JP5534502B2 - Protein having laccase activity, polynucleotide encoding the protein, method for producing the protein, and method for obtaining the polynucleotide - Google Patents

Description

  The present invention is a novel protein having a laccase activity obtained from environmental samples, particularly wood compost, a novel polynucleotide encoding the protein, a method for producing the protein, and the polynucleotide is easily obtained. Regarding the method.

  Laccase (also called polyphenol oxidase or urushiol oxidase) is a general term for phenol oxidase, which is widely present in microorganisms, fungi, plants, and the like, and is an enzyme that oxidizes phenolic compounds in the presence of oxygen. Laccase catalyzes lignin decomposing action, phenolic compounds such as urushiol and raccol, aromatic amines such as p-phenylenediamine, and oxidation reactions of proteins. This catalytic capacity can be applied to various chemical reactions. For example, treatment of waste liquid containing highly toxic phenolic compounds and aromatic amines, removal of lignin in pulp production processing, bio battery, biosensor, artificial lacquer production, synthesis of organic compounds, detoxification of toxic chemicals, To many industrial fields such as removal of bitter and astringent taste of food, production of adhesives, synthesis of concrete admixture, browning treatment of black tea, production of melanin for cosmetics, food gelling agent, clinical laboratory reagent, bleaching agent, etc. Is expected to be used.

  Patent Document 1 describes an alkaline laccase having substrate specificity that is derived from filamentous fungi and acts strongly on heterocyclic compounds or aromatic amines. In the laccase, the optimum pH is about 10, the optimum working temperature is 50 ° C., and the molecular weight is about 42,000 to 43,000.

  Patent Document 2 includes actinomycetes, which are catechol, resorcinol, hydroquinone, pyrogallol, cresol, guaiacol, p-phenylenediamine, p-toluidine, L-tyrosine, L-3,4-dihydroxyphenylalanine, L-ascorbine. Laccases with substrate specificity to oxidize acids are described. In the laccase, the optimum pH is about 4.5, the optimum reaction temperature is about 50 ° C., and the molecular weight is about 73,000.

  In Patent Document 3, 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), guaiacol, 4-hydroxyindole, 2,6-dimethoxyphenol, which is derived from imperfect fungi, Laccases that catalyze an oxidation reaction using catechol, pyrogallol and shilling aldazine as substrates are described. In the laccase, the optimum pH is 6.5 to 7.5, the optimum reaction temperature is 40 to 45 ° C., and the molecular weight is 70,000 to 80,000.

  Patent Document 4 describes a laccase derived from a microorganism belonging to the highly thermophilic bacterium of the genus Thermus, and having an activity that acts strongly on phenolic compounds and heterocyclic compounds in the presence of copper ions. In the laccase, the optimum pH is 5.0, the optimum reaction temperature is 92 ° C., and the molecular weight is 53 kDa.

JP 2004-208503 A JP 2002-171968 A JP 2004-41124 A JP 2006-158252 A

  Laccase and polyphenol oxidase are generally widespread in nature. As the laccase-producing bacteria, the above-mentioned filamentous fungi (Patent Document 1), actinomycetes (Patent Document 2), and incomplete bacteria (Patent Document 3) are known, but these fungi have a growth rate during culture. Very slow and generally difficult to mass culture. Moreover, in order to improve the productivity of these bacterium-derived laccases, genetic manipulation and production of mutants are required, but it is difficult to express them in prokaryotes having a high growth rate. Therefore, it has been very difficult to mass-produce laccase stably and inexpensively from these fungi.

When the laccase derived from the above-mentioned production bacteria is used in a wide variety of industrial fields, the laccase is desired to be structurally stable. For example, the laccases of Patent Documents 1 to 3 have an optimum reaction temperature of about 40 to 50 ° C. and are considered to have low thermal stability. For this reason, there are cases where it is unsuitable for use in the treatment of waste liquid or the removal of lignin in the pulp production process.
On the other hand, laccase produced by the highly thermophilic bacterium of the genus Thermus (Patent Document 4) has an optimum reaction temperature of about 90 ° C., and is considered to have high thermal stability.

  In order to obtain an enzyme with high heat stability, microorganisms (thermophilic Thermus spp.) Are collected mainly in a high temperature environment such as a hot spring. From the viewpoint of industrial use, if laccase can be obtained not only from a limited environment such as a hot spring, but also from microorganisms that inhabit various environments, laccase can be easily mass-produced.

  Accordingly, an object of the present invention is to obtain a protein having a novel laccase activity excellent in heat resistance and durability and a polynucleotide encoding the protein. Moreover, the method of producing the said protein and the method of acquiring the said polynucleotide simply are provided.

A wide variety of microorganisms inhabit environments such as soil and rivers. In the conventional genome analysis of microorganisms, genomic DNA is prepared through the isolation and culture process of a single bacterial species. On the other hand, in a new research field called metagenomics, DNA directly collected from a sample collected from the environment (environmental sample) is handled. In other words, in metagenomics, the genome (metagenome) is prepared from a wide variety of microbial populations without going through the isolation and culture process of a single bacterial species, and the heterogenomic metagenome is prepared. Screening is performed by incorporating into a host such as E. coli, or sequencing using the metagenome is used (metagenome analysis).
It is estimated that 99% or more of the bacteria living on the earth are microbial species that cannot be cultivated alone. Metagenomic analysis has made it possible to obtain genome information on difficult-to-culture bacteria, which was difficult with conventional methods. Therefore, metagenome analysis is expected as a method to elucidate the huge number of unknown microorganisms and unknown genes buried in the environment.

  As a result of intensive studies to provide a novel protein having laccase activity and a method for obtaining a novel polynucleotide encoding the protein, the present inventors have found that a compost sample containing wood as a component among environmental samples. As a result, it was found that a protein having laccase activity and a polynucleotide encoding the protein can be obtained very efficiently, and the present invention was completed.

That is, in order to achieve the above object, the following inventions [1] to [8] are provided.
[1] A protein having laccase activity and having any of the following amino acid sequences.
(A) the amino acid sequence shown in SEQ ID NO: 10 (b) SEQ ID NO: 11 amino acid sequence [2] indicating encoding a protein having laccase activity, a polynucleotide having any of the following nucleotide sequences.
(A) the nucleotide sequence shown in SEQ ID NO: 8 (b) nucleotide sequence shown in SEQ ID NO: 9 [3] of the polynucleotide according to [2], a recombinant vector comprising at least the coding region.
[4] A transformant comprising the recombinant vector according to [3] above.
[5] The transformant according to [4] above, wherein the recombinant vector is introduced into Escherichia coli.
[6] A method for producing a protein having laccase activity using the transformant according to [4] or [5].
[7] A protein having laccase activity derived from a microorganism having the following properties.
(1) Activity (substrate specificity): Oxidizes 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), syringaldazin, guaiacol, dimethoxyphenol (2) Optimal reaction temperature Around 80 ° C. (3) Optimal reaction around pH 5 (4) Temperature stability When held at pH 5 for 400 minutes, it is stable up to 60 ° C. (5) Molecular weight 54.6-55.3 kDa (SDS-PAGE)

According to the configuration of the above [1] to [7], a novel protein having laccase activity and a novel polynucleotide encoding the protein can be obtained. These are different from the conventionally known heat-resistant laccases, especially since almost no homology with the laccase described in Patent Document 4 was observed.
The protein and polynucleotide of the present invention are industrially advantageous proteins and polynucleotides because they can be mass-produced by cloning into a known host such as Escherichia coli. Moreover, since the protein which has the laccase activity of this invention is excellent in heat resistance and durability, it can be utilized also in the field | area which was difficult to utilize conventionally.

[8] the step of extracting the metagenomic DNA from the compost is an environmental sample, and have a PCR process for amplifying a polynucleotide encoding a protein having laccase activity from the metagenomic DNA, in the PCR process, SEQ ID NO: 12, A laccase for amplifying a polynucleotide encoding a protein having laccase activity using 13 primer pairs, a primer pair of SEQ ID NOs: 14 and 15, a primer pair of SEQ ID NOs: 16 and 17, and a primer pair of SEQ ID NOs: 18 and 19 A method for obtaining a polynucleotide encoding a protein having activity.
[9] The method for obtaining a polynucleotide encoding a protein having laccase activity according to [8] above, wherein the compost is wood compost containing wood as a component .

According to the configuration of [8] to [9] above, the novel protein having laccase activity and the novel polynucleotide encoding the protein do not specify the microorganism from which they are derived, but are heated by fermentation fever. It can be obtained efficiently and simply by direct screening from so-called metagenomic DNA obtained from compost, which can be an environmental sample. In particular, if the compost is wood compost, its acquisition is easy, so the method of the present invention is an industrially useful method.

It is a photograph which shows the agarose gel electrophoresis of the PCR product amplified using the metagenomic DNA as a template using the primer of sequence number 1-3. It is a photograph which shows the agarose gel electrophoresis of the PCR product amplified using metagenomic DNA as a template using the primer of sequence number 12-15. It is the figure which compared the amino acid sequence of mgLAC-1 and LCS_Trichodesmium. It is the figure which compared the amino acid sequence of mgLAC-2 and LCS_Sorangium. It is the figure which compared the amino acid sequence of mgLAC-1 and TthLAC. It is the figure which compared the amino acid sequence of mgLAC-2 and TthLAC. It is the photograph which showed the result of the acrylamide gel electrophoresis of the refined expression protein. It is the figure which compared the temperature dependence of three types of laccase. It is the figure which compared the pH dependence of three types of laccase. It is the figure which compared the high temperature durability of two types of laccase (mgLAC-1, mgLAC-2). It is the photograph which showed the result of the electrophoretic analysis of the PCR product amplified from five types of environmental samples using the primer which amplifies the partial arrangement | sequence of mgLac-1. It is the photograph which showed the result of the electrophoretic analysis of the PCR product amplified from five types of environmental samples using the primer which amplifies the partial arrangement | sequence of mgLac-2.

Embodiments of the present invention will be described below with reference to the drawings.
The present invention is a novel protein having laccase activity and a novel polynucleotide encoding the protein. In particular, in the present invention, a protein having laccase activity and a polynucleotide encoding the protein are obtained by directly adjusting a metagenome from an environmental sample collected from an environment such as soil or compost.

  The “laccase” referred to in the present embodiment refers to a protein having laccase activity. In addition, the “laccase gene” in the present embodiment refers to at least one selected from a polynucleotide encoding laccase, a polynucleotide encoding a protein having laccase activity, and a polynucleotide containing at least one of these. It is.

  The present invention also provides a recombinant vector containing the obtained novel polynucleotide, a transformant obtained by introducing the recombinant vector into a host, and a protein having laccase activity by expressing the transformant. Provide a way to do it.

(Environmental sample)
Examples of environmental samples include water collected from soil, compost, rivers, and the like. In the present invention, a sample collected from compost is exemplified as an environmental sample. “Compost” is a fertilizer obtained by naturally depositing and fermenting plant residues and fermenting them by microorganisms. In the present embodiment, in particular, a sample collected from a compost containing wood as a component as a plant-based residue (hereinafter referred to as “wood compost”) and a sample being fermented during compost production is shown as an environmental sample. “Containing wood as a component” refers to a state in which, for example, wood pieces (wood chips) obtained by chopping wood into flakes or chips of an appropriate size are included in an amount of about 30 to 50% by weight of compost. . However, the content ratio of the wood chip is not limited to this. In wood compost, examples of components other than wood chips include organic matter such as livestock manure, but are not limited thereto. The wood chip is prepared by pulverizing, for example, thinned wood, pruned wood, rooted wood, driftwood, etc. with a pulverizer. Sawdust discharged from the sawmill may be used.

  For example, wood chips laid on a ranch farm can be preferably used as wood compost. Wood compost collected from the pasture is contaminated with fermentative bacteria contained in livestock excreta. The fermenting bacteria may be artificially mixed into the wood chip. Fermentative bacteria fertilize cellulose and other wood fibers of wood chips and other contained hydrocarbons to compost the wood chips. For example, lactic acid bacteria, actinomycetes, acetic acid-producing bacteria, and yeast are known. Since it is thought that a wide variety of microorganisms are involved in the fermentation, the fermenting bacteria are not limited to the above-mentioned known microorganisms but also include unknown microorganisms. Wood compost usually has a high temperature of about 60 to 80 ° C. due to fermentation heat generated by the fermentation action of the fermenting bacteria.

  Thus, it is speculated that the wood compost has many microorganisms that oxidize and decompose wood components. That is, the environmental sample is not limited to wood compost as long as it can be a high temperature due to fermentation heat generation or a sample in which many microorganisms that oxidize and decompose wood components live. For example, humus made of accumulated fallen leaves can be an environmental sample.

(Extraction of metagenomic DNA)
Wood compost, an environmental sample, contains a wide variety of microorganisms. Metagenomics approaches the target genetic resources (laccases) by handling samples containing a wide variety of microorganisms without going through the isolation and culture process of a single bacterial species. In the approach, first, metagenomic DNA is purified from wood compost (metagenomic DNA extraction step).

  In analyzing genes in environmental samples, it is indispensable to recover a sufficient amount of highly pure DNA from wood compost. For the extraction of metagenomic DNA, a known DNA extraction method using a surfactant or an organic solvent can be used. In addition, the extract extracted from the environmental sample may contain a PCR reaction inhibitor such as humic acid. For this reason, an extraction system that can exclude substances that inhibit the subsequent PCR reaction should be used.

(Acquisition of a polynucleotide encoding a protein having laccase activity)
In order to amplify a target polynucleotide sequence from metagenomic DNA, for example, a primer pair is designed based on a base sequence or amino acid sequence specific to a known laccase gene base sequence. Sequence information can be obtained using known nucleic acid sequence databases such as DDBJ, EMBL, NCBI, and GenBank, and protein (amino acid) sequence databases.

  A “primer” is a base extension by a polymerase that is a polymerization enzyme that hybridizes with a single-stranded nucleic acid or nucleic acid fragment under a predetermined condition for initiation of enzymatic polymerization in a nucleic acid amplification method such as PCR. It is an oligonucleotide of several bases to several tens of bases that can induce a reaction. The primer is designed so as to amplify the desired region, for example, using primer design support software or the like, with the desired amplification region interposed therebetween. The number of bases of the primer must be long enough to specifically recognize the target nucleic acid. However, if the length is too long, a nonspecific 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. Is exemplified by oligonucleotides consisting of about 10 to 50 bases. The primer is synthesized by a technique such as chemical synthesis. The chemical synthesis can be performed by a known DNA synthesis method.

  By using the extracted metagenomic DNA as a template and performing the polymerase chain reaction (PCR) method using the designed primer pair, a known laccase gene (polynucleotide encoding a protein having laccase activity) and the gene (polynucleotide) ) Can be obtained (PCR step).

  Nucleic acid amplification conditions (denaturation temperature, annealing temperature and annealing time, cycle number, extension temperature and extension time) are appropriately determined depending on the designed primer length, GC content, and the like. As a polymerase used for PCR, a known polymerizing enzyme such as a DNA-dependent DNA polymerase such as Taq polymerase may be used.

  In order to confirm the PCR amplification product obtained by the PCR method described above, a PCR reaction solution is collected and subjected to electrophoresis. The electrophoresis may be performed using an appropriate buffer and gel. The electrophoresis time and voltage are set appropriately. After the electrophoresis, the PCR amplification product is detected by staining the PCR amplification product with a staining agent such as ethidium bromide at a predetermined concentration for a predetermined time and visualizing it by ultraviolet irradiation or the like. Since the base length of the known laccase gene is known, it is possible to estimate whether the PCR amplification product is a homolog of the known laccase gene by examining the size of the detected PCR amplification product.

  The target PCR amplification product is purified and cloned into a known appropriate vector such as pUC118, and the base sequence of the PCR amplification product is determined by a known method. For cloning, a known cloning technique may be used. The base sequence can be determined using a known sequence technique such as Maxam-Gilbert method or dideoxy method.

(Acquisition and amplification of a full-length fragment of a polynucleotide encoding a protein having laccase activity)
When acquiring a specific gene from an environmental sample, sequence information is also required for an unknown base sequence in the peripheral region of a polynucleotide sequence in which a part of the base sequence is decoded. Therefore, using the primer designed so that the base sequence adjacent to the region extends in the direction of the unknown polynucleotide sequence region from the decoded sequence region in the polynucleotide, the unknown polynucleotide sequence is obtained by PCR. To obtain the full-length laccase gene.
The primer is designed based on, for example, a sequence near the end in the polynucleotide sequence whose base sequence has been decoded. Using the extracted metagenomic DNA as a template, the band of the target DNA sequence is confirmed after amplification by PCR using the above primers, and after cloning into an appropriate vector as described above, the base sequence is determined.

  Based on the determined nucleotide sequence information, a specific primer for amplifying a full-length fragment of a polynucleotide encoding a protein having laccase activity is designed. By performing amplification by PCR using the extracted metagenomic DNA as a template and the above primers, the full-length fragment can be amplified.

(Recombinant vector and transformant)
The obtained full-length fragment is incorporated into an expression vector, and the resulting recombinant vector is introduced into an appropriate host cell to obtain a transformant. If the transformant is cultured, the transformant can produce laccase.

The expression vector is not particularly limited as long as it can incorporate foreign DNA and can replicate autonomously in a host cell. According to the present invention, a gene encoding the laccase of the present invention can be expressed in any host cell to produce laccase. For example, plasmid vectors (pET system, pUC system, pBR system, etc.), phage vectors (λgt10, λgt11, λZAP, etc.), cosmid vectors, virus vectors (vaccinia virus, baculovirus, etc.) and the like can be mentioned. it can.
The expression vector is incorporated so that the polynucleotide encoding the protein having the laccase activity of the present invention can express its function. At this time, the expression vector may contain other known base sequences necessary for the functional expression of the nucleic acid molecule, and the other known base sequences here are not particularly limited.
In particular, when E. coli is used as a host cell, in general, an expression vector can be composed of at least a promoter-operator region, a start codon, DNA encoding the polypeptide of the present invention, a stop codon, a terminator region, and a replicable unit. Specifically, when the host is Escherichia coli, it preferably comprises Trp promoter, lac promoter, recA promoter, .lambda.P _L promoter, lpp promoter, tac promoter, is intended to include the T7 promoter and the like are exemplified. Known terminator regions and replicable units can be used.

  For example, a nucleic acid molecule or the like can be inserted into an expression vector by, for example, cleaving a polynucleotide encoding laccase with an appropriate restriction enzyme and inserting and ligating it into a restriction enzyme site of the expression vector. It is not limited to. For ligation, a known method such as a method using DNA ligase can be used.

  The host cell used for the production of the transformant is not particularly limited as long as it is compatible with the expression vector and can be transformed, and any cell that can efficiently express laccase may be used. 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 DH5α, 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, yeasts such as Saccharomyces cerevisiae, insect cells such as Sf9 cells, animal cells such as CHO cells, COS-7 cells, and the like can be used.

  The transformant of the present invention can be prepared by introducing the above expression vector into a host cell. Transformation can be performed by a known technique such as a competent cell method by calcium treatment, an electroporation method, a calcium phosphate precipitation method, a liposome method, or a microinjection method.

(Production of protein with laccase activity)
The protein having the laccase activity of the present invention can be produced by culturing a transformant containing the expression vector prepared as described above in a known nutrient medium such as a polypeptone-yeast extract medium.

The medium to be used preferably contains a carbon source, an inorganic nitrogen source or an organic nitrogen source necessary for the growth of the host cell (transformant). Examples of the carbon source include glucose, dextran, soluble starch, and sucrose, and examples of the inorganic nitrogen source or organic nitrogen source include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, and casein. If desired, other nutrients such as inorganic salts (for example, calcium chloride / sodium dihydrogen phosphate / magnesium chloride), vitamins, antibiotics (for example, tetracycline, neomycin, ampicillin, kanamycin, etc.) may be contained. Although there is no restriction | limiting in particular also about a culture form, A liquid medium can be utilized suitably from a viewpoint of mass culture.
When the host cell is Escherichia coli, LB medium, M9 medium and the like are preferable media.

  The laccase present in the culture medium is obtained by centrifuging or filtering the culture to obtain a culture supernatant (filtrate), from which, for example, salting out, solvent precipitation, dialysis, ultrafiltration, gel filtration, Non-denaturing PAGE, SDS-PAGE, various chromatography (eg, gel filtration chromatography, ion exchange chromatography, hydroxylapatite chromatography, affinity chromatography, reversed phase high performance liquid chromatography, etc.), known isoelectric focusing, etc. It can be obtained by appropriately selecting a separation method. In particular, from the viewpoint of yield, it is preferable to use ion exchange chromatography.

  Laccase present in the cytoplasm is collected by centrifuging or filtering the culture, collecting the cells, suspending them in an appropriate buffer solution, for example, sonication, lysozyme treatment, freeze-thawing, osmotic shock, and surfactant treatment. After disrupting (dissolving) cells and organelle membranes, a soluble fraction can be obtained by centrifugation, filtration, etc., and the soluble fraction can be isolated and / or purified by treating in the same manner as described above. it can. You may refine | purify from an insoluble fraction other than the said soluble fraction.

For example, if a laccase to which a tag such as a histidine tag (His-tag) or a Flag tag (FLAG-tag) is added is produced in a transformant, a substance having an affinity for the tag (for example, Ni ²⁺ resin, tag Can be used to easily isolate and purify laccase produced by the transformant. That is, for example, the tag is cloned into an expression vector with an oligonucleotide encoding histidine linked to the C-terminal side of the laccase gene, and an appropriate host is transformed. The protein produced by culturing the transformant is a fusion protein having laccase and histidine. Therefore, the fusion protein can be purified by affinity chromatography using the tag.

  The activity of the laccase obtained by purification is based on the colorimetric method in which the enzyme solution and the substrate solution are mixed, and the amount of the product produced by the oxidation of the substrate as the reaction proceeds is measured at a specific absorbance. The amount of oxygen consumed by the enzyme per unit time with the progress of this can be measured by an oxygen electrode method or the like that measures with an oxygen electrode.

(Protein having laccase activity of the present invention and polynucleotide encoding the protein)
The protein having laccase activity of the present invention is preferably exemplified by the amino acid sequences shown in SEQ ID NO: 10 and SEQ ID NO: 11, but is not limited to a specific amino acid sequence, and has a structure similar to the protein. And functionally equivalent proteins. Accordingly, it is intended to include proteins modified by artificial mutagenesis or genetic recombination in addition to naturally occurring laccase. For example, the above-mentioned laccase has an amino acid sequence in which one or more alterations selected from deletion, substitution, insertion and addition of one or more amino acids have occurred in the amino acid sequence having the naturally-occurring laccase activity. And variants with similar biological functions may be included .

  A person skilled in the art can easily predict a modification that retains the enzymatic activity of the protein having the laccase activity of the present invention 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 have similar properties to each other. Examples of uncharged amino acids 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. In addition, 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 a 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.

Such a variant can be obtained by screening a protein having the activity from mutants generated by natural or artificial mutation. Alternatively, it can also be obtained by modifying a polynucleotide encoding laccase.
Moreover, about the modified body obtained in this way, it is good to confirm the presence or absence of laccase activity by the method mentioned above.

The protein having laccase activity of the present invention has an activity to oxidize 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), syringaldazine, guaiacol, and dimethoxyphenol as a substrate.
The optimum reaction temperature is around 80 ° C. and the optimum reaction pH is around 5. The temperature stability does not reach the 80% inactivation level up to 60 ° C. when held at pH 5 for 400 minutes, thus maintaining stable enzyme activity. The molecular weight measured by SDS-PAGE is 55.3-54.6 kDa.

The polynucleotide encoding the protein having laccase activity of the present invention is preferably exemplified by the nucleotide sequences shown in SEQ ID NO: 8 and SEQ ID NO: 9, but is not limited to a specific nucleotide sequence, and the laccase activity is not limited. As long as it encodes the retained protein, it is understood to include a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence encoding laccase.
Also included are base sequences in which one or more modifications selected from deletion, substitution, insertion and addition of one or more bases have occurred in the base sequence of a polynucleotide comprising a base sequence encoding a protein having laccase activity It is. Accordingly, preferred examples include those in which a base sequence encoding a His-Tag sequence is added to the 3 ′ or 5 ′ end of the base sequences of SEQ ID NO: 8 and SEQ ID NO: 9.
It is understood that the above-mentioned polynucleotide comprising a base sequence encoding a protein having laccase activity also includes a base sequence complementary to the base sequence within the scope of the present invention.

“Stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed . Scan stringency can be adjusted by appropriately changing the salt concentration and temperature, etc. during the hybridization reaction and washing.
Hybridization conditions are, for example, 6 × SSC, 0.5% SDS, and 30-5.
Examples are heating in a solution of 0% (preferably 50%) formamide at 37-50 ° C. (preferably 42 ° C.) for 16-20 hours. Examples of washing conditions include washing in a solution of 0.1 × SSC, 0.5% SDS at 68 ° C. for 5 minutes. However, neither hybridization conditions nor washing conditions are limited to such an embodiment.

  There is no restriction | limiting in particular as a method to modify | change a polynucleotide, 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. It can also be prepared by constructing a polynucleotide having a desired modification using a DNA synthesis method such as a conventional phosphoramidite method. Or it can obtain by making exonuclease act on the polynucleotide which has the base sequence of sequence number 8 and sequence number 9.

  The recombinant vector of the present invention includes at least a coding region among the polynucleotides encoding the protein having laccase activity described above, and the transformant of the present invention includes the recombinant vector.

[Example 1]
(Collecting environmental sample wood compost and extracting metagenomic DNA contained in wood compost)
As wood compost, farm compost containing many bacteria that oxidize and decompose wood components was collected from farms in Chiba City and used as environmental samples. Wood compost was collected three times, immediately after composting, one month after composting, and four to five months after composting (final compost).

In addition, the wood compost of this embodiment was created as follows.
Wood chips were produced by pulverizing thinned and pruned timber with a pulverizer. After mixing the wood chip and cow dung containing the fermenting bacteria so that the volume ratio is about 1: 1, the mixture is mixed once every 7 to 10 days using a compost turning machine, at 50 to 70 ° C. High temperature fermentation.

Sampling for extracting metagenomic DNA from wood compost was performed immediately after wood compost production, one month after wood compost production, and four to five months after wood compost production (final).
Metagenomic DNA was extracted from 2-6 g (wet weight) of wood compost sampled using PowerMax Soil DNA isolation Kit (manufactured by MO BIO Laboratories) according to the manufacturer's instructions. The extracted metagenomic DNA was finally dissolved in 5 mL of Tris buffer. By using this kit, it is possible to eliminate substances that inhibit the subsequent PCR reaction.
In addition, the metagenomic DNA was extracted from the environmental sample of cow dung (immediately after defecation) two months after the production of rice husk compost as a target sample.

[Example 2]
(Acquisition of a polynucleotide encoding a protein having laccase activity)
When using metagenomic DNA, first, a primer used in the PCR method is designed to amplify a target polynucleotide sequence. Specifically, based on the common amino acid sequence of known laccases, the CODEHOP program (
“CODEHOP (Consensus-Degenerate Hybrid Oligonucleotide Primer) PCR primer design” Timothy M. Rose, Jorja G. Henikoff and Steven Henikoff, Nucleic Acid Research, 2003, Vol. 31, No. 13, 3763-3766 (http: // degenerate primers were designed using blocks.fhcrc.org/blocks/codehop.html). The code hop program is PCR support tool software for designing primers for degenerate PCR used for cloning genes based on amino acid sequences.
Known laccase gene sequences entered into the program include Cyanothece (NCBI accession number ZP_01731625), Lyngbya (NCBI accession number ZP_01621366), Synechococcus (NCBI accession number ZP_01081498),
Thermus (NCBI accession number YP_005339) and Trichodesmium (NCBI accession number YP_720301) were used.
The following primers were designed by this program. The length of the PCR product amplified using these primers was expected to be about 900 bp.

5'-CCCTGCTGGAATGTTCTGGTAYCAYCCNCA-3 '(forward primer: SEQ ID NO: 1)
5'-CCAGATCCTCATGGTCCAGAATRTGRCARTG-3 '(reverse primer: SEQ ID NO: 2)
5'-GGATTGACATGTACGTGAAAGGGRTGRTCCAT-3 '(reverse primer: SEQ ID NO: 3)

Using Multiplex PCR Assay Kit (manufactured by Takara-Bio), using the metagenomic DNA extracted in Example 1 as a template, after heat denaturation reaction at 94 ° C. for 30 seconds, heat denaturation reaction at 94 ° C. for 30 seconds, 55 ° C. The PCR reaction was performed by repeating 40 cycles of 30 seconds of annealing reaction and 74 ° C. for 90 seconds of extension reaction. Thereafter, a final extension step was performed at 74 ° C. for 7 minutes to complete the reaction.
The results of analyzing the PCR product by agarose gel electrophoresis are shown in FIG. The environmental samples from which the template DNA of each lane is derived are as follows: lane 1 is immediately after preparation of wood compost, lane 2 is one month after preparation of wood compost, lane 3 is four to five months after preparation of wood compost (final), lane 4 2 months after the production of rice husk compost, Lane 5 used cow dung (immediately after defecation). Lane 6 is E. coli genomic DNA.
As a result, amplification of a band of about 900 bp could be confirmed in all three kinds of environmental samples. That is, in these environmental samples, it was recognized that a DNA fragment having high homology with laccase in known bacteria, that is, a DNA fragment that is a candidate for the laccase gene was amplified.

  The target DNA fragment (about 900 bp) was purified and cloned into a pUC118 vector (manufactured by Takara-Bio) by a known method, and then the nucleotide sequence was determined (results not shown).

Example 3
(Acquisition of a full-length fragment of a polynucleotide encoding a protein having laccase activity)
The full-length sequence of a known laccase gene homolog by using a primer designed so that the nucleotide sequence adjacent to the region extends in the direction of the unknown polynucleotide sequence region from the polynucleotide sequence region from which the base sequence was decoded It was determined.
The extracted metagenomic DNA is used as the template DNA, the primer is designed based on the sequence near the end of the polynucleotide sequence whose base sequence is decoded, and an unknown polynucleotide sequence is amplified using the inverse PCR method. did. A detailed procedure will be described below.

50 units each of restriction enzymes BamHI, HindIII, EcoRI, and SmaI (manufactured by NEB) were added to the extracted metagenomic DNA, and a restriction enzyme reaction was performed at 37 ° C. for 5 hours. 50 μL of the reaction solution attached to each restriction enzyme was used. The DNA in the solution after the reaction solution was purified using a DNA purification kit (GE-Healthcare).
Next, the purified DNA was dissolved in TE buffer to make 20 μL, and 20 μL of DNA Ligation Kit (Mighty Mix) (manufactured by Takara Bio Inc.) was added to this solution and kept at 16 ° C. overnight to allow self-ligation reaction. . After completion of the reaction, the ligated DNA was purified using a DNA purification kit (GE-Healthcare).

  The total amount of DNA contained in the purified sample was used as a template for the inverse PCR method. For the reaction solution used for inverse PCR, add PrimeSTAR GXL DNA Polymerase (Takara-Bio) and primers (final concentration 0.2 μM) as DNA polymerase to the template DNA, and make up to 50 μL with sterile distilled water. It was prepared by. The sequences of the primers are as follows. In this embodiment, two sets of primer pairs are used.

5′-CAGGTTATCCGGATTCTGCTGGTTTG-3 ′ (forward primer: SEQ ID NO: 4)
5′-GATAGCGACGAGTGGCACCCGTTCCATATC-3 ′ (reverse primer: SEQ ID NO: 5)

5′-CAAAGCGCGGATCGATCTGCCCATC-3 ′ (forward primer: SEQ ID NO: 6)
5′-AAGGTGATTGCGCGTAACGGTCAGC-3 ′ (reverse primer: SEQ ID NO: 7)

The reaction solution was subjected to a PCR reaction using a thermal cycler GeneAmp (registered trademark) PCR System 9700 (manufactured by Applied Biosystems).
The PCR reaction was a thermal denaturation reaction at 98 ° C. for 10 seconds, an annealing and extension reaction at 68 ° C. for 120 seconds for 25 cycles, and a final extension reaction at 68 ° C. for 10 minutes was performed.
The solution after the PCR 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 determined by a detection device and visual observation. As a result, a band of the target DNA sequence could be confirmed after amplification by the inverse PCR method (results are not shown).

  The resulting DNA fragment of interest was cloned into the pUC118 vector, and the nucleotide sequence was determined. This revealed a 5 'terminal sequence encoding the N-terminal region of two types of laccase genes (mgLac-1, mgLac-2) and a 3' terminal sequence encoding the C-terminal region. Thereby, the whole base sequence including the coding region of laccase was determined (mgLac-1: SEQ ID NO: 8, mgLac-2: SEQ ID NO: 9). The amino acid sequences of the proteins deduced from these base sequences are shown in SEQ ID NO: 10 (mgLAC-1) and SEQ ID NO: 11 (mgLAC-2).

Example 4
(Amplification of a full-length fragment of a polynucleotide encoding a protein having laccase activity)
In order to amplify a full-length fragment, first, the following specificities for the partial sequence of the obtained laccase gene homolog (5 ′ end sequence encoding the N-terminal region of laccase and 3 ′ end sequence encoding the C-terminal region) Specific primers were designed.

Primer that amplifies mgLac-1
5'-GAGCATATGGAAGATTCCAGACGGCATGG-3 '(forward primer: SEQ ID NO: 12)
5'-CTAAAGCTTCTCCACAACCTCGATGATGC-3 '(reverse primer: SEQ ID NO: 13)
Primer that amplifies mgLac-2
5'-GAGCATATGAATAGGAAGAGTTTGCAGAG-3 '(forward primer: SEQ ID NO: 14)
5'-CTAAAGCTTCTGCGCGGCACGCGTACGCT-3 '(reverse primer: SEQ ID NO: 15)

Using Multiplex PCR Assay Kit (manufactured by Takara Bio Inc.), 25 cycles of heat denaturation reaction at 98 ° C. for 10 seconds, annealing at 68 ° C. for 120 seconds and extension reaction using the metagenomic DNA extracted in Example 1 as a template The PCR reaction was performed. Then, the final extension process for 10 minutes was performed at 68 degreeC, and reaction was complete | finished.
The results of analyzing the PCR product by agarose gel electrophoresis are shown in FIG. In FIG. 2, lane 1 is mgLac-1, and lane 2 is mgLac-2. The PCR product was purified from an agarose gel and cloned into the pUC118 vector.

Example 5
(Homology search of new laccase gene)
From the results of homology search with the laccase protein of Trichodesmium erythraeum, the coding region of the laccase gene mgLac-1 is 1497 bp, the protein (mgLAC-1) deduced from the base sequence is 499 residues, the laccase gene mgLac- The coding region of 2 was 1485 bp, and it was revealed that the protein (mgLAC-2) deduced from the base sequence was 494 residues. Further, upstream of the coding region of the gene, a sequence characteristic of prokaryotic promoters such as an SD sequence, which is a translation signal specific to prokaryotes, was present around -12 bp.

  When the default (initial condition) parameters were used in the BLAST search to find a sequence group having high similarity, mgLAC-1 had 32% homology with Trichodesmium erythraeum laccase (LCS_Trichodesmium) at the amino acid level (FIG. 3), mgLAC− In 2, the homology of Sorangium cellulosum with laccase (LCS_Sorangium) was 56% at the amino acid level (FIG. 4).

  Moreover, the result of having compared the amino acid sequence with the laccase (TthLAC) of the hyperthermophilic thermus genus bacteria described in patent document 4 is shown in FIG. 5 (mgLAC-1) and FIG. 6 (mgLAC-2). Since the homology between mgLAC-1 and TthLAC was 29% at the amino acid level, and the homology between mgLAC-2 and TthLAC was 27% at the amino acid level, it was recognized that there was no amino acid sequence homology.

Example 6
(Construction of recombinant vectors and transformants)
In this embodiment, Escherichia coli for laccase expression was constructed as follows.
The laccase gene was cloned into the NdeI / HindIII site of pET22b (Novagen), which is an E. coli expression vector containing an oligonucleotide encoding a peptide consisting of 6-residue histidine. Cloning was performed using DNA Ligation kit (Takara Bio) according to the manufacturer's instructions.
The plasmids in which the novel laccase gene of the present invention has been cloned have the accession numbers NITE P-782 (mgLAC-1) and NITE P-783 (mgLAC-2) in 2009. Deposited on July 15th.
The plasmid thus obtained was transformed into Escherichia coli BL21 (DE3) strain (manufactured by Novagen) to obtain cells expressing the laccase gene.

Example 7
(Fusion protein construction and protein purification)
Escherichia coli into which the plasmid has been introduced is cultured in an LB liquid medium for 15 hours, and 50 mL of the culture solution is made up to 500 mL with an LB liquid medium, and cultured at 37 ° C. until the absorbance OD600 reaches about 0.6. IPTG (isopropio thio-β-galactoside) was added and the mixture was further cultured at 37 ° C. for 4 hours to induce and express the fusion protein (laccase and histidine).
The culture solution is centrifuged to collect E. coli, 200 μL of a solubilizing solution (50 mM sodium phosphate buffer (pH 7.0)) is added to the cells (wet weight 200 g) and suspended, and then the cells are sonicated. (Crushing condition: 30-s pulses; maximum power; high intensity; 0.5-s repeating duty cycle). The cell disruption solution was centrifuged at 15000 g for 10 minutes, and the supernatant was heat-treated at 95 ° C. for 3 minutes.

NaCl is added to 50 mM of the obtained supernatant, the enzyme solution is subjected to Ni ²⁺ column chromatography, the fusion protein is eluted with a solution containing 300 mM imidazole, the active fractions are collected, and 50 mM phosphate buffer (pH 7.4) is collected. Was dialyzed overnight as an external dialysis solution to obtain a purified laccase.
The enzyme solution thus obtained was dialyzed against 50 mM Tris-HCl buffer (pH 7.4). In this example, a batch method was employed, but purification can also be performed by a column method. If necessary, the purified laccase was concentrated with an ultrafiltration membrane. For confirmation of protein, 20 μL of solubilized solution was added to 20 μL of enzyme solution, and the sample was heat-treated at 95 ° C. for 3 minutes. Visualized. The result is shown in FIG.
Based on this result and the amino acid sequence deduced from the laccase genes mgLac-1 and mgLac-2, the molecular weight of these proteins (laccase) was calculated to be 54.6-55.3 kDa.

Example 8
(Measurement of laccase activity)
The laccase activity was measured by a colorimetric method using a 20 mM sodium acetate buffer solution having a pH of 5.0 containing 1 mM ABTS as a reaction solution. First, the reaction solution was kept at 30 ° C. for 10 minutes to achieve temperature equilibrium, and then the enzyme solution was added to start the reaction. The reaction volume was 100 μL. The activity of laccase was measured by measuring the increase in absorbance at 420 nm accompanying the oxidation of the substrate (ABTS) (ABTS molar absorbance coefficient: 36000 / M / cm).

The activities of three types of proteins having laccase activity (hereinafter simply referred to as laccase) (mgLAC-1, mgLAC-2, TthLAC) containing the enzyme of the present invention were compared. The measurement conditions for the activity were acidic conditions (pH 5.5) and neutral conditions (pH 7.5).
The molecular activity Kcat of an enzyme is a parameter that indicates how many substrates are catalyzed per minute per molecule of enzyme. For example, the molecular activity measurement results of the above three types of enzymes are as follows.

mgLAC-1: Kcat = 4 × 10 ³ / min (60 ° C., pH 5.5, 1 mM ABTS)
mgLAC-2: Kcat = 6 × 10 ³ / min (60 ° C., pH 5.5, 1 mM ABTS)
TthLAC: Kcat = 1 × 10 ³ / min (60 ° C., pH 7.5, 1 mM ABTS)

Table 1 shows the results of the relative activity when the activity of TthLAC is 1 among the three types of laccase.
As a result, it was confirmed that the purified protein showed activity, and thus it was confirmed that the laccase gene homologs mgLac-1 and mgLac-2 obtained in Example 4 encoded a protein having laccase activity. It was.
Further, it was found that the activity of mgLAC-1 and mgLAC-2 has an activity about 3 to 5 times higher than that of TthLAC under acidic conditions.

From the above, it was confirmed that the laccase (mgLAC-1, mgLAC-2) of the present invention oxidizes ABTS as a substrate.
Table 2 shows the results of examining the activity against other substrates. The activity of each substrate was measured according to the above-described method for measuring the activity of ABTS. Table 2 shows the relative activity when the activity of ABTS is 1.

  As a result, it was found that the laccase (mgLAC-1, mgLAC-2) of the present invention has an oxidizing action not only on ABTS but also on syringaldazine, guaiacol, and dimethoxyphenol.

The metal ion requirement of three kinds of proteins having the laccase activity including the enzyme of the present invention was examined.
The measurement was performed by a colorimetric method using a 20 mM sodium acetate buffer solution having a pH of 5.0 containing 1 mM ABTS and 0.2 mM various metal ions as a reaction solution. First, the reaction solution was kept at 50 ° C. for 10 minutes to achieve temperature equilibrium, and then the enzyme solution was added to start the reaction. The reaction volume was 100 μL. The increase in absorbance at 420 nm accompanying the oxidation of the substrate (ABTS) was measured. The results are shown in Table 3. Table 3 shows relative values when the required amount of CuCl ₂ is 1.

As a result, since the required amounts of CuCl ₂ and CuSO ₄ showed high values, it was recognized that the laccase (mgLAC-1, mgLAC-2) of the present invention requires copper ions for its activity.

Example 9
(Characteristic analysis of laccase (temperature dependence of activity))
The temperature dependence of the activity of three types of laccase containing the enzyme of the present invention (mgLAC-1, mgLAC-2, TthLAC) was measured in the temperature range of 30 ° C to 90 ° C according to the above colorimetric method. Enzyme solutions were prepared so that the enzyme amounts were mgLAC-1: 1500 ng / μL, mgLAC-2: 1000 ng / μL, and TthLAC: 6000 ng / μL, respectively. FIG. 8 shows the results of measuring the change in absorbance at 418 nm at each temperature by adding an enzyme solution to an activity measurement solution containing 20 mM sodium acetate buffer (pH 5.0), 0.1 mM copper sulfate, and 1 mM ABTS. From these results, it was found that the reaction rate of the three kinds of enzymes reached a maximum at about 80 ° C., and that the optimum temperature was 60 to 90 ° C., preferably 70 to 90 ° C., more preferably 75 to 85 ° C. FIG. 8 shows the relative activity when the reaction rate at 80 ° C. is 1. From the above, it was found that mgLAC-1 and mgLAC-2 react efficiently at high temperatures.

Example 10
(Characteristic analysis of laccase (pH dependence of activity))
The pH dependence of the activity of three types of laccase containing the enzyme of the present invention (mgLAC-1, gLAC-2, TthLAC) was measured in the range of pH 5.0 to 8.5 according to the above colorimetric method. Each enzyme solution was prepared in the same manner as in Example 9. The result of measuring the change in absorbance at 418 nm at 50 ° C. is shown in FIG. As a result, it was found that mgLAC-1 and mgLAC-2 had the maximum reaction rate at pH 5. Since these maintain about 80% of the maximum reaction rate up to around pH 5.5, the reaction pH is preferably around 5 to 5.5.
On the other hand, TthLAC had a maximum reaction rate around pH 7. FIG. 9 shows the relative activity when mgLAC-1 and mgLAC-2 have a reaction rate of pH 5 of 1, and TthLAC has a reaction rate of pH 7 of 1.

Example 11
(Characteristic analysis of laccase (high temperature durability))
It was examined how the enzyme activity changed when three types of laccase containing the enzyme of the present invention (mgLAC-1, gLAC-2, TthLAC) were exposed to high temperatures (60 ° C. and 80 ° C.).
FIG. 10 shows the change in relative activity over time when the enzyme activity immediately after exposure to high temperature is 1. The enzyme activity was measured according to Example 9 (pH 5.5). As a result, when heated to 60 ° C. (FIG. 10A), the relative activity hardly changes even after 400 minutes and does not reach the 80% inactivation level. On the other hand, when heated to 80 ° C. (FIG. 10B), the relative activity decreased to about 1/10 after 25 minutes. From this, it was found that the activity of the laccase of the present invention was stable up to 60 ° C. when held at pH 5.5 for 400 minutes.

Example 12
(Confirmation of genes contained in environmental samples)
Partial sequences of laccase genes (mgLac-1, mgLac-2) obtained in Example 3 using metagenomic DNA extracted from various environmental samples used for amplification of laccase gene homologues in Examples 1 and 2 as templates. Specific primers that amplify about 600 bp) were designed and PCR was performed.

Primers that amplify a partial sequence of mgLac-1 are as follows.
5'-TTCGAATTCAACCTGCCGGAGAACC-3 '(forward primer: SEQ ID NO: 16)
5′-CGAAGCGAATTTCTCGATGCACATCCAC-3 ′ (reverse primer: SEQ ID NO: 17)

Primers that amplify a partial sequence of mgLac-2 are as follows.
5′-CGAAGGATGCCGGGACGTTCTGGTT-3 ′ (forward primer: SEQ ID NO: 18)
5′-CATAGGTCACGTCCACGCCCTCCTT-3 ′ (reverse primer: SEQ ID NO: 19)

  Using 1 μL of a diluted solution (template solution) obtained by serial dilution of the sample (18 ng / μL) containing the metagenomic DNA extracted in Example 1 as a template, 98 ° C. using Multiplex PCR Assay Kit (manufactured by Takara-Bio) PCR reaction was carried out by repeating 60 cycles of heat denaturation reaction for 10 seconds, annealing at 68 ° C. for 30 seconds and extension reaction for 60 cycles. Thereafter, a final extension step was performed at 72 ° C. for 10 minutes to complete the reaction.

FIG. 11 (mgLac-1) and FIG. 12 (mgLac-2) show the results of electrophoretic analysis of PCR products amplified from five types of environmental samples. The arrow indicates the expected amplification size of the laccase gene. The environmental samples from which the template DNA of each lane is derived are as follows: lane 1 is immediately after preparation of wood compost, lane 2 is one month after preparation of wood compost, lane 3 is four to five months after preparation of wood compost (final), lane 4 2 months after the production of rice husk compost, Lane 5 used cow dung (immediately after defecation).
As a result, mgLac-1 1 (immediately after the production of wood compost) 2 (one month after wood compost production), sample no. 3 (wood compost final), mgLac-2 is sample no. Obtained from 1, 2 only. Sample No. 4 (chaff compost) and sample no. It was found that the laccase gene could not be obtained from 5 (cow manure).
From the above, as a result of intensive studies on the selection of environmental samples in isolating a new laccase gene, it can be said that the acquisition of a new laccase gene was successful by selecting wood chips.

  A protein having laccase activity of the present invention, a polynucleotide encoding the protein, a method for producing the protein, and a method for obtaining the polynucleotide include lignin removal, biobattery, Biosensor, artificial lacquer production, synthesis of organic compounds, detoxification of toxic chemicals, removal of bitter taste of food, production of adhesives, synthesis of concrete admixture, browning treatment of black tea, production of melanin for cosmetics, food It can be used in many industrial fields such as gelling agents, clinical test reagents, and bleaching agents.

NITE P-782
NITE P-783

Claims (9)

A protein having laccase activity and having any of the following amino acid sequences.
(A) the amino acid sequence shown in the amino acid sequence (b) SEQ ID NO: 11 shown in SEQ ID NO: 10 A polynucleotide encoding a protein having laccase activity and having any of the following base sequences.
(A) nucleotide sequence shown in nucleotide sequence (b) SEQ ID NO: 9 shown in SEQ ID NO: 8   A recombinant vector comprising at least a coding region of the polynucleotide according to claim 2.   A transformant comprising the recombinant vector according to claim 3.   The transformant according to claim 4, wherein the recombinant vector is introduced into Escherichia coli.   A method for producing a protein having laccase activity using the transformant according to claim 4. A protein having a laccase activity derived from a microorganism having the following properties.
(1) Activity (substrate specificity): Oxidizes 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), syringaldazin, guaiacol, dimethoxyphenol (2) Optimal reaction temperature Around 80 ° C. (3) Optimal reaction around pH 5 (4) Temperature stability When held at pH 5 for 400 minutes, it is stable up to 60 ° C. (5) Molecular weight 54.6-55.3 kDa (SDS-PAGE) Step of extracting a metagenomic DNA from compost is an environmental sample, and have a PCR process for amplifying a polynucleotide encoding a protein having laccase activity from the metagenomic DNA,
In the PCR step, a protein having laccase activity using a primer pair of SEQ ID NOs: 12 and 13, a primer pair of SEQ ID NOs: 14 and 15, a primer pair of SEQ ID NOs: 16 and 17, and a primer pair of SEQ ID NOs: 18 and 19 A method for obtaining a polynucleotide encoding a protein having a laccase activity for amplifying a polynucleotide encoding. The method for obtaining a polynucleotide encoding a protein having laccase activity according to claim 8, wherein the compost is wood compost containing wood as a component .