JP5791025B2 - Thermostable protein having laccase activity, nucleic acid molecule encoding the protein, and method for producing the protein - Google Patents

Description

  The present invention relates to a thermostable protein having laccase activity, a nucleic acid molecule encoding the protein, a method for producing the protein, and use of the protein.

  Laccase is a general term for phenol oxidase, also called polyphenol oxidase and urushiol oxidase, which has substrate oxidation activity and catalyzes reactions that oxidize o-, p-diphenol, p-phenylenediamine, ascorbic acid, etc. It is a multi copper enzyme. The catalytic ability of laccase is applicable to various chemical reactions. For example, treatment of waste liquid containing highly toxic phenolic compounds and aromatic amines, removal of ligrin in pulp production treatment, etc., electrode catalyst, artificial lacquer production, synthesis of organic compounds, browning treatment of black tea, melanin production for cosmetics, It is expected to be used in many industrial fields such as food gelling agents, clinical test reagents, and bleaching agents. Laccase is known to use an electrode as an electron donor, and is expected to be industrially used as an electrode catalyst for a bio battery or a biosensor by combining with an appropriate carbon electrode. When used as an electrocatalyst, it is required to exhibit enzyme activity in a room temperature range of approximately 25 ° C, which is a generally assumed usage environment, and to maintain durability that can be adapted to various usage environments. The In particular, the use of a protein having heat resistance and high stability also has an advantage of enabling use under more efficient conditions.

  Laccase is known to exist widely in microorganisms, fungi, plants and the like, and has been isolated from a wide variety of organisms. Among them, laccases that can retain activity at relatively high temperatures have been reported. Examples include laccase derived from E. coli (Non-patent Document 1), laccase derived from Bacillus genus (Patent Document 1), laccase derived from actinomycetes (Patent Document 2), and alkaline laccase derived from filamentous fungi (Patent Document 3). Specifically, when the residual activity after the laccase derived from Escherichia coli of Non-Patent Document 1 was subjected to heat treatment at various constant temperatures of 20 to 70 ° C. for 10 minutes, the activity at 25 ° C. at 70 ° C. was measured. It is described that it was confirmed that the activity was about 67% by comparison. Moreover, the laccase derived from the genus Bacillus disclosed in Patent Document 1 has an optimum temperature of 60 to 80 ° C, and in other temperature ranges, assuming that the activity at 60 to 80 ° C is 100, 40% at 20 ° C. At 30 ° C, it is about 60%. Further, it has been reported that the residual activity after heat treatment at various constant temperatures for 30 minutes is almost 100% at 70 ° C and about 50% at 80 ° C. And when the relative activity in each temperature of 30-75 degreeC was computed for the actinomycete laccase disclosed by patent document 2, the optimal temperature is about 50 degreeC, On the other hand, when it becomes 30 degrees C or less and 70 degrees C or more, it is 50. The activity is reduced to less than half of that at ℃. And after holding for 10 minutes at various temperatures of 20-95 ° C, the enzyme activity was measured, and it was stable up to 70 ° C. However, when the temperature exceeded 70 ° C, the activity decreased extremely, and at 80 ° C, Only about 10% shows residual activity. In addition, the alkaline laccase derived from filamentous fungi disclosed in Patent Document 3 was calculated to have a relative activity at 30 to 70 ° C., and the optimum temperature was about 50 ° C., whereas 50 ° C. at 30 ° C. and 70 ° C. The activity is reduced to less than half of that at ℃. When the enzyme activity was measured after holding at various temperatures of 0 to 90 ° C for 30 minutes, the residual activity was about 95% at 60 ° C, about 73% at 65 ° C, and about 56% or more at 70 ° C. It has been confirmed that It has been reported that when the temperature exceeds 70 ° C., the activity is remarkably reduced, and at 80 ° C., only about 20% of the residual activity is exhibited. As a matter of fact, all of these enzymes show heat resistance against heating in a short time at around 60 ° C., but the presence or absence of heat resistance to heat treatment at a higher temperature and for a long time has not been reported. Therefore, it is assumed that the activity cannot be sufficiently maintained depending on the use environment, and it does not satisfy the market demand from the viewpoint of durability.

  Furthermore, a laccase cloned from an environmental metagenomic library has been reported as a laccase having heat resistance (Patent Document 4). Patent Document 4 describes that the laccase has a heat resistance of 90% or more by treatment at 80 ° C. for 10 minutes. However, the presence or absence of heat resistance of the laccase for a long time, for example, 1 hour or more, has not been known. For this reason, the enzyme does not sufficiently meet the market demand from the viewpoint of durability.

As laccase having higher heat resistance, laccase derived from Thermus thermophilus has been reported (Patent Document 5). Thermus thermophilus, which is derived from the laccase, is a thermophilic bacterium and has an optimum temperature around 92 ° C. when the temperature dependency of the activity is measured. The enzyme solution was kept at 80 ° C. and the enzyme activity was measured at regular intervals. The half-life at 80 ° C. was 868 minutes (14 hours), indicating a much higher long-term heat resistance. However, 2,2'-azino-bis- (3-ethylbenzothiazoline-6-sulfonic acid (2,2'-azino-bis- (3-ethylbenzothiazoline-6-sulfonate) (ABTS) )))), The oxidation activity at 70 ° C is 47 μmol / min / mg protein, but it has been reported to decrease to 1 or less at 25 ° C. Although the enzyme itself has high heat resistance, It could not be activated unless the temperature was higher than 70 ℃, that is, a structure capable of exhibiting the highest activity at high temperature because it is an enzyme derived from a thermophile that grows in a high temperature environment of 70 ℃ or higher. The problem is that it can only be activated at high temperatures, so it is active at room temperature and is stable and substrate-specific under high temperatures. It was still desired to provide a laccase that could be used.

Japanese Patent Laid-Open No. 9-206071 JP 2002-171968 A JP 2004-208503 A JP 2009-201481 JP 2006-158252 A

Sue A. Roberts et al., “A Labile Regulatory Copper Iron Lies Near the T1 Copper Site in the Multicopper Oxidase CueO”, JOURNAL OF BIOLOGICAL CHEMISTRY, August 2003, 278, 34, 31958-63

  In order to solve the above problems, an object of the present invention is to provide a protein having laccase activity excellent in heat resistance in addition to activity in a normal temperature range. Another object of the present invention is to provide a nucleic acid molecule encoding the protein and to provide a technique for mass production of the protein by a genetic engineering technique using the nucleic acid molecule. Another object of the present invention is to provide a utilization technique utilizing the characteristics of the protein.

As a result of diligent studies to solve the above problems, the present inventors have selected a compost sample containing wood as a component from among environmental samples, so that laccase activity excellent in heat resistance in addition to activity in a normal temperature range It has been found that a protein having can be obtained. In addition, the present inventors have succeeded in obtaining a nucleic acid molecule encoding the protein, and found that the protein can be produced in large quantities by genetic engineering. Furthermore, a utilization technique utilizing the characteristics of the protein has been found, and the present invention has been completed based on these findings.

  That is, in order to achieve the above object, the following inventions [1] to [8] are provided.

[1] A protein having the following laccase activity (a) or (b), which shows activity in a temperature range of 25 to 85 ° C., and retains 70% or more even after heat treatment at 60 ° C. for 17 hours. A protein having laccase activity.
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 2 (b) at least one modification selected from deletion, substitution, insertion and addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 A protein comprising an amino acid sequence [2] A protein having the following laccase activity (c) or (d), showing activity in a temperature range of 25 to 85 ° C., and 70% by heat treatment at 60 ° C. for 17 hours A protein having laccase activity that retains the above activities.
(C) A protein comprising the amino acid sequence shown in SEQ ID NO: 3 (d) In the amino acid sequence shown in SEQ ID NO: 3, at least one modification selected from deletion, substitution, insertion and addition of one or more amino acids occurred Protein containing amino acid sequence [3] The protein of the present invention is recombinant

  According to the configurations [1] to [3], it is possible to provide a protein having a novel laccase activity. The protein having a novel laccase activity provided by the present invention exhibits laccase activity in a normal temperature range, has high heat resistance, and does not inactivate the activity even by a long-term heat treatment. In addition, it is substrate-specific and has a substrate catalytic ability equivalent to that of other low heat-resistant laccases. Therefore, a protein having a novel laccase activity can be applied to a technique requiring a substrate oxidation reaction in various industrial fields such as medical, food, and environmental fields. In particular, the fuel is required to have an activity under normal use conditions of about 25 ° C and durability that can stably function in various use environments such as long-term use and various high-temperature conditions. It is expected to be used as an electrode catalyst for batteries and biosensors. Furthermore, when the protein having the novel laccase activity of the present invention has a small molecular weight and is used as an electrode catalyst for a fuel cell or a biosensor, the amount of enzyme molecules supported per area of the conductive member can be increased. The density can be improved. Therefore, high performance of fuel cells and biosensors can be expected by utilizing the protein having the novel laccase activity of the present invention.

[4] A nucleic acid molecule encoding a protein having laccase activity of the present invention.
[5] The isolated nucleic acid molecule of the present invention comprises the following polynucleotide (i) or (ii).
(I) a polynucleotide comprising the base sequence represented by SEQ ID NO: 1 (ii) a polynucleotide that hybridizes with the polynucleotide comprising the base sequence represented by SEQ ID NO: 1 under stringent conditions [6] containing the nucleic acid molecule of the present invention Recombinant vector.
[7] A transformant containing the recombinant vector of the present invention.
[8] A production method for culturing the transformant of the present invention and collecting the protein having the laccase activity of the present invention from the obtained culture.

  According to the configuration of [4] to [8] above, it is possible to provide a nucleic acid molecule encoding a protein having laccase activity of the present invention, a recombinant vector, a transformant, and a method for producing laccase. Moreover, since the base sequence encoding the protein having the laccase activity of the present invention has been found, the enzyme can be mass-produced industrially at low cost by genetic engineering techniques. Thereby, the industrial utility value of the protein having the laccase activity of the present invention can be further improved.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an electrophoretogram showing the results of Example 1 in which a part of a nucleic acid molecule encoding a protein having laccase activity was cloned by PCR from metagenomic DNA derived from an environmental sample. It is an electrophoretic diagram which shows the result of Example 2 which acquired the full length sequence of the nucleic acid molecule which codes the protein which has the laccase activity of this invention using inverse PCR method. It is an amino acid sequence alignment which shows the result of Example 3 which performed the homology search about the amino acid sequence of the protein which has the laccase activity of this invention. FIG. 7 is an amino acid sequence alignment showing the results of Example 3 in which the amino acid sequence of a protein having laccase activity according to the present invention is compared with the amino acid sequence of a protein having known laccase activity. Panel A shows the laccase derived from Thermus thermophilus. Amino acid sequence, panel B, shows a comparison with the amino acid sequence of laccase from the environmental metagenome. It is an electrophoretic diagram which shows the result of Example 5 which confirmed the manufacture after transforming the nucleic acid molecule which codes the protein which has the laccase activity of this invention into colon_bacillus | E._coli, and expressing it in a colon_bacillus | E._coli cell. It is a graph which shows the result of Example 7 which confirmed the pH dependence of the laccase activity of the protein which has the laccase activity of this invention. It is a graph which shows the result of Example 8 which confirmed the heat resistance of the laccase activity of the protein which has the laccase activity of this invention. It is a graph which shows the result of Example 9 which confirmed the long-term heat resistance of the laccase activity of the protein which has laccase activity of this invention. It is a graph which shows the result of Example 10 which confirmed the temperature dependence of the laccase activity of the protein which has the laccase activity of this invention. The amino acid sequence showing the result of Example 11 in which the present inventors compared the amino acid sequence of the protein having laccase activity with the amino acid sequence of the protein having known laccase activity previously obtained from the environmental metagenome. Alignment, panel A shows the amino acid sequence of mgL AC-1 and panel B shows a comparison with the amino acid sequence of mgLAC-2. The temperature dependence of the laccase activity of the protein having laccase activity of the present invention was compared with a protein having a known laccase activity previously obtained from the environmental metagenome and a known laccase derived from Bacillus subtilis . It is a graph which shows the result of Example 12, Panel A shows mgLAC-1, Panel B shows mgLAC-2, Panel C shows a comparison with laccase derived from Bacillus subtili . The graph which shows the result of Example 15 which examined the rapid holonization method of this invention

  Hereinafter, the present invention will be described in detail.

1. The protein which has the laccase activity of this invention TECHNICAL FIELD This invention relates to the protein which has the laccase activity which has the following biological characteristics. The explanation is divided into physicochemical properties, primary structure of amino acid sequence, and origin. In the present specification, the protein having the laccase activity of the present invention may be referred to as “MELAC”, and the nucleic acid molecule encoding the protein may be referred to as “melac”.

1-1. Physicochemical properties 1-1-1. Laccase activity
MELAC is a protein having laccase activity. Here, laccase is an oxidase having a catalytic ability for a substrate oxidation reaction, and is a multi-copper oxidase in which four copper ions are bonded to the center of the catalyst. Substrate oxidation activity includes oxidation of o-, p-diphenol, p-phenylenediamine, ascorbic acid, etc., and its catalytic function is to extract electrons from the substrate (electron accepting site) and use the electrons to produce oxygen. It is demonstrated by reducing water to water. It is known that the reaction site with the electron donor (type I copper site) is different from the reaction site with the electron acceptor. Examples of the substrate include ABTS, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline (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, 2,6-dimethoxyphenol, p-phenylenediamine, propyl gallate and the like can be exemplified, and ABTS is particularly preferably used.

1-1-2. Heat-resistant
MELAC exhibits laccase activity at room temperature and has high heat resistance. Specifically, in addition to the activity in a normal temperature range of about 15 ° C. to about 35 ° C., the activity is also exhibited in a high temperature range exceeding 50 ° C. Therefore, stable activity is exhibited in a temperature range of about 25 to 85 ° C. The optimum temperature is particularly preferably 80 ° C. or higher. In other words, this means that the limit temperature of the durability of the enzyme is 80 ° C. or higher. Furthermore, MELAC is resistant to long-term heat treatment. For example, it is preferable to maintain an activity of 60% or more with respect to the activity in a normal temperature range even for a heat treatment at 80 ° C. or higher for about 1 hour. In particular, it is preferable to maintain an activity of 70% or more with respect to the activity in the normal temperature range even by a long-time heat treatment at 60 ° C. for up to 17 hours. Furthermore, it is preferable not to deactivate even by heat treatment at 80 ° C. for 17 hours or more. In particular, it is preferable that the activity can be maintained at 70% or more even by heating for 1 hour at 60 to 70 ° C. Here, the deactivation means that the protein is denatured and does not show activity, and it is preferable that 10 to 20% of the activity in the normal temperature range can be maintained even by heating treatment. Therefore, since MELAC can exhibit activity in a normal temperature range and has high heat resistance, it can be applied to a technique that requires a substrate oxidation reaction in various industrial fields such as medical, food, and environmental fields. In particular, fuel that is required to have durability that can stably perform its functions under general use conditions of about 25 ° C and in various use environments such as long-term use and various high-temperature conditions. It is expected to be used as an electrode catalyst for batteries and biosensors. Furthermore, even when MELAC is synthesized in large quantities by genetic engineering as a recombinant, the host-derived contaminating protein can be easily removed as an insoluble fraction by heat treatment. Therefore, there is an advantage that the degree of purification can be improved during purification, and a highly reliable enzyme can be produced.

1-1-3. Molecular weight Laccase may have different molecular weights due to the difference in its amino acid composition, but preferably it has a relatively small molecular weight, especially MELAC is about 47.5 kDa, which is a relatively small molecular weight for laccase. Have. Electrodes such as fuel cells and biosensors for which laccase is particularly expected to be applied are required to be configured by immobilizing a catalytic enzyme on a conductive member in high density and in large quantities from the viewpoint of improving output. Therefore, the low molecular weight substance can increase the amount of molecules supported per area of the conductive member, and the current density can be improved. Therefore, the use of MELAC can be expected to improve the performance of fuel cells and biosensors.

1-2. Amino acid sequence
MELAC is preferably exemplified by the one containing the amino acid sequence of SEQ ID NO: 2. Furthermore, as long as the physicochemical properties described above are retained, the amino acid sequence of SEQ ID NO: 2 may include an amino acid sequence having a modified site where a specific amino acid is modified. “Modification” means that a modification in which at least one of one or more amino acids is deleted, substituted, inserted, or added in the amino acid sequence of the protein that is the basis of the modification has occurred. "A modification comprising at least one of deletion, substitution, insertion and addition of one or a plurality of amino acids" means a known DNA recombination technique or a point mutation introduction method for a gene encoding a protein serving as a basis for the modification. Etc. means that as many amino acids as can be deleted, substituted, inserted or added are deleted, substituted, inserted or added, including combinations thereof. For example, such a variant may maintain homology of 70% or more, preferably 80% or more, more preferably 90% or more, at the amino acid level with respect to the amino acid sequence represented by SEQ ID NO: 2. it can. Specifically, for example, in the amino acid sequence shown in SEQ ID NO: 2 as an example of the amino acid sequence of MELAC, the 36th valine is replaced with isoleucine (V36I), or the 120th asparagine is replaced with aspartic acid ( N120D) is included. Those having both substitutions are preferably exemplified, and the amino acid sequence thereof is shown in SEQ ID NO: 3 of the Sequence Listing. Furthermore, as long as the physicochemical properties described above are retained, the amino acid sequence of SEQ ID NO: 3 may include an amino acid sequence having a modified site where a specific amino acid is modified.

  One skilled in the art can easily predict modifications that retain the enzymatic activity of MELAC upon modification of the amino acid sequence. Specifically, for example, in the case of amino acid substitution, an amino acid having properties similar to the amino acid before substitution in terms of polarity, charge, hydrophilicity, hydrophobicity, etc. can be substituted from the viewpoint of maintaining the protein structure. . Such substitutions are well known to those skilled in the art as conservative substitutions. For example, alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine and tryptophan are classified as nonpolar amino acids, and thus have similar properties to each other. Examples of the neutral neutral amino acid include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Examples of acidic amino acids include aspartic acid and glutamic acid. Examples of basic amino acids include lysine, arginine, and histidine. Amino acid substitutions within each of these groups are permissible as protein function is maintained. Further, for convenience such as subsequent purification and immobilization on a solid phase, those having His-tag, FLAG-tag, etc. added to the N- or C-terminus of the amino acid sequence are also preferably exemplified. Such Tag peptide can be introduced by a conventional method. Moreover, the cut | disconnected type which cut | disconnected the amino acid residue of the C terminal side or the N terminal side may be sufficient as long as the loss | disappearance of an enzyme activity is not caused. Furthermore, chemical modification such as glucosylation may be added.

  However, the form in which amino acids 1 to 34 of the amino acid sequence represented by SEQ ID NO: 2 as an example of MELAC are deleted does not show activity. Such a defective region does not include amino acids conserved in all multi-copper oxidases or amino acids involved in binding to copper. That is, it is considered that the region includes amino acids essential for MELAC-specific laccase activity expression, and amino acids 1 to 34 of the amino acid sequence represented by SEQ ID NO: 2 are preferably conserved.

1-3. Origin etc.
The origin of MELAC is not limited as long as it has the physicochemical properties of MELAC of the present invention described above. For example, it is an organism having the ability to produce MELAC having the above-mentioned physicochemical properties, and may be derived from any organism, in particular from bacteria. Preferably, it originates from organisms present in environmental samples such as compost. For example, it can be obtained by screening a protein having the above physicochemical properties from a protein expressed from metagenomic DNA prepared from an environmental sample.

  Furthermore, MELAC may be produced by genetic engineering techniques. For example, based on the sequence information of the amino acid sequence of MELAC disclosed in this specification, the MELAC of the present invention can be produced by genetic engineering. For example, MELAC capable of retaining the above-mentioned physicochemical properties can be expressed by a hybridization method using a DNA probe prepared based on a base sequence encoding part or all of the amino acid sequence shown in SEQ ID NO: 2 or 3. Nucleic acid molecules encoding MELAC of the present invention can be prepared from bacterial genomic DNA. Similarly, by PCR using a part of the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 or 3 as a primer, the genomic DNA of a bacterium capable of expressing MELAC capable of retaining the above physicochemical properties is used as a template. Nucleic acid molecules encoding MELACs of the invention can be prepared. Furthermore, a nucleic acid molecule encoding the MELAC of the present invention can be chemically synthesized using a conventional DNA synthesis method such as a phosphoramidite method. The MELAC of the present invention can be produced by the gene recombination technique described in detail below for the obtained nucleic acid molecule.

  Here, the genomic DNA used as a template for hybridization and PCR is a genomic DNA derived from a bacterium capable of expressing MELAC capable of retaining the above-mentioned physicochemical properties. Preferably, it is genomic DNA prepared from organisms present in environmental samples such as compost. At this time, DNA may be extracted and cloned directly from an environmental sample without separating and culturing organisms, but is not limited thereto.

  In the production of the MELAC of the present invention, the probe used when utilizing hybridization is an oligonucleotide containing a sequence complementary to the MELAC of the present invention and can be prepared based on a conventional method. For example, a chemical synthesis method based on the phosphoramidite method or the like, or a restriction enzyme fragment or the like can be used when a target nucleic acid has already been obtained. Examples of such a probe include an oligonucleotide consisting of 10 or more, preferably 15 or more, more preferably about 20 to 50 bases of the base sequence based on the base sequence of the nucleic acid molecule encoding MELAC of the present invention. Illustrated. The probe may be appropriately labeled as necessary, and examples of such a label include a radioisotope and a fluorescent dye.

  In addition, in the production of the MELAC of the present invention, a primer used when PCR is used is an oligonucleotide containing a sequence complementary to the MELAC of the present invention, and can be prepared based on a conventional method. For example, a chemical synthesis method based on the phosphoramidite method or the like, or a restriction enzyme fragment or the like can be used when a target nucleic acid has already been obtained. When preparing a primer based on a chemical synthesis method, it is designed based on the sequence information of the target nucleic acid prior to synthesis. The primer can be designed using, for example, primer design support software so as to amplify a desired region. After synthesis, the primer is purified by means such as HPLC. Moreover, when performing chemical synthesis, it is also possible to use a commercially available automatic synthesizer. Such a primer is designed on the basis of the base sequence of the nucleic acid molecule encoding MEL AC of the present invention and sandwiches a desired amplification region, and is 10 or more, preferably 15 or more, more preferably about 20 to 50 bases. An oligonucleotide consisting of

  Here, complementary means that the primer and the target nucleic acid can specifically bind according to the base pairing rule to form a stable double-stranded structure. Here, not only perfect complementarity, but only a few nucleobases fit along the base-pairing rule as long as the primer and target nucleic acid are sufficient to form a stable duplex structure with each other Even partial complementarity is acceptable. The number of bases must be long enough to specifically recognize the target nucleic acid, but if it is too long, a non-specific reaction is induced, which is not preferable. Accordingly, an appropriate length is determined depending on many factors such as target nucleic acid sequence information such as GC content, and hybridization reaction conditions such as reaction temperature and salt concentration in the reaction solution. It is 20-50 bases long.

  Furthermore, the MELAC of the present invention can also be produced by a chemical synthesis technique. For example, the amino acid sequence shown in SEQ ID NO: 2 or 3 can be prepared by synthesizing all or part of the amino acid sequence using a peptide synthesizer and reconstructing the resulting polypeptide under appropriate conditions. it can.

  In addition, in the amino acid sequence of SEQ ID NO: 2 or 3 showing an example of MELAC, a variant having a modified site in which a specific amino acid has been modified is selected from the mutants caused by natural or artificial mutation. It can be obtained by screening a protein having activity. Alternatively, it can also be obtained by modifying a nucleic acid molecule encoding MELAC. There is no restriction | limiting in particular as a method to modify | change a nucleic acid molecule, The mutagenesis technique for preparation of the modification protein well-known to those skilled in the art can be utilized. For example, a known mutagenesis technique such as a site-directed mutagenesis method, a PCR abrupt induction method that introduces a point mutation using PCR, or a transposon insertion mutagenesis method can be used. A commercially available mutagenesis kit (for example, QuikChange (registered trademark) Site-directed Mutagenesis Kit (manufactured by Stratagene), etc.) may be used, and DNA synthesis methods such as a conventional phosphoramidite method may be used, It can also be prepared by constructing nucleic acid molecules with the desired modifications.

2. Nucleic acid molecules encoding MELAC
Nucleic acid molecules encoding MELAC include those encoding all MELACs having the aforementioned physicochemical properties. For example, it is a polynucleotide encoding a protein containing the amino acid sequence described in SEQ ID NO: 2 or 3, and a specific example thereof is a polynucleotide containing the base sequence shown in SEQ ID NO: 1. Here, the polynucleotide in the present invention includes both DNA and RNA, and when it is DNA, it is not necessarily double-stranded if it is single-stranded.

  Furthermore, a nucleic acid molecule comprising a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of the base sequence shown in SEQ ID NO: 1 is also included in the present invention as long as it retains the aforementioned properties of MELAC. Such a polynucleotide can be prepared by using a known mutation introduction technique. For example, a known mutagenesis technique such as a site-directed mutagenesis method, a PCR abrupt induction method that introduces a point mutation using PCR, or a transposon insertion mutagenesis method can be used. A commercially available mutation introduction kit (for example, QuikChange (registered trademark) Site-directed Mutagenesis Kit (manufactured by Stratagene), etc.) may be used, and a DNA synthesis method such as a conventional phosphoramidite method may be used. It can be performed by constructing a nucleic acid molecule encoding MELAC with a desired modification, or can be obtained by allowing an exonuclease to act on a polynucleotide having the base sequence of SEQ ID NO: 1. Such polynucleotides include those in which one or more bases are deleted, substituted, added or inserted in the base sequence of SEQ ID NO: 1. Therefore, 3 ′ of the base sequence of SEQ ID NO: 1, or Preferred examples include those in which a base sequence encoding a His-Tag sequence is added to the 5 ′ end, however, the first to third amino acid sequences shown in SEQ ID NO: 2. For the base encoding the amino acid of 4, modification that causes mutation of the encoded amino acid is not preferable.

  Here, the stringent condition means that DNAs having the identity of 60% or more, preferably 70%, more preferably 80% or more, particularly preferably 90% or more in the base sequence are preferentially hybridized. The condition to obtain. The stringency can be adjusted by appropriately changing the salt concentration and temperature during the hybridization reaction and washing. For example, the conditions for Southern hybridization described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, and the like.

  More specifically, hybridization is exemplified in 50% (v / v) formamide, 5 × SSC at 42 ° C. for 16 hours. Here, 1 × SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0. Further, sodium dodecyl sulfate (SDS) may be contained in an amount of 0.1 to 1.0% (v / v), and denatured non-specific DNA may be contained in an amount of 25 to 100 ng per mL of the hybridization buffer. Examples of washing conditions include washing for 5 minutes at 5 ° C in 2X SSC, 0.1% SDS, and washing for 30 minutes to 4 hours at 65 ° C in 0.1X SSC, 0.1% SDS. Also, those skilled in the art can easily understand these equivalent conditions.

  The nucleic acid molecule encoding MELAC of the present invention can be prepared based on the base sequence of the nucleic acid molecule of the present invention. For example, by hybridization using a DNA probe prepared based on a part or all of the base sequence shown in SEQ ID NO: 1, the present DNA is obtained from bacterial genomic DNA capable of expressing laccase activity capable of retaining the above physicochemical properties. Nucleic acid molecules encoding MELACs of the invention can be prepared. Similarly, PCR using a part of the nucleotide sequence of SEQ ID NO: 1 as a primer also encodes the MELAC of the present invention using as a template a bacterial genomic DNA capable of expressing the laccase activity capable of retaining the above-mentioned physicochemical properties. Nucleic acid molecules can be prepared. In addition, as shown in Example 2, it was obtained by cloning genomic DNA of bacteria capable of expressing MELAC capable of retaining the above-mentioned physicochemical properties by PCR using primers constructed from known MELAC sequence information. can do. Furthermore, a nucleic acid molecule encoding the MELAC of the present invention can be chemically synthesized using a conventional DNA synthesis method such as a phosphoramidite method. Details are described above.

3. Recombinant Vector The recombinant vector of the present invention can be constructed by incorporating a nucleic acid molecule encoding the MELAC of the present invention into an appropriate vector. The vector that can be used is not particularly limited as long as it can incorporate foreign DNA and can replicate autonomously in a host cell. Thus, the vector contains a sequence of at least one restriction enzyme site into which a nucleic acid molecule encoding MELAC can be inserted or a site-specific recombination sequence. For example, plasmid vectors (pET system, pUC system, pBR system, etc.), phage vectors (λt10, λt11, λAP, etc.), cosmid vectors, virus vectors (vaccinia virus, baculovirus, etc.) and the like are included.

  The recombinant vector of the present invention is incorporated so that the nucleic acid molecule encoding MELAC can express its function. Therefore, other known base sequences necessary for the functional expression of the nucleic acid molecule may be included. For example, a promoter sequence, a leader sequence, a signal sequence, a ribosome binding sequence and the like can be mentioned. As the promoter sequence, for example, when the host is Escherichia coli, lac promoter, trp promoter and the like are preferably exemplified. However, the present invention is not limited to this, and a known promoter sequence can be used. Furthermore, the recombinant vector of the present invention can also include a marking sequence that can confer phenotypic selection in the host. Examples of such marking sequences include sequences encoding genes such as drug resistance and auxotrophy. Specifically, kanamycin resistance gene, chloramphenicol resistance gene, ampicillin resistance gene and the like are exemplified.

  The insertion of a nucleic acid molecule encoding MELAC into a vector includes, for example, a method in which the gene of the present invention is cleaved with an appropriate restriction enzyme and inserted into a restriction enzyme site or a multicloning site of an appropriate vector and ligated. Although it can be used, it is not limited to this. For ligation, a known method such as a method using DNA ligase can be used. Commercially available ligation kits such as DNA Ligation Kit (Takara-bio) can also be used.

4). Transformant The transformant of the present invention can be constructed by transforming an appropriate cell with a recombinant vector containing a nucleic acid molecule encoding MELAC of the present invention. Here, the host cell is not particularly limited as long as it can efficiently express the nucleic acid molecule encoding MELAC of the present invention. Prokaryotes can be preferably used, and in particular, E. coli can be used. In addition, Bacillus subtilis, Bacillus bacteria, Pseudomonas bacteria, and the like can also be used. As E. coli, for example, E. coli DH5 瘁 A E. coli BL21, E. coli JM109 and the like can be used. Furthermore, eukaryotic cells can be used without being limited to prokaryotes. For example, it is also possible to utilize yeasts such as Saccharomyces cerevisiae, insect cells such as Sf 9 cells, CHO cells, animal cells such as COS-7 cells. As a transformation method, a known method such as a calcium chloride method, an electroporation method, a liposome transfection method, a microinjection method, or the like can be used.

5. Manufacturing method of MELAC
MELAC is produced by culturing the above-described transformant of the present invention and collecting a protein having laccase activity from the obtained culture. That is, the method includes a culture step of culturing the transformant of the present invention described above and a recovery step of recovering the protein expressed in the culture step. As described above, MELAC can be mass-produced at low cost by expressing it in an appropriate host.

  The culturing step is carried out by inoculating the transformant of the present invention in an appropriate medium and culturing according to a conventional method. For the culture of the transformant of the present invention, the culture conditions may be selected in consideration of the nutritional physiological properties of the host cells. The medium to be used is not particularly limited as long as it contains nutrients that can be assimilated by the host cell and can efficiently express the protein in the transformant. Therefore, a medium containing a carbon source, a nitrogen source and other essential nutrients necessary for the growth of the host cell is preferable, regardless of whether it is a natural medium or a synthetic medium. Examples of the carbon source include glucose, dextran, starch, and the like, and examples of the nitrogen source include ammonium salts, nitrates, amino acids, peptone, and casein. As other nutrients, inorganic salts, vitamins, antibiotics, and the like can be included as desired. When the host cell is Escherichia coli, LB medium, M9 medium and the like can be suitably used. Moreover, although there is no restriction | limiting in particular also about a culture | cultivation form, A liquid medium can be utilized suitably from a viewpoint of mass culture.

  Selection of a host cell carrying the recombinant vector of the present invention can be performed by, for example, the presence or absence of expression of a marking sequence. For example, when a drug resistance gene is used as the marking sequence, it can be performed by culturing in a drug-containing medium corresponding to the drug resistance gene.

  The purification step may be performed by recovering, that is, isolating and purifying MELAC from the transformant culture obtained in the above-described culture step. Depending on the fraction in which the enzyme of the present invention is present, a technique according to a general protein isolation and purification method may be applied. Specifically, when MELAC is produced outside the host cell, the culture solution is used as it is, or the host cell is removed by means of centrifugation, filtration or the like to obtain a culture supernatant. Subsequently, the enzyme of the present invention can be isolated and purified by appropriately selecting a known protein purification method for the culture supernatant. For example, known isolation and purification techniques such as ammonium sulfate precipitation, dialysis, SDS-PAGE electrophoresis, gel filtration, various types of chromatography such as hydrophobicity, anion, cation, and affinity chromatography can be applied alone or in appropriate combination. . In particular, when affinity chromatography is used, it is preferable to express the enzyme of the present invention as a fusion protein with a tag peptide such as His Tag and use the affinity for such tag peptide. When MELAC is produced in the host cell, the host cell is recovered by means such as centrifugation and filtration of the culture. Subsequently, the host cells are disrupted by an enzymatic disruption method such as lysozyme treatment or a physical disruption method such as ultrasonic treatment, freeze-thawing, and osmotic shock. After crushing, the solubilized fraction is collected by means such as centrifugation or filtration. The obtained solubilized fraction can be isolated and purified by treating in the same manner as in the case where it can be produced extracellularly. Here, since MELAC has high heat resistance, it is useful and convenient to use heat treatment in combination with the aforementioned isolation and purification steps. The host cell and culture supernatant obtained from the culture contain various proteins derived from the host cell. However, the host cell-derived contaminating protein is denatured and condensed and precipitated by heat treatment. On the other hand, MELAC does not cause denaturation because it has heat resistance, so it can be easily separated from host-derived contaminating proteins by centrifugation or the like. In addition, even when the culture solution is used as it is or when a crude extract is used, other proteins are inactivated by heat treatment, so that it can be used as an enzyme solution containing only MELAC. Therefore, even when MELAC is produced by genetic engineering techniques, other proteins derived from the host can be easily removed. Therefore, there is an advantage that the degree of purification can be improved and a highly reliable enzyme can be produced.

  The performance of the isolated and purified protein can be confirmed by analyzing its physicochemical properties and sequence. For example, the method described in Examples 6 to 10 can be performed.

6). Use of MELAC
MELAC is a laccase that is activated at room temperature and has excellent heat resistance, and can therefore be used in various industrial fields. The mode of use will be described below, but is not limited thereto.

6-1. Enzyme electrode
MELAC can be used as a catalyst for enzyme electrodes. Preferably, it can be constructed by immobilizing the MELAC of the present invention on a conductive substrate connected to an external circuit, and the constructed electrode can be used for a fuel cell, a biosensor, or the like.

Examples of conductive substrates include carbon materials such as graphite and glassy carbon, metals or alloys such as aluminum, copper, gold, platinum, silver, nickel, and palladium, SnO ₂ , In ₂ O ₃ , WO ₃ , TiO _2, etc. A conventionally known conductive material such as a conductive oxide can be used. Moreover, you may comprise this with a single layer or a 2 or more types of laminated structure. The size and shape of the electrode are not particularly limited, and can be appropriately adjusted according to the purpose of use. Immobilization of the enzyme on the conductive substrate can be performed by a known method. For example, it is possible to use a carrier binding method that is immobilized through physical adsorption, ionic bond, or covalent bond. Further, a crosslinking method in which crosslinking is performed with a crosslinking reagent having a divalent functional group such as glutaraldehyde can be used. Furthermore, polysaccharides such as alginic acid and carrageenan, conductive polymers, redox polymers, polymers having a network structure such as photocrosslinkable polymers, and a comprehensive method in which they are sealed in a semipermeable membrane such as a dialysis membrane Can also be used. Moreover, you may use combining these.

6-2. Fuel cell
MELAC can be used to build fuel cells, and such fuel cells also form part of the present invention. The fuel cell of the present invention includes, for example, an anode electrode that performs an oxidation reaction and a cathode electrode that performs a reduction reaction, and includes an electrolyte layer that separates the anode and the cathode as necessary. The MELAC described in the item 1 is provided with an electrode fixed on a conductive base material connected to an external circuit. Alternatively, MELAC may be supplied in a form dissolved in an appropriate buffer. In immobilization, MELAC is preferably immobilized in the state of a holoenzyme containing a copper atom. However, it may be immobilized in the form of an apoenzyme and supplied as a separate layer or in a form dissolved in an appropriate buffer solution. In addition, other substances necessary for the expression of the catalytic activity of the enzyme may be supplied as a separate layer or dissolved in an appropriate buffer. And preferably, the electrode fixed on the electroconductive base material which connected MELAC to the external circuit is constructed | assembled as a cathode side electrode. Preferably, the anode side electrode is a carbon electrode, and is configured to directly exchange electrons between the carbon electrode and MELAC. Further, a redox substance may be interposed between the carbon electrode and MELAC. For example, an electrode on which an oxidoreductase such as glucose dehydrogenase is immobilized can be used as the anode side electrode. If necessary, an electron mediator that mediates enzyme reaction and electron transfer between electrodes is used. The mediator is not particularly limited, and examples thereof include quinones, cytochromes, viologens, phenazines, phenoxazines, phenothiazines, ferricyanides, ferredoxins, ferrocene and derivatives thereof.

  The fuel cell of the present invention utilizes MELAC which exhibits laccase activity in a normal temperature range, has high heat resistance, and particularly has excellent physicochemical properties that the activity is not deactivated even by long-term heat treatment. Therefore, even under normal operating conditions of about 25 ° C, a high-performance fuel cell with durability that can provide stable output under various usage environments such as long-term use and various high-temperature conditions is constructed. can do. Further, since MELAC is a low-molecular substance, the amount of enzyme molecules supported per area of the conductive member can be increased as compared with known laccases. As a result, the current density is improved and high power generation is possible, so that further improvement in performance of the fuel cell can be achieved.

6-3. Biosensor
MELAC can be used to build biosensors, and such biosensors also form part of the present invention. The biosensor of the present invention includes an electrode in which MELAC described in the section 6-1 is immobilized on a conductive base material connected to an external circuit. For example, this electrode is used as a working electrode and a counter electrode is provided. If necessary, from the viewpoint of improving the reliability of measurement accuracy, a three-electrode system provided with a reference electrode such as silver-silver chloride may be used. By comprising in this way, o-, p-diphenol, p-phenylenediamine, ascorbic acid and the like which are substrates for laccase can be detected.

  The measurement by the biosensor of the present invention is performed by bringing a measurement sample into contact with the sensor and detecting the current generated by the oxidation reaction of MELAC on the electrode and the substrate, thereby determining the presence or absence of the substrate in the sample. Alternatively, the concentration can be measured. At this time, by preparing a standard curve prepared in advance with a substrate solution having a standard concentration within the target concentration range, the substrate concentration can be determined based on the obtained current value. Here, as a sample, all samples in which the presence of a substrate is expected can be targeted. Examples include biological samples derived from organisms such as blood, urine, saliva, food samples, environmental samples, and the like, but are not limited thereto. Moreover, the sample which performed the appropriate process to these samples as needed may also be included. The biosensor of the present invention can be used for detection of all substances that can serve as laccase substrates. Furthermore, the biosensor of the present invention exhibits MELAC having excellent physicochemical properties that exhibit laccase activity in a normal temperature range, high heat resistance, and in particular, that the activity is not inactivated even by long-term heat treatment. Because it is used, it has a high durability that can stably perform its functions even under normal use conditions of about 25 ° C, and in various use environments such as long-term use and various high-temperature conditions. A biosensor with high performance can be constructed, and since MELAC is a low-molecular substance, the amount of enzyme molecules supported per area of the conductive member can be increased as compared with known laccases. Thereby, since the current density is improved, further enhancement of the performance of the biosensor can be achieved. Therefore, it can be used in various industrial fields such as medical, food and environmental fields.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, this invention is not limited to these Examples at all.

[Example 1] Cloning of laccase gene A part of a nucleic acid molecule encoding laccase was cloned from metagenomic DNA derived from an environmental sample using PCR.

1. Primer design In order to obtain a laccase gene, a degenerate primer for PCR amplification was designed with reference to the amino acid sequence of the existing laccase. Use Known laccase gene sequence, Thermus thermophilus (NCBI accession number YP_005339 , Cyanothece sp. (NCBI accession number ZP_01731625, Synechococcus sp. (NCBI accession number ZP_01081498, Lyngbya sp. NCBI accession number ZP_01621366, the Trichodesmium erythraeum (NCBI accession number YP_720301 It was.
The sequence information of the designed primer is as follows.
Forward primer sequence 5'-CCCTGCTGGAATGTTCTGOLE_LINK3GTAYCAYCCNCA-3 '(SEQ ID NO: 4)
Reverse primer sequence 5'-OLE_LINK3CCAGATCCTCATGGTCCAGAATRTGRCARTG-3 '(SEQ ID NO: 5)

  When using the above primers, the length of the target DNA sequence to be amplified was expected to be about 1000 bp.

2. Preparation of template DNA Metagenomic DNA from an environmental sample was used as a template. Here, as an environmental sample, wood compost containing a large amount of bacteria that oxidatively degrade wood components collected from a pasture in Midori Ward, Chiba City was used. Specifically, thinned wood and pruned wood are crushed with a pulverizer to produce wood chips. After mixing the wood chips and cow dung containing fermentative bacteria so that the volume ratio is 1: 1, compost The mixture was mixed at a rate of 7 to 10 days using a turner and fermented at a high temperature of 50 to 70 ° C. Sampling to extract metagenomic DNA from wood compost was performed immediately after wood chip production, wood compost after 1 month of fermentation, and 4-5 months after (final). The extracted DNA solution of the compost sample used as a template was prepared according to the procedure described in the attached document using a 2 to 6 g (wet weight) compost sample as a starting material and using DNA Isolation Kit (MO-BIO). did. The extracted DNA was finally dissolved in the eluate attached to the 5 ml kit.
In addition, the metagenomic DNA was similarly extracted from the environmental samples of the rice husk compost fermented for two months and cow dung (immediately after defecation) as the target samples.

3. PCR amplification reaction PCR amplification was performed using the forward and reverse primers prepared in 1 above and the template DNA prepared in 2 above. PCR reaction was performed using Multiplex PCR Assay Kit (manufactured by Takara-Bio) according to the instructions attached to the reagent. Specific PCR reaction temperature conditions are as follows.
(1) Thermal denaturation step: 94 ° C, 30 seconds (2) Thermal denaturation step: 94 ° C, 30 seconds (3) Annealing step: 55 ° C, 30 seconds (4) Extension step: 74 ° C, 90 seconds (5) Final Extension process: 74 ° C., 7 minutes After the thermal denaturation in the step (1), the steps (2) to (4) were repeated 40 cycles, and finally the reaction was terminated by the final extension in the step (5).

  The PCR product was analyzed and purified by agarose gel electrophoresis, and the target DNA fragment was cloned into pUC118 vector (Takara-Bio), and the nucleotide sequence was determined. The results of this agarose gel electrophoresis are shown in FIG. In the figure, lane 1 shows the result of using a wood compost sample (17 ng) immediately after chip production as an environmental sample. Lane 2 shows the result of using wood compost (20 ng) after 1 month of fermentation, and Lane 3 shows the result of using wood compost (15 ng) after 4 to 5 months of fermentation. Lane 4 shows the results of using rice husk compost (17 ng) for 2 months of fermentation, and Lane 5 shows the results of using cow manure compost (18 ng). Lane 6 shows the result of using E. coli genomic DNA (20 ng). As a result, amplification of a band of about 1000 bp was confirmed in all three kinds of environmental samples. As a result, it was recognized that a DNA fragment presumed to be a laccase gene was amplified, and the following experiment was conducted.

[Example 2] Acquisition of full-length gene The full-length sequence of the DNA fragment obtained in Example 1 was obtained using the inverse PCR method. When a useful gene or the like is obtained from a sample such as environmental DNA as in the present invention, it is necessary to amplify an unknown DNA sequence in the peripheral region of the DNA sequence that is known by decoding. For this purpose, an inverse PCR method is used to amplify an unknown DNA sequence using a primer designed to elongate from the known DNA sequence region in the direction of the unknown DNA sequence region adjacent to the region. The full-length gene was obtained.

1. Preparation of template DNA Metagenomic DNA extracted from wood compost after 1 month of fermentation was added to restriction enzymes Bam HI, Hind III, Kpn I, Nde I, Not I, Pst I, Sac I, Sal I, Sma I, Sph I, 50 units of Xba I and Xho I were added, respectively, and a restriction enzyme reaction was performed at 37 ° C. for 5 hours. At this time, the dose of the reaction solution was 50 μl, and the ones attached to the restriction enzymes were used. From the solution after the reaction, DNA in the OLE_LINK2 solution was purified using an OLE_LINK2DNA purification kit (manufactured by GE-Healthcare). Next, dissolve the purified DNA in TE buffer to make 20 μl, add 20 μl of DNA Ligation Kit (Mighty Mix) (Takara Bio) to this solution, and incubate overnight at 16 ° C. for self-ligation reaction. I let you. After completion of the reaction, the reaction product was purified using a DNA purification kit (manufactured by GE-Healthcare). The target DNA sequence was amplified using the whole amount of the purified sample as a template for inverse PCR.

2. Primer design Primers were designed to amplify an unknown DNA sequence in the peripheral region of a DNA sequence that became known by decoding the DNA fragment obtained in Example 1. The sequence information of the designed primer is as follows.
Forward primer sequence 5′-AGTCGTACGTGAAGGTTTCGCCGGG-3 ′ (SEQ ID NO: 6)
Reverse primer sequence 5′-GGTATGATGGGCCATATTCACCTCACTCCC-3 ′ (SEQ ID NO: 7)

3. Inverse PCR reaction The total amount of the reaction product prepared in 1 above was subjected to a PCR reaction. Then, use PrimeSTAR GXL DNA Polymerase (Takara-Bio) as the DNA polymerase, add the primer (final concentration 0.2 μM) according to the attached material, and make up to 50 μl with sterile distilled water. It was prepared by. Subsequently, inverse PCR was performed on the prepared reaction solution using a thermal cycler GeneAmp (registered trademark) PCR System 9700 (Applied Biosystems). Specific PCR reaction temperature conditions are as follows.
(1) Thermal denaturation step: 98 ° C., 10 seconds (2) Annealing and extension step: 68 ° C., 120 seconds (3) Final extension step: 68 ° C., 10 minutes Steps (1) and (2) are repeated 25 cycles, Finally, the reaction was terminated by the final extension in step (3).

  In order to analyze and purify the PCR product, the solution after the amplification reaction was subjected to agarose gel electrophoresis. After electrophoresis, the presence or absence of a band corresponding to the target DNA sequence on the gel was detected. The results of this agarose electrophoresis are shown in FIG. As a result, a band thought to be derived from the target DNA sequence could be confirmed after amplification by the inverse PCR method.

  The obtained target DNA fragment was cloned into pUC118 Vector, and then the base sequence of the DNA fragment was determined. By this sequencing, the 5′-end sequence encoding the N-terminal region of one protein presumed to be laccase from the base sequence information of the DNA amplified fragment using environmental DNA treated with the restriction enzyme SacI as a template, and the C-terminal The 3 'terminal sequence encoding the region could be revealed. In this way, after obtaining the virtual 5 ′ and virtual 3 ′ base sequences by inverse PCR, 5 ′ and 3 ′ primers were prepared based on this, and the entire gene was cloned again. The nucleotide sequence including the coding region of the protein was determined by determining the sequence and obtaining the full length of the gene. The entire base sequence of the gene determined here and the amino acid sequence of the protein deduced from this base sequence are shown as SEQ ID NOS: 1 and 2, respectively. As a result of sequencing, it was revealed that the coding region was 1254 bp, the protein deduced from the base sequence was 417 residues, and the estimated molecular weight was 47.5 kDa.

[Example 3] Homology search The amino acid sequence of the protein presumed to be laccase obtained in Example 2 was subjected to a homology search and an amino acid sequence alignment with a protein having a known substrate oxidation activity.

1. Homology search The homology search was performed by a BLAST search, which is widely used to find sequences with high similarity. When the default or initial condition parameters were used as the search conditions, the highest score was the oxidoreductase (SEQ ID NO: 8) derived from the prokaryote Sorangium cellulosum (AC CESSION YP_001611435). As shown in FIG. 3, the homology was about 46% (Identities = 196/422) at the amino acid level.

2. Sequence alignment The alignment with the amino acid sequence of the existing heat-resistant laccase was performed. The results are shown in FIG. Panel A shows the comparison with the amino acid sequence (SEQ ID NO: 9) of the thermus thermophilus-derived laccase disclosed in Patent Document 5 (Japanese Patent Laid-Open No. 2006-158252) presented as a prior art document as an existing heat-resistant laccase. Indicates. Hereinafter, the laccase derived from the Thermus thermophilus may be referred to as “TthLAC”. Panel B shows a comparison with the amino acid sequence (SEQ ID NO: 10) of laccase derived from environmental metagenomic DNA disclosed in Patent Document 4 (Japanese Patent Laid-Open No. 2009-201481) presented as a prior art document. Hereinafter, the environmental metagenomic DNA-derived laccase may be abbreviated as “SLAC”. As a result, the homology with the existing laccase from Thermos thermophilus is about 29% at the amino acid level (Identities = 135/462), and the homology with the existing laccase from the environmental metagenomic DNA is 20% at the amino acid level ( Identities = 73/360), and it was found that there was almost no homology with existing laccases.

[Example 4] Amplification of full-length gene The gene presumed to be laccase obtained in Example 2 was amplified.

1. Primer design Specific primers for the partial sequence of the gene presumed to be laccase obtained in Example 2 (5 'terminal sequence encoding the N-terminal region of laccase and 3' terminal sequence encoding the C-terminal region) Designed. Specifically, the sequence information of the primers designed here is as follows.
Forward primer sequence 5′-GAAGGAGATATACAT-ATGGAGCTCGAGGCGCGCGTCAC-3 ′ (SEQ ID NO: 11)
Reverse primer sequence 5′-GAGTGCGGCCGCAAG-GGGAGTGAGGTGAATATGGCCCA-3 ′ (SEQ ID NO: 12)

2. PCR amplification reaction The DNA fragment obtained in Example 2 was used as a template, and the PCR was carried out using the above primers. PCR reaction was performed using PrimeSTAR GXL DNA Polymerase (Takara-Bio) according to Example 2. Specific PCR reaction temperature conditions are as follows.
(1) Thermal denaturation step: 98 ° C., 10 seconds (2) Annealing and extension step: 68 ° C., 120 seconds (3) Final extension step: 68 ° C., 10 minutes Steps (1) and (2) are repeated 25 cycles, Finally, the reaction was terminated by the final extension in step (3). The obtained PCR product was analyzed and purified by agarose gel electrophoresis and used in the following experiments.

[Example 5] Production of transformant and production of recombinant protein The DNA fragment obtained in Example 4 was transformed into E. coli and expressed in E. coli cells to produce a recombinant protein.

1. Construction of E. coli protein expression plasmid The DNA fragment obtained in Example 4 was cloned into the multi-cloning site of pET22b Vector (between Nde I and Hin dIII), an E. coli expression vector, an In-Fusion Advantage PCR cloning kit. And Clontech). In SEQ ID NO 11 and SEQ ID NO: 12, shows between the homologous sequences in the vector to be added to the ends of the insert (15 bases) and terminal sequence of the insert a hyphen. At this time, the peptide containing histidine on the C-terminal side (LAAALEHHHHHH (SEQ ID NO: 13): HHHHHH is a His tag) is obtained by cloning into the NdeI / HindIII site of pET22b except for the stop codon at the C-terminal of the laccase gene. A fusion protein expression plasmid was constructed, transformed into Escherichia coli DH5α strain, and the gene transfer plasmid was selected.

2. Protein expression The E. coli BL21 (DE3) pLysS strain transformed with the protein expression plasmid selected above is cultured, and IPTG (isopropyl thio-β-galactoside) is added to induce the fusion protein. And expressed. Specifically, Escherichia coli was cultured at 37 ° C. until the absorbance OD ₆₀₀ reached about 0.2, and further 0.1 mM IPTG and 0.5 mM CuCl ₂ were added and cultured at 20 ° C. for 20 hours. After culturing, E. coli was collected by centrifuging the culture solution and frozen until the next experiment.

3. Purification of protein The cells expressing the protein were suspended in 10 mM Tris-HCl, 1 mM EDTA, pH 7.4, 0.4% surfactant (Brij58) was added, and the mixture was left on ice for 30 minutes. Next, the cells were crushed with an ultrasonic cell crusher, the insoluble matter was removed by centrifugation, and the cell lysate was collected. A solubilized solution (sample preparation solution for electrophoresis containing SDS) was added to the obtained cell lysate and heat-treated at 95 ° C. Subsequently, in order to confirm the expression of the protein, this cell lysate was subjected to electrophoresis on an acrylamide gel (10-20% PAGEL, manufactured by Ato), and the protein was visualized by a CBB staining method (manufactured by Wako Pure Chemical Industries). . In addition, the supernatant obtained by centrifuging this cell disruption solution at 40,000 × g for 30 minutes was similarly subjected to electrophoresis to visualize the protein.

  The results are shown in FIG. Lane 1 shows the electrophoresis result of the supernatant obtained by centrifuging the cell disruption solution, and Lane 2 shows the supernatant obtained by centrifuging the cell disruption solution.

Next, purification was performed with a metal affinity carrier for purification of histidine tag fusion protein (TALON, manufactured by Clontech). Specifically, an appropriate amount of support is packed in an open column, pre-washed with 20 mM sodium phosphate, 5 mM imidazole, and 0.5 M NaCl solution, and the final concentration of the cell lysate obtained above is 0.5 M. In this manner, NaCl was added and applied to the column. Subsequently, after washing with 20 mM sodium phosphate, 10 mM imidazole, 0.5 M NaCl solution, the protein was eluted with 20 mM sodium phosphate, 150 mM imidazole, 0.5 M NaCl solution. After elution, the eluate was dialyzed overnight using 25 mM Tris-HCl (pH 7.4) buffer and 1 mM CuCl ₂ as external solutions. Thereby, removal of salts (such as imidazole and NaCl) used for elution and copper ion can be taken into the laccase to make holo.

  Subsequently, the eluate after dialysis was subjected to electrophoresis on an acrylamide gel (10-20% e-PAGEL, manufactured by Ato Corporation) in the same manner as described above, and the protein was visualized by the CBB staining method (manufactured by Wako Pure Chemical Industries, Ltd.). . As a control, a solution that passed through the column after the cell disruption solution was applied to the column was also subjected to electrophoresis in the same manner.

  The results are shown in FIG. Lane 3 shows the solution passing through the column after applying the cell lysate to the column, and lane 4 shows the result of the eluate after dialysis. A single band of the protein was confirmed in Lane 4, and the purification of the protein was successfully completed, and this was used as a purified protein solution in the following experiments.

[Example 6] Activity confirmation 1-laccase activity The presence or absence of laccase activity of a protein presumed to be the laccase purified in Example 5 was confirmed.

  The protein solution purified in Example 5 was confirmed for the presence or absence of laccase activity. The laccase activity was measured by a colorimetric method using ABTS as a substrate and measuring the increase in absorbance at 419 nm due to oxidation thereof. Specifically, a 20 mM sodium acetate buffer solution having a pH of 5.0 containing 1 mM ABTS was used as a reaction solution. And the protein solution refine | purified in Example 5 was added to this reaction liquid, and the variation | change_quantity per unit time of the light absorbency of 419 nm accompanying the oxidation of ABTS was measured. By calculating the amount of ABTS oxidized with a molar absorbance coefficient of ABTS of 36,000 / M / cm, the amount of enzyme that oxidizes 1 μmol of ABTS per minute was determined.

  As a result, it was confirmed by measurement at 25 ° C. that the laccase activity of 103 μmol / min / mg protein was exhibited, and it was found that the purified protein had laccase activity. The laccase activity confirmed here is a known protein having laccase activity reported to have a heat resistance of about 60 ° C. (non-patent document 1 disclosed in the section of prior art document, disclosed in Patent Documents 1 to 3) Laccase) (20-200 μmol / min / mg protein)), the protein obtained here proved to be an industrially available laccase from the standpoint of catalytic ability. . Since the laccase activity was confirmed, the purified protein was named “MELAC” and the gene encoding it was named “melac”.

[Example 7] Activity confirmation 2-pH dependence
The pH dependence of the laccase activity of MELAC was confirmed.

  The pH dependence of the laccase activity of MELAC purified in Example 5 was confirmed in the range of pH 3.5-10. As buffers, citric acid was used at pH 3.5 to 5.5, phosphoric acid was used at pH 5 to 7, tris-hydrochloric acid was used at pH 6.5 to 9, and glycine-NaOH was used at pH 8.5 to 10. The laccase activity was measured according to the colorimetric method of Example 6, and the amount of change in absorbance at 419 nm at 25 ° C. when each pH buffer solution was used as a reaction solution was measured.

  The results are shown in FIG. From these results, it was found that MELAC has an optimum pH in a weakly acidic region.

[Example 8] Activity confirmation 3-heat resistance
The heat resistance of laccase activity of MELAC was confirmed.

  The heat resistance of the laccase activity of MELAC purified in Example 5 was confirmed in the range of 60 to 85 ° C. Purified MELAC was dissolved at a concentration of 1 mg / ml in 25 mM Tris-HCl pH7.4 buffer and heated at 60 ° C, 65 ° C, 70 ° C, 75 ° C, 80 ° C or 85 ° C for 60 minutes, respectively. . The residual activity after the heat treatment was measured according to Example 6 by an oxidation reaction using a citrate buffer containing 1 mM ABTS as a substrate as a reaction solution.

The results are shown in FIG. In this figure, the laccase activity at 25 ° C. where heat treatment was not performed was taken as 1, and the activity at the heat treatment temperature was shown as a relative value. From this result, compared to the activity without heat treatment (25 ° C), 60% or more activity can be maintained even at 60 ° C, 65 ° C, 70 ° C, 75 ° C, or 80 ° C, and 85 ° C In the treatment with, 50% or more of the activity was retained. As a result, it was found that MELAC has excellent heat resistance without significantly decreasing the activity even after heat treatment at 80 ° C. for 60 minutes.
As a control, MELAC was not added and only the reaction solution was treated at 85 ° C., and the laccase activity was measured in the same manner. Therefore, it can be understood that the activity remaining after heating is not derived from the reaction solution but is entirely derived from MELAC.

[Example 9] Activity confirmation 4-long-term heat resistance In Example 8, the heat resistance of MELAC laccase activity by heating for 60 minutes was confirmed. In this example, MELAC laccase when the heating time was further increased. The heat resistance of the activity was confirmed.

The long-term heat resistance of the laccase activity of MELAC purified in Example 5 was confirmed in the range of 60 to 80 ° C. Purified MELAC was dissolved in 25 mM Tris-HCl pH 7.4 buffer solution at a concentration of 1 mg / ml and heated at 60 ° C., 70 ° C., and 80 ° C. for 17 hours, respectively. The residual activity after long-term heat treatment was measured according to Example 6 by an oxidation reaction using a citrate buffer containing 1 mM ABTS as a substrate as a reaction solution.

  The results are shown in FIG. In this figure, the laccase activity at 25 ° C. where heat treatment was not performed was taken as 1, and the activity at the heat treatment temperature was shown as a relative value. From this result, it was confirmed that 70% or more of the activity was maintained even at the treatment at 60 ° C. as compared with the case without the heat treatment (25 ° C.). Further, when the treatment was performed at 70 ° C. or 80 ° C., the activity decreased to about 20%, but the activity did not become zero. As a result, it was shown that MELAC has excellent heat resistance without a significant decrease in activity even after heat treatment at 60 ° C. for 17 hours.

From the results of Examples 8 and 9, it has been found that a heat reduction of about 20% is recognized by the heat treatment at 60 ° C. regardless of the heating time. In view of the results, about 20% which is reduced by the heat treatment. It can be expected that this activity is caused by the fact that a part of the molecule having an unstable structure is contained in the enzyme solution and the molecule is deactivated even by a short heat treatment. Based on this expectation, it is considered that the decrease in activity can be eliminated if unstable molecules can be removed by increasing the degree of purification of MELAC, and further improvement in heat resistance can be expected by improving the degree of purification.

[Example 10] Activity confirmation 5-temperature dependence
The temperature dependence of laccase activity of MELAC was investigated.

  The temperature dependence of the laccase activity of MELAC purified in Example 5 was measured in the temperature range of 30 to 80 ° C. according to the colorimetric method described in Example 6. Specifically, the enzyme solution was added to an activity measurement solution containing 20 mM sodium acetate buffer (pH 5.0), 0.1 mM copper sulfate, and 1 mM ABTS. Subsequently, the change in absorbance at 419 nm at each temperature was measured for 3 minutes, and the amount of change in absorbance per minute was calculated.

  The results are shown in FIG. From this result, it was found that MEL AC has an optimum temperature above 80 ° C.

[Example 11] Comparison with known laccase 1-amino acid sequence
Similar to MELAC, MELAC was compared with laccase isolated from metagenomic DNA derived from wood compost at the amino acid sequence level.

  The inventors of the present application have previously isolated two proteins having laccase activity from a metagenome derived from wood compost as in the present application (Japanese Patent Application No. 2009-241586). Alignment with these amino acid sequences was performed.

  The results are shown in FIG. In FIG. 10, panel A is a comparison with the amino acid sequence of the protein named “mgLAC-1” (SEQ ID NO: 14) in the above specification, and panel B is the amino acid of the protein named “mgLAC-2”. Comparison with the sequence (SEQ ID NO: 15). Homology with “mgLAC-1” is about 22% (Identities = 112/499) at the amino acid level, and homology with “mgLAC-2” is about 19% (Identities = 91/476) at the amino acid level. there were. The MELAC isolated in the present invention was found to have almost no homology with any of them.

  In addition, MELAC showed no activity at all in the truncated form in which amino acids 1 to 34 of the amino acid sequence shown in SEQ ID NO: 2 were deleted. Such a defective region does not include amino acids conserved in all multi-copper oxidases or amino acids involved in binding to copper. When the primary analysis of the amino acid of SEQ ID NO: 2 is explained, the amino acid sequence conserved in all the multi-copper oxidases is the 66th aspartic acid, and the amino acid involved in the binding to copper is the 55th amino acid. Histidine (2), 57th histidine (3), 97th histidine (3), 353rd histidine (1), 355th histidine (2), 398th histidine (3), The 399th cysteine (1), the 400th histidine (3), the 404th histidine (1), and the 410th methionine (1) are estimated. The numbers 1, 2, and 3 after the copper-binding amino acid residue indicate type I copper, type II copper, and type III copper ligand, respectively. The primary analysis of such amino acids was estimated based on the description of Kataoka K, JOURNAL OF BIOLOGICAL CHEMISTRY, May 2009, Vol. 284, No. 21, pages 14405-13. That is, this region is considered to contain amino acids essential for MELAC-specific laccase activity expression. And this area | region has low homology with mgLAC-1 and mgLAC-2, From this knowledge, it is recognized that it is an area | region which is related to the activity unique to MELAC which is not mgLAC-1 and mgLAC-2.

[Example 12] Comparison with known laccase 2-Temperature dependence
Similar to MELAC, MELAC was compared with laccase isolated from a metagenome derived from wood compost and laccase derived from Bacillus subtili from the viewpoint of temperature dependence.

Regarding the temperature dependence of MELAC laccase, mgLAC-1 and mgLAC-2 examined in Example 11 and laccase derived from Bacillus subtili (Martins LO et al., JOURNAL OF BIOLOGICAL CHEMISTRY, May 2002, Vol. 277, No. 21, pp. 18849-18859). The temperature dependence was measured in the same manner as in Example 10. Hereinafter, the laccase derived from Bacillus subtilis may be referred to as “CotA”.

  The results are shown in FIG. In FIG. 11, panel A shows the results for mgLAC-1, panel B shows the results for mgLAC-2, and panel C shows the results for CotA. As a result, it was found that the optimum temperature for mgLAC-1 was around 55 ° C, the optimum temperature for mgLAC-2 was 80 ° C or higher, and the optimum temperature for CotA was around 70 ° C. Therefore, MELAC was found to have the same heat resistance as mgLAC-2 and higher heat resistance than mgLAC-1 and CotA.

[Example 13] Comparison with known laccase 3-Molecular weight
MELAC was compared in terms of molecular weight with known laccases such as laccase isolated from metagenomic DNA derived from wood compost, similar to MELAC.

Here, the molecular weights were compared because the laccases derived from mgLAC-1, mgLAC-2, CotA and Myrothecium verrucaria examined in Example 11 (Koikeda S
Others, JOURNAL OF BIOLOGICAL CHEMISTRY, September 1993, 268, 25, 18801-9, laccase derived from E. coli (non-patent document 1 presented as prior art document (Sue A. Roberts et al.) , JOURNAL OF BIOLOGICAL CHEMISTRY, August 2003, Vol. 278, No. 34, Nos. 31958-63)), SLAC and TthLAC examined in Example 3. The results are shown in Table 1. The laccase derived from Myrothecium verrucaria may be referred to as “M.verBOD” and the laccase derived from E. coli may be referred to as “CueO”.

  From Table 1, it was confirmed that MELAC had a molecular weight smaller than that of a protein exhibiting similar laccase activity, and SLAC was found to be the only laccase having a molecular weight smaller than that of MELAC. When the molecular weight is small, particularly when used as an enzyme electrode, it is advantageous because the amount of enzyme molecules supported per area of the conductive member can be increased. In other words, the current density can be improved by increasing the loading, and MELAC is expected to be applied to fuel cells and biosensors.

[Example 14] Search for MELAC subspecies A subspecies having an activity equivalent to that of MELAC whose activity was confirmed in Examples 6 to 10 was searched.

  In the same manner as in Example 4, the full length of the laccase gene was PCR-amplified using the metagenomic DNA from the environmental sample used in Example 1 (compost collected from Midori Ward, Chiba City) as a template, and 16 amplified products were obtained. Obtained. Subsequently, as a result of determining the base sequence of the amplified product, it was found that there were subspecies with different amino acids at two positions when viewed from the gene sequence. Specifically, in the amino acid sequence of SEQ ID NO: 2, the 36th valine is substituted with isoleucine (V36I), and the 120th asparagine is substituted with aspartic acid (N120D). This was named “MELAC-2” and its amino acid sequence is shown in SEQ ID NO: 3.

  Subsequently, the enzyme activity of OLE_LINK6MELAC-2 and the thermostable OLE_LINK6 were examined, and it was confirmed that it had the same physicochemical properties as MELAC. From these results, it was found that MELAC subspecies enzymes differing in several amino acids exist in environmental samples.

[Example 15] MELAC rapid holophoresis method The enzyme purified in Example 5 was examined for a rapid holophoresis method.

Laccase is a multi-copper enzyme containing a copper atom, and it is essential for its activity to have a holo form in which copper is coordinated to the active center. Actually, MELAC purified as in Example 5 has laccase activity, but 100% does not necessarily have a holo shape incorporating copper ions in its active center. Therefore, it was examined whether or not the holo efficiency could be improved by mixing and heating MELAC and copper. Specifically, in Example 5, an enzyme (apo-type) purified without containing copper in the final dialysate was obtained, and 25 mM Tris-HCl pH 7 containing 1 mM CuCl ₂ at an enzyme concentration of 1 mg / ml. Mixed with .4 buffer. The mixture was heated at 60 ° C. for 60 minutes, 120 minutes, and 360 minutes, and then placed in ice, and the laccase activity of each reaction solution was examined in the same manner as in Example 6.

  The results are shown in FIG. It can be understood that the percentage of holo-type enzyme molecules incorporating OLE_LINK5 copper ion OLE_LINK5 is small without heat treatment. On the other hand, it was found that by performing the heat treatment conditions of about 60 ° C. for several hours, the holoformation rate gradually improved, and the heat treatment for 6 hours improved more than 4 times compared to the case of 2 hours.

  The present invention relates to a protein having a novel laccase activity, a nucleic acid molecule encoding the same, and use thereof, and can be used in all fields where utilization of laccase activity is required, particularly in the medical, food and environmental fields. It can be used in various industrial fields such as the electrochemical field.

Claims (8)

A protein having the following laccase activity (a) or (b), which exhibits activity in a temperature range of 25 to 85 ° C. and retains 70% or more of the activity even after heat treatment at 60 ° C. for 17 hours A protein having
(A) for the amino acid sequence shown in (b) a protein SEQ ID NO: 2 comprising the amino acid sequence shown in SEQ ID NO: 2, a protein comprising an amino acid sequence having at least 90% identity A protein having a laccase activity of (c) or (d) below, which exhibits activity in a temperature range of 25 to 85 ° C. and retains 70% or more of the activity even after heat treatment at 60 ° C. for 17 hours A protein having
(C) for the amino acid sequence shown in Protein (d) SEQ ID NO: 3 comprising the amino acid sequence shown in SEQ ID NO: 3, a protein comprising an amino acid sequence having at least 90% identity   The protein according to claim 1 or 2, which is a recombinant.   The nucleic acid molecule which codes the protein as described in any one of Claims 1-3. The nucleic acid molecule according to claim 4, which comprises the following polynucleotide (i) or (ii).
(I) a polynucleotide comprising the base sequence represented by SEQ ID NO: 1 (ii) a polynucleotide which hybridizes with the polynucleotide comprising the base sequence represented by SEQ ID NO: 1 under stringent conditions   A recombinant vector containing the nucleic acid molecule according to claim 4 or 5.   A transformant containing the recombinant vector according to claim 6.   The method for producing a protein having laccase activity according to any one of claims 1 to 3, wherein the transformant according to claim 7 is cultured, and a protein having laccase activity is collected from the obtained culture.