solution (A).

[0228] The enzyme solution (A) was fractionated with ammonium sulfate and dialyzed against 20 mM Tris-HCl (pH 7.6) buffer to perform buffer exchange, thereby obtaining a purified enzyme solution (D).

[0229] The supernatant (S), filtrate (F), ammonium sulfate fractionated enzyme solution (A) and purified enzyme solution (D) of the enzymes WT, mut1, mut2 and mut3 produced by recombinant Escherichia coli BL21(pKL-CueO), BL21(pKL-CueO1), BL21(pKL-CueO2) and BL21(pKL-CueO3), respectively, were represented by SDS (Sodium dodecyl sulfate)-polyacrylamide gel electrophoresis (SDS-PAGE). Figure 3 The results are shown. Bands were confirmed at positions corresponding to the molecular weight of CueO. It was also confirmed that the enzyme was purified in the purified enzyme solution (D).

[0230] (Experiment 5: Determination of ABTS oxidation activity)

[0231] The ABTS oxidation activity (activity for oxidizing ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid))) per 1 mg of protein in the purified enzyme solutions of WT, mut1, mut2, and mut3 prepared in Experiment 4) was measured by the following procedure.

[0232] Method for determining the amount of protein in the purified enzyme solution: The amount of protein per unit of the purified enzyme solution was quantified using the Bradford method. The purified enzyme solution and Quick Start Bradford 1×Dye Reagent (manufactured by BIO-RAD, trade name) were thoroughly mixed, and the difference in absorbance at a wavelength of 595 nm from the background containing no protein was measured by a spectrophotometer. The protein concentration was determined based on a standard straight line drawn by the absorbance of a standard sample (standard) with a significant protein concentration.

[0233] The purified enzyme solution was diluted with a dilution buffer (20 mM Tris-HCl (pH 7.0), 0.02% Tween 20) to prepare an enzyme dilution solution. A solution prepared by dissolving 10 mM ABTS in 50 mM acetate buffer (pH 5.0) at 30°C was used as a substrate solution, and the enzyme dilution solution was added to a multiwell plate, and the change in absorbance at 420 nm after 0 to 600 seconds was measured using a spectrophotometer (Multiskan GO, manufactured by Thermo Fisher Scientific).

[0234] The molar absorptivity of ABTS at 420 nm is ε 420 = 36,000, and the activity value was calculated. The unit of activity (U) is the amount of enzyme that oxidizes 1 μmol of ABTS in 1 minute. (Reference: Japanese Patent Application Publication No. 2010-183857)

[0235] The following table shows the measurement results of ABTS oxidation activity per 1 mg enzyme protein of WT, mut1, mut2 and mut3 and the ratio of ABTS oxidation activity of mut1, mut2 and mut3 to WT. The specific activity of ABTS oxidation activity of mut3 is 14 times that of WT and mut1 and 2.1 times that of mut2, which is significantly high.

[0236]

Table 2

[0237]

[0238] (Experiment 6: Effect of copper addition on ABTS oxidation activity)

[0239] The dilution buffer in Experiment 5 did not contain copper. In order to investigate the effect of copper addition on the ABTS oxidation activity of each enzyme, 20 mM Tris-HCl (pH 7.0), 0.02% Tween 20, and 4 mM CuSO 4 were used as the dilution buffer, and the specific activity of ABTS oxidation activity of WT, mut1, mut2, and mut3 was measured in the same procedure as Experiment 5, except that the purified enzyme solution was diluted and incubated at 30°C for 5 minutes before adding the substrate.

[0240] The results are shown below. Cu- represents the specific activity in Experiment 5 using a dilution buffer that does not contain copper, and Cu+ represents the specific activity in this experiment using a dilution buffer that contains copper. In addition, for each enzyme, the ratio of the specific activity of Cu+ to the specific activity of Cu- is shown.

[0241] WT showed that copper addition was required for activation. On the other hand, mut3 had high ABTS oxidation activity when no copper was added, but showed a tendency that the ABTS oxidation activity was suppressed when copper was added.

[0242]

Table 3

[0243]

[0244] (Experiment 7: Thermal stability test)

[0245] The specific activity of ABTS oxidation activity of WT, mut1, mut2 and mut3 was determined by following the same procedures as in Experiment 5, except that an enzyme dilution was used which was incubated at 60°C, 70°C or 80°C after preparation and cooled on ice for 5 minutes after sampling every 15 minutes.

[0246] The ratio of the specific activity measured using the enzyme dilution incubated at each temperature for a predetermined time to the specific activity measured using the enzyme dilution not incubated at each temperature was taken as the residual activity (%).

[0247] The results are shown in Figure 4A (Cultivation temperature 60°C), Figure 4B (Cultivation temperature 70°C) and Figure 4C (Cultivation temperature: 80°C) Regarding thermal stability, there was no substantial difference between mut3 and WT.

[0248] (Experiment 8: Storage Test)

[0249] 60% glycerol preparations (liquids) of WT, mut1, mut2, and mut3 were prepared by the following procedure: Glycerol was added to each purified enzyme solution at a weight ratio of 4:6, and the mixture was used as a 60% glycerol preparation (liquid).

[0250] 60% glycerol preparations of each enzyme were stored at 4°C, 25°C, and 40°C for 7 days. The specific activity of ABTS oxidation activity of the samples before and after 7 days of storage was measured in the same procedure as in Experiment 5. The ratio of the specific activity after 7 days of storage at each temperature to the specific activity before storage at each temperature was taken as the residual activity (%).

[0251] The results are shown in Figure 5A (WT), Figure 5B (mut1), Figure 5C (mut2) and Figure 5D (mut3) It was confirmed that the storage stability of mut1 and mut3 was slightly improved compared with WT.

[0252] <Example 2>

[0253] (Experiment 1: Preparation of plasmid for expressing D360K / G304X mutant)

[0254] Using the D360K mutant (mut1) expression plasmid pKL-mCueO1 constructed in Experiment 3 of Example 1 as a template, a plasmid into which the G304X mutation (X is K, N, R, V, L, S, Y, C, D or F) was introduced was constructed by the following steps.

[0255] In the case of introducing G304K, the primers ClaI-mCueO-F (sequence number 7) and mCueO-R2 (sequence number 10), mCueO-F2 (sequence number 11) and HindIII-CueO-R (sequence number 6) described in Experiment 3 of Example 1 were used to perform PCR with pKL-mCueO1 as a template to amplify two fragments. Next, ClaI-mCueO-F (sequence number 7) and HindIII-CueO-R (sequence number 6) were used to perform PCR with the mixture of the two amplified fragments as a template to produce a DNA fragment of the mutant CueO-D360K / G304K mutation introduction sequence. The PCR used the same conditions as those of Experiment 2 of Example 1. The obtained DNA fragment was digested with ClaI and HindIII, and inserted into the ClaI-HindIII gap of the plasmid pKL-CueO to construct a plasmid for expressing the D360K / G304K mutant.

[0256] When G304 is replaced by N, R, V, L, S, Y, C, D or F, primers ClaI-mCueO-F (sequence number 7) and mCueO-R2-N (sequence number 15), mCueO-F2-N (sequence number 16) and HindIII-CueO-R (sequence number 6) are used to perform PCR with pKL-mCueO1 as a template to amplify two fragments. Next, PCR is performed using ClaI-mCueO-F (sequence number 7) and HindIII-CueO-R (sequence number 6) with the mixture of the two amplified fragments as a template to produce a DNA fragment of the mutant CueO-D360K / G304X mutation introduction sequence. The PCR uses the same conditions as in Experiment 2 of Example 1. The obtained DNA fragment was digested with ClaI and HindIII and inserted into the ClaI-HindIII gap of plasmid pKL-CueO, thereby constructing a plasmid for expressing the D360K / G304X mutant.

[0257] ClaI-mCueO-F (SEQ ID NO: 7):

[0258] 5'-TGGGGTATCGATGATGTTCCGGTGATCG-3'

[0259] mCueO-R2-N (SEQ ID NO: 15) The underlined part is the mutation introduction site (N is a mixture of A, T, G and C):

[0260] 5'-GCAATCGCCATC NN CATCTGGCTGACC-3'

[0261] mCueO-F2-N (SEQ ID NO: 16) The underlined part is the mutation introduction site (N is a mixture of A, T, G and C):

[0262] 5'-GGTCAGCCAGATG NN GATGGCGATTGC-3'

[0263] HindIII-CueO-R (SEQ ID NO: 6):

[0264] 5'-CCCAAGCTTTTATACCGTAAACCCTAACATCATCCCGTATCTT C-3'

[0265] Each of the plasmids constructed above was sequenced, and the plasmids in which the introduction of the predetermined amino acid substitution was confirmed were used in the following Experiment 2.

[0266] (Experiment 2: Preparation of CueO protein)

[0267] The plasmids constructed and sequenced in Experiment 1 were respectively introduced into Escherichia coli BL21 strain to obtain recombinant Escherichia coli.

[0268] The recombinant Escherichia coli was cultured in a 2×LB Lennox medium (trade name, manufactured by Nacalai Tesque) containing 5 mM CuSO 4 ·5H 2 O and 2% lactose at 30° C. for 72 hours with stirring.

[0269] The culture solution was heat treated at 62°C for 2 hours and then ultrasonically disrupted. The ultrasonically disrupted product was centrifuged to recover the supernatant.

[0270] The protein amount and ABTS oxidation activity in the supernatant were measured in the same manner as in Experiment 5 of Example 1 to determine the specific activity.

[0271] For comparison, the recombinant E. coli BL21(pKL-CueO), BL21(pKL-CueO1), BL21(pKL-CueO2) and BL21(pKL-CueO3) obtained in Example 1 were cultured under the same conditions, and the culture solution was subjected to heat treatment, ultrasonic disruption and centrifugation to obtain a supernatant. The protein amount and ABTS oxidation activity of the supernatant were also measured in the same manner to determine the specific activity.

[0272] The following table shows the ratio of the specific activity of the supernatant obtained from the recombinant Escherichia coli expressing each mutant plasmid to the specific activity (WT) of the supernatant obtained from the recombinant Escherichia coli BL21 (pKL-CueO).

[0273] It was confirmed that the D360K / G304X double mutants all had ABTS oxidation activity exceeding that of the wild type WT and mut1 (D360K). In particular, mut4 (D360K / G304K), mut9 (D360K / G304N), and mut12 (D360K / G304R) had higher activity than mut3 (D360K / G304K / D373A / Q374A) confirmed to have high activity in Example 1. Sequence number 22 represents the amino acid sequence of mut4 to mut13 including the signal peptide.

[0274]

Table 4

[0275]

[0276] <Example 3>

[0277] (Experiment 1: Preparation of plasmid for expressing D360S / G304X mutant)

[0278] Construction of plasmid for expression of D360S mutant

[0279] Design PCR primers ClaI-mCueO-F (sequence number 7), mCueO-R1S (sequence number 24), mCueO-F1S (sequence number 25), and HindIII-CueO-R (sequence number 6). Use the designed primers and pKL-CueO as a template to amplify the two fragments by PCR. Next, use ClaI-mCueO-F (sequence number 7) and HindIII-CueO-R (sequence number 6) to perform PCR using the mixture of the two amplified fragments as a template to produce a DNA fragment of the mutant CueO-D360S mutation introduction sequence. PCR uses the same conditions as in Experiment 2 of Example 1. The obtained DNA fragment is digested with ClaI and HindIII, and inserted into the ClaI-HindIII gap of the plasmid pKL-CueO, thereby constructing the plasmid pKL-mCueO-D360S. The base sequences of the primers used are shown below.

[0280] ClaI-mCueO-F (SEQ ID NO: 7):

[0281] 5'-TGGGGTATCGATGATGTTCCGGTGATCG-3'

[0282] mCueO-R1S (SEQ ID NO: 24): The underline indicates the site of mutation introduction

[0283] 5'-GCATCCCCATCAT CGA GAGCATCGGGTCC-3'

[0284] mCueO-F1S (SEQ ID NO: 25): The underline indicates the site of mutation introduction

[0285] 5'-GGACCCGATGCTC TCG ATGATGGGGATGC-3'

[0286] HindIII-CueO-R (SEQ ID NO: 6):

[0287] 5'-CCCAAGCTTTTATACCGTAAACCCTAACATCATCCCGTATCTT C-3'

[0288] In the case of replacing G304, primers ClaI-mCueO-F (sequence number 7) and mCueO-R2-N (sequence number 15), mCueO-F2-N (sequence number 16) and HindIII-CueO-R (sequence number 6) were used to perform PCR with pKL-mCueO-D360S as a template to amplify two fragments. Next, ClaI-mCueO-F (sequence number 7) and HindIII-CueO-R (sequence number 6) were used to perform PCR with the mixture of the two amplified fragments as a template to produce a DNA fragment of the mutant CueO-D360S / G304X mutation introduction sequence. The PCR used the same conditions as those of Experiment 2 in Example 1. The obtained DNA fragment was digested with ClaI and HindIII and inserted into the ClaI-HindIII gap of the plasmid pKL-CueO, thereby constructing a plasmid for expressing the D360S / G304X mutant.

[0289] ClaI-mCueO-F (SEQ ID NO: 7):

[0290] 5'-TGGGGTATCGATGATGTTCCGGTGATCG-3'

[0291] mCueO-R2-N (SEQ ID NO: 15) The underlined part is the mutation introduction site (N is a mixture of A, T, G and C):

[0292] 5'-GCAATCGCCATC NN CATCTGGCTGACC-3'

[0293] mCueO-F2-N (SEQ ID NO: 16) The underlined part is the mutation introduction site (N is a mixture of A, T, G and C):

[0294] 5'-GGTCAGCCAGATG NN GATGGCGATTGC-3'

[0295] HindIII-CueO-R (SEQ ID NO: 6):

[0296] 5'-CCCAAGCTTTTATACCGTAAACCCTAACATCATCCCGTATCTT C-3'

[0297] Each of the plasmids constructed above was sequenced, and the plasmids in which the introduction of the predetermined amino acid substitution was confirmed were used in the following Experiment 2.

[0298] (Experiment 2: Preparation of CueO protein)

[0299] According to the conditions and steps described in Experiment 2 of Example 2, the plasmids constructed and sequenced in Experiment 1 were introduced into the Escherichia coli BL21 strain to obtain recombinant Escherichia coli and recombinant Escherichia coli BL21 expressing wild-type CueO (pKL-CueO), and the protein amount and ABTS oxidation activity in the culture supernatant were measured.

[0300] The following table shows the ratio of the specific activity of the supernatant obtained from the recombinant Escherichia coli based on the plasmid for expressing each mutant to the specific activity (WT) of the supernatant obtained from the recombinant Escherichia coli BL21 (pKL-CueO).

[0301] It was confirmed that all the D360S / G304X double mutants had ABTS oxidation activity exceeding that of the wild type WT. In particular, mut15 (D360S / G304V), mut16 (D360S / G304P), mut19 (D360S / G304T), mut20 (D360S / G304N), mut22 (D360S / G304R), and mut23 (D360S / G304H) had high activity. Sequence number 23 represents the amino acid sequence of mut14 to mut24 including the signal peptide.

[0302]

Table 5

[0303]

Claims

1. A protein comprising the amino acid sequence of [1] or [2]: [1] In the amino acid sequence of SEQ ID NO: 14, the amino acid sequence after the introduction of the mutations (a) and (b) comprises at least the amino acid sequence of the region from positions 29 to 516: (a) D360 is replaced by an amino acid other than D; (b) one or more selected from the group consisting of G304 being replaced by an amino acid other than G, D373 being replaced by an amino acid other than D, and Q374 being replaced by an amino acid other than Q, [2] an amino acid sequence having a sequence identity of 90% or more to the amino acid sequence of [1], and having the same amino acid residues at positions 360, 304, 373 and 374 as those of the amino acid sequence of [1], The protein has laccase activity.

2. The protein according to claim 1, wherein The mutation of (a) is that D360 is replaced by K or S, The mutation of (b) includes replacement of G304 by K, N, R, V, L, S, Y, C, D, F, P, T, A or H.

3. The protein according to claim 2, wherein The mutation of (a) is that D360 is replaced by K, The mutation of (b) includes replacement of G304 by K, N, R, V, L, S, Y, C, D or F.

4. The protein according to claim 3, wherein The mutation of (b) includes replacement of G304 by K, N or R.

5. The protein according to claim 2, wherein The mutation of (a) is that D360 is replaced by S, The mutation of (b) includes replacement of G304 by V, P, T, N, R, H, A, L, F, C or D.

6. The protein according to claim 5, wherein The mutation of (b) includes replacement of G304 by V, P, T, N, R or H.

7. The protein according to any one of claims 2 to 6, wherein The mutation of (b) further includes substitution of D373 by A and substitution of Q374 by A.

8. The protein according to any one of claims 1 to 7, wherein The amino acid sequence of [2] contains the same region as the region corresponding to positions 299 to 398 of the amino acid sequence of SEQ ID NO: 14 in the amino acid sequence of [1]. 9 . A polynucleotide comprising a base sequence encoding the amino acid sequence of the protein according to claim 1 .

10. A vector or a DNA fragment comprising the polynucleotide according to claim 9.

11. A transformed cell which retains the vector or DNA fragment according to claim 10.

12. A method for oxidizing a laccase substrate compound, comprising oxidizing the laccase substrate compound in a reaction solution comprising the protein according to any one of claims 1 to 8 and the laccase substrate compound.

13. The method of oxidizing a laccase substrate compound according to claim 12, wherein: The method does not include adding copper ions to the reaction solution. 14 . A biofuel cell comprising a cathode-side electrode comprising the protein according to claim 1 .

15. The biofuel cell according to claim 14, wherein The biofuel cell further comprises: an anode-side electrode; and An electrolyte layer to which copper ions are not added is disposed between the cathode-side electrode and the anode-side electrode.

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